Stable aqueous anti-TFPI antibody formulation

Abstract

The present invention relates to the field of pharmaceutical formulations of antibodies. In particular, the present invention relates to stable liquid antibody formulations and pharmaceutical formulations and uses thereof. The present invention is exemplified by an aqueous formulation of an anti-tissue factor pathway inhibitor (TFPI) antibody.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 934,781, filed on November 13, 2019, and U.S. Provisional Application No. 63 / 081,409, filed on September 22, 2020, which are hereby incorporated by reference in their entireties.

[0003] Reference to a sequence listing

[0004] This application is filed electronically via EFS-Web and includes a sequence listing submitted electronically in .txt format. The .txt file contains a sequence listing titled "PC72541A_Seq_Listing_ST25.txt", which was created on October 13, 2020 and has a size of 42,550 bytes. The sequence listing contained in the .txt file is part of this specification and is hereby incorporated by reference in its entirety.

[0005] Field of the Invention

[0006] The present invention relates to the field of pharmaceutical preparations of antibodies. In particular, the present invention relates to stable liquid antibody preparations and pharmaceutical preparations and uses thereof.

[0007] background

[0008] Antibody formulations intended for therapeutic or preventive use require stabilizers to prevent loss of activity or structural integrity of the protein due to denaturation, deamidation, oxidation, or aggregation over a period of time during storage and transportation prior to use. These problems are exacerbated at high concentrations of antibody typically desired for therapeutic administration. A large number of formulation options are available, but no one method or system is suitable for all proteins (see, e.g., Wang et al., J. Pharm Sci. 96:1-26(2007)).

[0009] The main goal in the development of antibody preparations is to maintain the efficacy of antibodies, solubility, stability and antigen binding thereof. In order to keep the antibody biologically active, the preparation must maintain the conformational integrity of the core of at least the antigen-binding amino acids of the antibody. It is also particularly desirable to avoid aggregates and particles in the solution, which may require aseptic filtration and limit the route of administration before being used for intravenous or subcutaneous injection. Salt, surfactant, pH and tonicity agents (such as sugar) can be used to overcome aggregation or denaturation problems. The preparation of antibody preparations requires careful selection of these factors, etc., to avoid the denaturation and loss of antigen binding activity of the protein. Therefore, a stable aqueous antibody preparation is needed, which stably supports the high concentration of biologically active antibodies in the solution, and is suitable for parenteral administration, including intravenous, intraocular, intravitreal, intraarterial, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraossial, intraperitoneal, intradermal or subcutaneous injection.

[0010] Tissue factor pathway inhibitor (TFPI) antibodies have been shown to be useful for treating and preventing coagulation deficiencies or bleeding disorders, such as hemophilia A and B (see, e.g., US 2017 / 0073428). Although liquid antibody formulations are known in the art (see, e.g., WO2006 / 096491, WO 2010 / 032220, WO2013 / 186719, US 2009 / 0110681, US 2017 / 0360929, and US 2018 / 0000933), there is a need to provide stable aqueous formulations of TFPI antibodies to meet the medical needs of patients with coagulation deficiencies or bleeding disorders.

[0011] All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes, to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be so incorporated by reference. In the event that the incorporated literature and similar materials differ or conflict with this application (including, but not limited to, defined terms, term usage, described techniques, etc.), this application shall prevail. SUMMARY OF THE INVENTION

[0013] Provided are stable aqueous pharmaceutical formulations with extended shelf life comprising tissue factor pathway inhibitor (TFPI) antibodies (also referred to as anti-TFPI antibodies). Formulations of the invention with high TFPI antibody concentrations are demonstrated to be stable (e.g., with low levels of % HMMS (high molecular mass species), % LMMS (low molecular mass species), % fragments, and oxidation), and are therefore suitable for parenteral administration.

[0014] Preparations of antibodies (including antigen binding fragments thereof) that bind to tissue factor pathway inhibitor (TFPI) are disclosed and exemplified herein. Using only routine experimentation, those skilled in the art will recognize or be able to ascertain many equivalents to the specific embodiments of the invention described herein. Such equivalents are contemplated to be covered by the following embodiment (E).

[0015] E1. A formulation comprising: about 15 mg / mL to about 250 mg / mL of an antibody that specifically binds to tissue factor pathway inhibitor (TFPI), a buffer, a polyol, a surfactant, and a chelating agent, wherein the formulation has a pH of about 5.0 to about 6.0.

[0016] E2. A formulation comprising: about 15 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), a buffer, a polyol, a surfactant, and a chelating agent, wherein the formulation has a pH of about 5.0 to about 6.0, and wherein according to the numbering of SEQ ID NO:2, the epitope comprises residues Ile105, Arg107, and Leu131.

[0017] E3. A formulation as described in any one of E1-E2, wherein the buffer is selected from: acetate, succinate, gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate, imidazole, tris, phosphate and mixtures thereof.

[0018] E4. A formulation as described in E3, wherein the buffer is histidine.

[0019] E5. A formulation as described in E3, wherein the buffer is succinate.

[0020] E6. An aqueous formulation as described in any one of E1-E5, wherein the concentration of the buffer is about 0.1 mM to about 100 mM.

[0021] E7. A formulation as described in E6, wherein the concentration of the buffer is about 1 mM to about 40 mM or about 10 mM to about 40 mM.

[0022] E8. A formulation as described in E7, wherein the concentration of the buffer is about 10 mM, about 20 mM or about 40 mM.

[0023] E9. A formulation as described in E8, wherein the concentration of the buffer is about 20 mM.

[0024] E10. A formulation as described in any one of E1 to E9, wherein the polyol is selected from mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, fructose, glucose, mannose, sucrose, sorbitol, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble dextran or a mixture thereof.

[0025] E11. A formulation as described in E10, wherein the polyol is sucrose or trehalose.

[0026] E12. A formulation as described in any of E1-E11, wherein the concentration of the polyol is about 1 mg / mL to about 300 mg / mL.

[0027] E13. A formulation as described in E12, wherein the concentration of the polyol is about 1 mg / mL to about 120 mg / mL, about 50 mg / mL to about 120 mg / mL, or about 60 mg / mL to about 110 mg / mL.

[0028] E14. A formulation as described in E13, wherein the concentration of the polyol is about 64 mg / mL, about 85 mg / mL or about 106 mg / mL.

[0029] E15. A formulation as described in E1-E14, wherein the concentration of the polyol is about 85 mg / mL.

[0030] E16. A formulation as described in any one of E1 to E15, wherein the surfactant is selected from the group consisting of polysorbate, poloxamer, triton, sodium dodecyl sulfate, sodium laurel sulfate, sodium octyl glucoside, lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, stearoyl-sulfobetaine, lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, stearoyl-sarcosine, linoleyl-betaine, myristyl-betaine, cetyl-betaine, lauroamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl midopropyl)-betaine, myristamidopropyl-betaine, palmitopropyl-betaine, isostearamidopropyl-betaine, myristamidopropyl-dimethylamine, palmitopropyl-dimethylamine, isostearamidopropyl-dimethylamine, sodium methyl cocoyl taurate, disodium methyl oleyl-taurate, dihydroxypropyl PEG 5 linoleyl ammonium chloride, polyethylene glycol, polypropylene glycol, polysorbate 20, polysorbate 21, polysorbate 40, polysorbate 60, polysorbate 61, polysorbate 65, polysorbate 80, polysorbate 81, polysorbate 85, PEG3350 and mixtures thereof.

[0031] E17. A formulation as described in E16, wherein the surfactant is polysorbate 80.

[0032] E18. A formulation as described in any one of E1-E17, wherein the concentration of the surfactant is about 0.01 mg / mL to about 10 mg / mL, about 0.05 mg / mL to about 5 mg / mL, about 0.1 mg / mL to about 1 mg / mL, or about 0.1 mg / ml to about 0.5 mg / mL.

[0033] E19. A formulation as described in E18, wherein the concentration of the surfactant is about 0.1 mg / ml, about 0.2 mg / mL or about 0.3 mg / mL.

[0034] E20. A formulation as described in E19, wherein the concentration of the surfactant is about 0.2 mg / mL.

[0035] E21. A formulation as described in any one of E1-E20, wherein the formulation does not contain any surfactant (eg, polysorbate 80).

[0036] E22. A formulation as described in any one of E1 to E21, wherein the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid 5 (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)ethylene glycol ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid, and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine) and N-(trihydroxymethylmethyl)-10 glycine (tricine), glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine (trien), disodium edetate dihydrate (or disodium EDTA dihydrate or disodium EDTA salt), calcium EDTA oxalate, malate, citric acid, citric acid monohydrate, and trisodium citrate-dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine, methionine, peptides, polypeptides and proteins, and mixtures thereof.

[0037] E23. A formulation as described in E22, wherein the chelating agent is edetate disodium dihydrate.

[0038] E24. A formulation as described in any one of E1-E23, wherein the concentration of the chelating agent is about 0.01 mg / mL to about 50 mg / mL, about 0.01 mg / mL to about 10 mg / mL, about 0.01 mg / mL to about 1 mg / mL, or about 0.02 mg / mL to about 0.08 mg / mL.

[0039] E25. A formulation as described in E24, wherein the concentration of the chelating agent is about 0.038 mg / mL, about 0.05 mg / mL or about 0.063 mg / mL.

[0040] E26. A formulation as described in E25, wherein the concentration of the chelating agent is about 0.05 mg / mL.

[0041] E27. A formulation as described in any one of E1-E26, wherein the formulation has a pH of about 5.0 to 6.6 or about 5.2 to 6.4.

[0042] E28. A formulation as described in E27, wherein the formulation has a pH of about 5.2, 5.8 or 6.4.

[0043] E29. A formulation as described in E27, wherein the formulation has a pH of about 5.5 or 5.8.

[0044] E30. A formulation as described in any one of E1-E29, wherein the antibody does not bind to Kunitz domain 1 (K1) of TFPI.

[0045] E31. The formulation of any one of E2-E30, wherein according to the numbering of SEQ ID NO: 2, the epitope further comprises residues Cys106, Gly108, Cys130, Leu131 and Gly132.

[0046] E32. The formulation of any one of E2-E31, wherein according to the numbering of SEQ ID NO: 2, the epitope further comprises Asp102, Arg112, Tyr127, Gly129, Met134 and Glu138.

[0047] E33. A formulation as described in any one of E2-E32, wherein, according to the numbering of SEQ ID NO: 2, the epitope does not include: E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126 and L140.

[0048] E34. A formulation as described in any one of E2-E32, wherein according to the numbering of SEQ ID NO: 2, the epitope does not include: D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126 and G128.

[0049] E35. A formulation as described in any one of E1-E34, wherein the antibody comprises a heavy chain variable region (VH), wherein the heavy chain variable region (VH) comprises:

[0050] (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13;

[0051] (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and

[0052] (c) VH complementarity determining region 3 (CDR-H3), which comprises the amino acid sequence of SEQ ID NO:15.

[0053] E36. A formulation as described in any one of E1-E35, wherein the antibody comprises a VH comprising an amino acid sequence that is at least 90%, at least 95% or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 16, 18 and 20.

[0054] E37. The formulation of any one of E1-E36, wherein the antibody comprises a VH comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 16, 18 and 20.

[0055] E38. A formulation as described in any one of E1-E37, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:16.

[0056] E39. A formulation as described in any one of E1-E37, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO:18.

[0057] E40. The formulation of any one of E1-E37, wherein the antibody comprises a VH comprising the amino acid sequence of SEQ ID NO: 20.

[0058] E41. A formulation as described in any one of E1-E40, wherein the antibody comprises a light chain variable region (VL), wherein the light chain variable region (VL) comprises:

[0059] (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8;

[0060] (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and

[0061] (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10.

[0062] E42. A formulation as described in any one of E1-E41, wherein the antibody comprises a VL comprising an amino acid sequence that is at least 90%, at least 95% or at least 99% identical to SEQ ID NO:11.

[0063] E43. A formulation as described in any one of E1-E42, wherein the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:11.

[0064] E44. The formulation of any one of E1-E43, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:17.

[0065] E45. The formulation of any one of E1-E43, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:19.

[0066] E46. The formulation of any one of E1-E43, wherein the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21.

[0067] E47. The formulation of any one of E1-E46, wherein the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0068] E48. A formulation as described in any one of E1-E47, wherein the antibody comprises:

[0069] (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO:13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO:14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO:15, and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10.

[0070] E49. The formulation of E48, wherein the antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO:18 and a VL comprising the amino acid sequence of SEQ ID NO:11.

[0071] E50. A formulation as described in any one of E1-E49, wherein the antibody comprises a VH sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122329.

[0072] E51. The formulation of any one of E1-E50, wherein the antibody comprises a VL sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122328.

[0073] E52. A formulation as described in any one of E1 to E51, wherein the antibody comprises a sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122329 and a sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122328.

[0074] E53. The formulation of any one of E1-E52, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0075] E54. The formulation of E48, wherein the antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO:16 and a VL comprising the amino acid sequence of SEQ ID NO:11.

[0076] E55. The formulation of E54, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0077] E56. The formulation of E48, wherein the antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO:20 and a VL comprising the amino acid sequence of SEQ ID NO:11.

[0078] E57. The formulation of E56, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO:21, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO:12.

[0079] E58. The formulation of any one of E1, E3-E29, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 22.

[0080] E59. The formulation of any one of E1, E3-E29, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 24.

[0081] E60. A formulation as described in any one of E1 to E59, wherein the antibody has a serum half-life of at least 25 hours, at least 29 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 50 hours, at least 55 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 75 hours, at least 80 hours, at least 85 hours, at least 90 hours, at least 95 hours, at least 100 hours, at least 105 hours, at least 110 hours, at least 115 hours, at least 120 hours or at least 125 hours.

[0082] E61. A formulation as described in any one of E1-E60, wherein the antibody has a molecular weight of about 5x10-7 M to about 5x10 -11 The binding affinity (K D ).

[0083] E62. A formulation as described in any of E1-E61, wherein the antibody has a subcutaneous (SC) bioavailability of at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or at least 99% relative to the intravenous bioavailability.

[0084] E63. A formulation as described in any of E1-E62, wherein the concentration of the antibody is about 20 mg / mL, 25 mg / mL, 50 mg / mL, 75 mg / mL, 100 mg / ml, 125 mg / ml, 150 mg / mL, 175 mg / mL, 200 mg / ml, 225 mg / ml or 250 mg / mL.

[0085] E64. An aqueous formulation comprising: about 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), about 20 mM succinate or histidine buffer; about 85 mg / mL sucrose or trehalose; about 0.2 mg / mL polysorbate 80 or polysorbate 20; about 0.05 mg / mL edetate disodium dihydrate or ethylenediaminetetraacetic acid (EDTA); wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and wherein the formulation has a pH of about 5.5 or 5.8.

[0086] E65. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and wherein the preparation has a pH of 5.8.

[0087] E66. A pharmaceutical formulation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12; and wherein the formulation has a pH of 5.8.

[0088] E67. A pharmaceutical formulation comprising: about 50 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 22; and wherein the formulation has a pH of 5.8.

[0089] E68. A pharmaceutical formulation comprising: about 50 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 24; and wherein the formulation has a pH of 5.8.

[0090] E69. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 10 mM histidine buffer, 64 mg / mL sucrose, 0.1 mg / mL polysorbate 80, 0.038 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and wherein the preparation has a pH of 6.4.

[0091] E70. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 40 mM histidine buffer, 106 mg / mL sucrose, 0.3 mg / mL polysorbate 80, 0.063 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and wherein the preparation has a pH of 5.2.

[0092] E71. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 10 mM histidine buffer, 64 mg / mL sucrose, 0.1 mg / mL polysorbate 80, 0.038 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and wherein the preparation has a pH of 5.2.

[0093] E72. A pharmaceutical formulation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 18 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 11; and wherein the formulation has a pH of 5.8.

[0094] E73. A pharmaceutical formulation as described in E72, wherein the formulation does not contain a surfactant (eg, polysorbate 80).

[0095] E74. A formulation as described in any of E1-E73, wherein the formulation has a shelf life of at least about 1 month, about 3 months, about 4 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 60 months (e.g., at about -20°C, at about 5°C, at about 25°C, or at about 40°C).

[0096] E75. A formulation as described in any one of E1-E74, wherein the formulation is an aqueous formulation.

[0097] E76. A formulation as described in any one of E1-E75, wherein the formulation is a pharmaceutical formulation.

[0098] E77. A method of shortening bleeding time, comprising administering to a subject in need thereof a therapeutically effective amount of a formulation as described in any one of E1-E76.

[0099] E78. A method for treating or preventing a coagulation defect or a bleeding disorder, comprising administering to a subject in need thereof a therapeutically effective amount of a formulation as described in any one of E1-E76.

[0100] E79. A method for treating or preventing hemophilia A, B or C, comprising administering to a subject in need thereof a therapeutically effective amount of a formulation as described in any one of E1-E76.

[0101] E80. A method of treating or preventing von Willebrand's disease (vWD), comprising administering to a subject in need thereof a therapeutically effective amount of a formulation as described in any one of E1-E76.

[0102] E81. A method for reducing the activity of TFPI, comprising administering to a subject in need thereof a therapeutically effective amount of a formulation as described in any one of E1-E76.

[0103] E82. The method as described in any one of E77-E81, wherein the subject suffers from or is susceptible to a coagulation defect or a bleeding disorder.

[0104] E83. A method as described in any of E77-E82, wherein the subject has or is susceptible to hemophilia A, B or C.

[0105] E84. A method as described in any of E77-E83, wherein the subject has or is susceptible to hemophilia A or B.

[0106] E85. The method as described in any one of E77-E82, wherein the subject suffers from or is susceptible to von Willebrand's disease (vWD).

[0107] E86. The method as described in any one of E77-E82, wherein the subject suffers from or is susceptible to a platelet disorder.

[0108] E87. The method as described in any of E77-82, wherein the subject suffers from or is susceptible to Factor VII deficiency.

[0109] E88. The method as described in any one of E77-E82, wherein the subject suffers from or is susceptible to factor XI deficiency.

[0110] E89. The method as described in any one of E77-E88, further comprising administering a coagulant to the subject.

[0111] E90. The method as described in E89, wherein the coagulation agent is selected from Factor Vila, Factor VIII, Factor IX, tranexamic acid and a bypass agent (eg, FEIBA).

[0112] E91. Use of a formulation of any of E1-E76 in a method as described in any of E77-E90.

[0113] E92. A formulation as described in any one of E1-E76, for use in a method as described in any one of E77-E90.

[0114] E93. A formulation for use in the method as described in any one of E77-E90, comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12, and wherein the formulation has a pH of 5.8.

[0115] E94. A pharmaceutical formulation consisting of: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12; and wherein the formulation has a pH of 5.8.

[0116] Details

[0117] Disclosed herein are stable aqueous pharmaceutical formulations with extended shelf life comprising tissue factor pathway inhibitor (TFPI) antibodies. The present application is based on the discovery that a formulation comprising a histidine buffer and having a pH of 5.8 stably supports high concentrations of TFPI antibodies.

[0118] Specifically, anti-TFPI antibodies stored in histidine buffered aqueous formulations at pH 5.8 were demonstrated to have low levels of degradation as measured by % high molecular weight species using size exclusion high performance liquid chromatography (SE-HPLC) (see Table 1). For example, under the expected storage conditions of 5±3°C, anti-TFPI antibodies were stable for up to 24 months, with little to no degradation observed (%HMMS ranged from 0.5 to 0.9). Although some degradation was observed under heat stress storage conditions of 40°C / 75% RH (%HMMS ranged from 0.9 to 1.2 at 1 month (i.e., 4 weeks) and from 1.5 to 2.4 at 3 months (i.e., 12 weeks)), this degradation was less than that observed for other IgG1 antibodies, such as anti-VEGF antibodies (see Table 9; data for anti-VEGF antibodies obtained from Table 11 of US 2018 / 0000933). Specifically, the amount of high molecular mass species (%HMMS) for the anti-VEGF antibody stored in a histidine buffer formulation with pH 5.8 at 40°C for 1 month (i.e., 4 weeks) was 3.1 (compared to 0.9-1.2 for the anti-TFPI antibody), and the amount of high molecular mass species (%HMMS) for the anti-VEGF antibody stored in a histidine buffer formulation with pH 5.8 at 40°C for 1 month (i.e., 4 weeks) was 2.6 (compared to 0.9-1.2 for the anti-TFPI antibody), and the amount of high molecular mass species (%HMMS) for the anti-VEGF antibody stored in a histidine buffer formulation with pH 5.8 at 40°C for 1 month (i.e., 4 weeks) was 1.2 (compared to 0.9-1.2 for the anti-TFPI antibody), and the amount of high molecular mass species (%HMMS) for the anti-VEGF antibody stored in a histidine buffer formulation with pH 5.8 at 40°C for 1 month (i.e., 4 weeks) was 2.6 (compared to 0.9-1.2 for the anti-TFPI antibody). The amount of high molecular mass substances (%HMMS) of the anti-VEGF antibody stored for 3 months (i.e., 12 weeks) in a histidine buffer formulation at pH 5.8 was 5.6 (compared to 1.5-2.4 for the anti-TFPI antibody). Thus, the histidine buffer formulation at pH 5.8 delayed degradation, reduced aggregate formation, and improved stability of the anti-TFPI antibody compared to the anti-VEGF antibody. This result was surprising and unexpected.

[0119] Thus, in one aspect, a formulation is provided, comprising: about 15 mg / mL to about 250 mg / mL of a tissue factor pathway inhibitor (TFPI) antibody, a buffer, a polyol, a surfactant, a chelating agent, and wherein the formulation has a pH of about 5.0 to about 6.0. For example, in some embodiments, a formulation is provided, comprising: about 15 mg / ml to about 250 mg / ml of a TFPI antibody (e.g., an anti-TFPI antibody), about 1 mM to about 40 mM of a buffer (e.g., a histidine buffer), about 1 mg / ml to about 300 mg / ml of a polyol (e.g., sucrose), about 0.01 mg / ml to about 10 mg / ml of a surfactant (e.g., polysorbate 80), about 0.01 mg / ml to about 50.0 mg / ml of a chelating agent (e.g., disodium diacetate dihydrate), wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the antibody concentration is about 15 g / ml to about 250 g / ml. In other embodiments, the antibody concentration is about 100 mg / mL, about 15 mg / mL, about 150 mg / mL, or 158 mg / mL.

[0120] General techniques

[0121] The practice of the present invention will employ, unless otherwise indicated, conventional techniques of molecular biology (including recombinant techniques), microbiology, cell biology, biochemistry, and immunology, which are within the skill of the art. Such techniques are fully explained in the literature, such as, Molecular Cloning: A Laboratory Manual, 2nd Edition (Sambrook et al., 1989) Cold Spring Harbor Press; Oligonucleotide Synthesis (MJ Gait, ed., 1984); Methods in Molecular Biology, Humana Press; Cell Biology: A Laboratory Notebook (JE Cellis, ed., 1998) Academic Press; Animal Cell Culture (RI Freshney, ed., 1987); Introduction to Cell and Tissue Culture (JP Mather and PE Roberts, 1998) Plenum Press; Cell and Tissue Culture: Laboratory Procedures (A. Doyle, JB Griffiths, and DG Newell, eds., 1993-1998) J. Wiley and Sons; Methods in Enzymology (Academic Press, Inc.); Handbook of Experimental Immunology (DM Weir and CC Blackwell, eds.); Gene Transfer Vectors for Mammalian Cells (JM Miller and MP Calos, eds., 1987); Current Protocols in Molecular Biology (FM Ausubel et al., eds., 1987); PCR: The Polymerase Chain Reaction, (Mullis et al., eds., 1994); Current Protocols in Immunology (JEColigan et al., eds., 1991); Short Protocols in Molecular Biology (Wiley and Sons, 1999); Immunobiology (CA Janeway and P. Travers, 1997); Antibodies (P. Finch, 1997); Antibodies: a practical approach (D. Catty., eds., IRL Press, 1988-1989); Monoclonal antibodies: a practical approach (P. Shepherd and C. Dean, eds., Oxford University Press, 2000); Using antibodies: alaboratory manual (E. Harlow and D. Lane (Cold Spring Harbor Laboratory Press, 1999)); The Antibodies (M. Zanetti and JD Capra, eds., Harwood AcademicPublishers, 1995).

[0122] definition

[0123] Unless otherwise indicated, the following terms should be understood to have the following meanings: The term "isolated molecule" or "purified molecule" (wherein the molecule is, for example, a polypeptide, polynucleotide or antibody) is a molecule that, by virtue of its origin or source of derivation: (1) is free of naturally associated components that accompany it in its native state, (2) is substantially free of other molecules from the same species, (3) is expressed by cells from a different species, or (4) does not exist in nature. Thus, a molecule that is chemically synthesized or expressed in a cell system different from the cell of its natural origin will be "isolated" from its naturally associated components. A molecule can also be rendered substantially free of naturally associated components by separation using purification techniques well known in the art. Molecular purity or homogeneity can be determined by a number of means well known in the art. For example, the purity of a polypeptide sample can be determined using polyacrylamide gel electrophoresis and gel staining to visualize the polypeptide using techniques well known in the art. For some purposes, higher resolution can be provided by using HPLC or other means well known in the art of purification.

[0124] As used herein, when it refers to an antibody, the term "preparation" is intended to describe a preparation of the antibody in a form that permits the biological activity of the antibody to be effective.

[0125] The term "pharmaceutical composition" or "pharmaceutical formulation" refers to a preparation of an antibody in combination with a pharmaceutically acceptable carrier / excipient. As used herein, "aqueous formulation" refers to a formulation containing water as a component.

[0126] "Pharmaceutically acceptable carriers / excipients" (vehicles, additives) are those that can be safely administered to a subject to provide an effective dose of the active ingredient used. As used herein, the term "excipient" or "carrier" refers to an inert substance that is commonly used as a diluent, vehicle, preservative, binder, or stabilizer for a drug. Compositions comprising such carriers are prepared by well-known conventional methods (see, e.g., Remington's Pharmaceutical Sciences, 18th edition, A. Gennaro, ed., Mack Publishing Co., Easton, PA, 1990; and Remington, The Science and Practice of Pharmacy 20th edition. Mack Publishing, 2000). As used herein, the term "diluent" refers to a pharmaceutically acceptable (safe and non-toxic for administration to humans) solvent, and can be used to prepare an aqueous formulation as described herein. Exemplary diluents include, but are not limited to, sterile water and bacteriostatic water for injection (BWFI).

[0127] "Antibodies" are immunoglobulin molecules that are able to specifically bind to targets, such as carbohydrates, polynucleotides, lipids, polypeptides, etc., through at least one antigen recognition site located in the variable region of the immunoglobulin molecule. As used herein, unless otherwise indicated, the term encompasses not only complete polyclonal or monoclonal antibodies, but also any antigen binding fragments / parts thereof that compete with complete antibodies for specific binding, fusion proteins comprising antigen binding portions, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites. Antigen binding portions include, for example, Fab, Fab', F(ab')2, Fd, Fv, domain antibodies (dAb, e.g., shark and camel antibodies), fragments including complementary determining regions (CDRs), single-chain variable fragment antibodies (scFv), maxibodies, minibodies, intrabodies, double antibodies, three antibodies, four antibodies (tetrabodies), v-NARs and double-scFv, and polypeptides containing at least a portion of immunoglobulins sufficient to confer specific antigen binding to the polypeptide. Antibodies include antibodies of any class, such as IgG, IgA or IgM (or its subclass), and the antibodies need not be of any particular class. Depending on the antibody amino acid sequence of its heavy chain constant region, immunoglobulins can be designated as different classes. There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG and IgM, and several of them can be further divided into subclasses (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The heavy chain constant regions corresponding to different classes of immunoglobulins are referred to as α, δ, ε, γ and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known.

[0128] The "variable region" of an antibody refers to the variable region of an antibody light chain alone or in combination or the variable region of an antibody heavy chain. As known in the art, the variable regions of heavy and light chains are each composed of four framework regions (FR) connected by three complementary determining regions (CDRs) (also referred to as hypervariable regions), and participate in the formation of the antigen binding site of an antibody. If a variant of a subject variable region is desired (particularly in an amino acid residue outside CDR (i.e., in a framework region) with a substitution), then appropriate amino acid substitutions can be identified by comparing the subject variable region with the variable region of other antibodies (which contain CDR1 and CDR2 sequences in the same canonical category as the subject variable region), preferably conservative amino acid substitutions (Chothia and Lesk, J Mol Biol 196 (4): 901-917, 1987).

[0129] In certain embodiments, the identification of residues of the clear delineation of CDR and the binding site comprising the antibody is completed by resolving the structure of the antibody and / or resolving the structure of the antibody-ligand complex. In certain embodiments, this can be completed by any of the various techniques known to those skilled in the art (such as X-ray crystallography). In certain embodiments, various analytical methods can be used to identify or estimate CDR regions. In certain embodiments, various analytical methods can be used to identify or estimate CDR regions. Examples of such methods include but are not limited to Kabat definition, Chothia definition, AbM definition, contact definition and conformational definition.

[0130] The Kabat definition is a standard for numbering residues in antibodies and is often used to identify CDR regions. See, e.g., Johnson & Wu, 2000, Nucleic Acids Res., 28: 214-8. The Chothia definition is similar to the Kabat definition, but the Chothia definition takes into account the position of certain structural loop regions. See, e.g., Chothia et al., 1986, J. Mol. Biol., 196: 901-17; Chothia et al., 1989, Nature, 342: 877-83. The AbM definition uses an integrated computer program produced by the Oxford Molecular Group that models antibody structures. See, e.g., Martin et al., 1989, Proc Natl Acad Sci (USA), 86: 9268-9272; "AbM™, A Computer Program for Modeling Variable Regions of Antibodies," Oxford, UK; Oxford Molecular, Ltd. The AbM definition uses a combination of knowledge databases and ab initio computational methods to model the tertiary structure of an antibody from the primary sequence, such as those described by Samudrala et al., 1999, "Ab Initio Protein Structure Prediction Using a Combined Hierarchical Approach," in PROTEINS, Structure, Function and Genetics Suppl., 3:194-198. The contact definition is based on analysis of available complex crystal structures. See, e.g., MacCallum et al., 1996, J. Mol. Biol., 5:732-45. In another approach referred to herein as "conformational definition" of CDRs, the positions of CDRs can be identified as residues that make enthalpic contributions to antigen binding. See, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166. Still other CDR boundary definitions may not strictly follow one of the above methods, but will still overlap at least a portion of the Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that a particular residue or group of residues does not significantly affect antigen binding. As used herein, a CDR may refer to a CDR defined by any method known in the art, including a combination of methods. The methods used herein may utilize CDRs defined according to any of these methods.For any given embodiment containing more than one CDR, the CDRs may be defined according to any of the Kabat, Chothia, extension, AbM, contact and / or conformational definitions.

[0131] As known in the art, a "constant region" of an antibody refers to the constant region of the antibody light chain or the constant region of the antibody heavy chain, alone or in combination.

[0132] As used herein, "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, i.e., each antibody constituting the population is identical, except for a possible naturally occurring mutation that may be present in a small amount. Monoclonal antibodies are highly specific, directed against a single antigenic site. In addition, in contrast to polyclonal antibody preparations that typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on an antigen. The modifier "monoclonal" indicates that the characteristic of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibodies to be produced by any ad hoc method. For example, the monoclonal antibody to be used according to the present invention can be prepared by the hybridoma method first described by Kohler and Milstein, 1975, Nature 256: 495, or can be prepared by a recombinant DNA method such as described in U.S. Patent No. 4,816,567. Monoclonal antibodies can also be separated from a phage library generated using, for example, McCafferty et al., 1990, Nature 348:552-554 described technology.

[0133] "Human antibody" is an antibody having an amino acid sequence corresponding to an antibody produced by a person and / or having been prepared using any technique for preparing human antibodies as disclosed herein. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. As used herein, the term "human antibody" is intended to include antibodies having variable regions and constant regions derived from human germline immunoglobulin sequences. This definition of human antibody includes antibodies comprising at least one human heavy chain polypeptide or at least one human light chain polypeptide. The human antibody of the present invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR and particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from another mammalian species, such as the germline of a mouse, have been transplanted to human framework sequences.

[0134] The term "chimeric antibody" means an antibody in which the variable region sequence is derived from one species and the constant region sequence is derived from another species, such as an antibody in which the variable region sequence is derived from a mouse antibody and the constant region sequence is derived from a human antibody.

[0135] As used herein, "humanized" antibody refers to the form of non-human (e.g., mouse) antibodies, which are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding sequences of antibodies), which contain minimal sequences derived from non-human immunoglobulins. Preferably, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the residues from the complementary determining regions (CDRs) of the receptor are replaced with residues from non-human species (donor antibodies), such as mouse, rat, or rabbit CDRs with desired specificity, affinity, and ability. In some cases, the Fv framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not found in receptor antibodies or introduced CDR or framework sequences, but include the residues to further refine and optimize antibody performance. In general, humanized antibodies include at least one and usually substantially all of two variable domains, wherein all or substantially all CDR regions correspond to those of non-human immunoglobulins, and all or substantially all FR regions are those of human immunoglobulin consensus sequences. Humanized antibodies also optimally include at least a portion of an immunoglobulin constant region or domain (Fc), typically a constant region or domain (Fc) of a human immunoglobulin. Antibodies preferably have an Fc region modified as described in WO 99 / 58572. Other forms of humanized antibodies have one or more CDRs (CDR L1, CDR L2, CDR L3, CDR H1, CDR H2, or CDR H3) that are changed relative to the original antibody, which are also referred to as "derived from" one or more CDRs from one or more CDRs of the original antibody.

[0136] There are 4 general steps to humanize a monoclonal antibody. These are: (1) determining the nucleotide and predicted amino acid sequences of the starting antibody light and heavy chain variable domains, (2) designing the humanized antibody, i.e., deciding which antibody framework regions to use during the humanization process, (3) the actual humanization method / technique, and (4) transfection and expression of the humanized antibody. See, e.g., U.S. Pat. Nos. 4,816,567; 5,807,715; 5,866,692; 6,331,415; 5,530,101; 5,693,761; 5,693,762; 5,585,089; and 6,180,370.

[0137] Many "humanized" antibody molecules comprising antigen binding sites derived from non-human immunoglobulins have been described, including chimeric antibodies having rodent or modified rodent V regions fused to human constant domains and their associated complementary determining regions (CDRs). See, e.g., Winter et al. Nature 349: 293-299 (1991), Lobuglio et al. Proc. Nat. Acad. Sci. USA 86: 4220-4224 (1989), Shaw et al. J Immunol. 138: 4534-4538 (1987), and Brown et al. Cancer Res. 47: 3577-3583 (1987). Other references describe rodent CDRs that are implanted into human supporting framework regions (FRs) before fusion with appropriate human antibody constant domains. See, e.g., Riechmann et al. Nature 332: 323-327 (1988), Verhoeyen et al. Science 239: 1534-1536 (1988), and Jones et al. Nature 321: 522-525 (1986). Another reference describes rodent CDRs supported by recombinantly veneered rodent framework regions. See, e.g., European Patent Publication No. 0519596. These "humanized" molecules are designed to minimize undesirable immune responses to rodent anti-human antibody molecules, which limits the duration and effectiveness of therapeutic applications of those parts in human receptors. For example, the antibody constant region can be engineered so that it is immunologically inert (e.g., does not trigger complement lysis). See, e.g., PCT Publication No. WO99 / 58572; UK Patent Application No. 9809951.8. Other methods of humanizing antibodies that may also be used are disclosed by Daugherty et al., Nucl. Acids Res. 19: 2471-2476 (1991) and in U.S. Pat. Nos. 6,180,377; 6,054,297; 5,997,867; 5,866,692; 6,210,671; and 6,350,861; and in PCT Publication No. WO 01 / 27160.

[0138] As used herein, the term "recombinant antibody" is intended to include all antibodies prepared, expressed, generated or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated or prepared from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes, and such recombinant human antibodies may be subjected to in vitro mutagenesis.

[0139] The term "epitope" refers to a portion of a molecule that can be recognized and bound by an antibody at one or more antibody antigen binding regions. An epitope is often composed of surface groupings of a molecule such as amino acids or sugar side chains, and has specific three-dimensional structural features and specific charge characteristics. In some embodiments, the epitope may be a protein epitope. A protein epitope may be linear or conformational. In a linear epitope, all points of interaction between a protein and an interacting molecule (such as an antibody) exist linearly along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" comprises non-adjacent polypeptides (or amino acids) within an antigenic protein, to which antibodies specific for the epitope bind. As used herein, the term "antigenic epitope" is defined as a portion of an antigen to which an antibody can specifically bind as determined by any method well known in the art (e.g., by conventional immunoassays). Once the desired epitope on an antigen is determined, an antibody to the epitope may be generated, for example, using the techniques described in this specification. Alternatively, during the discovery process, the generation and characterization of antibodies may elucidate information about the desired epitope. Based on this information, antibodies may be competitively screened for binding to the same epitope. One way to accomplish this is to perform competition and cross-competition studies to find antibodies that compete or cross-compete with each other for binding to TFPI, e.g., the antibodies compete for binding to the antigen.

[0140] The term "antagonist antibody" refers to an antibody that binds to a target and prevents or reduces the biological effect of the target. In some embodiments, the term may refer to an antibody that reduces the biological function of the target to which it binds, such as TFPI.

[0141] An antibody that "preferentially binds" or "specifically binds" (used interchangeably herein) an epitope is a term well understood in the art, and methods of determining such specific or preferential binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferential binding" if it reacts or associates with a particular cell or substance more frequently, more rapidly, for a longer duration and / or with a greater affinity than it reacts or associates with alternative cells or substances. An antibody "specifically binds" or "preferentially binds" a target if it binds to the target with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other substances. For example, an antibody that specifically or preferentially binds to an epitope of TFPI is an antibody that binds to that epitope sequence with greater affinity, avidity, more readily, and / or for a longer duration than it binds to other sequences. It is also understood by reading this definition that, for example, an antibody (or portion or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Thus, "specific binding" or "preferential binding" does not necessarily require (although it can include) exclusive binding.

[0142] Various assay formats can be used to select antibodies or peptides that specifically bind to a target molecule. For example, solid phase ELISA immunoassays, immunoprecipitation, Biacore TM (GE Healthcare, Piscataway, NJ), KinExA, fluorescence activated cell sorting (FACS), Octet™ (ForteBio, Inc., Menlo Park, CA) and Western blot analysis are many assays that can be used to identify antibodies that specifically react with an antigen or receptor or its ligand binding portion (which specifically binds to a cognate ligand or binding partner). Typically, a specific or selective reaction will be at least twice the background signal or noise, more typically more than 10 times the background, more typically more than 50 times the background, more typically more than 100 times the background, yet more typically more than 500 times the background, even more typically more than 1000 times the background, and even more typically more than 10,000 times the background. In addition, when the equilibrium dissociation constant (K D )≤7 nM, the antibody is said to “specifically bind” to the antigen.

[0143] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules (eg, an antibody or fragment thereof and an antigen). The term "binding affinity" is used to describe a monovalent interaction (intrinsic activity).

[0144] The binding affinity between two molecules (e.g., an antibody or fragment thereof and an antigen) through a monovalent interaction can be measured by the dissociation constant (K D ) to quantify. In turn, K can be determined by measuring the kinetics of complex formation and dissociation using, for example, the surface plasmon resonance (SPR) method (Biacore). D The rate constants corresponding to the association and dissociation of the monovalent complex are called the association rate constants k and k, respectively. a (or k on ) and the dissociation rate constant k d (or k off ). D Through the equation K D =k d / k a The value of the dissociation constant can be determined directly by well-known methods, and can even be calculated for a mixture of complexes by methods such as those shown in Caceci et al. (1984, Byte 9:340-362). For example, K DA dual filter nitrocellulose filter binding assay, such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci. USA 90: 5428-5432) can be used to establish. Other standard assays for assessing the binding capacity of ligands (such as antibodies to target antigens) are known in the art, including, for example, ELISA, Western blots, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere herein. The binding kinetics and binding affinity of antibodies can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR) (e.g., by using Biacore TM system or KinExA).

[0145] An antibody that specifically binds to its target may have high affinity (i.e., exhibit a low K as discussed above). D ) binds to its target and can bind other non-target molecules with lower affinity. For example, an antibody can bind 1×10 -6 M or higher (preferably 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, even more preferably 1×10 -2 M or higher) K D Binding to Non-Target Molecules. Preferably, an antibody of the invention is capable of binding its target with an affinity that is at least 2, 10, 50, 100, 200, 500, 1,000 or 10,000 times or more greater than its binding affinity to another non-TFPI molecule.

[0146] As used herein, "immunospecific" binding of an antibody refers to the antigen-specific binding interaction that occurs between the antigen binding site of the antibody and the specific antigen recognized by the antibody (i.e., the antibody reacts with the protein in an ELISA or other immunoassay and does not detectably react with unrelated proteins).

[0147] As used herein with respect to antibodies, the term "competition" means that the first antibody or its antigen-binding portion binds to an epitope in a manner very similar to the binding of the second antibody or its antigen-binding portion, such that the result of the first antibody binding to its cognate epitope in the presence of the second antibody is detectably reduced compared to the binding of the first antibody in the absence of the second antibody. Alternative situations in which the binding of the second antibody to its epitope can also be detectably reduced in the presence of the first antibody are possible, but not necessarily so. That is, the first antibody can inhibit the second antibody from binding to its epitope while the second antibody does not inhibit the first antibody from binding to its corresponding epitope. However, when each antibody detectably inhibits the binding of another antibody to its cognate epitope or ligand, whether to the same, greater or lesser extent, it is considered that the antibodies "cross-compete" with each other to bind to their respective epitopes. The present invention encompasses competing and cross-competing antibodies. Regardless of the mechanism by which such competition or cross-competition occurs (e.g., steric hindrance, conformational changes, or binding to a common epitope or portion thereof), based on the teachings provided herein, the skilled person will understand that such competing and / or cross-competing antibodies are encompassed and can be used in the methods disclosed herein.

[0148] As used herein, the term "tissue factor pathway inhibitor or TFPI" refers to any form of TFPI and variants thereof that retain at least a portion of the activity of TFPI. TFPI is a multivalent Kunitz domain containing a protease inhibitor. Exemplary sequences of human, mouse, cynomolgus monkey, rabbit and rat TFPI are provided in Table 20. Human TFPI is an extracellular glycoprotein that has two major forms, TFPI-α and TFPI-β. TFPIα, which is a glycosylated protein of 276 amino acids (MW 43kD), is the largest form of TFPI and consists of three Kunitz-like domains and a basic carboxyl-terminal region. Variable shearing produces TFPI-β, which contains Kunitz domain 1 (K1) and Kunitz domain 2 (K2), but contains an alternative C-terminal portion lacking Kunitz domain 3 (K3) and a basic region. TFPI-β is anchored to the cell membrane by post-translational modification with a glycosylphosphatidylinositol (GPI) anchor.

[0149] The main targets of TFPI are the proteases Factor Xa (FXa) and Factor VIIa (FVIIa), which are key factors in the initial stages of the coagulation cascade. Biochemical analysis has revealed that K2 is an inhibitor of FXa, while K1 inhibits the FVIIa-tissue factor complex. The role of K3 is unclear, as it does not appear to have direct protease inhibitory activity, but may serve as a recognition site for cofactor protein S. The C-terminal domain unique to TFPI-α may be involved in the recognition of prothrombinase on the platelet surface.

[0150] Kunitz domain 1 (K1) corresponds to amino acid residues 26-76 of SEQ ID NO: 2, and Kunitz domain 2 (K2) corresponds to residues 91 to 147 of SEQ ID NO: 2. K1 and K2 domains from other TFPI homologs, isoforms, variants or fragments can be identified by sequence alignment or structural alignment against SEQ ID NO: 2.

[0151] TFPI of the present disclosure includes any naturally occurring form of TFPI, which can be derived from any suitable organism. For example, TFPI can be mammalian TFPI, such as human, mouse, rat, non-human primate, cattle, sheep, dog, cat or pig TFPI. In certain embodiments, TFPI is human TFPI. TFPI can be a mature form of TFPI (i.e., a TFPI protein that has undergone post-translational processing in a suitable cell). For example, this mature TFPI protein can be glycosylated.

[0152] TFPI of the present disclosure includes any functional fragment or variant derived from naturally occurring TFPI. A functional fragment of TFPI can be any component or portion of TFPI that retains the activity of TFPI (such as the ability to inhibit factor Xa (FXa), inhibit the activity of the FVIIa-tissue factor complex, and / or function as a negative regulator of coagulation or hemostasis). For example, a functional fragment may include a Kunitz domain, such as the K1 domain, the K2 domain, or both the K1 and K2 domains of TFPI.

[0153] Functional variants may comprise one or more mutations compared to naturally occurring TFPI and still retain the activity of naturally occurring TFPI, such as the ability to inhibit factor Xa (FXa) or the ability to inhibit the activity of the FVIIa-tissue factor complex. For example, the variant may have various degrees of sequence identity to SEQ ID NO: 1, 2, 3, 4, 5, 6 or 7, such as at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity to the sequence set forth in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6 or SEQ ID NO: 7.

[0154] The TPFI fragments, variants, isoforms and homologs of the present invention should maintain important epitope residues (such as Ile105, Arg107 and Leu131 if TFPI-23 and TFPI-24 antibodies are used). In addition, TFPI may contain 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more surface accessible residues of the K2 domain of TFPI. Surface accessible residues are residues with a relative accessibility greater than 40%.

[0155] For example, for the K2 domain of TFPI (see, e.g., SEQ ID NO: 2), the following amino acid residues have a relative accessibility of greater than 40%: 94-95, 98, 100-110, 118-121, 123-124, 131, 134, 138-142, and 144- 145. TFPI may comprise 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more of these residues, such as a TFPI fragment comprising 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more of these residues.

[0156] Specific amino acid residue positions in TFPI are numbered according to SEQ ID NO:2 (human TFPIα K1K2K3). However, the present invention is not limited to SEQ ID NO:2. Corresponding residues from other TFPI homologs, isoforms, variants or fragments can be identified according to sequence alignments or structural alignments known in the art. For example, the alignment can be done manually or using well-known sequence alignment programs, such as ClustalW2 or "BLAST 2 Sequences", using default parameters. For example, Arg107 of SEQ ID NO:2 corresponds to Arg104 of mouse TFPI K1K2 (SEQ ID NO:4).

[0157] As used herein, a "TFPI antagonist antibody" (interchangeably referred to as a "TFPI antibody" or "anti-TFPI antibody") refers to an antibody that is capable of binding to TFPI and inhibiting TFPI biological activity and / or downstream pathways mediated by TFPI signaling. TFPI antagonist antibodies encompass antibodies that block, antagonize, inhibit or reduce (including significantly) TFPI biological activity, including downstream pathways mediated by TFPI signaling, such as ligand binding and / or eliciting a cellular response to TFPI. For the purposes of the present invention, it will be expressly understood that the term "TFPI antagonist antibody" encompasses all previously identified terms, titles, and functional states and features, whereby TFPI itself, TFPI biological activity (including but not limited to its ability to mediate any aspect of blood coagulation) or the consequences of the biological activity are substantially ineffective, reduced or neutralized to any meaningful extent. In some embodiments, a TFPI antagonist antibody binds to TFPI and prevents TFPI from binding to and / or inhibiting the tissue factor (TF) / factor VIIa complex. In other embodiments, a TFPI antibody binds to TFPI and prevents TFPI from binding to factor Xa and / or inhibiting factor Xa. Examples of TFPI antagonist antibodies are provided herein.

[0158] An "effective amount" of a drug, formulation, compound, or pharmaceutical composition is an amount sufficient to achieve a beneficial or desired result, including a clinical result, such as a reduction or decrease in a target pathological condition. An effective amount can be administered in one or more administrations. For purposes of the present invention, an effective amount of a drug, compound, or pharmaceutical composition is an amount sufficient to treat, improve, or reduce the intensity of a target pathological condition. As understood in a clinical context, an effective amount of a drug, compound, or pharmaceutical composition may or may not be achieved in conjunction with another drug, compound, or pharmaceutical composition. Thus, an "effective amount" may be considered in the context of administering one or more therapeutic agents, and a single agent may be considered to be administered in an effective amount if the desired result may be achieved or obtained in conjunction with one or more other agents.

[0159] A "therapeutically effective amount" means an amount effective, at the dosage and for the period of time necessary, to achieve the desired therapeutic result, which in the context of an anti-TFPI antibody includes treatment or prophylactic prevention of a target pathological condition, e.g., a bleeding disorder such as hemophilia A, B, or C. It should be noted that dosage values ​​may vary with the severity of the condition to be alleviated. It should be further understood that for any particular subject, the specific dosage regimen should be adjusted over time according to the individual needs and the professional judgment of the person administering or supervising the administration of the composition, and that the dosages shown herein are exemplary only and are not intended to limit the scope or practice of the claimed compositions. Likewise, a therapeutically effective amount of an antibody or antibody portion may vary according to factors such as the disease state, age, sex, and weight of the individual, the ability of the antibody or antibody portion to elicit a desired response in the individual, and the desired route of administration of the antibody formulation. A therapeutically effective amount is also an amount in which the therapeutically beneficial effects far outweigh any toxic or deleterious effects of the antibody or antibody portion.

[0160] The therapeutically effective amount of an anti-TFPI antibody is described in, for example, PCT / IB2019 / 058597 filed on October 9, 2019 (which claims priority to US 62 / 802,401 and US 62 / 744,481), which is incorporated herein in its entirety. In some embodiments, the therapeutically effective amount includes an initial dose of about 300 mg, followed by a subsequent weekly (ie, weekly) dose of about 150 mg. In some embodiments, the therapeutically effective amount includes an initial dose of about 150 mg and a subsequent weekly (ie, weekly) dose of about 150 mg. In some embodiments, the therapeutically effective amount includes an initial dose of about 300 mg and a subsequent weekly (ie, weekly) dose of about 300 mg. In some embodiments, the therapeutically effective amount includes an initial dose of about 450 mg and a subsequent weekly (ie, weekly) dose of about 450 mg.

[0161] As used herein, the term "treatment" refers to therapeutic treatment as well as prophylactic or preventative measures, wherein the goal is to prevent or slow down (lessen) the targeted pathological condition (e.g., any condition that would benefit from treatment with an antibody. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose the mammal to the disorder in question. Those in need of treatment include those already having the condition as well as those prone to having the condition or those in which the condition is to be prevented. As used herein, "treatment" is an approach to obtaining beneficial or desired clinical results, including, but not limited to, one or more of the following: including lessening of severity, alleviation of one or more symptoms associated with a pathological condition.

[0162] As used herein, for the purpose of treatment, the term "subject" or "individual" includes any subject, and is preferably a subject of a pathological condition (e.g., hemophilia) that needs to be treated. For the purpose of prevention, the subject is any subject, and is preferably a subject at risk or prone to developing a targeted pathological condition. The term "subject" is intended to include living organisms, e.g., prokaryotes and eukaryotes. Examples of subjects include mammals, such as humans, dogs, cows, horses, pigs, sheep, goats, cats, mice, rabbits, rats, and transgenic non-human animals. In a specific embodiment of the present invention, the subject is a human.

[0163] As used herein, the terms "polynucleotide" or "nucleic acid" used interchangeably herein mean a polymeric form of nucleotides, ribonucleotides or deoxynucleotides, or a modified form of either type of nucleotide, and may be in single-stranded or double-stranded form. Unless otherwise indicated, a "polynucleotide" or "nucleic acid" sequence encompasses its complement. As used herein, the term "isolated polynucleotide" or "isolated nucleic acid" means a polynucleotide of genomic, cDNA, or synthetic origin, or some combination thereof, which, by virtue of its origin or source of derivation, has one to three of the following: (1) is unrelated to all or a portion of a polynucleotide to which the "isolated polynucleotide" is found in nature, (2) is operably linked to a polynucleotide to which it is not linked in nature, or (3) does not occur as part of a larger sequence in nature.

[0164] References to "about" values ​​or parameters herein include (and describe) embodiments for the value or parameter itself. For example, a description referring to "about X" includes a description of "X". Numerical ranges include numbers that define the range. In general, the term "about" refers to the indicated value of a variable and all values ​​of the variable that are within the experimental error of the indicated value (e.g., within a 95% confidence interval of the mean) or within 10% of the indicated value (whichever is greater).

[0165] When aspects or embodiments of the invention are described in terms of Markush groups or other alternative groupings, the invention encompasses not only the entire group listed as a whole, but also each member of the group individually and all possible subgroups of the main group, as well as the main group without one or more group members. The invention also contemplates the explicit exclusion of any one or more group members in the claimed invention.

[0166] When introducing elements of the present invention or preferred embodiments thereof, the articles "a", "an", "the" and "said" are intended to refer to one or more of the elements. The terms "comprising", "comprise", "comprises", "including" and "having" are intended to include, and mean that there may be additional elements other than the listed elements. It should be understood that wherever the language "comprising" is used herein to describe an embodiment, additional similar embodiments described in terms of "consisting of" and / or "consisting essentially of" are also provided.

[0167] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the invention belongs. In the event of a conflict, the present specification (including definitions) will prevail. Unless the context requires otherwise, a singular term shall include a plural term, and a plural term shall include a singular term.

[0168] Exemplary methods and materials are described herein, although those similar or equivalent to those described herein can also be used in the practice or testing of the present invention. The materials, methods, and examples are illustrative only and are not intended to be limiting.

[0169] Anti-TFPI antibody preparations

[0170] The present application is based on the surprising and unexpected observation that formulations comprising a histidine buffer and having a pH of 5.8 delay degradation, reduce aggregate formation, and improve stability of anti-TFPI antibodies compared to other IgG1 antibodies, such as anti-VEGF antibodies.

[0171] Thus, in one aspect, provided herein is a formulation comprising: about 15 mg / mL to about 250 mg / mL of a tissue factor pathway inhibitor (TFPI) antibody, a buffer, a polyol, a surfactant, a chelating agent, and wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the formulation comprises: about 15 mg / mL to about 250 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), a buffer, a polyol, a surfactant, a chelating agent, wherein the formulation has a pH of about 5.0 to about 6.0, and wherein according to the numbering of SEQ ID NO: 2, the epitope comprises residues Ile105, Arg107, and Leu131. The formulations described herein have an extended shelf life, preferably at least about 1 month, about 4 months, about 6 months, about 12 months, about 24 months, about 36 months, about 48 months, or about 60 months (e.g., at about -70°C, at about -20°C, at about 5°C, at about 25°C, or at about 40°C).

[0172] In some embodiments, the formulation comprises at least one TFPI antagonist antibody (i.e., anti-TFPI antibody). Exemplary TFPI antagonist antibodies include, but are not limited to, those described in WO 2017 / 029583, WO 2010 / 017196, WO 2011 / 109452, WO 2014 / 144577, WO 2010 / 072687, WO 2012 / 001087, WO 2014 / 140240, and WO 2015 / 007880 (each of which is incorporated herein by reference in its entirety).

[0173] In some embodiments, the TFPI antagonist antibody is selected from TFPI 106 (also known as PF-06741086), TFPI-23, TFPI-107, concizumab (also known as mAb-2021, hz4F36), 2A8 and 2A8-200 (see, e.g., US20170073428).

[0174] In some embodiments, more than one antibody may be present. There may be at least 1, at least 2, at least 3, at least 4, at least 5 or more different antibodies. Typically, two or more different antibodies have complementary activities that do not negatively affect each other. One or more antibodies may also be used in combination with other substances used to enhance and / or supplement the effectiveness of the antibodies.

[0175] In some embodiments, the antibody specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), wherein the epitope comprises residues Ile105, Arg107, and Leu131 (numbering according to SEQ ID NO: 2). In some embodiments, the anti-TFPI antibody does not bind to Kunitz domain 1 (K1) of TFPI. In some embodiments, the epitope further comprises residues Cys106, Gly108, Cys130, Leu131, and Gly132 (numbering according to SEQ ID NO: 2). In some embodiments, the epitope further comprises Asp102, Arg112, Tyr127, Gly129, Met134, and Glu138 (numbering according to SEQ ID NO: 2). In some embodiments, the epitope does not include: E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140 (according to the numbering of SEQ ID NO: 2). In some embodiments, the epitope does not include: D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128 (according to the numbering of SEQ ID NO: 2).

[0176] In some embodiments, the antibody comprises a heavy chain variable region (VH) comprising:

[0177] (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13;

[0178] (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and

[0179] (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO:15.

[0180] In some embodiments, the antibody comprises a VH comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to an amino acid sequence selected from SEQ ID NOs: 16, 18, and 20. In some embodiments, the antibody comprises a VH comprising an amino acid sequence selected from SEQ ID NOs: 16, 18, and 20. In some embodiments, the antibody comprises a VH comprising an amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a VH comprising an amino acid sequence of SEQ ID NO: 18. In some embodiments, the antibody comprises a VH comprising an amino acid sequence of SEQ ID NO: 20.

[0181] In some embodiments, the antibody comprises a light chain variable region (VL) comprising:

[0182] (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10.

[0183] In some embodiments, the antibody comprises a VL comprising an amino acid sequence that is at least 90%, at least 95%, or at least 99% identical to SEQ ID NO: 11. In some embodiments, the antibody comprises a VL comprising the amino acid sequence of SEQ ID NO:11.

[0184] In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 17. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 19. In some embodiments, the antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO: 21. In some embodiments, the antibody comprises a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0185] In some embodiments, the antibody comprises:

[0186] (i) a heavy chain variable region (VH) comprising: (a) a CDR-H1 comprising the amino acid sequence of SEQ ID NO: 13, (b) a CDR-H2 comprising the amino acid sequence of SEQ ID NO: 14, and (c) a CDR-H3 comprising the amino acid sequence of SEQ ID NO: 15, and

[0187] (ii) a light chain variable region (VL), comprising: (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO:8, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO:9, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO:10.

[0188] In some embodiments, the antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 18 and a VL comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 19, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0189] Exemplary antibodies of the invention were deposited at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA, on July 22, 2015. The plasmid vector mAb-TFPI-106VH with ATCC Accession No. PTA-122329 comprises a DNA insert encoding the heavy chain variable region of antibody TFPI-106, and the plasmid vector mAb-TFPI-106VL with ATCC Accession No. PTA-122328 comprises a DNA insert encoding the light chain variable region of antibody TFPI-106.

[0190] In some embodiments, the antibody comprises: a VH comprising the amino acid sequence of SEQ ID NO: 16 and a VL comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 17, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0191] In some embodiments, the antibody or antigen-binding fragment thereof comprises: a VH comprising the amino acid sequence of SEQ ID NO: 20 and a VL comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 21, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 12.

[0192] In some embodiments, the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 23, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 22. In some embodiments, the antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 25, and comprises: a light chain comprising the amino acid sequence of SEQ ID NO: 24.

[0193] In some embodiments, the antibody has a serum half-life of at least 25 hours, at least 29 hours, at least 30 hours, at least 35 hours, at least 40 hours, at least 50 hours, at least 55 hours, at least 60 hours, at least 65 hours, at least 70 hours, at least 75 hours, at least 80 hours, at least 85 hours, at least 90 hours, at least 95 hours, at least 100 hours, at least 105 hours, at least 110 hours, at least 115 hours, at least 120 hours, or at least 125 hours. In some embodiments, the antibody has a serum half-life of at least 25 hours, at least 29 hours, or at least 30 hours. In some embodiments, the antibody has a serum half-life of at least 29 hours. In some embodiments, the antibody has a serum half-life of at least 30 hours. In some embodiments, the antibody has a serum half-life of at least 115 hours, at least 120 hours, or at least 125 hours.

[0194] In some embodiments, the antibody has a -7 M to about 5x10 -11 The binding affinity (K D In some embodiments, the antibody has a -8 M to about 1x10 -10 K of M (0.1 to 10 nm) D In some embodiments, the antibody has a K D In some embodiments, the antibody does not have a K in the low pM range (i.e., ≤100 pM). D In some aspects, K D Measured by surface plasmon resonance. In some aspects, surface plasmon resonance can be measured using Biacore. In some aspects, SPR can be measured using Biacore with captured antibody and solution phase human TFPI.

[0195] In some aspects, the subcutaneous bioavailability of the antibody can be at least 10% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 15% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 20% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 25% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 27% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 30% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 35% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 40% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 50% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 60% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 70% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 80% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 90% relative to intravenous bioavailability. In some aspects, the subcutaneous bioavailability of the antibody can be at least 99% relative to intravenous bioavailability.

[0196] The antibody can be present in the formulation at a concentration ranging from about 0.1 mg / mL to about 250 mg / mL, about 15 mg / mL to 250 mg / mL, about 20 mg / mL to about 175 mg / mL, or about 25 mg / mL to about 160 mg / mL. For example, in some embodiments, the concentration of the antibody is about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, about 6 mg / mL, about 6.5 mg / mL, about 7 mg / mL, about 7.5 mg / mL, about 8 mg / mL, about 8.5 mg / mL, about 9 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL mg / mL, about 70 mg / mL, about 80 mg / mL, about 90 mg / mL, about 100 mg / mL, about 101 mg / mL, about 102 mg / mL, about 102.5 mg / mL, about 103 mg / mL, about 103.5 mg / mL, about 104 mg / mL, about 104.5 mg / mL, about 105 mg / mL, about 105.5 mg / mL, about 106 mg / mL, about 106.about 106 mg / mL, about 107 mg / mL, about 107.5 mg / mL, about 108 mg / mL, about 108.5 mg / mL, about 109 mg / mL, about 109.5 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, about 150 mg / mL, about 151 mg / mL, about 152 mg / mL, about 153 mg / mL, about 154 mg / mL, about 155 mg / mL, about 156 mg / mL, about 157 mg / mL, about 158 ​​mg / mL, about 159 mg / mL, about 160 mg / mL, about 170 mg / mL, about 171 mg / mL, about 172 mg / mL mg / mL, about 190 mg / mL, about 200 mg / mL, about 210 mg / mL, about 220 mg / mL, about 230 mg / mL, about 240 mg / mL, or about 250 mg / mL. In some embodiments, the concentration of the antibody in the formulation is about 100 mg / mL, about 115 mg / mL, about 150 mg / mL, or about 158 ​​mg / mL. In some embodiments, the concentration of the antibody in the formulation is about 150 mg / mL. In some embodiments, the concentration of the antibody in the formulation is about 155 mg / mL. In some embodiments, the concentration of the antibody in the formulation is about 158 ​​mg / mL. .

[0197] According to the invention, a buffer (eg, histidine or succinate buffer) provides a formulation with a pH close to physiological pH for reducing the risk of painful or allergic side effects of injection, and provides enhanced antibody stability and resistance to aggregation, oxidation and fragmentation.

[0198] Buffer can be, for example, but not limited to, acetate, succinate (disodium succinate hexahydrate), gluconate, citrate, histidine, acetic acid, phosphate, phosphoric acid, ascorbate, tartaric acid, maleic acid, glycine, lactate, lactic acid, ascorbic acid, imidazole, bicarbonate and carbonic acid, succinic acid, sodium benzoate, benzoic acid, gluconate, edetate, acetate, malate, imidazole, tris, phosphate and mixtures thereof. In some embodiments, the buffer is histidine or succinate. In some embodiments, the succinate buffer comprises disodium succinate hexahydrate (alkaline form) and / or succinic acid or mixtures thereof. Preferably, the buffer is histidine, wherein the histidine buffer comprises L-histidine, L-histidine monohydrochloride (also referred to as L-histidine monohydrochloride monohydrate and / or L-histidine hydrochloride monohydrate) or mixtures thereof.

[0199] The concentration of the buffer may range from about 0.1 millimolar (mM) to about 100 mM. Preferably, the concentration of the buffer is from about 0.5 mM to about 50 mM, more preferably from about 1 mM to about 30 mM, and more preferably from about 1 mM to about 25 mM. Preferably, the concentration of the buffer is about 1 mM, about 2 mM, about 3 mM, about 4 mM, about 5 mM, about 6 mM, about 7 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, about 15 mM, about 16 mM, about 17 mM, about 18 mM, about 19 mM, about 20 mM, about 21 mM, about 22 mM, about 23 mM, about 24 mM, about 25 mM, about 30 mM, about 35 mM, about 40 mM, about 45 mM, about 50 mM, about 55 mM, about 60 mM, about 65 mM, about 70 mM, about 75 mM, about 80 mM, about 85 mM, about 90 mM, about 95 mM or about 100 mM. In some embodiments, the buffer is histidine at a concentration of about 20 mM. In some embodiments, the buffer is succinate at a concentration of about 20 mM.

[0200] The concentration of the buffer can also range from about 0.01 mg / mL to about 30 mg / mL, about 0.1 mg / mL to about 5 mg / mL, or about 0.5 mg / mL to about 4 mg / mL. For example, the concentration of the buffer is about 0.01 mg / mL, 0.02 mg / mL, 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, 0.08 mg / mL, 0.09 mg / mL, about 0.10 mg / mL, 0.11 mg / mL, 0.12 mg / mL, 0.13 mg / mL, about 0.14 mg / mL, mL, about 0.15 mg / mL, about 0.16 mg / mL, about 0.17 mg / mL, 0.18 mg / mL, 0.19 mg / mL, about 0.20 mg / mL, about 0.25 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL, about 1.0 mg / mL mL, about 2.0 mg / mL, about 3.0 mg / mL, about 4.0 mg / mL, about 5.0 mg / mL, about 6.0 mg / mL, about 7.0 mg / mL, about 8.0 mg / mL, about 9.0 mg / mL, about 10.0 mg / mL, about 11.0 mg / mL, about 12.0 mg / mL, about 13.0 mg / mL, about 14.0 mg / mL, about 15.0 mg / mL, about 16.0 mg / mL In some embodiments, the preparation comprises about 0.5-5.0 mg / mL histidine buffer. In some embodiments, the buffer is a histidine buffer comprising about 0.1-1.5 mg / mL L-histidine and about 1-3 mg / mL L-histidine monohydrochloride. In some embodiments, the buffer is a histidine buffer comprising 1.12 mg / mL L-histidine and 2.67 mg / mL L-histidine monohydrochloride. In some embodiments, the buffer is a succinate buffer comprising about 0.5-5.0 mg / mL disodium succinate hexahydrate and about 0.1-1.0 mg / mL succinic acid. In some embodiments, the buffer is a succinate buffer comprising about 4.08 mg / mL disodium succinate hexahydrate and about 0.58 mg / mL succinic acid.In some embodiments, the buffer is a succinate buffer comprising about 2.362 mg / mL succinic acid.

[0201] In some embodiments, such as but not limited to, the polyol can have a molecular weight less than about 600kD (e.g., in the range of about 120-about 400kD), and contain multiple hydroxyls, including sugars (e.g., reducing and non-reducing sugars or mixtures thereof, sugars or carbohydrates), sugar alcohols, sugar acids or their salts or mixtures. Examples of non-reducing sugars include but are not limited to sucrose, trehalose and mixtures thereof. In some embodiments, the polyol is mannitol, trehalose, sorbitol, erythritol, isomaltose, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol or mixtures thereof. In other embodiments, such as but not limited to, the polyol can be a monosaccharide, a disaccharide or a polysaccharide, or any of the aforementioned mixtures. For example, but not limited to, the sugar or carbohydrate can be fructose, glucose, mannose, sucrose, sorbitol, xylose, lactose, maltose, sucrose, dextran, pullulan, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble dextran or a mixture thereof.

[0202] In some embodiments, the polyol is selected from mannitol, trehalose, sorbitol, erythritol, isomalt, lactitol, maltitol, xylitol, glycerol, lactitol, propylene glycol, polyethylene glycol, inositol, fructose, glucose, mannose, sucrose, sorbitol, xylose, lactose, maltose, sucrose, dextran, amylopectin, dextrin, cyclodextrin, soluble starch, hydroxyethyl starch, water-soluble glucan or its mixture. In some embodiments, the polyol is sucrose or trehalose. In some embodiments, the polyol is sucrose.

[0203] The concentration of the polyol in the formulation ranges from about 1 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, or about 1 mg / mL to about 120 mg / mL. Preferably, the concentration of the polyol in the formulation is from about 50 mg / mL to about 120 mg / mL, about 60 mg / mL to about 110 mg / mL, or about 80 mg / mL to about 90 mg / mL). For example, the concentration of the polyol in the formulation is about 0.5 mg / mL, about 1 mg / mL, about 2 mg / mL, about 2.5 mg / mL, about 3 mg / mL, about 3.5 mg / mL, about 4 mg / mL, about 4.5 mg / mL, about 5 mg / mL, about 5.5 mg / mL, about 6 mg / mL, about 6.5 mg / mL, about 7 mg / mL, about 7.5 mg / mL, about 8 mg / mL, about 8.5 mg / mL, about 9 mg / mL, about 9.5 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL about 65 mg / mL, about 70 mg / mL, about 75 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL 138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, or about 150 mg / mL.

[0204] In some embodiments, the polyol is sucrose at a concentration of about 1 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, or about 1 mg / mL to about 120 mg / mL. Preferably, the concentration of sucrose in the formulation is about 50 mg / mL to about 120 mg / mL, about 60 mg / mL to about 110 mg / mL, or about 80 mg / mL to about 90 mg / mL. In some embodiments, the concentration of sucrose in the formulation is about 85 mg / mL. In some embodiments, the concentration of sucrose in the formulation is about 84 mg / mL. In some embodiments, the concentration of sucrose in the formulation is about 90 mg / mL. In some embodiments, the polyol is trehalose at a concentration of about 84 mg / mL, about 85 mg / mL, or about 90 mg / mL.

[0205] As used in the present invention, surfactants can change the surface tension of liquid antibody formulations. In certain embodiments, the surfactant reduces the surface tension of liquid antibody formulations. In other embodiments, the surfactant can help improve the stability of any antibody in the formulation. The surfactant can also reduce the aggregation of formulated antibodies (e.g., during transportation and storage) and / or minimize the formation of particles in the formulation and / or reduce adsorption (e.g., adsorption to a container). For example, the surfactant can also improve the stability of antibodies during and after the freeze / thaw cycle. For example, but not limited to, the surfactant can be polysorbate, poloxamer, triton, sodium dodecyl sulfate, sodium laurel sulfate, polysorbate 60, polysorbate 70, polysorbate 100, polysorbate 200, polysorbate 300, polysorbate 400, polysorbate 500, polysorbate 600, polysorbate 700, polysorbate 800, polysorbate 900, polysorbate 1 ... sulfate), sodium octyl glucoside, lauryl sulfobetaine, myristyl sulfobetaine, linoleyl sulfobetaine, stearoyl sulfobetaine, lauryl sarcosine, myristyl sarcosine, linoleyl sarcosine, stearoyl sarcosine, linoleyl betaine, myristyl betaine, cetyl betaine, lauroamidopropyl betaine, cocamidopropyl betaine, linoleamidopropyl The surfactant may be PEG-500, PEG-600, PEG-700, PEG-800, PEG-900, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000, PEG-1000

[0206] The concentration of the surfactant typically ranges from about 0.01 mg / mL to about 10 mg / mL, about 0.01 mg / mL to about 5.0 mg / mL, about 0.01 mg / mL to about 2.0 mg / mL, about 0.01 mg / mL to about 1.5 mg / mL, about 0.01 mg / mL to about 1.0 mg / mL, about 0.01 mg / mL to about 0.5 mg / mL, about 0.01 mg / mL to about 0.4 mg / mL, about 0.01 mg / mL to about 0.3 mg / mL, about 0.01 mg / mL to about 0.2 mg / mL, about 0.01 mg / mL to about 0.15 mg / mL, about 0.01 mg / mL to about 0.1 mg / mL, about 0.01 mg / mL to about 0.05 mg / mL, about 0.1 mg / mL to about 1 mg / mL, about 0.1 mg / mL to about 0.5 mg / mL, or about 0.1 mg / mL to about 0.3 mg / mL. Further preferably, the concentration of the surfactant is about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.1 mg / mL, about 0.15 mg / mL, about 0.2 mg / mL, about 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, about 0.8 mg / mL, about 0.9 mg / mL or about 1 mg / mL.

[0207] In some embodiments, the polysorbate is polysorbate 80 at a concentration ranging from about 0.1 mg / mL to about 0.3 mg / mL (eg, 0.2 mg / mL).

[0208] Chelating agents as used in the present invention reduce the formation of reduced oxygen species, reduce the formation of acidic species (e.g., deamidation), reduce antibody aggregation, and / or reduce antibody fragmentation, and / or reduce antibody oxidation in the formulations of the present invention. For example, the chelating agent can be a multidentate ligand that forms at least one bond (e.g., covalent, ionic or other) with a metal ion and acts as a stabilizer to complex with substances that might otherwise promote instability.

[0209] In some embodiments, the chelating agent can be selected from aminopolycarboxylic acids, hydroxyaminocarboxylic acids, N-substituted glycine, 2-(2-amino-2-oxyethyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, nicotinamide and deoxycholate and mixtures thereof. In some embodiments, the chelating agent is selected from ethylenediaminetetraacetic acid (EDTA), diethylenetriaminepentaacetic acid (DET), 5-(2-amino-2-oxyethyl)aminoethanesulfonic acid (BES), deferoxamine (DEF), citric acid, nicotinamide and deoxycholate and mixtures thereof. (DTPA), nitrilotriacetic acid (NTA), N-2-acetamido-2-iminodiacetic acid (ADA), bis(aminoethyl)glycol ether, N,N,N',N'-tetraacetic acid (EGTA), trans-diaminocyclohexanetetraacetic acid (DCTA), glutamic acid, and aspartic acid, N-hydroxyethyliminodiacetic acid (HIMDA), N,N-bis-hydroxyethylglycine (bicine) and N-(trihydroxymethylmethyl)10glycine (tricine), glycylglycine, sodium deoxycholate, ethylenediamine, propylenediamine, diethylenetriamine, triethylenetetramine (trien), disodium edetate dihydrate (or disodium EDTA dihydrate or disodium EDTA), calcium EDTA oxalate, malate, citric acid, citric acid monohydrate, and trisodium citrate-dihydrate, 8-hydroxyquinolate, amino acids, histidine, cysteine, methionine, peptides, polypeptides and proteins, and mixtures thereof. In some embodiments, the chelating agent is selected from salts of EDTA, including but not limited to dipotassium edetate, disodium edetate, disodium calcium edetate, sodium edetate, trisodium edetate and potassium edetate; and a suitable salt of deferoxamine (DEF) is deferoxamine mesylate (DFM), or a mixture thereof. Where possible, the chelating agent used in the present invention may exist as a free acid or free base form or a salt form of the compound, and may also exist as an anhydrous, solvated or hydrated form of the compound or the corresponding salt.

[0210] In some embodiments, the chelating agent is EDTA. In some embodiments, the chelating agent is a salt of EDTA. In some embodiments, the chelating agent is edetate disodium dihydrate.

[0211] The concentration of the chelating agent typically ranges from about 0.01 mg / mL to about 50 mg / mL, from about 0.1 mg / mL to about 10.0 mg / mL, from about 5 mg / mL to about 15.0 mg / mL, from about 0.01 mg / mL to about 1.0 mg / mL, from about 0.02 mg / mL to about 0.5 mg / mL, from about 0.025 mg / mL to about 0.075 mg / mL. Further preferably, the concentration of the chelating agent typically ranges from about 0.01 mM to about 2.0 mM, from about 0.01 mM to about 1.5 mM, from about 0.01 mM to about 0.5 mM, from about 0.01 mM to about 0.4 mM, from about 0.01 mM to about 0.3 mM, from about 0.01 mM to about 0.2 mM, from about 0.01 mM to about 0.15 mM, from about 0.01 mM to about 0.1 mM, from about 0.0 1mM to about 0.09mM, about 0.01mM to about 0.08mM, about 0.01mM to about 0.07mM, about 0.01mM to about 0.06mM, about 0.01mM to about 0.05mM, about 0.01mM to about 0.04mM, about 0.01mM to about 0.03mM, about 0.01mM to about 0.02mM, about 0.02 or about 0.05mM to about 0.01mM. Preferably, the concentration of the chelating agent can be about 0.01 mg / mL, about 0.02 mg / mL, about 0.025 mg / mL, about 0.03 mg / mL, about 0.04 mg / mL, about 0.05 mg / mL, about 0.06 mg / mL, about 0.07 mg / mL, about 0.075 mg / mL, about 0.08 mg / mL, about 0.09 mg / mL, about 0.10 mg / mL or about 0.20 mg / mL. Further preferably, the concentration of the chelating agent is about 0.025 mg / mL, about 0.03 mg / mL, about 0.035 mg / mL, about 0.04 mg / mL, about 0.045 mg / mL, about 0.05 mg / mL, about 0.055 mg / mL, about 0.06 mg / mL, about 0.065 mg / mL, about 0.07 mg / mL or about 0.075 mg / mL. Most preferably, the concentration of the chelating agent is about 0.05 mg / mL.

[0212] In some embodiments, the chelating agent is edetate disodium dihydrate at a concentration of about 0.05 mg / mL. In some embodiments, the chelating agent is ethylenediaminetetraacetic acid (EDTA) at a concentration of about 0.05 mg / mL.

[0213] In some embodiments, the pH may be in the range of about pH 5.0 to about 6.6, preferably about pH 5.0 to 6.5 or about 5.0 to 6.0, and most preferably pH 5.2 to 5.8. In some embodiments, the pH of the formulation of the present invention may be in the range selected from any one of about pH 5.2, 5.3, 5.4, 5.5 or 5.6 and about any one of pH 6.5, 6.4, 6.3, 6.2, 6.1, 6.0, 5.9, 5.8 or 5.7. In some embodiments, the pH can be selected from a pH value of about any one of pH 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5. In some embodiments, the pH is pH 5.8 + / - 0.5, and in some embodiments, the pH is pH 5.8 + / - 0.3.

[0214] In some embodiments, the formulation may contain a preservative. Preferably, the preservative is selected from phenol, m-cresol, benzyl alcohol, benzalkonium chloride, benzethonium chloride, phenoxyethanol and methyl paraben.

[0215] The concentration of the preservative typically ranges from about 0.001 mg / mL to about 50 mg / mL, about 0.005 mg / mL to about 15.0 mg / mL, about 0.008 mg / mL to about 12.0 mg / mL, or about 0.01 mg / mL to about 10.0 mg / mL. Preferably, the concentration of the preservative can be about 0.1 mg / mL, 0.2 mg / mL, 0.3 mg / mL, about 0.4 mg / mL, about 0.5 mg / mL, about 0.6 mg / mL, about 0.7 mg / mL, 0.8 mg / mL, 0.9 mg / mL about 1.0 mg / mL, 2.0 mg / mL, 3.0 mg / mL, about 4.0 mg / mL, about 5.0 mg / mL, about 6.0 mg / mL, about 7.0 mg / mL, 8.0 mg / mL, 9.0 mg / mL, about 9.1 mg / mL, about 9.2 mg / mL, 9.3 mg / mL, 9.4 mg / mL, 9.5 mg / mL, 9.6 mg / mL, 9.7 mg / mL, 9.8 mg / mL, 9.9 mg / mL, 10.0 mg / mL. Most preferably, the concentration of the preservative is about 0.1 mg / mL or 9.0 mg / mL.

[0216] In some embodiments, the formulation is free of preservatives.

[0217] In some embodiments, the antibody can be selected from monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, ScFv, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single chains (ScFv), mutants thereof, fusion proteins comprising antibody portions (e.g., domain antibodies), humanized antibodies, human antibodies, and any other modified configurations of immunoglobulin molecules comprising antigen recognition sites of desired specificity, including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. The antibody can be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody is a human antibody. Preferably, the antibody is isolated, and further preferably, it is substantially pure. When the antibody is an antibody fragment, this preferably retains the functional characteristics of the original antibody, i.e., ligand binding and / or antagonist or agonist activity.

[0218] In some embodiments, the antibody heavy chain constant region may be from any type of constant region, such as IgG, IgM, IgD, IgA and IgE; and any isotype, such as IgG1, IgG2, IgG3 and IgG4. Preferably, the antibody is an IgG1 antibody.

[0219] According to a further aspect of the invention, a formulation is provided comprising or consisting of: about 15 mg / ml to about 250 mg / ml of a tissue factor pathway inhibitor (TFPI) antibody; about 1 mM to about 100 mM of a buffer; about 1 mg / ml to about 300 mg / ml of a polyol; about 0.01 mg / ml to about 10 mg / ml of a surfactant; about 0.01 mg / ml to about 50.0 mg / ml of a chelating agent; and wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the antibody comprises: (i) a heavy chain variable region (VH), comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO:13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO:14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO:15, and (ii) a light chain variable region (VL), comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the anti-TFPI antibody comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 18 and a VL region comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or marstacimab). In some embodiments, the buffer is a histidine buffer, the polyol is sucrose or trehalose, the surfactant is a polysorbate (e.g., polysorbate 80), and / or the chelating agent is EDTA or edetate disodium dihydrate. In some embodiments, the buffer is a histidine buffer, the polyol is sucrose, the surfactant is polysorbate 80, and the chelating agent is edetate disodium dihydrate.

[0220] In some embodiments, formulations of anti-TFPI antibody drug substances disclosed herein can be stored in sterilized ethylene vinyl acetate (EVA) bags having ethylene vinyl acetate mono-material (EVAM) product contact surfaces. In some embodiments, formulations of anti-TFPI antibody drug substances disclosed herein can be stored in sterilized high-density polyethylene bottles (HDPE). In some embodiments, formulations of anti-TFPI antibody drug substances disclosed herein can be stored in stainless steel containers. In some embodiments, formulations of anti-TFPI antibody drug products disclosed herein can be stored in sterilized vials or pre-filled syringes (PFS). In some embodiments, formulations of anti-TFPI antibody drug products disclosed herein can be stored in pre-filled pens (PFP).

[0221] According to a further aspect of the invention, a formulation is provided comprising or consisting of: about 100 mg / mL, about 110 mg / mL, about 115 mg / mL, about 120 mg / mL, about 130 mg / mL, about 140 mg / mL, about 150 mg / mL, about 158 ​​mg / mL, about 160 mg / mL or about 200 mg / mL of a tissue factor pathway inhibitor antibody (e.g., a human anti-TFPI antibody); about 1 mM to about 100 mM buffer; about 1 mg / mL to about 300 mg / mL of a polyol; about 0.01 mg / mL to about 10 mg / mL of a surfactant; about 0.01 mg / mL to about 1.0 mg / mL of a chelator; and wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the antibody comprises: (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO:13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO:14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO:15, and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or matasinumab).

[0222] According to a further aspect of the invention, a formulation is provided comprising or consisting of: about 50 mg / mL to about 160 mg / mL of a tissue factor pathway inhibitor antibody (e.g., a human anti-TFPI antibody); about 20 mM of a buffer; about 1 mg / ml to about 120 mg / mL of a polyol; about 0.1 mg / mL to about 0.3 mg / mL of a surfactant; about 0.025 mg / mL to about 0.075 mg / mL of a chelator; and wherein the formulation has a pH of about 5.0 to about 6.0. In some embodiments, the antibody comprises: (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 15, and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO: 8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO: 9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO: 10. In some embodiments, the anti-TFPI antibody comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 18 and a VL region comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or matasinumab). In some embodiments, the buffer is a histidine buffer, the polyol is sucrose, the chelating agent is edetate disodium dihydrate, and / or the surfactant is polysorbate 80. In some embodiments, the concentration of the antibody is 100 mg / mL, 115 mg / mL, 150 mg / mL, or 158 mg / mL.

[0223] In some embodiments, an aqueous formulation is provided comprising or consisting of: about 150 mg / mL of a tissue factor pathway inhibitor antibody (e.g., a human anti-TFPI antibody); about 20 mM histidine buffer; about 85 mg / mL of sucrose; about 0.2 mg / mL of polysorbate 80; about 0.05 mg / mL of edetate disodium dihydrate; and wherein the formulation has a pH of about 5.8. In some embodiments, the antibody comprises: (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO:13; (b) a VH complementarity determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO:14; and (c) a VH complementarity determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO:15, and (ii) a light chain variable region (VL) comprising: (a) a VL complementarity determining region 1 (CDR-L1) comprising the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) comprising the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the anti-TFPI antibody comprises: a VH region comprising the amino acid sequence of SEQ ID NO: 18 and a VL region comprising the amino acid sequence of SEQ ID NO: 11. In some embodiments, the anti-TFPI antibody comprises: a heavy chain comprising the amino acid sequence of SEQ ID NO: 19 and a light chain comprising the amino acid sequence of SEQ ID NO: 12. In some embodiments, the anti-TFPI antibody is TFPI-106 (also known as PF-06741086 or matasinumab).

[0224] In some embodiments, the formulations as described herein have a shelf life of at least or more than about 1 month, about 3 months, about 6 months, about 12 months, about 18 months, about 24 months, about 30 months, about 36 months, about 42 months, about 48 months, or about 60 months (e.g., at -20°C, at 5°C, 25°C, or 40°C). For example, in some embodiments, the formulations of the invention have a shelf life of at least about 1 month, about 3 months, about 6 months, 7 months, 8 months, 9 months, 10 months, 11 months, 12 months, 13 months, 14 months, 15 months, 16 months, 17 months, 18 months, 19 months, 20 months, 21 months, 22 months, 23 months, 24 months, 25 months, 26 months, 27 months, 28 months, 29 months, 30 months, 31 months, 32 months, 33 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, 60 months, 61 months, 62 months, 63 months, 64 months, 65 months, 66 months, 67 months, 68 months, 69 months, 70 months, 71 months, 72 months, 73 months, 74 months, 75 months, 76 months, 77 months, 78 months, 79 months 3 months, 34 months, 35 months, 36 months, 37 months, 38 months, 39 months, 40 months, 41 months, 42 months, 43 months, 44 months, 45 months, 46 months, 47 months, 48 ​​months, 49 months, 50 months, 51 months, 52 months, 53 months, 54 months, 55 months, 56 months, 57 months, 58 months, 59 months, or 60 months (e.g., at -20°C, at 5°C, 25°C, or 40°C). In some embodiments, the formulations described herein have a shelf life of at least about 24 months at 5±3°C.

[0225] In some embodiments, the formulations as described herein have less than about 7% HMMS (e.g., as measured by size exclusion HPLC) for up to 1 month, 2 months, 3 months, 4 months, 5 months, or 6 months at 40°C / 75% RH. In some embodiments, the formulations as described herein have less than about 3% HMMS (e.g., as measured by size exclusion HPLC) for up to 1 month, 2 months, or 3 months. In some embodiments, the formulations as described herein have less than about 2% HMMS (e.g., as measured by size exclusion HPLC) for up to 1 month.

[0226] In some embodiments, the formulations as described herein have less than about 2% HMMS (e.g., as measured by size exclusion HPLC) at 5±3°C for up to 24 months. In some embodiments, the formulations as described herein have less than about 1% HMMS (e.g., as measured by size exclusion HPLC) at 5±3°C for up to 1 month, 2 months, 3 months, 4 months, 5 months, 6 months, 12 months, 18 months, or 24 months. In some embodiments, the formulations as described herein have less than about 2% fragments (e.g., as measured by reducing capillary gel electrophoresis) at 5±3°C for up to 24 months.

[0227] Unless otherwise stated, the concentrations listed herein are at ambient conditions, ie, at 25°C and atmospheric pressure.

[0228] Methods of using anti-TFPI antibody preparations

[0229] The formulations described herein can be used for a variety of applications, including but not limited to therapeutic treatment methods. The treatment method comprises administering a formulation of the invention to a subject in need thereof.

[0230] Exemplary therapeutic uses of the formulations of the present invention include shortening bleeding time in a subject in need thereof, treating or preventing coagulation defects or blood disorders (e.g., hemophilia A, hemophilia B, hemophilia C, von Willebrand Disease (vWD), factor VII deficiency, factor VIII, factor IX or factor XI deficiency), treating or preventing thrombocytopenia and treating or preventing platelet disorders (abnormal platelet function or number). The formulations described herein may also be used to treat uncontrolled bleeding (indications, uncontrolled bleeding in trauma and hemorrhagic stroke). The formulations described herein may also be used for prophylactic treatment (e.g., treating or preventing bleeding prior to surgery).

[0231] In particular, the formulations described herein can be used to treat coagulation deficiencies or coagulation defects or coagulation disorders. For example, the formulations described herein can be used to reduce or inhibit the interaction of TFPI with FXa, or reduce TFPI-dependent inhibition of TF / FVIIa / FXa activity.

[0232] Therefore, in some embodiments, the subject suffers from or is susceptible to coagulation defects or blood disorders, such as the following: In some embodiments, the subject suffers from or is susceptible to hemophilia A, B or C. In some embodiments, the subject suffers from or is susceptible to hemophilia A or B. In some embodiments, the subject suffers from or is susceptible to hemophilia A, and has neutralizing antibodies (i.e., inhibitors) for coagulation factor VIII. In some embodiments, the subject suffers from or is susceptible to hemophilia B, and has neutralizing antibodies (i.e., inhibitors) for coagulation factor IX. In some embodiments, the subject suffers from or is susceptible to hemophilia C, and has neutralizing antibodies (i.e., inhibitors) for coagulation factor XI. In some embodiments, the subject suffers from or is susceptible to von Willebrand's disease (vWD). In some embodiments, the subject suffers from or is susceptible to platelet disorders. In some embodiments, the subject suffers from or is susceptible to coagulation factor VII deficiency. In some embodiments, the subject suffers from or is susceptible to coagulation factor XI deficiency.

[0233] The formulations comprising the TFPI antagonist antibodies or antigen binding portions described herein can be used in combination with coagulants. The present invention provides separate, simultaneous or sequential administration of the antibodies of the present invention and coagulants. In some embodiments, the formulations described herein further comprise a coagulant. Examples of coagulants include, but are not limited to, factor VIIa, factor VIII, factor IX, tranexamic acid, and bypass agents (e.g., anti-inhibitor coagulation complex or FEIBA).

[0234] In some embodiments, there is provided use of a formulation of the present invention for the preparation of a medicament for shortening bleeding time.

[0235] In some embodiments, the preparation of the present invention can be directly administered into the bloodstream, muscle, tissue, fat or the internal organs of the subject. Suitable modes for parenteral administration include intravenous, intraocular, intravitreal, intraarterial, intraperitoneal, intrathecal, intraventricular, intraurethral, ​​intrasternal, intracranial, intramuscular, intraossial, intradermal and subcutaneous. Suitable devices for parenteral administration include needles (including microneedles, microinjections (microprojection), soluble needles and other micropore formation technologies) syringes, needle-free syringes and infusion techniques. In some embodiments, the preparation of the present invention is administered intravenously or subcutaneously to the subject. In some embodiments, the preparation of the present invention is administered subcutaneously to the subject. In some embodiments, the preparation of the present invention is administered intravenously to the subject.

[0236] In some embodiments, the administration mode of the preparation of the present invention includes once a week, once every two weeks, once every three weeks, once every four weeks, once every five weeks, once every six weeks, once every seven weeks, once every eight weeks, once every nine weeks, once every ten weeks, once every fifteen weeks, once every twenty weeks, once every twenty-five weeks, or once every twenty-six weeks. In some embodiments, the preparation described herein is administered once a month, once every two months, once every three months, once every four months, once every five months, or once every six months. In some embodiments, the preparation described herein is administered every day or weekly (i.e., once a week). The progress of this therapy is easily monitored by conventional techniques and determinations. The dosage administration regimen can vary over time.

[0237] In some embodiments, the formulations described herein are administered with an initial antibody dose of about 300 mg, followed by subsequent weekly (i.e., weekly) doses of about 150 mg. In some embodiments, the formulations described herein are administered weekly (i.e., weekly) with an antibody dose of about 150 mg. In some embodiments, the formulations described herein are administered weekly (i.e., weekly) with an antibody dose of about 300 mg. In some embodiments, the formulations described herein are administered weekly (i.e., weekly) with an antibody dose of about 450 mg.

[0238] For the purposes of the present invention, the appropriate dosage of the drug will depend on the antibody employed, the type and severity of the condition to be treated, the administration of the agent for preventive or therapeutic purposes, previous therapy, the patient's clinical history and response to the agent, and the discretion of the attending physician. Typically, the clinician will administer the drug until a dosage is reached that achieves the desired result. The dosage can be determined empirically.

[0239] The following examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way. Indeed, various modifications of the present invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. Example

[0240] Example 1. Stability study of anti-TFPI antibodies

[0241] This example illustrates the stability of formulations containing anti-TFPI antibodies in high density polyethylene (HDPE) bottles, vials, and prefilled syringes.

[0242] 100 mg / mL, 115 mg / mL and 150 mg / mL of anti-TFPI antibody were prepared in 20 mM histidine, 85 mg / mL sucrose, 0.05 mg / mL edetate disodium dihydrate, 0.2 mg / mL polysorbate 80 (pH 5.8). In this example, the anti-TFPI antibody used has a heavy chain variable region having an amino acid sequence of SEQ ID NO: 18 and a light chain variable region having an amino acid sequence of SEQ ID NO: 11.

[0243] 115 mg / mL of anti-TFPI antibody was filled into HDPE bottles and stored at -20±5°C and 5±3°C for a duration of at least 3 months (ie 12 weeks).

[0244] 100 g / mL and 150 g / mL of the anti-TFPI antibody were filled into Type I glass vials, sealed with coated stoppers, capped with aluminum seals, and stored in an inverted orientation at 5±3°C, 25°C / 60% relative humidity (RH), and 40°C / 75% relative humidity (RH) for a duration of at least 3 months (i.e., 12 weeks). In addition, 150 mg / mL of the anti-TFPI antibody was filled into 2.25 mL and 1 mL Type I glass prefilled syringes (PFS), stoppered with coated plungers, and stored in a horizontal orientation at 5±3°C, 25°C / 60% RH, and 40°C / 75% RH for a duration of at least 3 months (i.e., 12 weeks).

[0245] The samples are then analyzed for quality attributes that are typically used to monitor protein degradation. For example, the analysis consists of size exclusion high performance liquid chromatography (SE-HPLC) for evaluation of high molecular weight species (HMMS), reducing and non-reducing capillary gel electrophoresis (CGE) for evaluation of purity and fragmentation, and imaging capillary isoelectric focusing (iCE) for evaluation of charge heterogeneity, such as acidic, basic, and major species. The analysis also includes the use of inhibition assays to evaluate the relative efficacy of anti-TFPI antibodies.

[0246] Specific analytical procedures, including pharmacopoeial and non-pharmacopoeial methods, were used to evaluate the characteristics, purity, and relative efficacy of anti-TFPI antibodies. A description of the analytical procedures is provided below. The results are summarized in Tables 1-8.

[0247] Appearance (clarity and color) : The clarity and colour of the drug substance were evaluated according to the current European Pharmacopoeia procedures, Ph. Eur. 2.2.1 and Ph. Eur. 2.2.2, respectively.

[0248] pH : According to current USP procedures <791> The pH of the drug substance was analyzed.

[0249] Protein concentration by UV : Using a spectrophotometer, the absorbance at 280 nm is used to determine the concentration of the test sample. The specific absorption coefficient or absorbance (a280) of PF-06741086 (given below) is used for concentration calculations.

[0250]

[0251] Imaging Capillary Isoelectric Focusing (iCE):The imaging capillary isoelectric focusing (iCE) method is used to evaluate charge heterogeneity. iCE separates protein species based on their charge differences in a pH gradient (generated by ampholytes under the influence of an electric field) and is used to monitor product charge heterogeneity. Protein charge species are focused within the capillary under a DC voltage and detected at 280 nm using full capillary imaging.

[0252] Size Exclusion HPLC (SE-HPLC) : SE-HPLC method was used to determine product purity. The test sample was diluted and injected into a size exclusion column. The content of high molecular weight species (HMMS) and monomers was reported as the total area percentage of all protein-related peaks.

[0253] Reduced capillary gel electrophoresis (CGE) : This method is used to determine the purity of reduced proteins. The sample is denatured with SDS and heated in the presence of a reducing agent. The protein is reduced to heavy and light chains, which are separated by electrophoresis in a capillary containing a sieving medium and detected using UV spectroscopy. The separation allows for quantitative resolution of heavy and light chains and size-related impurities. Purity is reported as a total percentage of heavy and light chains.

[0254] Non-reducing capillary gel electrophoresis (CGE) : This method is used to determine the purity of intact proteins. The sample is denatured with SDS and heated in the presence of an alkylating agent. The proteins are separated by electrophoresis in capillaries containing a sieving medium and detected using UV spectroscopy. The separation allows for quantitative resolution of intact IgG as well as size-related impurities. Purity is reported as a percentage of IgG.

[0255] Lysyl endoproteinase (Lys-C) mapping :This method is used to determine the percent oxidation at the Fc-peptide containing methionine residues in anti-TFPI antibodies. Anti-TFPI antibody samples are digested into peptide fragments using lysyl endoproteinase (Lys-C). The resulting peptide fragments are separated by reverse phase HPLC. The separation of Fc-peptide and oxidized Fc-peptide allows the determination and monitoring of the percent oxidation of anti-TFPI antibodies.

[0256] Biological activity by inhibition assay : The inhibition assay method was used to evaluate the in vitro biological activity of PF-06741086. The inhibition assay showed that PF-06741086 was able to inhibit TFPI:FXa complex formation, resulting in an increase in free human coagulation factor Xa (FXa), which in turn made free FXa available for coagulation activity.

[0257] TFPI is added to a microtiter plate. Dilutions of the PF-06741086 reference material and the test sample are added to the microtiter plate to allow binding of the antibody to its target. FXa is then added to the microtiter plate and binds to available TFPI that is not bound by PF-06741086, and forms a TFPI:FXa complex. Free FXa is detected using the chromogenic substrate Spectrozyme FXa. After addition of the substrate, the colorimetric response is measured spectrophotometrically.

[0258] The method of data analysis includes nonlinear curve fitting and evaluation of the parallelism between the standard curve and the sample curve. The sample efficacy is determined by the shift of the IC50 between the parallel curves. The specific activity of the sample (expressed in units per PF-06741086) is calculated by multiplying the relative efficacy value by the number of units assigned to one milligram (mg) of the reference material.

[0259] Table 1

[0260]

[0261] NMT = Not More Than

[0262] NS = Not Scheduled.

[0263] Table 2

[0264]

[0265] NS = Not Scheduled.

[0266] Table 3

[0267]

[0268] NMT = Not More Than

[0269] NS = Not Scheduled.

[0270] Table 4

[0271]

[0272] NS = Not Scheduled.

[0273] Table 5

[0274]

[0275] NS = Not Scheduled.

[0276] Table 6

[0277]

[0278] NS = Not Scheduled.

[0279] Table 7

[0280]

[0281] NS = Not Scheduled.

[0282] Table 8

[0283]

[0284] NS = Not Scheduled.

[0285] Data from this study showed that high concentrations (100 mg / mL to 150 mg / mL) of anti-TFPI antibodies were stable in a 20 mM histidine, 85 mg / mL sucrose, 0.05 mg / mL edetate disodium dihydrate, 0.2 mg / mL polysorbate 80 (pH 5.8) formulation. The results also showed that different container / closure systems (HDPE bottles, vials, PFS) did not affect the stability of the anti-TFPI antibodies.

[0286] The expected storage condition of 5±3°C, accelerated storage condition of 25°C / 60% RH, and heat stress storage condition of 40°C / 75% RH were evaluated in the study. Under long-term storage condition of 5±3°C, the anti-TFPI antibody was shown to be stable for up to 24 months, with no significant differences observed for any product quality attributes.

[0287] A slight increase in % HMMS was observed after 3 months at 25°C / 60% RH and 1 month at 40°C / 75% RH. Degradation was observed at 3 months at 40°C / 75% RH, but the magnitude was less than that seen with other IgG1 antibodies, such as anti-VEGF antibodies. Table 9 compares the % HMMS data for anti-TFPI and anti-VEGF antibodies at 40°C / 75% RH. A qualified product-specific SE-HPLC method was used to quantify the % HMMS for each antibody.

[0288] Table 9

[0289]

[0290] * Data for anti-VEGF antibodies obtained from Table 11 of US2018 / 0000933.

[0291] Despite observed degradation, anti-TFPI antibodies maintained activity for up to 24 months at 25°C / 60% RH and 12 months at 40°C / 75% RH in a 20 mM histidine, 85 mg / mL sucrose, 0.05 mg / mL edetate disodium dihydrate, 0.2 mg / mL polysorbate 80 (pH 5.8) formulation.

[0292] Example 2. Study on the robustness and stability of anti-TFPI antibody formulations

[0293] This example illustrates the stability of 150 mg / mL anti-TFPI antibody in a target formulation (control formulation) and anti-TFPI antibodies formulated with high or low excipient levels and pH. The formulations evaluated are shown in Table 10. The concentration of each component was confirmed by analytical testing. In this example, the anti-TFPI antibody (TFPI-106) used had a heavy chain variable region having the amino acid sequence of SEQ ID NO: 18 and a light chain variable region having the amino acid sequence of SEQ ID NO: 11.

[0294] Table 10: Excipients and pH ranges evaluated in formulation robustness studies

[0295]

[0296] 1 Each formulation contained 150 mg / ml anti-TFPI antibody.

[0297] The formulations were stored for 6 months under the expected storage conditions of 5 ± 3 ° C, the accelerated storage conditions of 25 ° C / 60% RH, and the heat stress storage conditions of 40 ° C / 75% RH. The quality attributes of the samples were then analyzed using the methods described herein, which are generally used to monitor protein degradation. For example, the analysis consists of: size exclusion high performance liquid chromatography (SE-HPLC) for evaluating high molecular weight substances (HMMS), reduced and non-reduced capillary gel electrophoresis (CGE) for evaluating purity and fragmentation, and imaging capillary isoelectric focusing (iCE) for evaluating charge heterogeneity, such as acidity, alkalinity, and main substances. The analysis also includes the use of inhibition assays to evaluate the relative efficacy of anti-TFPI antibodies. Specific analytical procedures, including pharmacopoeial and non-pharmacopoeial methods, are used to evaluate the characteristics, purity, and relative efficacy of anti-TFPI antibodies. The results are summarized in Tables 11-19.

[0298] Table 11. pH results for 6 months at 5°C, 25°C, and 40°C

[0299]

[0300] Table 12. Protein concentration results by UV for up to 6 months at 5°C, 25°C and 40°C

[0301]

[0302] Table 13. SE-HPLC results for up to 6 months at 5°C, 25°C, and 40°C

[0303]

[0304] Table 14. Non-reduced CGE results for 6-month data at 5°C, 25°C, and 40°C

[0305]

[0306] Table 15. CGE results for the reduction of data for up to 6 months at 5°C, 25°C, and 40°C

[0307]

[0308] Table 16. iCE results for 6 months of data at 5°C, 25°C, and 40°C

[0309]

[0310] Table 17. Methionine oxidation results for 6 months at 5°C, 25°C, and 40°C

[0311]

[0312] Table 18. Appearance Results for 6 Months at 5°C, 25°C and 40°C**

[0313]

[0314] *EFVP: Essentially free of visible particles

[0315] ** Clarity and colour evaluated according to current European Pharmacopoeia procedures Ph. Eur. 2.2.1 and Ph. Eur. 2.2.2 respectively

[0316] † Ref. II, Ref. III, and Ref. IV are equal to 6 NTU, 18 NTU, and 30 NTU, respectively.

[0317] Table 19. Inhibition assay results for up to 6 months of data at 5°C, 25°C, and 40°C

[0318]

[0319] The data shown in Tables 11-19 and described below demonstrate that anti-TFPI antibody formulations containing either high excipients or low excipients or pH levels were stable after 6 months under both real-time stability conditions.

[0320] Appearance

[0321] After 6 months storage at 2-8°C and 25°C (Table 18), no significant changes in color, clarity and visible particles were observed for most formulations (AE). After 6 months storage at 2-8°C and 25°C, a significant increase in visible particles was observed for formulation F without PS80.

[0322] After 6 months storage at 40°C, significant changes in solution color were observed in all formulations (AF). Based on previous stability studies, this result after storage under accelerated stress conditions was expected. These effects were most significant for formulations C and E, which contained higher excipient concentrations relative to the target formulation. No major changes in solution clarity were observed. For formulations AE, visible particles were occasionally observed, however, these may be attributed to sample handling. After 6 months at 40°C, a significant increase in visible particles was observed for formulation F without PS80, although it was noted that the increase was not as dramatic as for the 2-8°C and 25°C storage conditions. This finding highlights the importance of including PS80 in drug formulations.

[0323] pH

[0324] After 6 months storage at 2-8°C, 25°C and 40°C (Table 11), no significant changes in pH were observed in all formulations (AF).

[0325] Protein concentration by UV

[0326] After 6 months storage at 2-8°C, 25°C, and 40°C (Table 12), protein concentration remained within the 150 mg / mL ± 15 mg / mL specification. Slight increases in protein concentration were observed for most formulations and storage conditions, which may be attributed to assay variability.

[0327] Size Exclusion Chromatography (SE-HPLC)

[0328] After 6 months storage at 2-8°C (Table 13), no significant changes in %HMMS and %monomer were observed for all formulations (AF). Note that during storage at 2-8°C, peaks corresponding to low molecular weight species were not consistently observed in all formulations.

[0329] After 6 months storage at 25° C., a slight increase of 1-2% in both %HMMS and %LMMS and a corresponding decrease in %Monomer were observed for all formulations (AF). All %HMMS results were below the acceptance criteria of 5%.

[0330] After 6 months storage at 40°C, significant increases in %HMMS and %LMMS and corresponding decreases in %monomer were observed for all formulations (AF). Formulations B (low excipient / high pH) and C (high excipient / low pH) showed the greatest decrease in %monomer, approximately 20%. Based on previous stability studies, these results under accelerated stress conditions were expected.

[0331] Non-reducing capillary gel electrophoresis (nrCGE)

[0332] After 6 months storage at 2-8°C (Table 14), no significant changes in %IgG, %Fragments and %Others were observed for all formulations (AF).

[0333] After 6 months storage at 25°C, a slight increase of 2-3% fragments and a corresponding decrease in % IgG was observed for all formulations (AF). No significant changes in % Others were observed.

[0334] After storage at 40°C for 6 months, a significant increase of >25% fragments and a corresponding decrease in % IgG were observed for all formulations (AF). The largest increases of >30% fragments were observed for formulations B (low excipient / high pH) and C (high excipient / low pH). This result is consistent with the SE-HPLC results. Slight increases in % others were observed in all formulations.

[0335] Reduced Capillary Gel Electrophoresis (rCGE)

[0336] After 6 months storage at 2-8°C (Table 15), no significant changes were observed for % Heavy + Light Chain (HC+LC), % Fragments and % Others for all formulations (AF).

[0337] After 6 months storage at 25°C, a slight increase of 1-2% fragments with a corresponding decrease in % HC+LC was observed for all formulations (AF). Only for formulations B and E was an additional slight increase of 1% observed.

[0338] After 6 months storage at 40°C, a significant increase of approximately 10% fragments and a corresponding decrease in % HC+LC were observed for all formulations (AF). The largest increase in % fragments was observed for formulation C (high excipient / low pH). Most formulations (A, C, D, and F) showed a slight increase of 2-3% others. In contrast, a significant increase of 7-8% in % others was observed for formulations B and E.

[0339] Isoelectric focusing capillary electrophoresis (iCE)

[0340] After 6 months storage at 2-8°C (Table 16), a slight increase in % acidic substances and a corresponding decrease in % major substances were observed in all formulations (AF), while no significant changes in % basic substances were observed.

[0341] After 6 months storage at 25°C, a significant increase of 10-20% acidic material and a corresponding decrease in % major material was observed in all formulations. A slight increase of 2-3% basic material was observed in most formulations. High pH formulations B and E showed the largest increase in % acidic material, about 18%. This finding can be confirmed by 9 This is explained by the well-described pH-dependence of asparagine deamidation.

[0342] After 6 months storage at 40°C, a significant increase of about 60% acidic material and a corresponding decrease in % major material was observed in all formulations. The largest increase in % acidic material was observed for formulation E, which can be explained by the pH-dependency of asparagine deamidation. Comparing the samples at T=0 and T=6 months, a net decrease in % basic material was observed in all formulations.

[0343] Methionine oxidation

[0344] After 6 months storage at 2-8°C (Table 17), no significant changes in %MetOx were observed in all formulations (AF). After 6 months storage at 25°C, a slight increase of 1-2% MetOx was observed in all formulations (AF). After 6 months storage at 40°C, a significant increase of approximately 10% MetOX was observed in all formulations. The largest increase in %MetOX, approximately 20%, was observed for Formulation C (high excipient / low pH).

[0345] Inhibition bioassay

[0346] After 6 months storage at 2-8°C and 25°C (Table 19), no significant changes in % relative efficacy were observed in all formulations. After 6 months storage at 40°C, a decreasing trend in relative efficacy was observed for formulations A, C, D and E, however, all results remained within the acceptable standards.

[0347] Thus, despite observed degradation, anti-TFPI antibodies maintained activity for 6 months under accelerated storage conditions of 25°C / 60% RH and heat stress storage conditions of 40°C / 75% RH in all formulations (Table 19).

[0348] Agitation research

[0349] Agitation studies were performed by filling 1 mL of Formulation AF into 1 mL long syringes with staked needles. The syringes were then subjected to 48 hours of agitation stress. Agitation studies were performed at ambient room temperature (~22°C) with the syringes placed in a horizontal orientation and agitated using an orbital shaker set at 300 rpm. Time zero (T0) controls were not exposed to shock stress.

[0350] After stirring at 300 rpm for 48 hours, no significant changes were observed in all formulations (AF) by SE-HPLC, nrCGE, rCGE, iCE and methionine oxidation. For formulation F without PS80, a significant increase in visible particles was observed, confirming the importance of PS80 in DP formulations. No changes in appearance were observed for most formulations (AE).

[0351] in conclusion

[0352] The goal of this study was to establish a design space and show that product quality was robust to changes in formulation composition and agitation. After 6 months of storage, for most formulations (AE), changes in formulation had minimal effect on DP stability at the expected storage temperature of 2-8°C. For formulations (F) without PS80, a significant increase in visible and subvisible particles was observed, confirming the importance of PS80 in DP formulations. At 25°C and 40°C, changes in product quality began to be observed at 3 months at 25°C and 1 month at 40°C, which was expected based on previous stability studies under accelerated stress conditions. Results from 40°C storage showed that protein aggregation reached a maximum at the corners of the design space (B and C), and deamidation reached a maximum at high pH (B and E). All formulations showed robustness to agitation stress, except for formulation F without PS80. These results show the robustness of DP to changes in excipient levels beyond expectations in the process. The formulations evaluated were comparable to formulations containing target excipient levels (control formulation A), thus demonstrating the robustness of the formulations.

[0353] Table 20: Sequence

[0354]

[0355]

[0356]

[0357]

[0358] The various features and embodiments of the present invention mentioned in the above various parts are applicable to other parts with necessary changes when appropriate. Therefore, the features specified in one part can be combined with the features specified in other parts when appropriate. All references cited herein, including the serial registration numbers of patents, patent applications, papers, textbooks and quotations, and the references cited therein are incorporated by reference in their entirety. When one or more of the documents and similar materials incorporated therein are different or contradictory from the present application (including, but not limited to the defined terms, term uses, described technologies, etc.), the present application shall prevail.

[0359] We seek protection for:

[0360]

[0361]

[0362]

[0363]

[0364]

Claims

1. A preparation comprising: 100 mg / mL to 175 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 10 mM to 40 mM histidine buffer; 60 mg / mL to 110 mg / mL sucrose; 0.1 mg / mL to 0.5 mg / mL polysorbate 80 (PS80); 0.01 mg / mL to 1.0 mg / mL of edetate disodium dihydrate or ethylenediaminetetraacetic acid (EDTA); and wherein the formulation has a pH of 5.2 to 6.4, wherein the antibody comprises: (i) a heavy chain variable region (VH) comprising: (a) a VH complementarity determining region 1 (CDR-H1) consisting of the amino acid sequence of SEQ ID NO: 13; (b) a VH complementarity determining region 2 (CDR-H2) consisting of the amino acid sequence of SEQ ID NO: 14; and (c) a VH complementarity determining region 3 (CDR-H3) consisting of the amino acid sequence of SEQ ID NO: 15, and (ii) a light chain variable region (VL), comprising: (a) a VL complementarity determining region 1 (CDR-L1) consisting of the amino acid sequence of SEQ ID NO:8; (b) a VL complementarity determining region 2 (CDR-L2) consisting of the amino acid sequence of SEQ ID NO:9; and (c) a VL complementarity determining region 3 (CDR-L3) consisting of the amino acid sequence of SEQ ID NO:

10.

2. The formulation of claim 1, wherein the concentration of the antibody is about 100 mg / mL, about 115 mg / mL, about 150 mg / mL, or about 158 ​​mg / mL.

3. The formulation of any one of claims 1 to 2, wherein the antibody comprises: a VH consisting of the amino acid sequence of SEQ ID NO: 18 and a VL consisting of the amino acid sequence of SEQ ID NO:

11.

4. The formulation of any one of claims 1 to 2, wherein the antibody comprises a VH sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122329 and a VL sequence encoded by an insert present in a plasmid deposited under ATCC Accession No. PTA-122328.

5. The formulation of any one of claims 1 to 2, wherein the antibody comprises: a heavy chain consisting of the amino acid sequence of SEQ ID NO: 19, and comprises: a light chain consisting of the amino acid sequence of SEQ ID NO:

12.

6. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain variable region consisting of the amino acid sequence of SEQ ID NO: 18 and a light chain variable region consisting of the amino acid sequence of SEQ ID NO: 11; and wherein the preparation has a pH of 5.

8.

7. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain consisting of the amino acid sequence of SEQ ID NO: 19, and comprises: a light chain consisting of the amino acid sequence of SEQ ID NO: 12; and wherein the preparation has a pH of 5.

8.

8. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain consisting of the amino acid sequence of SEQ ID NO: 17, and comprises: a light chain consisting of the amino acid sequence of SEQ ID NO: 12; and wherein the preparation has a pH of 5.

8.

9. A pharmaceutical preparation comprising: 150 mg / mL of an antibody that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), 20 mM histidine buffer, 85 mg / mL sucrose, 0.2 mg / mL polysorbate 80, and 0.05 mg / mL edetate disodium dihydrate, wherein the antibody comprises: a heavy chain consisting of the amino acid sequence of SEQ ID NO: 21, and comprises: a light chain consisting of the amino acid sequence of SEQ ID NO: 12; and wherein the preparation has a pH of 5.

8.

10. The formulation of any one of claims 1 to 2 and 6 to 9, wherein the formulation has a shelf life of at least about 24 months at 5±3°C.

11. The formulation of any one of claims 1 to 2 and 6-9, wherein the formulation has less than about 7% high molecular mass species (HMMS) as measured by size exclusion high performance liquid chromatography (HPLC) at 40°C / 75% relative humidity (RH) for up to 1 month, 2 months, 3 months, 4 months, 5 months or 6 months.

12. The formulation of any one of claims 1 to 2 and 6-9, wherein the formulation has less than about 3% HMMS as measured by size exclusion HPLC at 40°C for up to 1 month, 2 months or 3 months.

13. The formulation of any one of claims 1 to 2 and 6-9, wherein the formulation has less than about 2% HMMS as measured by size exclusion HPLC at 40°C for up to 1 month.

14. Use of a formulation according to any one of claims 1 to 13 for the preparation of a medicament for shortening bleeding time in a subject in need thereof.

15. Use of a formulation according to any one of claims 1 to 13 in the preparation of a medicament for treating or preventing a coagulation defect or a bleeding disorder in a subject in need thereof.

16. Use of a formulation according to any one of claims 1 to 13 in the preparation of a medicament for treating or preventing hemophilia A, B or C in a subject in need thereof.

17. Use of a formulation according to any one of claims 1 to 13 in the preparation of a medicament for treating or preventing von Willebrand's disease (vWD) in a subject in need thereof.

18. Use of a formulation according to any one of claims 1 to 13 for the preparation of a medicament for reducing the activity of TFPI in a subject in need thereof.

19. The use according to any one of claims 14 to 18, wherein the formulation is administered to the subject subcutaneously or intravenously.

20. The use according to any one of claims 14 to 18, wherein the subject is a human.

21. The use of claim 15, wherein the formulation is used to prepare a medicament for treating a bleeding disorder in a subject.

22. The use of claim 16, wherein the formulation is used to prepare a medicament for treating hemophilia A or B in a subject.

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