Tissue factor pathway inhibitor antibodies and their use

Abstract

Antibodies and antigen-binding fragments thereof that specifically bind and inhibit the activity of TFPI are provided. Such antibodies and fragments are useful for treating bleeding disorders and shortening clotting times. The present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope in Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI).

Description

[Technical Field]

[0001] This invention relates to an antibody that binds to tissue factor pathway inhibitors (TFPIs). [Background technology]

[0002] Hemophilia A and B are X-linked genetic disorders resulting from functional deficiencies in plasma proteins factor VIII (FVIII) or factor IX (FIX), respectively. The clinical severity of hemophilia is related to the residual level of coagulation factor activity. Factor activity of less than 1% is associated with a severe phenotype, moderate hemophilia is associated with factor activity of 2% to 5%, and mild hemophilia is associated with factor activity of 5% to 40%.

[0003] The standard of care for these disorders is replacement of deficient coagulation factors via intravenous infusion. Replacement factors are generally recombinant proteins such as Xyntha (factor VIII) or BeneFIX (FIX), although plasma-derived products of varying purities are still used. Treatment with replacement factors can be temporary, treating bleeding on demand when it occurs, or prophylactic, preventing bleeding by maintaining factor levels within a protective range. There is significant evidence that prophylactic treatment prevents bleeding and associated joint damage, a major pathological condition in hemophilia patients. Effective prophylactic treatment requires intravenous injections of factors 3-4 times weekly, which can lead to compliance difficulties and a reduced quality of life. The cost of treatment is also high, due to the complexity of manufacturing coagulation factors. Furthermore, a significant number of patients—up to 32%—with severe hemophilia A develop neutralizing antibodies against administered factors, which are perceived as heterologous proteins by patients with mutations in their own genes. These patients require alternative treatments such as bypass factors or factor VIIa (NovoSeven).

[0004] An alternative therapy involves bypassing the need for supplemental factors by strengthening the intact extrinsic pathway. While hemophilia patients possess some ability to stop bleeding through their intact extrinsic pathway, this is insufficient to block massive bleeding or prevent spontaneous bleeding. The extrinsic pathway is rapidly blocked by tissue factor pathway inhibitors (TFPIs), making it inadequate for protection.

[0005] While antibodies that bind to human TFPI are disclosed in WO2010 / 017196 (Bayer), WO2011 / 109452 (Bayer), WO2014 / 144577 (Bayer), WO2010 / 072687 (Novo Nordisk), WO2012 / 001087 (Novo Nordisk), WO2014 / 140240 (Novo Nordisk), and WO2015 / 007880 (Novo Nordisk), these do not result in the antibodies of the present invention that possess the properties to make them novel potential therapeutic agents for hemophilia. [Overview of the Initiative] [Problems that the invention aims to solve]

[0006] Products that reduce the frequency of coagulation factor administration, decrease the amount of factors used, enable alternative delivery routes (e.g., subcutaneous), and provide prophylactic protection with a lower risk of producing neutralizing antibodies, address a significant unmet need for patients with hemophilia. [Means for solving the problem]

[0007] Antibodies (and their antigen-binding fragments) that bind to tissue factor pathway inhibitors (TFPIs) are disclosed and illustrated herein.

[0008] Those skilled in the art will expect to be able to recognize or confirm many equivalents of the specific embodiments of the invention described herein simply by using conventional experiments. Such equivalents are intended to be encompassed by the following embodiment (E). E1. An isolated antibody or its antigen-binding fragment that specifically binds to an epitope in the Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), wherein the epitope comprises residues Ile105, Arg107, and Leu131 as numbered in Sequence ID No. 2. E2. The antibody or antigen-binding fragment according to Embodiment 1, which does not bind to the Kunitz domain 1 (K1) of TFPI. E3. The antibody or antigen-binding fragment according to Embodiment 1 or 2, wherein the epitope further comprises one or more residues selected from the group consisting of Cys106, Gly108, Cys130, Leu131, and Gly132, as numbered in SEQ ID NO: 2. E4. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 3, wherein the epitope further comprises residues Cys106, Gly108, Cys130, Leu131, and Gly132 as numbered in SEQ ID NO: 2. E5. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 4, wherein the epitope further comprises one or more residues selected from the group consisting of Asp102, Arg112, Tyr127, Gly129, Met134, and Glu138, as numbered in SEQ ID NO: 2. E6. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 5, wherein the epitopes further comprise Asp102, Arg112, Tyr127, Gly129, Met134, and Glu138, as numbered in SEQ ID NO: 2. E7. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 6, wherein the epitope does not contain one or more residues selected from the group consisting of E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140 as numbered in SEQ ID NO: 2. E8. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 7, wherein the epitope does not include E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140 as numbered in SEQ ID NO: 2. E9. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 6, wherein the epitope does not contain one or more residues selected from the group consisting of D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128 as numbered in SEQ ID NO: 2. E10. An antibody or antigen-binding fragment according to any one of embodiments 1 to 6 and 9, wherein the epitope does not include D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128 as numbered in SEQ ID NO: 2. E11. The antibody or antigen-binding fragment according to any one of Embodiments 1 to 10, wherein the epitope comprises one or more residues selected from the group consisting of Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Asn133, Met134, and Glu138 (as numbered in SEQ ID NO: 2), and the epitope residue has a non-zero change in buried surface area (BSA) due to interaction with the antibody or antigen-binding fragment. E12. The antibody or antigen-binding fragment according to Embodiment 11, wherein the epitopes include Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Asn133, Met134, and Glu138 (as numbered in SEQ ID NO: 2). E13. The antibody or antigen-binding fragment according to any one of Embodiments 1 to 12, wherein the epitope comprises one or more residues selected from the group consisting of Asp102, Arg107, Arg112, Tyr127, and Leu131 (as numbered in Sequence ID No. 2), and the epitope residue is involved in hydrogen bonding with a residue derived from the antibody or antigen-binding fragment. E14. The antibody or antigen-binding fragment according to Embodiment 13, wherein the epitopes include Asp102, Arg107, Arg112, Tyr127, and Leu131 (as numbered in SEQ ID NO: 2). E15. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 14, wherein the epitope comprises one or more contact residues selected from the group consisting of Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, and Glu138 (as numbered in SEQ ID NO: 2). E16. The antibody or antigen-binding fragment according to Embodiment 15, wherein the epitopes include Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, and Glu138 (as numbered in SEQ ID NO: 2). E17. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 16, comprising the following heavy (H)-chain and light (L)-chain paratope residues (numbered by Kabat) having a non-zero change in BSA due to interaction with TFPI: H33 Ala, H58 Tyr, H95 Leu, H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 Leu, H100A Ser, L29 Ala, L31 Tyr, L91 Tyr, L95A Ser, and L95B Gly. E18. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 17, comprising a contact residue (numbered by Kabat) where (a) H47 is Trp or Tyr, (b) H58 is Tyr, (c) L91 is Tyr or Arg, and (d) L96 is Gly or Asn. E19. (a) H33 is Ala, Asn, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val; (b) H47 is Trp or Tyr; (c) H50 is Ala, Arg, Gly, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val; (d) H51 is Ile, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val; (e) H52 is Ser, Ala, A (f)H56 is Ser, Arg, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, (g)H58 is Tyr, (h)H95 is Leu, Gln, Ile, Phe, or Tyr, (i)H96 is Gly, Ala, Arg, Asn, Asp, Gln, Ile, Lys, Met, Phe, Pro, (j)H97 is Ser, Thr, or Val, and (k)H98 is Thr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (l)H99 is Ser, Ala, Gly, Phe, or Pro, and (m)H100 is Leu, Arg, His, Ile (n)H100A is Ser, Ala, Arg, Asn, Asp, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, or Trp, (o)L29 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val, (p)L31 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys,(q)L91 is Tyr or Arg, (r)L95A is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (s)L95B is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (t)L95C is Ser The antibody or antigen-binding fragment according to any one of Embodiments 1 to 18, comprising a contact residue (numbered by Kabat) which is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, wherein (u)L93 may comprise a residue which is Tyr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, and (v)L96 may comprise a residue which is Gly or Asn. E20. (a) H33 is Ala or Val, (b) H47 is Trp, (c) H50 is Ala, (d) H51 is Ile, (e) H52 is Ser, Arg, Lys, Phe, or Tyr, (f) H56 is Ser, Arg, or Lys, (g) H58 is Tyr, (h) H95 is Leu, (i) H96 is Gly, Ala, Arg, Asn, Lys, Pro, Ser, or Val, (j) H97 is Ala, (k) H98 is Thr, His, Ile, Leu, Met, Phe, or Tyr, (l) H99 is Ser, (m) H100 is Leu, Phe, Trp, or Tyr, (n) H The antibody or antigen-binding fragment according to any one of Embodiments 1 to 18, comprising a residue (numbered by Kabat) where 100A is Ser, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, or Trp, (o)L29 is Ala, (p)L31 is Tyr, (q)L91 is Tyr, (r)L95A is Ser, Phe, Trp, or Tyr, (s)L95B is Gly, (t)L95C is Ser, Arg, Asn, Gln, Glu, Ile, Leu, Lys, Met, Phe, Trp, Tyr, or Val, and (u)L93 is Ser, and (v)L96 is Gly. E21. (a) H33 is Ala, Val, His, or Phe, (b) H47 is Trp or Tyr, (c) H50 is Ala, Thr, Ser, or Phe, (d) H51 is Ile, Arg, Lys, or Pro, (e) H52 is Ser, Phe, Arg, or Tyr, (f) H56 is Ser, Lys, Tyr, or Phe, ( g)H58 is Tyr, (h)H95 is Leu, Ile, Gln, or Phe, (i)H96 is Gly, Arg, Asn, or Lys, (j)H97 is Ala, Leu, Tyr, or Ile, (k)H98 is Thr, Tyr, Phe, or His, (l)H99 is Ser, Pro, Ala, or Phe, (m)H100 is The antibody or antigen-binding fragment according to any one of Embodiments 1 to 18, comprising a contact residue (numbered by Kabat) where (n)H100A is Ser, Arg, Leu, or Trp, (o)L29 is Ala, Glu, Asp, or Gln, (p)L31 is Tyr, Glu, Asp, or Trp, (q)L91 is Tyr or Arg, (r)L95A is Ser, Phe, Tyr, or His, (s)L95B is Gly, Glu, Asp, or Pro, (t)L95C is Ser, Trp, Tyr, or Phe, (u)L93 is Ser, Glu, Asp, or His, and (v)L96 is Gly or Asn. E22. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 18, comprising the following contact residues (numbered by Kabat): H33 Ala, H47 Trp, H50 Ala, H51 Ile, H52 Ser, H56 Ser, H58 Tyr, H95 Leu, H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 Leu, H100A Ser, L29 Ala, L31 Tyr, L91 Tyr, L95A Ser, L95B Gly, and L95C Ser, and also comprising the following residues: L93 Ser and L96 Gly. E23. (a) Heavy chain variable region (VH) complementarity-determining region 1 (CDR-H1) containing the amino acid sequence of SEQ ID NO: 38, (b) VH complementarity determination region 2 (CDR-H2) containing the amino acid sequence of SEQ ID NO: 39, and (c) VH complementarity determination region 3 (CDR-H3) containing the amino acid sequence of SEQ ID NO: 40 An antibody or antigen-binding fragment according to any one of Embodiments 1 to 22, comprising VH containing the antibody. E24. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 22, comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of Sequence ID No. 41. E25. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 24, comprising a human VH3 framework sequence. E26. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 24, comprising a human VH1 framework sequence. E27. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 24, comprising a human VH5 framework sequence. E28. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 24, comprising a VH framework sequence of human germline IGHV3-23 or IGHV1-69. E29. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 24, comprising a VH framework sequence of human germline IGHV3-7. E30. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 24, comprising a human VH germline consensus framework sequence. E31. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 30, comprising a VH having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 63, and 65. E32. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 31, comprising a VH containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 41, 63, and 65. E33. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 32, comprising a VH containing the amino acid sequence of SEQ ID NO: 41. E34. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 32, comprising a VH containing the amino acid sequence of SEQ ID NO: 63. E35. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 32, comprising a VH containing the amino acid sequence of SEQ ID NO: 65. E36. (a) Light chain variable region (VL) complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO: 33, (b) VL complementarity determination region 2 (CDR-L2) containing the amino acid sequence of SEQ ID NO: 34, and (c) VL complementarity determination region 3 (CDR-L3) containing the amino acid sequence of SEQ ID NO: 35 An antibody or antigen-binding fragment according to any one of Embodiments 1 to 35, comprising a VL containing the antibody or antigen-binding fragment thereof. E37. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 35, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of Sequence ID No. 36. E38. Human V κ An antibody or antigen-binding fragment according to any one of Embodiments 1 to 37, comprising a framework sequence. E39. Human V λ An antibody or antigen-binding fragment according to any one of Embodiments 1 to 37, comprising a framework sequence. E40. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 37, comprising a VL framework sequence of human germline IGKV3-20. E41. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 37, comprising a VL framework sequence of human germline IGKV1-39. E42. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 37, comprising a human VL germline consensus framework sequence. E43. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 42, comprising a VL having at least 90% identical amino acid sequence to SEQ ID NO: 36. E44. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 43, comprising a VL containing the amino acid sequence of SEQ ID NO: 36. E45. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 44, comprising a heavy chain constant region (CH) having at least 90% identical amino acid sequence to SEQ ID NO: 20. E46. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 45, comprising a CH containing the amino acid sequence of SEQ ID NO: 20. E47. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 46, comprising a light chain constant region (CL) having at least 90% identical amino acid sequence to SEQ ID NO: 26. E48. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 47, comprising a CL containing the amino acid sequence of SEQ ID NO: 26. E49. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 48, comprising an Fc domain. E50. The antibody or antigen-binding fragment according to Embodiment 49, wherein the Fc domain is the Fc domain of IgA. E51. The antibody or antigen-binding fragment according to Embodiment 50, wherein IgA is IgA1 or IgA2. E52. The antibody or antigen-binding fragment according to Embodiment 49, wherein the Fc domain is the Fc domain of IgD. E53. The antibody or antigen-binding fragment according to Embodiment 49, wherein the Fc domain is the Fc domain of IgE. E54. The antibody or antigen-binding fragment according to Embodiment 49, wherein the Fc domain is the Fc domain of IgM. E55. The antibody or antigen-binding fragment according to Embodiment 49, wherein the Fc domain is the Fc domain of IgG. E56. The antibody or antigen-binding fragment according to Embodiment 55, wherein IgG is IgG1, IgG2, IgG3, or IgG4. E57. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 56, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 42. E58. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 56, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 64. E59. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 56, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 66. E60. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 59, comprising a light chain containing the amino acid sequence of SEQ ID NO: 37. E61. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 60, comprising a VH sequence encoded by an insert present in a plasmid deposited under ATCC accession number PTA-122329. E62. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 61, comprising a VL sequence encoded by an insert present in a plasmid deposited under ATCC accession number PTA-122328. E63. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 4, wherein the epitope further comprises one or more residues selected from the group consisting of Glu100, Glu101, Asp102, Gly104, and Tyr109, as numbered in SEQ ID NO: 2. E64. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63, wherein the epitopes further comprise Glu100, Glu101, Asp102, Gly104, and Tyr109 as numbered in SEQ ID NO: 2. E65. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 64, wherein the epitope does not contain one or more residues selected from the group consisting of P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140 (numbered according to SEQ ID NO: 2). E66. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 65, wherein the epitopes do not include P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140 (numbered according to SEQ ID NO: 2). E67. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 64, wherein the epitope does not contain one or more residues selected from the group consisting of D31, D32, P34, C35, K36, P103, K126, Y127, G128 (numbered according to SEQ ID NO: 2). E68. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4, 63 to 64, and 67, wherein the epitope does not include D31, D32, P34, C35, K36, P103, K126, Y127, G128 (numbered according to Sequence ID No. 2). E69. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 68, comprising the following residues (numbered by Kabat): H33 Ala, H35 Gln, H52 Ser, H53 Asn, H55 Arg, H56 Ser, H95 Phe, H96 Leu, H97 His, H99 Ser, H101 Asp, L31 Met, L32 Tyr, L34 His, L36 Tyr, L50 Arg, L91 Trp, and L96 Tyr. E70. (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 48, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 49, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 50 An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 69, comprising VH containing the antibody or antigen-binding fragment thereof. E71. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 69, comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 51. E72. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 71, comprising a human VH3, VH1, or VH5 framework sequence. E73. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 72, comprising a VH framework sequence of human germline IGHV3-23 or IGHV1-69. E74. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 72, comprising a VH framework sequence of human germline IGHV3-7. E75. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 71, comprising a human VH germline consensus framework sequence. E76. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 75, comprising a VH having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 69, 51, and 79. E77. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 76, comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 67, 69, 51, and 79. E78. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 77, comprising a VH containing the amino acid sequence of SEQ ID NO: 67. E79. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 77, comprising a VH containing the amino acid sequence of SEQ ID NO: 69. E80. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 77, comprising a VH containing the amino acid sequence of SEQ ID NO: 51. E81. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 77, comprising a heavy chain variable region (VH) containing the amino acid sequence of SEQ ID NO: 79. E82. (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 43, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 44, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 45 An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 81, comprising a VL containing the antibody or antigen-binding fragment thereof. E83. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 81, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 46. E84. Human V κ or V λ An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 83, comprising a framework sequence. E85. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 84, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39. E86. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 83, comprising a human VL germline consensus framework sequence. E87. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 86, comprising a VL having an amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 46, 71, 73, 75, and 77. E88. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 87, comprising a VL containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 71, 73, 75, and 77. E89. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 88, comprising a VL containing the amino acid sequence of SEQ ID NO: 46. E90. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 88, comprising a VL containing the amino acid sequence of SEQ ID NO: 71. E91. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 88, comprising a VL containing the amino acid sequence of SEQ ID NO: 73. E92. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 88, comprising a VL containing the amino acid sequence of SEQ ID NO: 75. E93. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 88, comprising a VL containing the amino acid sequence of SEQ ID NO: 77. E94. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 93, comprising a CH having an amino acid sequence at least 90% identical to SEQ ID NO: 20. E95. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 94, comprising a CH containing the amino acid sequence of SEQ ID NO: 20. E96. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 95, comprising a CL having an amino acid sequence at least 90% identical to SEQ ID NO: 26. E97. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 96, comprising a CL containing the amino acid sequence of SEQ ID NO: 26. E98. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 97, comprising an Fc domain. E99. The antibody or antigen-binding fragment according to Embodiment 98, wherein the Fc domain is the Fc domain of IgA. E100. The antibody or antigen-binding fragment thereof according to Embodiment 99, wherein IgA is IgA1 or IgA2. E101. The antibody or antigen-binding fragment according to Embodiment 98, wherein the Fc domain is the Fc domain of IgD, IgE, or IgM. E102. The antibody or antigen-binding fragment according to Embodiment 98, wherein the Fc domain is the Fc domain of IgG. E103. The antibody or antigen-binding fragment thereof according to Embodiment 102, wherein IgG is IgG1, IgG2, IgG3, or IgG4. E104. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 103, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 52. E105. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 103, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 68. E106. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 103, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 70. E107. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 103, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 80. E108. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 107, comprising a light chain containing the amino acid sequence of SEQ ID NO: 47. E109. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 107, comprising a light chain containing the amino acid sequence of SEQ ID NO: 72. E110. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 107, comprising a light chain containing the amino acid sequence of SEQ ID NO: 74. E111. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 107, comprising a light chain containing the amino acid sequence of SEQ ID NO: 76. E112. An antibody or antigen-binding fragment according to any one of embodiments 1 to 4 and 63 to 107, comprising a light chain containing the amino acid sequence of SEQ ID NO: 78. E113. An isolated antibody or its antigen-binding fragment that specifically binds to an epitope in the Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), wherein the epitope comprises the residues Glu101, Pro103, Tyr109, Thr111, Ser119, Gln121, Glu123, Arg124, Lys126, and Leu140, as numbered in SEQ ID NO: 2. E114. The antibody or antigen-binding fragment according to Embodiment 113, which does not bind to the Kunitz domain 1 (K1) of TFPI. E115. The antibody or antigen-binding fragment according to Embodiment 113 or 114, wherein the epitope does not contain one or more residues selected from the group consisting of E100, D102, R107, Y113, F114, N116, Q118, and C122 (numbered according to SEQ ID NO: 2). E116. An antibody or antigen-binding fragment according to any one of embodiments 113 to 115, wherein the epitope does not include E100, D102, R107, Y113, F114, N116, Q118, and C122 (numbered according to SEQ ID NO: 2). E117. The antibody or antigen-binding fragment according to Embodiment 113 or 114, wherein the epitope does not contain one or more residues selected from the group consisting of D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127, and G128 (numbered according to SEQ ID NO: 2). E118. An antibody or antigen-binding fragment according to any one of embodiments 113 to 114 and 117, wherein the epitope does not include D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127, and G128 (numbered according to SEQ ID NO: 2). E119. The antibody or antigen-binding fragment according to Embodiments 113 to 118, comprising the following residues (as numbered by Kabat): H50 Asp, H57 Thr, H58 Leu, H59 Tyr, H61 Gln, H98 Asp, H99 Tyr, H100 Asp, L30 His, L50 Trp, L92 Tyr, L93 Thr, L94 Thr, and L96 Tyr. E120. (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 87, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 88, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 89 An antibody or antigen-binding fragment according to any one of embodiments 113 to 119, comprising VH containing the antibody or antigen-binding fragment thereof. E121. An antibody or antigen-binding fragment according to any one of embodiments 113 to 119, comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 90. E122. An antibody or antigen-binding fragment according to any one of Embodiments 113 to 121, comprising a human VH3, VH1, or VH5 framework sequence. E123. An antibody or antigen-binding fragment according to any one of Embodiments 113 to 122, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7. E124. An antibody or antigen-binding fragment according to any one of Embodiments 113 to 121, comprising a human VH germline consensus framework sequence. E125. An antibody or antigen-binding fragment according to any one of embodiments 113 to 124, comprising a VH having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 90, 95, 97, 99, 101, 103, 105, and 107. E126. An antibody or antigen-binding fragment according to any one of embodiments 113 to 125, comprising a VH having an amino acid sequence selected from the group consisting of SEQ ID NOs: 90, 95, 97, 99, 101, 103, 105, and 107. E127. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 90. E128. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 95. E129. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 97. E130. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 99. E131. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 101. E132. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 103. E133. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 105. E134. An antibody or antigen-binding fragment according to any one of embodiments 113 to 126, comprising a VH containing the amino acid sequence of SEQ ID NO: 107. E135. (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 81, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 82, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 83 An antibody or antigen-binding fragment according to any one of embodiments 113 to 134, comprising a VL containing the antibody or antigen-binding fragment thereof. E136. An antibody or antigen-binding fragment according to any one of Embodiments 113 to 134, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of Sequence ID No. 84. E137. Human V κ or V λ An antibody or antigen-binding fragment according to any one of embodiments 113 to 136, comprising a framework sequence. E138. An antibody or antigen-binding fragment according to any one of Embodiments 113 to 137, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39. E139. An antibody or antigen-binding fragment according to any one of embodiments 113 to 136, comprising a human VL germline consensus framework sequence. E140. An antibody or antigen-binding fragment according to any one of embodiments 113 to 139, comprising a VL having an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 109, and 111. E141. An antibody or antigen-binding fragment according to any one of embodiments 113 to 140, comprising a VL containing an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 109, and 111. E142. An antibody or antigen-binding fragment according to any one of embodiments 113 to 141, comprising a VL containing the amino acid sequence of SEQ ID NO: 84. E143. An antibody or antigen-binding fragment according to any one of embodiments 113 to 141, comprising a VL containing the amino acid sequence of SEQ ID NO: 109. E144. An antibody or antigen-binding fragment according to any one of embodiments 113 to 141, comprising a VL containing the amino acid sequence of SEQ ID NO: 111. E145. An antibody or antigen-binding fragment according to any one of embodiments 113 to 144, comprising a CH having an amino acid sequence at least 90% identical to that of SEQ ID NO: 20. E146. An antibody or antigen-binding fragment according to any one of embodiments 113 to 145, comprising a CH containing the amino acid sequence of SEQ ID NO: 20. E147. An antibody or antigen-binding fragment according to any one of embodiments 113 to 144, comprising a CH having an amino acid sequence at least 90% identical to that of SEQ ID NO: 91. E148. An antibody or antigen-binding fragment according to any one of embodiments 113 to 144 and 147, comprising a CH containing the amino acid sequence of SEQ ID NO: 91. E149. An antibody or antigen-binding fragment according to any one of embodiments 113 to 148, comprising a CL having an amino acid sequence at least 90% identical to that of SEQ ID NO: 14. E150. An antibody or antigen-binding fragment according to any one of embodiments 113 to 149, comprising a CL containing the amino acid sequence of SEQ ID NO: 14. E151. An antibody or antigen-binding fragment according to any one of embodiments 113 to 148, comprising a CL having an amino acid sequence at least 90% identical to that of SEQ ID NO: 85. E152. An antibody or antigen-binding fragment according to any one of embodiments 113 to 148 and 151, comprising a CL containing the amino acid sequence of SEQ ID NO: 85. E153. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 152, comprising an Fc domain. E154. The antibody or antigen-binding fragment according to Embodiment 153, wherein the Fc domain is the Fc domain of IgA (e.g., IgA1 or IgA2). E155. The antibody or antigen-binding fragment according to Embodiment 153, wherein the Fc domain is the Fc domain of IgD, IgE, or IgM. E156. The antibody or antigen-binding fragment according to Embodiment 153, wherein the Fc domain is the Fc domain of IgG. E157. The antibody or antigen-binding fragment thereof according to Embodiment 156, wherein IgG is IgG1, IgG2, IgG3, or IgG4. E158. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 92. E159. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 94. E160. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 96. E161. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 98. E162. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 100. E163. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 102. E164. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 104. E165. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 106. E166. An antibody or antigen-binding fragment according to any one of embodiments 113 to 157, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 108. E167. An antibody or antigen-binding fragment according to any one of embodiments 113 to 166, comprising a light chain containing the amino acid sequence of SEQ ID NO: 86. E168. An antibody or antigen-binding fragment according to any one of embodiments 113 to 166, comprising a light chain containing the amino acid sequence of SEQ ID NO: 93. E169. An antibody or antigen-binding fragment according to any one of embodiments 113 to 166, comprising a light chain containing the amino acid sequence of SEQ ID NO: 110. E170. An antibody or antigen-binding fragment according to any one of embodiments 113 to 166, comprising a light chain containing the amino acid sequence of SEQ ID NO: 112. E171. (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 16, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 17, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 18 An isolated antibody or its antigen-binding fragment that specifically binds to the Kunitz domain 2 (K2) of TFPI, including VH. E172. An isolated antibody or its antigen-binding fragment containing the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 19, which specifically binds to the K2 domain of TFPI. E173. (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 10, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 11, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 12 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including a VL containing [specific component]. E174. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, containing the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 13. E175. (i) (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 16, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 17, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 18 VH including, as (ii) (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 10, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 11, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 12 VL including An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including [the specified component]. E176. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, containing the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 19, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 13. E177. An antibody or antigen-binding fragment according to any one of Embodiments 171 to 176, comprising a human VH3, VH1, or VH5 framework sequence. E178. An antibody or antigen-binding fragment according to any one of Embodiments 171 to 177, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7. E179. An antibody or antigen-binding fragment according to any one of embodiments 171 to 176, comprising a human VH germline consensus framework sequence. E180. An antibody or antigen-binding fragment according to any one of embodiments 171 to 179, comprising a VH having at least 90% the same amino acid sequence as SEQ ID NO: 19. E181. An antibody or antigen-binding fragment according to any one of embodiments 171 to 180, comprising a VH containing the amino acid sequence of SEQ ID NO: 19. E182. Human V κ or V λ An antibody or antigen-binding fragment according to any one of embodiments 171 to 181, comprising a framework sequence. E183. An antibody or antigen-binding fragment according to any one of Embodiments 171 to 182, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39. E184. An antibody or antigen-binding fragment according to any one of Embodiments 171 to 181, comprising a human VL germline consensus framework sequence. E185. An antibody or antigen-binding fragment according to any one of embodiments 171 to 184, comprising a VL having at least 90% identical amino acid sequence to SEQ ID NO: 13. E186. An antibody or antigen-binding fragment according to any one of embodiments 171 to 185, comprising a VL containing the amino acid sequence of SEQ ID NO: 13. E187. An antibody or antigen-binding fragment according to any one of embodiments 171 to 186, comprising a CH having an amino acid sequence at least 90% identical to that of SEQ ID NO: 20. E188. An antibody or antigen-binding fragment according to any one of embodiments 171 to 187, comprising a CH containing the amino acid sequence of SEQ ID NO: 20. E189. An antibody or antigen-binding fragment according to any one of embodiments 171 to 188, comprising a CL having an amino acid sequence at least 90% identical to SEQ ID NO: 14. E190. An antibody or antigen-binding fragment according to any one of embodiments 171 to 189, comprising a CL containing the amino acid sequence of SEQ ID NO: 14. E191. An antibody or antigen-binding fragment according to any one of embodiments 171 to 190, comprising an Fc domain. E192. The antibody or antigen-binding fragment according to Embodiment 191, wherein the Fc domain is the Fc domain of IgA (e.g., IgA1 or IgA2), IgD, IgE, or IgM. E193. The antibody or antigen-binding fragment according to Embodiment 191, wherein the Fc domain is the Fc domain of IgG. E194. The antibody or antigen-binding fragment thereof according to Embodiment 193, wherein IgG is IgG1, IgG2, IgG3, or IgG4. E195. An antibody or antigen-binding fragment according to any one of embodiments 171 to 194, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 21. E196. An antibody or antigen-binding fragment according to any one of embodiments 171 to 195, comprising a light chain containing the amino acid sequence of SEQ ID NO: 15. E197. (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 28, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 29, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 30 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including VH. E198. An isolated antibody or its antigen-binding fragment containing the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 31, which specifically binds to the K2 domain of TFPI. E199. (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 22, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 23, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 24 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including a VL containing [specific component]. E200. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, containing the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 25. E201. (i) (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28, (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29, and (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30 comprising VH, and (ii) 0](a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22, (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23, and (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24 comprising VL An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI. E202. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 31, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 25. E203. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 202, comprising a human VH3, VH1, or VH5 framework sequence. E204. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 203, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7. E205. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 202, comprising a human VH germline consensus framework sequence. E206. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 205, comprising a VH comprising an amino acid sequence that is at least 90% identical to SEQ ID NO: 31. E207. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 206, comprising a VH comprising the amino acid sequence of SEQ ID NO: 31. E208. Human V κ or V λAn antibody or antigen-binding fragment according to any one of embodiments 197 to 207, comprising a framework sequence. E209. An antibody or antigen-binding fragment according to any one of Embodiments 197 to 208, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39. E210. An antibody or antigen-binding fragment according to any one of Embodiments 197 to 207, comprising a human VL germline consensus framework sequence. E211. An antibody or antigen-binding fragment according to any one of embodiments 197 to 210, comprising a VL having at least 90% the same amino acid sequence as SEQ ID NO: 25. E212. An antibody or antigen-binding fragment according to any one of embodiments 197 to 211, comprising a VL containing the amino acid sequence of SEQ ID NO: 25. E213. An antibody or antigen-binding fragment according to any one of embodiments 197 to 212, comprising a CH having an amino acid sequence at least 90% identical to that of SEQ ID NO: 20. E214. An antibody or antigen-binding fragment according to any one of embodiments 197 to 213, comprising a CH containing the amino acid sequence of SEQ ID NO: 20. E215. An antibody or antigen-binding fragment according to any one of embodiments 197 to 214, comprising a CL having an amino acid sequence at least 90% identical to that of SEQ ID NO: 26. E216. An antibody or antigen-binding fragment according to any one of embodiments 197 to 215, comprising a CL containing the amino acid sequence of SEQ ID NO: 26. E217. An antibody or antigen-binding fragment according to any one of embodiments 197 to 216, comprising an Fc domain. E218. The antibody or antigen-binding fragment according to Embodiment 217, wherein the Fc domain is the Fc domain of IgA (e.g., IgA1 or IgA2), IgD, IgE, or IgM. E219. The antibody or antigen-binding fragment according to Embodiment 217, wherein the Fc domain is the Fc domain of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). E220. An antibody or antigen-binding fragment according to any one of embodiments 197 to 219, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 32. E221. An antibody or antigen-binding fragment according to any one of Embodiments 197 to 220, comprising a light chain containing the amino acid sequence of Sequence ID No. 27. E222. (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 58, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 59, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 60 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including a heavy chain variable region (VH). E223. An isolated antibody or its antigen-binding fragment containing the CDR-H1, CDR-H2, and CDR-H3 sequences of Sequence ID No. 61, which specifically binds to the K2 domain of TFPI. E224. (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 53, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 54, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 55 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including a VL containing [specific component]. E225. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, containing the CDR-L1, CDR-L2, and CDR-L3 sequences of Sequence ID No. 56. E226. (i) (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 58, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 59, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 60 VH including, as (ii) (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 53, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 54, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 55 VL including An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including [the specified component]. E227. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 61, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 56. E228. An antibody or antigen-binding fragment according to any one of Embodiments 222 to 227, comprising a human VH3, VH1, or VH5 framework sequence. E229. An antibody or antigen-binding fragment according to any one of Embodiments 222 to 228, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7. E230. An antibody or antigen-binding fragment according to any one of Embodiments 222 to 227, comprising a human VH germline consensus framework sequence. E231. An antibody or antigen-binding fragment according to any one of embodiments 222 to 230, comprising a VH having at least 90% the same amino acid sequence as SEQ ID NO: 61. E232. An antibody or antigen-binding fragment according to any one of embodiments 222 to 231, comprising a VH containing the amino acid sequence of SEQ ID NO: 61. E233. Human V κ or V λ An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 232, comprising a framework sequence. E234. An antibody or antigen-binding fragment according to any one of Embodiments 222 to 233, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39. E235. An antibody or antigen-binding fragment according to any one of Embodiments 222 to 232, comprising a human VL germline consensus framework sequence. E236. An antibody or antigen-binding fragment according to any one of embodiments 222 to 235, comprising a VL having at least 90% the same amino acid sequence as SEQ ID NO: 56. E237. An antibody or antigen-binding fragment according to any one of embodiments 222 to 236, comprising a VL containing the amino acid sequence of SEQ ID NO: 56. E238. An antibody or antigen-binding fragment according to any one of embodiments 222 to 237, comprising a CH having an amino acid sequence at least 90% identical to that of SEQ ID NO: 20. E239. An antibody or antigen-binding fragment according to any one of embodiments 222 to 238, comprising a CH containing the amino acid sequence of SEQ ID NO: 20. E240. An antibody or antigen-binding fragment according to any one of embodiments 222 to 239, comprising a CL having an amino acid sequence at least 90% identical to that of SEQ ID NO: 26. E241. An antibody or antigen-binding fragment according to any one of embodiments 222 to 240, comprising a CL containing the amino acid sequence of SEQ ID NO: 26. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 241, comprising an E242.Fc domain. E243. The antibody or antigen-binding fragment according to Embodiment 242, wherein the Fc domain is the Fc domain of IgA (e.g., IgA1 or IgA2), IgD, IgE, or IgM. E244. The antibody or antigen-binding fragment according to Embodiment 242, wherein the Fc domain is the Fc domain of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). E245. An antibody or antigen-binding fragment according to any one of embodiments 222 to 244, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 62. E246. An antibody or antigen-binding fragment according to any one of embodiments 222 to 245, comprising a light chain containing the amino acid sequence of Sequence ID No. 57. E247. (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 118, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 119, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 120 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including VH. E248. An isolated antibody or its antigen-binding fragment containing the CDR-H1, CDR-H2, and CDR-H3 sequences of Sequence ID No. 121, which specifically binds to the K2 domain of TFPI. E249. (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 113, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 114, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 115 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including a VL containing [specific component]. E250. An isolated antibody or its antigen-binding fragment containing the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 116, which specifically binds to the K2 domain of TFPI. E251. (i) (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 118, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 119, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 120 VH including, as (ii) (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 113, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 114, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 115 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including a VL containing [specific component]. E252. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 121, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 116. E253. An antibody or antigen-binding fragment according to any one of Embodiments 247 to 252, comprising a human VH3, VH1, or VH5 framework sequence. E254. An antibody or antigen-binding fragment according to any one of Embodiments 247 to 253, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7. E255. An antibody or antigen-binding fragment according to any one of Embodiments 247 to 252, comprising a human VH germline consensus framework sequence. E256. An antibody or antigen-binding fragment according to any one of embodiments 247 to 255, comprising a VH having at least 90% identical amino acid sequence to SEQ ID NO: 121. E257. An antibody or antigen-binding fragment according to any one of embodiments 247 to 256, comprising a VH containing the amino acid sequence of SEQ ID NO: 121. E258. Human V κ or V λ An antibody or antigen-binding fragment according to any one of embodiments 247 to 257, comprising a framework sequence. E259. An antibody or antigen-binding fragment according to any one of Embodiments 247 to 258, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39. E260. An antibody or antigen-binding fragment according to any one of Embodiments 247 to 257, comprising a human VL germline consensus framework sequence. E261. An antibody or antigen-binding fragment according to any one of embodiments 247 to 260, comprising a VL having at least 90% the same amino acid sequence as SEQ ID NO: 116. E262. An antibody or antigen-binding fragment according to any one of embodiments 247 to 261, comprising a VL containing the amino acid sequence of SEQ ID NO: 116. E263. An antibody or antigen-binding fragment according to any one of embodiments 247 to 262, comprising a CH having an amino acid sequence at least 90% identical to that of SEQ ID NO: 91. E264. An antibody or antigen-binding fragment according to any one of embodiments 247 to 263, comprising a CH containing the amino acid sequence of SEQ ID NO: 91. E265. An antibody or antigen-binding fragment according to any one of embodiments 247 to 264, comprising a CL having an amino acid sequence at least 90% identical to that of SEQ ID NO: 85. E266. An antibody or antigen-binding fragment according to any one of embodiments 247 to 265, comprising a CL containing the amino acid sequence of SEQ ID NO: 85. E267. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 266, comprising an Fc domain. E268. The antibody or antigen-binding fragment according to Embodiment 267, wherein the Fc domain is the Fc domain of IgA (e.g., IgA1 or IgA2), IgD, IgE, or IgM. E269. The antibody or antigen-binding fragment according to Embodiment 267, wherein the Fc domain is the Fc domain of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). E270. An antibody or antigen-binding fragment according to any one of embodiments 247 to 269, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 122. E271. An antibody or antigen-binding fragment according to any one of embodiments 247 to 270, comprising a light chain containing the amino acid sequence of SEQ ID NO: 117. E272. (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 128, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 129, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 130 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including VH. E273. An isolated antibody or its antigen-binding fragment containing the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 131, which specifically binds to the K2 domain of TFPI. E274. (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 123, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 124, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 125 An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including a VL containing [specific component]. E275. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, containing the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 126. E276. (i) (a) CDR-H1 containing the amino acid sequence of SEQ ID NO: 128, (b) CDR-H2 containing the amino acid sequence of SEQ ID NO: 129, and (c) CDR-H3 containing the amino acid sequence of SEQ ID NO: 130 VH including, as (ii) (a) CDR-L1 containing the amino acid sequence of SEQ ID NO: 123, (b) CDR-L2 containing the amino acid sequence of SEQ ID NO: 124, and (c) CDR-L3 containing the amino acid sequence of SEQ ID NO: 125 VL including An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, including [the specified component]. E277. An isolated antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, comprising the CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 131, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 126. E278. An antibody or antigen-binding fragment according to any one of Embodiments 272 to 277, comprising a human VH3, VH1, or VH5 framework sequence. E279. An antibody or antigen-binding fragment according to any one of Embodiments 272 to 278, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7. E280. An antibody or antigen-binding fragment according to any one of Embodiments 272 to 277, comprising a human VH germline consensus framework sequence. E281. An antibody or antigen-binding fragment according to any one of embodiments 272 to 280, comprising a VH having at least 90% identical amino acid sequence to SEQ ID NO: 131. E282. An antibody or antigen-binding fragment according to any one of embodiments 272 to 281, comprising a VH containing the amino acid sequence of SEQ ID NO: 131. E283. Human V κ or V λ An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 282, comprising a framework sequence. E284. An antibody or antigen-binding fragment according to any one of Embodiments 272 to 283, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39. E285. An antibody or antigen-binding fragment according to any one of Embodiments 272 to 282, comprising a human VL germline consensus framework sequence. E286. An antibody or antigen-binding fragment according to any one of embodiments 272 to 285, comprising a VL having at least 90% the same amino acid sequence as SEQ ID NO: 126. E287. An antibody or antigen-binding fragment according to any one of embodiments 272 to 286, comprising a VL containing the amino acid sequence of SEQ ID NO: 126. E288. An antibody or antigen-binding fragment according to any one of embodiments 272 to 287, comprising a CH having an amino acid sequence at least 90% identical to that of SEQ ID NO: 91. E289. An antibody or antigen-binding fragment according to any one of embodiments 272 to 288, comprising a CH containing the amino acid sequence of SEQ ID NO: 91. E290. An antibody or antigen-binding fragment according to any one of embodiments 272 to 289, comprising a CL having an amino acid sequence at least 90% identical to that of SEQ ID NO: 85. E291. An antibody or antigen-binding fragment according to any one of embodiments 272 to 290, comprising a CL containing the amino acid sequence of SEQ ID NO: 85. E292. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 291, comprising an Fc domain. E293. The antibody or antigen-binding fragment according to Embodiment 292, wherein the Fc domain is the Fc domain of IgA (e.g., IgA1 or IgA2), IgD, IgE, or IgM. E294. The antibody or antigen-binding fragment according to Embodiment 292, wherein the Fc domain is the Fc domain of IgG (e.g., IgG1, IgG2, IgG3, or IgG4). E295. An antibody or antigen-binding fragment according to any one of embodiments 272 to 294, comprising a heavy chain containing the amino acid sequence of SEQ ID NO: 132. E296. An antibody or antigen-binding fragment according to any one of embodiments 272 to 295, comprising a light chain containing the amino acid sequence of SEQ ID NO: 127. E297. An antibody or antigen-binding fragment that specifically binds to the K2 domain of TFPI, competing for binding to TFPI with the antibody or antigen-binding fragment described in any one of Embodiments 1 to 296. E298. TFPI-3, TFPI-21, TFPI-23, TFPI-24, TFPI-26, TFPI-106, TFPI-107, TFPI-108, TFPI-109, TFPI-110, TFPI-111, TFPI-112, TFPI-113, TFPI-114, TFPI-115, TFPI-118, TFPI-119, TFPI-122, TFPI-123, TFPI-126, 4D8.b1, mu-hu 4D8 Chimera, 4D8-Vk1.0xVH1.0, 4D8-Vk1.0xVH1.1, 4D8-Vk1.0xVH1.2, 4D8-Vk1.0xVH1.3, 4D8-Vk1. 0xVH1.4, 4D8-Vk1.0xVH1.5, 4D8-Vk1.0xVH1.6, 4D8-Vk1.1xVH1.0, 4D8-Vk1.1xVH1.1, 4D8-Vk1 An antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, competing for binding to TFPI with an antibody selected from the group consisting of .1xVH1.2, 4D8-Vk1.1xVH1.3, 4D8-Vk1.1xVH1.4, 4D8-Vk1.1xVH1.5, 4D8-Vk1.1xVH1.6, hz4D8, 6B7.c5, and 7A4.D9. E299. An antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, competing for binding to TFPI with an antibody selected from the group consisting of TFPI-23, TFPI-24, TFPI-106, and TFPI-118. E300. An antibody or antigen-binding fragment thereof according to Embodiment 299, which competes with TFPI-23 or TFPI-106 for binding to TFPI. E301. The antibody or antigen-binding fragment thereof according to Embodiment 299, which competes with TFPI-24 or TFPI-118 for binding to TFPI. E302. An antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, competing with antibody 4D8 for binding to TFPI. E303. An antibody or antigen-binding fragment that binds to the same TFPI epitope as the antibody or antigen-binding fragment described in any one of Embodiments 1 to 296, and that specifically binds to the K2 domain of TFPI. E304. TFPI-3, TFPI-21, TFPI-23, TFPI-24, TFPI-26, TFPI-106, TFPI-107, TFPI-108, TFPI-109, TFPI-110, TFPI-111, TFPI-112, TFPI-113, TFPI-114, TFPI-115, TFPI-118, TFPI-119, TFPI-122, TFPI-123, TFPI-126, 4D8.b1, mu-hu 4D8 Chimera, 4D8-Vk1.0xVH1.0, 4D8-Vk1.0xVH1.1, 4D8-Vk1.0xVH1.2, 4D8-Vk1.0xVH1.3, 4D8-Vk1. 0xVH1.4, 4D8-Vk1.0xVH1.5, 4D8-Vk1.0xVH1.6, 4D8-Vk1.1xVH1.0, 4D8-Vk1.1xVH1.1, 4D8-Vk1. An antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, and binds to the same TFPI epitope as an antibody selected from the group consisting of 1xVH1.2, 4D8-Vk1.1xVH1.3, 4D8-Vk1.1xVH1.4, 4D8-Vk1.1xVH1.5, 4D8-Vk1.1xVH1.6, hz4D8, 6B7.c5, and 7A4.D9. E305. An antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, and binds to the same TFPI epitope as an antibody selected from the group consisting of TFPI-23, TFPI-24, TFPI-106, and TFPI-118. E306. The antibody or antigen-binding fragment thereof according to Embodiment 305, which binds to the same TFPI epitope as TFPI-23 or TFPI-106. E307. The antibody or antigen-binding fragment thereof according to Embodiment 305, which binds to the same TFPI epitope as TFPI-24 or TFPI-118. E308. An antibody or its antigen-binding fragment that specifically binds to the K2 domain of TFPI, and which binds to the same TFPI epitope as antibody 4D8. E309. An antibody or antigen-binding fragment according to any one of embodiments 297 to 308, which does not bind to the K1 domain of TFPI. E310. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 309, which is an Fc fusion protein, a monobody, a maxibody, a bifunctional antibody, scFab, scFv, a peptidebody, or any of the antigen-binding fragments described above. E311. Approximately 1×10 -7 M ~ approx. 1×10 -12 An antibody or antigen-binding fragment according to any one of Embodiments 1 to 310, which binds to TFPI at a binding affinity (Kd) value of M. E312. Approximately 5×10 -7 M ~ approx. 5×10 -11 An antibody or antigen-binding fragment according to any one of Embodiments 1 to 311, which binds to TFPI at a binding affinity (Kd) value of M. E313. Approximately 1 x 10 -8 M ~ approx. 1×10 -10 An antibody or antigen-binding fragment according to any one of Embodiments 1 to 312, which binds to TFPI at a binding affinity (Kd) value of M. E314. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 313, which reduces clotting time when measured by a plasma-based diluted prothrombin time (dPT) assay, (ii) reduces clotting time in whole blood when measured by thromboelastography or rotational thromboelastometry, (iii) increases thrombin production, (iv) increases FXa activity in the presence of TFPI, or (v) any combination thereof. E315. The antibody or antigen-binding fragment thereof according to Embodiment 314, which reduces clotting time when measured by a plasma-based diluted prothrombin time assay. E316. The antibody or antigen-binding fragment according to Embodiment 315, wherein the reduction in coagulation time, as measured by a plasma-based diluted prothrombin time assay, is dose-dependent. E317. The antibody or antigen-binding fragment according to Embodiment 314, which reduces the clotting time in whole blood when measured by thromboelastography or rotational thromboelastometry. E318. The antibody or antigen-binding fragment according to Embodiment 317, wherein the reduction in coagulation time, as measured by thromboelastography or rotational thromboelastometry, is dose-dependent. E319. An antibody or antigen-binding fragment according to Embodiment 314 that increases thrombin production. E320. The antibody or antigen-binding fragment according to Embodiment 319, wherein the increase in thrombin production is dose-dependent. E321. The antibody or antigen-binding fragment according to Embodiment 314, which increases FXa activity in the presence of TFPI. E322. The antibody or antigen-binding fragment according to Embodiment 321, wherein the increase in FXa activity in the presence of TFPI is dose-dependent. E323. The antibody or antigen-binding fragment thereof according to Embodiment 322, which enhances platelet accumulation in the presence of TFPI. E324. The antibody or antigen-binding fragment according to Embodiment 323, wherein the enhancement of platelet accumulation in the presence of TFPI is dose-dependent. E325. The antibody or antigen-binding fragment thereof according to Embodiment 324, which increases fibrin production in the presence of TFPI. E326. The antibody or antigen-binding fragment according to Embodiment 325, wherein the increase in fibrin production in the presence of TFPI is dose-dependent. E327. The antibody or antigen-binding fragment according to Embodiment 314, wherein a reduction in clotting time in whole blood is determined using whole blood obtained from a human patient with severe hemophilia A. E328. The antibody or antigen-binding fragment according to Embodiment 314, wherein a reduction in clotting time in whole blood is determined using whole blood obtained from a human patient having severe hemophilia A and inhibitory antibodies against human factor VIII. E329. The antibody or antigen-binding fragment according to Embodiment 314, wherein a reduction in clotting time in whole blood is determined using whole blood obtained from a human patient with moderate hemophilia A. E330. The antibody or antigen-binding fragment according to Embodiment 314, wherein a reduction in clotting time in whole blood is determined using whole blood obtained from a human patient with severe hemophilia B. E331. The antibody or antigen-binding fragment according to Embodiment 314, wherein a reduction in clotting time in whole blood is determined using whole blood obtained from a human patient having severe hemophilia B and inhibitory antibodies against human factor IX. E332. The antibody or antigen-binding fragment according to Embodiment 314, wherein a reduction in clotting time in whole blood is determined using whole blood obtained from a human patient with moderate hemophilia B. E333. The antibody or antigen-binding fragment according to Embodiment 314, wherein the reduction in clotting time, as measured by a dPT assay, is determined using plasma obtained from a human patient with severe hemophilia A. E334. The antibody or antigen-binding fragment according to Embodiment 314, wherein the reduction in clotting time, as measured by a dPT assay, is determined using plasma obtained from a human patient with severe hemophilia A and inhibitory antibodies against human factor VIII. E335. The antibody or antigen-binding fragment according to Embodiment 314, wherein the reduction in clotting time, as measured by a dPT assay, is determined using plasma obtained from a human patient with moderate hemophilia A. E336. The antibody or antigen-binding fragment according to Embodiment 314, wherein the reduction in clotting time, as measured by a dPT assay, is determined using plasma obtained from a human patient with severe hemophilia B. E337. The antibody or antigen-binding fragment according to Embodiment 314, wherein the reduction in clotting time, as measured by a dPT assay, is determined using plasma obtained from a human patient with severe hemophilia B and inhibitory antibodies against human factor IX. E338. The antibody or antigen-binding fragment according to Embodiment 314, wherein the reduction in clotting time, as measured by a dPT assay, is determined using plasma obtained from a human patient with moderate hemophilia B. E339. The antibody or antigen-binding fragment according to Embodiment 314, wherein increased thrombin production is determined using plasma obtained from a human patient with severe hemophilia A. E340. The antibody or antigen-binding fragment according to Embodiment 314, wherein increased thrombin production is determined using plasma obtained from a human patient having severe hemophilia A and inhibitory antibodies against human factor VIII. E341. The antibody or antigen-binding fragment according to Embodiment 314, wherein increased thrombin production is determined using plasma obtained from a human patient with moderate hemophilia A. E342. The antibody or antigen-binding fragment according to Embodiment 314, wherein increased thrombin production is determined using plasma obtained from a human patient with severe hemophilia B. E343. The antibody or antigen-binding fragment according to Embodiment 314, wherein increased thrombin production is determined using plasma obtained from a human patient having severe hemophilia B and inhibitory antibodies against human factor IX. E344. The antibody or antigen-binding fragment according to Embodiment 314, wherein increased thrombin production is determined using plasma obtained from a human patient with moderate hemophilia B. E345. The antibody or antigen-binding fragment according to Embodiment 314, wherein a 100 nM antibody or antigen-binding fragment thereof is at least as effective in reducing the clotting time of whole blood obtained from a human patient with severe hemophilia A as recombinant human factor VIII in an amount sufficient to achieve 5% of normal coagulation activity. E346. The antibody or antigen-binding fragment according to Embodiment 314, wherein a 100 nM antibody or antigen-binding fragment thereof is at least equally effective in increasing peak thrombin production in platelet-rich plasma obtained from a human patient with severe hemophilia A, in an amount sufficient to achieve 5% of normal coagulation activity with recombinant human factor VIII. E347. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 346, wherein the TFPI is human TFPI. E348. An antibody or antigen-binding fragment according to any one of Embodiments 1 to 347, wherein TFPI comprises residues 91-147 of SEQ ID NO: 2. E349. An isolated nucleic acid molecule or nucleic acid molecule comprising one or more nucleotide sequences encoding an antibody or antigen-binding fragment thereof as described in any one of Embodiments 1 to 348. E350. An isolated nucleic acid molecule encoding an antibody or its antigen-binding fragment that specifically binds to TFPI, wherein the nucleic acid sequence is selected from the group consisting of the nucleic acid sequence of SEQ ID NO: 175, SEQ ID NO: 176, SEQ ID NO: 177, SEQ ID NO: 178, the nucleic acid sequence of the insert of the vector deposited as mAb-TFPI-106VL under ATCC accession number PTA-122328, and the nucleic acid sequence of the insert of the vector deposited as mAb-TFPI-106VH under ATCC accession number PTA-122329. E351. A vector comprising the nucleic acid molecule described in Embodiments 349 and 350. E352. A host cell containing a nucleic acid molecule as described in Embodiment 349 or 350, or a vector as described in Embodiment 351. E353. A host cell according to embodiment 352, which is a mammalian cell. E354. Host cells according to Embodiment 353, which are CHO cells, HEK-293 cells, or Sp2.0 cells. E355. A method for producing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to any one of Embodiments 352 to 354 under conditions in which the antibody or antigen-binding fragment is expressed by the host cell. E356. The method according to Embodiment 355, further comprising isolating an antibody or an antigen-binding fragment thereof. E357. An antibody or its antigen-binding fragment obtained by the method described in Embodiment 355 or 356. E358. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof as described in any one of Embodiments 1 to 347 and 357, and a pharmaceutically acceptable carrier or excipient. E359. A method for reducing the activity of a tissue factor pathway inhibitor (TFPI), comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof described in any one of Embodiments 1 to 347 and 357 or a pharmaceutical composition described in Embodiment 358. E360. A method for shortening bleeding time, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 347 and 357 or a pharmaceutical composition according to embodiment 358. E361. The method according to Embodiment 358 or 360, wherein the subject is a human. E362. The method according to any one of embodiments 359 to 361, wherein the subject is suffering from or is susceptible to blood coagulation disorders. E363. The method according to any one of embodiments 359 to 361, wherein the subject is suffering from or is susceptible to platelet disorders. E364. The method according to any one of embodiments 359 to 361, wherein the subject has or is susceptible to hemophilia A, B, or C. E365. The method according to any one of embodiments 359 to 361, wherein the subject has or is susceptible to hemophilia A or B. E366. The method according to any one of embodiments 359 to 361, wherein the subject has or is susceptible to von Willebrand disease (vWD). E367. The method according to Embodiment 360, further comprising administering a therapeutically effective dose of FVII. E368. The method according to embodiment 367, which increases thrombin production in the presence of TFPI. E369. The method according to any one of Embodiments 359 to 368, comprising intravenous administration of an antibody or an antigen-binding fragment thereof or a pharmaceutical composition. E370. The method according to any one of Embodiments 359 to 368, comprising subcutaneous administration of an antibody or an antigen-binding fragment thereof or a pharmaceutical composition. E371. The method according to any one of Embodiments 359 to 368, wherein the antibody or its antigen-binding fragment or pharmaceutical composition is administered once every 3 days, once every 4 days, once every 5 days, once every 6 days, once a week, or twice a week. E372. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 347 and 357 for use as a pharmaceutical, or a pharmaceutical composition according to embodiment 358. E373. An antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 347 and 357, or a pharmaceutical composition according to Embodiment 358, for use in reducing the activity of TFPI in a subject. E374. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 347 and 357, or a pharmaceutical composition according to embodiment 358, for use in shortening bleeding time in a subject. E375. An antibody or antigen-binding fragment or pharmaceutical composition according to any one of embodiments 372 to 374, wherein the subject is a human. E376. An antibody or antigen-binding fragment or pharmaceutical composition according to any one of embodiments 372 to 375, wherein the subject is suffering from or susceptible to blood coagulation disorders. E377. An antibody or antigen-binding fragment or pharmaceutical composition according to any one of Embodiments 372 to 375, wherein the subject has or is susceptible to hemophilia A, B, or C. E378. An antibody or antigen-binding fragment or pharmaceutical composition according to any one of embodiments 372 to 375, wherein the subject has or is susceptible to hemophilia A or B. E379. An antibody or antigen-binding fragment or pharmaceutical composition according to any one of embodiments 372 to 375, wherein the subject is suffering from or susceptible to von Willebrand disease (vWD). E380. An antibody or antigen-binding fragment or pharmaceutical composition according to any one of embodiments 372 to 375, wherein the subject is suffering from or susceptible to platelet disorders. E381. Use of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 348 and 357, or the pharmaceutical composition according to embodiment 358, to reduce the activity of TFPI in a subject. E382. Use of an antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 348 and 357 or a pharmaceutical composition according to Embodiment 358 in the manufacture of a pharmaceutical for reducing the activity of TFPI in a target. E383. Use of an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 348 and 357, or the pharmaceutical composition according to embodiment 358, to shorten bleeding time in a subject. E384. Use of an antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 348 and 357 or a pharmaceutical composition according to Embodiment 358 in the manufacture of a pharmaceutical for shortening bleeding time in a subject. E385. The use according to any one of Embodiments 381 to 384, wherein the subject is a human. E386. Use according to any one of embodiments 381 to 384, wherein the subject is suffering from or is susceptible to blood coagulation disorders. E387. Use according to any one of Embodiments 381 to 384, wherein the subject has or is susceptible to hemophilia A, B, or C. E388. Use according to any one of Embodiments 381 to 384, wherein the subject has or is susceptible to hemophilia A or B. E389. Use according to any one of embodiments 381 to 384, wherein the subject has or is susceptible to von Willebrand disease (vWD). E390. Use according to any one of embodiments 381 to 384, wherein the subject has or is susceptible to platelet disorders. [Brief explanation of the drawing]

[0009] [Figure 1A]This diagram shows the cocrystal structures of various anti-TFPI antibodies and the K2 domain of TFPI. "R&D" or "R&D Fab" refers to the antibody Mab2974 manufactured by R&D Systems. The Novo2021 antibody is also known as "hz4F36". "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 1B] This diagram shows the cocrystal structures of various anti-TFPI antibodies and the K2 domain of TFPI. "R&D" or "R&D Fab" refers to the antibody Mab2974 manufactured by R&D Systems. The Novo2021 antibody is also known as "hz4F36". "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 1C] This diagram shows the cocrystal structures of various anti-TFPI antibodies and the K2 domain of TFPI. "R&D" or "R&D Fab" refers to the antibody Mab2974 manufactured by R&D Systems. The Novo2021 antibody is also known as "hz4F36". "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 1D] This diagram shows the cocrystal structures of various anti-TFPI antibodies and the K2 domain of TFPI. "R&D" or "R&D Fab" refers to the antibody Mab2974 manufactured by R&D Systems. The Novo2021 antibody is also known as "hz4F36". "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 1E] This diagram shows the cocrystal structures of various anti-TFPI antibodies and the K2 domain of TFPI. "R&D" or "R&D Fab" refers to the antibody Mab2974 manufactured by R&D Systems. The Novo2021 antibody is also known as "hz4F36". "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 1F]This diagram shows the cocrystal structures of various anti-TFPI antibodies and the K2 domain of TFPI. In particular, as shown in Figure 1F, the exemplary antibodies disclosed herein, TFPI-23, TFPI-24, and 4D8, all bind to non-overlapping epitopes of the K2 domain compared to other reference antibodies. TFPI-106 binds to the same site as TFPI-23, and TFPI-118 binds to the same site as TFPI-24. "R&D" or "R&D Fab" refers to the antibody Mab2974 manufactured by R&D Systems. The Novo2021 antibody is also called "hz4F36". "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 2A] This diagram illustrates the interaction between epitope residues in the K2 domain of TFPI and paratope residues derived from various anti-TFPI antibodies. "R&D" or "R&D Fab" refers to Mab2974 manufactured by R&D Systems. "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 2B] This diagram illustrates the interaction between epitope residues in the K2 domain of TFPI and paratope residues derived from various anti-TFPI antibodies. "R&D" or "R&D Fab" refers to Mab2974 manufactured by R&D Systems. "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 2C] This diagram illustrates the interaction between epitope residues in the K2 domain of TFPI and paratope residues derived from various anti-TFPI antibodies. "R&D" or "R&D Fab" refers to Mab2974 manufactured by R&D Systems. "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 2D] This diagram illustrates the interaction between epitope residues in the K2 domain of TFPI and paratope residues derived from various anti-TFPI antibodies. "R&D" or "R&D Fab" refers to Mab2974 manufactured by R&D Systems. "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 2E]This diagram illustrates the interaction between epitope residues in the K2 domain of TFPI and paratope residues derived from various anti-TFPI antibodies. "R&D" or "R&D Fab" refers to Mab2974 manufactured by R&D Systems. "Clone 23" refers to TFPI-23, and "Clone 24" refers to TFPI-24. [Figure 3A] This figure shows the in vivo efficacy of various anti-TFPI antibodies in a mouse injury model. Figure 3A shows the duration of reduced bleeding in hemophilia A factor VIII-deficient (FVIII- / -) mice after administration of 2A8-200 (used as the reference antibody in this study). The antibody was administered intravenously to hemophilia A mice (FVIII- / -) at the indicated time point (hours (h)) before injury at 6 mg / kg. The total volume of blood loss (μL) was then measured after tail amputation. Hemophilia A mice treated with saline served as a control. All measurements are presented as mean ± SEM. *=P<0.05. FVIII+ / + (wild-type) mice received saline. n=5 / group. [Figure 3B] This figure shows the in vivo efficacy of various anti-TFPI antibodies in a mouse injury model. Figure 3B shows the duration of reduced bleeding in hemophilia A factor VIII-deficient (FVIII- / -) mice after administration of the 2A8 antibody (used as the reference antibody in this study). The antibody was administered intravenously to hemophilia A mice (FVIII- / -) at the indicated time point (hours (h)) before injury at 6 mg / kg. The total volume of blood loss (μL) was then measured after tail amputation. Hemophilia A mice treated with saline served as a control. All measurements are presented as mean ± SEM. *=P<0.05. FVIII+ / + (wild-type) mice received saline. n=5 / group. [Figure 3C]This figure shows the in vivo efficacy of various anti-TFPI antibodies in a mouse injury model. Figure 3C shows the duration of effect in hemophilia A mice after administration of TFPI 4D8 (control), TFPI-21, TFPI-23, and TFPI-24 antibodies. Antibodies were administered intravenously to hemophilia A mice (FVIII- / -) at the indicated time point (hours (h)) before injury at 6 mg / kg. The total volume of blood loss (μL) was then measured after tail amputation. Hemophilia A mice treated with saline served as controls. All measurements are presented as mean ± SEM. *=P<0.05. FVIII+ / + (wild-type) mice received saline. n=5 / group. [Figure 3D] This figure shows the in vivo efficacy of various anti-TFPI antibodies in a mouse injury model. Figure 3D shows the duration of efficacy in hemophilia A mice after administration of TFPI-106 antibody. The antibody was administered intravenously to hemophilia A mice (FVIII- / -) at the indicated time point (hours (h)) before injury at 6 mg / kg. The total volume of blood loss (μL) was then measured after tail amputation. Hemophilia A mice treated with saline served as a control. All measurements are presented as mean ± SEM. *=P<0.05. FVIII+ / + (wild-type) mice received saline. n=5 / group. [Figure 3E] This figure shows the in vivo efficacy of various anti-TFPI antibodies in a mouse injury model. Figure 3E shows the duration of TFPI-118 antibody administration in hemophilia A mice. The antibody was administered intravenously to hemophilia A mice (FVIII- / -) at the indicated time point (hours (h)) before injury at 6 mg / kg. The total volume of blood loss (μL) was then measured after tail amputation. Hemophilia A mice treated with saline served as controls. All measurements are presented as mean ± SEM. *=P<0.05. FVIII+ / + (wild-type) mice received saline. n=5 / group. [Figure 4]This figure shows the duration of bleeding after tail amputation in hemophilia B mice administered with TFPI-106 antibody. The antibody was administered intravenously to hemophilia B mice at the indicated time (hours (h)) before injury at 6 mg / kg. The total volume of blood loss (μL) was then measured after tail amputation. Hemophilia A mice treated with saline served as a control. All measurements are presented as mean ± SEM. *=P<0.05. n=4~5 / group. [Figure 5A] Figure 5 shows micrographs of in vivo microscopy (IVM) observations, including panels A and B, each containing six panels (Figure 5A, 1-6 and Figure 5B, 1-6), demonstrating the detection of TFPI in vivo within platelet thrombi and along the endothelium at vascular injury sites in wild-type mice. Figure 5A shows the enlargement of platelet thrombi at injury sites when detected using Dylight649-labeled CD42c, which binds to GP1bβ on platelets. The presence or absence of platelets is demonstrated by the fluorescence signals detected in panel 1 (0 sec), panel 2 (15 sec), panel 3 (30 sec), panel 4 (60 sec), panel 5 (90 sec), and panel 6 (120 sec). Alexa488-labeled negative control IgG was also administered, but no fluorescence was detected. [Figure 5B]Figure 5 includes panels A and B, each containing six panels (Figure 5A, 1-6 and Figure 5B, 1-6), and shows micrographs of in vivo in vivo microscopic observations (IVM) demonstrating the detection of TFPI within platelet thrombi and along the endothelium at vascular injury sites in wild-type mice. Figure 5B includes panels 1-6 and shows micrographs of IVM demonstrating the presence of TFPI within platelet thrombi and along the endothelium after laser-induced vascular injury in wild-type mice. Alexa488-labeled TFPI (green signal shown in gray) is not detected at 0 seconds (Figure 5B, panel 1), and a faint signal can be seen at 15 seconds (Figure 5B, panel 2). At 30 seconds (Figure 5B, panel 3), the green fluorescence signal increases, and a faint red signal (Dylight649-labeled CD42c) can be seen, indicating the detection of platelet accumulation at almost the same site where TFPI is detected. Figure 5B, panel 4 (60 seconds) shows strong green and red fluorescence signals (both light gray, where the red fluorescence can be seen to the left of the vascular injury site and the green signal is mainly detected toward the right side of the injury site), demonstrating that platelet accumulation and TFPI are both detected at the injury site. Figure 5B, panel 5 shows both red (platelet) and green (TFPI) fluorescence signals at the injury site up to 60 seconds, with both signals greater than at 30 seconds. Figure 5B, panel 6 shows a decrease in both the red (platelet) and green (TFPI) signals, both still detectable at the injury site at 120 seconds. [Figure 6A]This graph shows the hemostatic effect of TFPI-106 in hemophilia A mice after laser-induced vascular injury, assessed using IVM. The amount of platelet thrombus is expressed as the area under the curve (AUC) (*=P<0.005). Figure 6A shows platelet accumulation at the injury site in wild-type mice (WT) 0.5 hours after injury when mice received only a saline control, compared to the absence of platelet accumulation in hemophilia A mice 0.5 hours after administration of saline. Platelet accumulation was detected in hemophilia A mice at 0.5 hours after administration of recombinant factor VIII (rFVIII) or TFPI-106. The thrombotic effect was still detected at 168 hours in hemophilia A mice administered with TFPI-106. [Figure 6B] This graph shows the hemostatic effect of TFPI-106 in hemophilia A mice after laser-induced vascular injury, assessed using IVM. Fibrin production is expressed as the area under the curve (AUC) (*=P<0.005). Figure 6B shows fibrin production at the injury site in wild-type mice (WT) 0.5 hours after injury when mice received only a saline control, compared to the absence of detectable fibrin production at 0.5 hours in hemophilia A mice administered with saline. Fibrin production was detected in hemophilia A mice at 0.5 hours after administration of recombinant factor VIII (rFVIII) or TFPI-106. The fibrin production effect was still detectable at 16 hours in hemophilia A mice administered with TFPI-106. [Figure 7] This figure shows a graph illustrating the effect of administration of TFPI-106 and recombinant factor VIIa (rFVIIa) on the thrombin production assay (TGA) in severe hemophilia A plasma in the presence of 1 pm tissue factor and 4 μM phospholipid. The graph shows thrombograms of hemophilia A plasma containing TFPI-106 alone (16 μg / ml) and in combination with rFVIIa (20 μg / ml, 2 μg / ml, or 0.2 μg / ml). Thrombograms of hemophilia A plasma and non-hemophilic plasma without either TFPI-106 or rFVIIa are also shown. [Figure 8A] This figure shows thrombograms illustrating the effect of 1 pM tissue factor and 4 μM phospholipid on thrombin production in hemophilia A plasma in the presence of TFPI-106 with or without rFVIIa, or in the presence of rFVIIa alone. Non-hemophilia plasma is included as a control. [Figure 8B] This figure shows thrombograms illustrating the effect of 3 Bethesda units (3 BU) of inhibitor on thrombin production in citrate-poor platelet-deficient hemophilia A plasma in the presence of TFPI-106 with or without rFVIIa, or in the presence of rFVIIa alone. Non-hemophilic plasma is included as a control. Control non-hemophilic plasma supplemented with TFPI-106 (16 μg / ml) is also included. [Figure 8C] This graph shows the effect of 3 Bethesda units (3 BU) of inhibitor on thrombin production in citrate-poor platelet-deficient hemophilia B plasma in the presence of TFPI-106 with or without rFVIIa, or in the presence of rFVIIa alone. Non-hemophilic plasma is included as a control. Control non-hemophilic plasma supplemented with TFPI-106 (16 μg / ml) is also included. [Figure 9A] This figure shows the effect on blood loss in hemophilia A mice administered the antibody TFPI-106 (6 mg / kg) immediately after tail amputation, compared to a control group administered recombinant factor VIII (200 units / kg) separately. [Figure 9B] This figure shows the effect on blood loss in hemophilia A mice administered three different doses of the antibody TFPI-106 (1, 3, and 6 mg / kg) two minutes after tail amputation, compared to a control group separately administered recombinant factor VIII (200 units / kg). [Figure 10A] This figure shows the effects of different concentrations of TFPI106 compared to recombinant factor VIII on the clotting time of whole blood from human patients with severe hemophilia A. [Figure 10B] This figure shows the effects of different concentrations of TFPI106 compared to recombinant factor VIII on peak thrombin production in platelet-rich plasma derived from human patients with severe hemophilia A. [Figure 11A] Figure showing the effect of different concentrations of TFPI106 on the clotting time of whole blood from human patients with severe hemophilia A and inhibitors of FVIII. [Figure 11B] Figure showing the effect of different concentrations of TFPI106 on peak thrombin generation in platelet-rich plasma from human patients with severe hemophilia A and inhibitors of FVIII. [Figure 11C] Figure showing the effect of different concentrations of TFPI106 on the clotting time of platelet-poor plasma from human patients with severe hemophilia A and inhibitors of FVIII. [Figure 12A] Figure showing the effect of different concentrations of TFPI106 compared to recombinant factor VIII on the clotting time of whole blood from human patients with moderate hemophilia A. [Figure 12B] Figure showing the effect of different concentrations of TFPI106 compared to recombinant factor VIII on peak thrombin generation in platelet-rich plasma from human patients with moderate hemophilia A. [Figure 12C] Figure showing the effect of different concentrations of TFPI106 on the clotting time of platelet-poor plasma from human patients with moderate hemophilia A. [Figure 13A] Figure showing the effect of different concentrations of TFPI106 compared to recombinant factor IX on the clotting time of whole blood from human patients with moderate hemophilia B. [Figure 13B] Figure showing the effect of different concentrations of TFPI106 compared to recombinant factor IX on peak thrombin generation in platelet-rich plasma from human patients with moderate hemophilia B. [Figure 13C] Figure showing the effect of different concentrations of TFPI106 on the clotting time of platelet-poor plasma from human patients with moderate hemophilia B. [Figure 14A] Figure showing the effect of different concentrations of TFPI106 compared to recombinant factor VIII on the clotting time of whole blood from multiple human patients with hemophilia A. [Figure 14B]This figure shows the effects of different concentrations of TFPI106 compared to recombinant factor VIII on peak thrombin production in platelet-rich plasma derived from multiple human patients with hemophilia A. [Figure 14C] This figure shows the effect of different concentrations of TFPI106 on clotting time in platelet-poor plasma from multiple human patients with hemophilia B. [Modes for carrying out the invention]

[0010] 1. Overview As described above, hemophilia patients possess some ability to stop bleeding through their own intact extrinsic pathway, but this pathway is rapidly blocked by tissue factor pathway inhibitors (TFPIs) and therefore insufficient to provide protection. Blocking / neutralizing TFPI inhibition in these patients can compensate for insufficient FXa production and normalize the bleeding diathesis. Accordingly, antibodies and their antigen-binding fragments that specifically bind to TFPI and inhibit its activity are disclosed and exemplified herein.

[0011] 2.Definition "Reducing TFPI activity" means that the antibody or its antigen-binding fragment can (i) reduce clotting time compared to clotting time in the absence of the antibody, for example, when measured by a plasma-based diluted prothrombin time assay; (ii) reduce clotting time in whole blood compared to clotting time in the absence of the antibody, for example, when measured by thromboelastography or rotational thromboelastometry; (iii) increase thrombin production; (iv) increase FXa in the presence of TFPI; (v) enhance platelet accumulation in the presence of TFPI; (vi) increase fibrin production in the presence of TFPI; or (vii) any combination thereof. The inhibitory activity of the antibody or antigen-binding fragment may, but does not have to be, dose-dependent (e.g., causing a dose-dependent reduction in clotting time when measured by a plasma-based diluted prothrombin time assay).

[0012] Furthermore, cocrystal structures of the anti-TFPI antibody and the Kunitz domain 2 (K2 domain) of TFPI were obtained as disclosed and illustrated herein. Structural analysis shows that the exemplary antibody of the present invention recognizes a unique epitope of TFPI compared to other publicly disclosed TFPI antibodies (these were used as reference antibodies in the examples). For example, as shown in Figures 1A-1F and 2A-2E, TFPI-23, TFPI-24, and 4D8 antibodies bind to non-overlapping sites in the K2 domain of TFPI compared to several reference TFPI antibodies (R&D(Mab2479)Fab, Novo2021(also known as hz4F36)Fab, 2A8Fab).

[0013] Therefore, in certain embodiments, the antibodies (and antigen-binding fragments) disclosed herein recognize a unique epitope of TFPI located in the K2 domain of TFPI. Based on cocrystal structure and computational alanine scan, this epitope contains three residues crucial for antibody-antigen interaction: Ile105, Arg107, and Leu131 (as shown in the human TFPI numbering sequence number 2). Mutation of these three residues to alanine results in loss of antibody binding. For example, the antibody TFPI-23 and its variants (e.g., TFPI-106 and TFPI-107) all recognize this epitope.

[0014] In certain embodiments, recognition of key epitope residues disclosed herein allows antibodies (and their antigen-binding fragments) to reduce the activity of TFPI. In particular, the crystalline structure shows that the K2 domain of TFPI adopts a cone-shaped structure, with the tip of the cone (specifically Arg107) bound to FXa. Both TFPI-23 and TFPI-24 recognize the tip of this cone-shaped region and block TFPI from binding to FXa. Antibody 4D8 recognizes a different epitope in the K2 domain. Although it does not directly interact with the residue at the tip of the cone, 4D8 nevertheless blocks TFPI from binding to FXa. Table 15 summarizes the non-overlapping epitope residues recognized by exemplary antibodies disclosed herein compared to other publicly known TFPI antibodies.

[0015] Furthermore, in certain embodiments, the antibodies and their antigen-binding fragments disclosed herein have demonstrated desirable pharmacological activity and pharmacokinetic properties for the treatment of coagulation factor deficiencies (such as hemophilia) and for reducing bleeding time.

[0016] Antibodies that “preferentially bind” or “specifically bind” (as used interchangeably herein) to an epitope are well understood terms in the art, and methods for 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, rapidly, for a longer duration and / or with greater affinity than it does with alternative cells or substances. An antibody “specifically binds” or “preferentially binds” if it binds to its target with greater affinity, binding activity, readily and / or for a longer duration than it binds to other substances. An antibody “specifically binds” or “preferentially binds” if it binds to its target in a sample with greater affinity, binding activity, readily and / or for a longer duration than it binds to other substances present in the sample. For example, an antibody that specifically or preferentially binds to a TFPI epitope is an antibody that binds to this epitope with greater affinity, binding activity, ease, and / or duration than it binds to other TFPI or non-TFPI epitopes. It can also be understood from reading this definition that, for example, an antibody (or partial or epitope) that specifically or preferentially binds to a first target may or may not specifically or preferentially bind to a second target. Therefore, "specific binding" or "preferential binding" does not necessarily require exclusive binding (although it may include exclusive binding). Generally, but not necessarily, references to binding imply preferential binding. "Specific binding" or "preferential binding" includes compounds that recognize and bind to specific molecules in a sample, but substantially do not recognize or bind to other molecules, such as proteins, nucleic acids, and antibodies. For example, an antibody or peptide receptor that recognizes and binds to a congeneral ligand or binding partner in a sample (e.g., an anti-TFPI antibody that binds to TFPI) but substantially does not recognize or bind to other molecules in the sample is specifically binding to its congeneral ligand or binding partner.Therefore, under the specified assay conditions, the designated binding site (e.g., an antibody or its antigen-binding site, or a receptor or its ligand-binding site) preferentially binds to the specific target molecule and does not bind in significant amounts to other components present in the test sample.

[0017] 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® (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet® (ForteBio, Inc., Menlo Park, CA), and Western blot analysis are among the many assays that can be used to identify antibodies that specifically react with an antigen or receptor, or their ligand-binding moiety that specifically binds to a homologous ligand or binding partner. Typically, specific or selective reactions are at least twice the background signal or noise, more typically more than 10 times the background, even more typically more than 50 times the background, more typically more typically more than 100 times the background, even more typically more typically more than 500 times the background, even more typically more typically more than 1000 times the background, even more typically more typically more than 10,000 times the background, and even more typically more than 10,000 times the background. Also, the equilibrium dissociation constant (K) D When the antibody's strength is 7 nM or less, the antibody is said to "specifically bind" to the antigen.

[0018] The term "binding affinity" is used herein as a measure of the strength of a non-covalent interaction between two molecules, for example, an antibody or a fragment thereof, and an antigen. The term "binding affinity" is used to describe monovalent interactions (intrinsic activity).

[0019] The binding affinity between two molecules, for example, an antibody or a fragment thereof, and an antigen, due to monovalent interactions is determined by the dissociation constant (K). D This can be quantified by determining the following: K DThis can be determined, for example, by measuring the dynamics of complex formation and dissociation using surface plasmon resonance (SPR) spectroscopy (Biacore). The rate constants corresponding to the association and dissociation of monovalent complexes are, respectively, the association rate constant k. a (or k on ) and the dissociation rate constant k d (or k off ) is called K D is, formula K D =k d / k a by k a and k d This is related to the following. The value of the dissociation constant can be directly determined by well-known methods, and can even be calculated for complex mixtures by methods such as those shown by Caceci et al. (1984, Byte, 9:340~362). For example, K D This can be established using a double-filter nitrocellulose filter binding assay, such as that disclosed by Wong & Lohman (1993, Proc. Natl. Acad. Sci., USA, 90:5428~5432). Other standard assays for evaluating the binding ability of ligands, such as antibodies, to target antigens are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry analysis, as well as other assays exemplified elsewhere herein. Antibody binding kinetics and binding affinity can also be assessed using standard assays known in the art, such as surface plasmon resonance (SPR) using the Biacore® system, or KinExA.

[0020] A competitive binding assay can be performed, in which the binding of an antibody to an antigen is compared to the binding of the target by another ligand of that target, such as another antibody or soluble receptor that binds the target differently. The concentration at which 50% inhibition occurs is K i It is publicly known as. Under ideal conditions, K i is, K D It is equivalent to K. i The value is never K D It does not become less than Ki The measurement of, conveniently, K D can be substituted to provide an upper limit of.

[0021] According to the above definition, the comparison of binding affinities associated with different intermolecular interactions, e.g., the binding affinities of different antibodies to a given antigen, can be compared by comparing the K D values of the individual antibody / antigen complexes. The K D values of an antibody or other binding partner can be determined using methods well established in the art. One way to determine K D is by using surface plasmon resonance, typically by using a biosensor system such as a Biacore® system.

[0022] Similarly, the specificity of an interaction can be assessed by determining and comparing the K D value of the interaction of interest, e.g., the specific interaction between an antibody and an antigen, with the K D value of the interaction of a control antibody known not to bind to an off-target, e.g., TFPI.

[0023] An antibody that specifically binds its target can bind to its target with high affinity, i.e., exhibit a low K D as discussed above, and can bind to other non-target molecules with lower affinity. For example, an antibody can bind to a non-target molecule with a K -6 of 1×10 -5 M or more, more preferably 1×10 -4 M or more, more preferably 1×10 -3 M or more, more preferably 1×10 -2 M or more, even more preferably 1×10 D M or more. The antibodies of the present invention can bind to their target with an affinity that is preferably at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold, or 10,000-fold, or more, greater than their affinity for binding to another non-TFPI molecule.

[0024] Generally, TFPI antibodies need to bind to TFPI with high affinity to effectively reduce its activity. However, if the antibody's binding affinity is too high, the antibody can be rapidly internalized and degraded by host cells. This can potentially lead to a short half-life and repeated injections. For example, the antibody TFPI-23 exhibits a lower binding affinity (Kd) compared to TFPI-24, and under certain circumstances, it appears more desirable for clinical use because it has a lower internalized rate and a longer half-life. Therefore, 5 × 10 -7 M ~ approx. 5×10 -11 M, in particular, about 1 × 10 -8 M ~ approx. 1×10 -10 The binding affinity (Kd) of M is generally desirable, especially for treating chronic conditions requiring repeated injections (e.g., hemophilia). Without wishing to be bound by any particular theory, this affinity range is thought to strike a balance between (i) the binding affinity required to effectively inhibit TFPI activity and (ii) a longer half-life and reduced internal antibody transfer.

[0025] Specific amino acid residue positions in TFPI are numbered according to Sequence ID No. 2 (human TFPIα K1K2K3). However, the present invention is not limited to Sequence ID No. 2. Corresponding residues from other TFPI homologs, isoforms, variants, or fragments can be identified according to sequence alignment or structural alignment known in the art. For example, alignment can be performed manually or using a well-known sequence alignment program such as ClustalW2 or "BLAST2 Sequences" with default parameters. For example, Arg107 in Sequence ID No. 2 corresponds to Arg104 in mouse TFPI K1K2 (Sequence ID No. 4).

[0026] Antigen-binding fragments of antibodies refer to fragments of a full-length antibody that retain the ability to specifically bind to an antigen (preferably with substantially the same binding affinity). Examples of antigen-binding fragments include (i) Fab fragments, monovalent fragments consisting of VL, VH, CL, and CH1 domains; (ii) F(ab')2 fragments, bivalent fragments containing two Fab fragments linked by disulfide crosslinking at the hinge region; (iii) Fd fragments consisting of VH and CH1 domains; (iv) Fv fragments consisting of VL and VH domains of a single arm of the antibody; (v) dAb fragments consisting of a VH domain (Ward et al., (1989) Nature, 341:544~546); and (vi) isolation complementarity-determining regions (CDRs), disulfide-linked Fv (dsFv), anti-idiotype (anti-Id) antibodies, and intrabodies. Furthermore, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined by a synthetic linker using recombination, which allows them to be constructed as a single protein chain (known as single-chain Fv (scFv)) where the VL and VH regions pair up to form a monovalent molecule; see, for example, Bird et al., Science, 242:423~426 (1988) and Huston et al., Proc. Natl. Acad. Sci. USA, 85:5879~5883 (1988). Other forms of single-chain antibodies, such as diabodies, are also included. Diabody is a bivalent, bispecific antibody expressed using a linker, in which the VH and VL domains are on a single polypeptide chain, but the linker is too short to allow pairing between the two domains on the same chain, thereby forcing the domains to pair with complementary domains on another chain, creating two antigen-binding sites (see, for example, Holliger et al., Proc. Natl. Acad. Sci. USA, 90:6444~6448 (1993); Poljak et al., 1994, Structure, 2:1121~1123).

[0027] An antibody "variable domain" refers, either alone or in combination, to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH). As is well known in the art, the variable regions of the heavy and light chains each consist of four framework regions (FRs) connected by three complementarity-determining regions (CDRs), and contribute to the formation of the antibody's antigen-binding site.

[0028] Residues in variable domains are numbered according to Kabat, a numbering scheme used for the heavy-chain or light-chain variable domains of antibody edits. See Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering scheme, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to the shortening or insertion of FR or CDR in the variable domain. For example, a heavy-chain variable domain may contain a single amino acid insert after H2 residue 52 (residue 52a according to Kabat) and an inserted residue after heavy-chain FR residue 82 (e.g., residues 82a, 82b, and 82c according to Kabat). The Kabat numbering of residues can be determined for a given antibody by alignment of the antibody sequence with a "standard" Kabat-numbered sequence in a region of homology. Various algorithms for assigning Kabat numbering are available. Unless otherwise noted, the algorithm implemented in the 2012 release of Abysis (www.abysis.org) is used herein to assign Kabat numbering to variable regions.

[0029] Specific amino acid residue positions within the antibody (such as paratope residues disclosed herein) are also numbered by Kabat.

[0030] The “complementarity-determining region” (CDR) can be identified by Kabat, Chothia, accumulation by both Kabat and Chothia, AbM, contact, and / or conformational definitions, or by any method of CDR determination known in the art. See, for example, Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th edition (hypervariable regions); Chothia et al., 1989, Nature, 342:877-883 (structural loop structures). The AbM definition of CDR is a compromise between Kabat and Chothia, using Oxford Molecular’s ​​AbM antibody modeling software (Accelrys®). The “contact” definition of CDR is based on observed antigen contact as shown in MacCallum et al., 1996, J.Mol.Biol., 262:732-745. The "conformation" definition of a CDR is based on residues that contribute enthalpy to antigen binding (see, for example, Makabe et al., 2008, Journal of Biological Chemistry, 283:1156-1166). Further CDR boundary definitions may not strictly follow one of the above methods, but nevertheless they will overlap with at least some Kabat CDRs, although they may be shortened or lengthened based on predictions or experimental findings that certain residues or groups of residues, or even the entire CDR, do not significantly impact antigen binding. As used herein, a CDR may refer to a CDR defined by any method known in the art, including combinations of methods.

[0031] In the examples (see Tables 3 and 4), the CDR is defined as follows (Kabat numbering, H: heavy chain, L: light chain): CDR-H1:H26-H35B, CDR-H2:H50-H65, CDR-H3:H95-H102 CDR-L1:L24-L34, CDR-L2:L50-L56, CDR-L3:L89-L97

[0032] "Framework" (FR) residues are antibody variable domain residues other than CDR residues. The VH or VL domain framework includes four framework subregions, FR1, FR2, FR3, and FR4, interspersed with CDRs in the following structure: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. In examples (see Tables 3 and 4), the FR residues are as follows (Kabat numbering, H: heavy chain, L: light chain):

[0033] [Table 1] Includes.

[0034] An "epitope" refers to an area or region of an antigen (Ag) to which an antibody specifically binds, such as an area or region containing residues that interact with an antibody (Ab). Epitopes can be linear or conformal. In linear epitopes, all interaction points between a protein and an interacting molecule (such as an antibody) are linearly aligned along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformal epitope" contains discontinuous polypeptides (or amino acids) within the antigen protein to which an epitope-specific antibody binds. As used herein, the term "epitope" is defined as the portion of an antigen to which an antibody can specifically bind when determined by any method well known in the art, such as a conventional immunoassay. Alternatively, during the discovery process, generating and characterizing antibodies can elucidate information about desired epitopes. From this information, it is then possible to competitively screen antibodies for binding to the same epitope. The method to achieve this involves conducting competitive and cross-competition studies to identify antibodies that compete with or cross-compete for binding to TFPI. That is, antibodies compete for binding to the antigen so that they compete for binding to the antigen-binding site of the anti-TFPI antibody of this disclosure.

[0035] The term "paratope" is derived from the above definition of "epitope" by reversing the perspective, and refers to an area or region of an antibody molecule involved in antigen binding, such as an area or region containing residues that interact with the antigen. Paratopes can be linear or three-dimensional (e.g., discontinuous residues in a CDR).

[0036] The epitope / paratope of a given antibody / antigen binding pair can be defined and characterized at different levels of detail using various experimental and computational epitope mapping methods. Experimental methods include mutagenesis, X-ray crystallography, nuclear magnetic resonance (NMR) spectroscopy, hydrogen / deuterium exchange mass spectrometry (HX-MS), and various competitive binding methods. Since each method utilizes a unique principle, the description of an epitope is closely related to the method by which it is determined. Therefore, the epitope / paratope of a given antibody / antigen binding pair will be defined differently depending on the mapping method used.

[0037] At its most detailed level, the epitopes / paratopes of Ag-Ab interactions can be defined by information about the spatial coordinates that define the atomic contacts present in the Ag-Ab interaction, and their relative contributions to bond thermodynamics. At one level, an epitope / paratope residue can be characterized by the spatial coordinates that define the atomic contacts between Ag and Ab. In one embodiment, an epitope / paratope residue can be defined by a specific criterion, e.g., the interatomic distance between Ab and Ag (e.g., a distance equal to or less than 4 Å from the heavy atom of the congenital antibody and the heavy atom of the antigen ("contact" residue)). In another embodiment, an epitope / paratope residue can be characterized as being involved in hydrogen bonding interactions with congenital antibodies / antigens, or with water molecules also hydrogen-bonded to congenital antibodies / antigens (water-mediated hydrogen bonds). In yet another embodiment, an epitope / paratope residue can be characterized as forming a salt bridge with residues of congenital antibodies / antigens. In yet another embodiment, epitope / paratope residues can be characterized as residues that have a non-zero change in buried surface area (BSA) due to interaction with a congeneral antibody / antigen. At a less detailed level, epitopes / paratopes can be characterized, for example, through function by competitive binding with other Abs. Epitopes / paratopes can also be generally defined by containing amino acid residues in which substitution by another amino acid alters the properties of the Ab-Ag interaction (e.g., alanine scanning).

[0038] In terms of the X-ray-derived crystal structure, defined by the spatial coordinates of an antibody, for example, a complex between one Fab fragment or two Fab fragments and their antigen, unless otherwise specified, an epitope residue refers to a TFPI residue that (i) has a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 Å from a heavy atom of a congenital antibody (also called a "contact" residue), (ii) is involved in hydrogen bonding with a residue of the congenital antibody or with a water molecule also hydrogen-bonded to the congenital antibody (water-mediated hydrogen bonding), (iii) is involved in salt bridging to a residue of the congenital antibody, and / or (iv) has a non-zero change in buried surface area (BSA) due to interaction with the congenital antibody. Generally, a cutoff is imposed on the BSA to avoid including residues with minimal interaction. Therefore, unless otherwise specified, an epitope residue under category (iv) is one that has a distance of 20 Å 2 A paratope residue is selected if it has a BSA greater than or equal to 20 Å, or if it is involved in electrostatic interactions when the antibody binds to TFPI. Similarly, in terms of the crystal structure derived by X-ray, unless otherwise specified or inconsistent with the context, a paratope residue refers to an antibody residue that (i) has a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 Å from the heavy atom of TFPI (also called a "contact" residue), (ii) is involved in hydrogen bonding with a TFPI residue or with a water molecule that is also hydrogen-bonded to TFPI (water-mediated hydrogen bonding), (iii) is involved in salt bridging to a TFPI residue, and / or (iv) has a non-zero change in buried surface area (BSA) due to interaction with TFPI. Again, unless otherwise specified, a paratope residue under category (iv) is one that has a BSA greater than or equal to 20 Å 2 If the BSA has the above characteristics, or if the antibody is involved in electrostatic interactions when it binds to TFPI, it is selected.

[0039] The fact that the description and definition of an epitope depend on the epitope mapping method used and can be obtained at different levels of detail means that comparisons of epitopes for different Abs on the same Ag can similarly be made at different levels of detail. For example, epitopes described at the amino acid level, or determined from, for example, X-ray structure, are said to be identical if they contain the same set of amino acid residues. If there are no shared amino acid residues in an epitope, it is said to be unique. Epitopes characterized by competitive binding are said to be overlapping if the binding of the corresponding antibodies is mutually exclusive, i.e., the binding of one antibody excludes the simultaneous or consecutive binding of another antibody, and if the antigen can adapt to the binding of both corresponding antibodies simultaneously, it is said to be unique.

[0040] The epitopes and paratopes of a given antibody / antigen pair can be identified by conventional methods. For example, the common location of an epitope can be determined by assessing the ability of the antibody to bind to different fragments or variant TFPI polypeptides, as previously described more fully elsewhere in this specification. Specific residues in the TFPI that come into contact with specific residues in the antibody can also be determined using conventional methods, such as those described in the examples. For example, the antibody / antigen complex can be crystallized. The crystal structure can be determined and used to identify specific sites of interaction between the antibody and antigen.

[0041] When used herein in relation to antibodies, the term “competing” means that the binding of a first antibody or its antigen-binding moiety to an antigen reduces the subsequent binding of a second antibody or its antigen-binding moiety to the same antigen. Generally, the binding of the first antibody results in steric hindrance, conformational change, or binding to a common epitope (or moiety thereof), resulting in reduced binding of the second antibody to the same antigen. Standard competition assays can be used to determine whether two antibodies compete with each other. One suitable assay for antibody competition involves the use of Biacore technology, which uses surface plasmon resonance (SPR) technology and can typically measure the degree of interaction using a biosensor system (such as the BIACORE® system). For example, SPR can be used in an in vitro competitive binding inhibition assay to determine the ability of one antibody to inhibit the binding of a second antibody. Another assay for measuring antibody competition uses ELISA-based methods. Furthermore, a high-throughput process for “binning” antibodies based on their competition is described in International Patent Application WO2003 / 48731. Competition exists when one antibody (or fragment) reduces the binding of another antibody (or fragment) to TFPI. For example, a sequential binding competition assay can be performed by adding different antibodies sequentially. The first antibody may be added to reach near-saturation binding. Then, the second antibody is added. If the binding of the second antibody to TFPI is not detected, or is significantly reduced compared to a parallel assay in the absence of the first antibody (which can be set to 100%) (e.g., a reduction of at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, or at least about 90%), then the two antibodies are considered to be competing with each other. An exemplary antibody competition assay (and overlapping epitope analysis) using SPR is provided in Example 6.

[0042] The anti-TFPI antibodies of this disclosure may have the ability to compete or cross-compete with other antibodies of this disclosure for binding to TFPI as described herein. For example, the antibodies of this disclosure may compete or cross-compete with the antibodies of this disclosure for binding to TFPI or a suitable fragment or variant of TFPI to which the antibodies of this disclosure are bound.

[0043] In other words, if a first anti-TFPI antibody competes with a second antibody for binding to TFPI, but does not compete when the second antibody is initially bound to TFPI, it is considered to "compete" with the second antibody (also known as unidirectional competition). If an antibody competes with another antibody regardless of which antibody is initially bound to TFPI, that antibody "cross-competes" with the other antibody for binding to TFPI. Such competing or cross-competing antibodies can be identified based on their ability to compete / cross-compete with known antibodies of this disclosure in standard binding assays. For example, competition / cross-competition can be demonstrated using SPR, ELISA assays, or flow cytometry using the Biacore® system. Such competition / cross-competition may suggest that the two antibodies bind to the same, overlapping, or similar epitopes.

[0044] Accordingly, the anti-TFPI antibodies of this disclosure can be identified by a method including a binding assay that assesses whether the test antibody can compete / cross-compete with the reference antibodies of this disclosure (e.g., TFPI-3, TFPI-21, TFPI-23, TFPI-24, TFPI-26, TFPI-106, TFPI-107, TFPI-108, TFPI-109, TFPI-110, TFPI-111, TFPI-112, TFPI-113, TFPI-114, 4D8, 6B7.c5, 7A4.D9) for the binding site of a target molecule.

[0045] An "Fc fusion" protein is a protein in which one or more polypeptides are operably linked to an Fc polypeptide. Fc fusion combines the Fc region of an immunoglobulin with a fusion partner.

[0046] The binding affinity of an antibody can be expressed as a Kd value, which refers to the dissociation rate of a particular antigen-antibody interaction. Kd is the ratio of the dissociation rate, also called the "off-rate (koff)," to the association rate or "on-rate (kon)." Thus, Kd is equal to koff / kon and expressed as molar concentration (M), with a smaller Kd indicating stronger binding affinity. The Kd value of an antibody can be determined using methods well established in the art. One exemplary method for measuring Kd is surface plasmon resonance (SPR), typically using a biosensor system such as the BIACORE® system. BIAcore kinetic analysis involves analyzing the binding and dissociation of an antigen from a chip on which molecules (e.g., molecules containing epitope-binding domains) are immobilized on the surface. Another method for determining the Kd of an antibody is typically by using biolayer interferometry with OCTET® technology (Octet QKe system, ForteBio). Alternatively or additionally, the KinExA® (kinetic exclusion assay) assay, available from Sapidyne Instruments (Boise, Id.), may also be used.

[0047] The term "therapeutic dose" means a sufficient amount of anti-TFPI antibody or fragment thereof, or a combination containing such antibody or fragment thereof, to achieve the intended purpose, such as increased clotting or, in the case of hemophilia, reduced clotting time, or to provide a measurable in vivo benefit to the target population. The exact amount will depend on a number of factors, including, but not limited to, the components and physical measurements of the therapeutic composition, the intended patient population, and individual patient considerations, and can be determined by a person skilled in the art.

[0048] The term “synergistic therapeutic dose” refers to the amount of anti-TFPI antibody or its antigen-binding fragment that, when provided with a second therapeutic agent, such as factor VIIa (FVIIa), produces a measurable benefit (e.g., reduced clotting time, reduced bleeding time, increased fibrin production, enhanced platelet accumulation, etc.) greater than the additive measurable effect of each therapeutic agent or antibody administered alone.

[0049] The term "treatment" includes prophylactic and / or therapeutic treatments. A treatment is considered prophylactic if it is administered before the clinical manifestation of a condition. Therapeutic treatments include, for example, remission or reduction of the severity of a disease, or reduction of the duration of a disease.

[0050] The term "approximately," when used here, refers to a range of + / - 10% of the value.

[0051] 3. Anti-TFPI antibody Antibodies (and their antigen-binding fragments) that bind to tissue factor pathway inhibitors (TFPIs) are disclosed and illustrated herein. The antibodies and antibody fragments bind to unique epitopes of TFPI. In certain embodiments, recognition of a specific epitope residue in TFPI allows the antibodies (and their antigen-binding fragments) to reduce the activity of TFPI. Furthermore, in certain embodiments, the antibodies (and their antigen-binding fragments) disclosed herein have demonstrated desirable pharmacological activity and pharmacokinetic properties for the treatment of coagulation factor deficiencies and for reducing bleeding time.

[0052] A. Tissue factor pathway inhibitors (TFPIs) TFPI is a polyvalent Kunitz domain containing a protease inhibitor. Exemplary sequences of human, mouse, cynomolgus monkey, rabbit, and rat TFPI are provided in Table 2.

[0053] Human TFPI is an extracellular glycoprotein with two dominant forms: TFPI-alpha and TFPI-beta. TFPI-alpha is a 276-amino acid glycosylated protein (MW 43kD), the largest form of TFPI, consisting of three Kunitz-like domains and a basic carboxyl-terminal region. Alternative splicing produces TFPI-beta, which contains Kunitz domain 1 (K1) and Kunitz domain 2 (K2), but lacks Kunitz domain 3 (K3) and a basic region, and contains an alternative C-terminal region. TFPI-beta is anchored to the cell membrane by post-translational modification with a glycosylphosphatidylinositol (GPI) anchor.

[0054] The primary targets of TFPI are proteases factor Xa (FXa) and factor VIIa (FVIIa), which are crucial initiations of the coagulation cascade. Biochemical analysis 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 it may function as a recognition site for cofactor protein S. The C-terminal domain unique to TFPI-alpha may be involved in the recognition of prothrombinase on the platelet surface.

[0055] Kunitz domain 1 (K1) corresponds to amino acid residues 26-76 of SEQ ID NO: 2, and Kunitz domain 2 (K2) corresponds to residues 91-147 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 with respect to SEQ ID NO: 2.

[0056] The TFPIs of this disclosure include any naturally occurring forms of TFPIs that may originate from any suitable organism. For example, TFPIs may be mammalian TFPIs, such as human, mouse, rat, non-human primate, cattle, sheep, dog, cat, or pig TFPIs. In certain embodiments, TFPIs are human TFPIs. TFPIs may be mature forms of TFPIs (i.e., proteins that have undergone post-translational processing in a suitable cell). Such mature TFPI proteins may be glycosylated, for example.

[0057] The TFPIs of this disclosure include any functional fragments or variants derived from naturally occurring TFPIs. Functional fragments of TFPIs may be any site or portion of TFPI that retains TFPI activity, 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, functional fragments may include the Kunitz domain of TFPI, such as the K1 domain, the K2 domain, or both the K1 and K2 domains.

[0058] Functional variants may contain one or more mutations compared to naturally occurring TFPI and may still retain the activity of naturally occurring TFPI, such as the ability to inhibit factor Xa (FXa) or the activity of the FVIIa-tissue factor complex. For example, a variant may have varying degrees of sequence identity with SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7, for example, at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identity with the sequences listed in SEQ ID NOs: 1, 2, 3, 4, 5, 6, or 7.

[0059] The TPFI fragments, variants, isoforms, and homologs of the present invention should retain the important epitope residues described herein (such as Ile105, Arg107, and Leu13 when TFPI-23 and TFPI-24 antibodies are used). Furthermore, TFPI may contain 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more surface-accessible residues of the TFPI K2 domain. Surface-accessible residues are residues with relative accessibility of more than 40%.

[0060] For example, regarding the K2 domain of TFPI (see, for example, SEQ ID NO: 2), the following amino acid residues have a relative proximity of more than 40%: 94-95, 98, 100-110, 118-121, 123-124, 131, 134, 138-142, and 144-145. TFPI may contain 5 or more of these residues, 8 or more, 10 or more, 12 or more, or 15 or more of these residues, for example, fragments of TFPI containing 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more of these residues.

[0061] B. Anti-TFPI antibody The antibody or antigen-binding fragment of the present invention can specifically bind to the K2 domain of TFPI, inhibit its interaction with FXa, and / or reduce the activity of TFPI.

[0062] TFPI-23 and variants In one embodiment, the present invention includes the antibody TFPI-23 and variants of TFPI-23 that have been prepared to increase the content of human framework germline residues ("germlining"). For example, TFPI-106 includes mutations from H1Q to E and H5V to L (Kabat numbering), and TFPI-107 includes mutations from H1Q to E, H5V to L, and H94I to K (Kabat numbering). With respect to the object of the present invention, the TFPI-23 parent antibody and the TFPI-106 germline variant are interchangeable in their epitope and paratope residue interactions.

[0063] In one embodiment, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope in the Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), wherein the epitope comprises residues Ile105, Arg107, and Leu131, as numbered in SEQ ID NO: 2. In a particular embodiment, the antibody or antigen-binding fragment does not bind to the Kunitz domain 1 (K1) of TFPI.

[0064] As disclosed and illustrated herein, a unique epitope in the K2 domain of TFPI was discovered based on cocrystal structure and computational alanine scan. In particular, the crystal structure shows that the K2 domain of TFPI adopts a cone-shaped structure, with the tip of the cone (particularly Arg107) bound to FXa. TFPI-23, TFPI-24, and their variants all recognize residues near the tip of this cone-shaped region and block the binding of TFPI to FXa. Therefore, antibodies that recognize epitope residues located near the tip of the cone are particularly useful for inhibiting TFPI activity.

[0065] In certain embodiments, the present invention discloses a TFPI epitope comprising three residues important for antibody-antigen interaction: Ile105, Arg107, and Leu131 (as numbered in human TFPI as shown in SEQ ID NO: 2). Mutation of these three residues to alanine results in the loss of binding by TFPI-23, TFPI-24, and their variants. See Table 28, which summarizes the alanine scan results.

[0066] Additional TPFI residues have also been identified as being involved in antibody binding, but these residues can be mutated to alanine without significant destabilizing effects. See Table 28. Thus, in certain embodiments, the TFPI epitope further comprises one or more residues selected from the group consisting of Cys106, Gly108, Cys130, Gly132 (as numbered in Sequence ID No. 2), and any combination thereof. These epitope residues are recognized by TFPI-23, TFPI-24, and their variants. See Table 27, which shows common epitope residues shared by TFPI-23 and TFPI-24.

[0067] In certain embodiments, the epitope further comprises one or more residues selected from the group consisting of Asp102, Arg112, Tyr127, Gly129, Met134, Glu138 (as numbered in Sequence ID No. 2), and any combination thereof. These epitope residues are recognized by TFPI-23 and its variants (e.g., TFPI-106, TFPI-107), but not by TFPI-24 (and its variants). See Table 27.

[0068] In certain embodiments, the epitope does not contain one or more residues selected from the group consisting of E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, L140 (numbered by SEQ ID NO: 2), and any combination thereof. See Table 27. According to WO201007269 (Novo Nordisk), the reference antibody 4F36 recognizes epitopes including E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140.

[0069] In certain embodiments, the epitope does not contain one or more residues selected from the group consisting of D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, G128 (numbered by Sequence ID No. 2), and any combination thereof. See Table 27. According to Table 27, reference antibodies 2A8 and 2A8-200 recognize epitopes including D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128.

[0070] In certain embodiments, an epitope may refer to one or more TFPI "contact" residues (having a heavy atom (i.e., a non-hydrogen atom) within a distance of 4 Å from a heavy atom of the congener antibody) and include one or more residues selected from the group consisting of Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, Glu138 (as numbered in SEQ ID NO: 2), and any combination thereof. See Table 29B.

[0071] In certain embodiments, an epitope may refer to one or more TFPI residues involved in hydrogen bonding with antibody residues or with water molecules also hydrogen-bonded to a congener antibody (water-mediated hydrogen bonding), and includes one or more residues selected from the group consisting of Asp102, Arg107, Arg112, Tyr127, and Leu131 (as numbered in SEQ ID NO: 2), and any combination thereof. These epitope residues are involved in hydrogen bonding with congener antibodies. See Table 29B.

[0072] In certain embodiments, an epitope may refer to a residue that exhibits a non-zero change in buried surface area (BSA) due to interaction with a congener antibody, and may include one or more residues selected from the group consisting of Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Asn133, Met134, Glu138 (as numbered in SEQ ID NO: 2), and any combination thereof. See Table 29B for these.

[0073] Any combination of these different categories of epitope residues is also included by the present invention.

[0074] In a particular embodiment, the epitope comprises at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least twelve, at least thirteen of the epitope residues described above, or all of them, or any combination of various categories of the epitope residues described above.

[0075] The paratope residues of TFPI-23 (and its variants) may refer to the following contact residues (within 4 Å of the TFPI epitope residue): H33 Ala, H47 Trp, H50 Ala, H51 Ile, H52 Ser, H56 Ser, H58 Tyr, H95 Leu, H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 Leu, H100A Ser, L29 Ala, L31 Tyr, L91 Tyr, L95A Ser, L95B Gly, and L95C Ser, and may also refer to L93 Ser and L96 Gly (numbered by Kabat). L93 Ser (4.07 Å) and L96 Gly (4.03 Å) are optional because, although their distance is slightly over 4 Å, they are close enough to be rounded to 4 Å.

[0076] It should be noted that the above contact residues are the original residues derived from the TFPI-23 antibody. However, based on structural analysis and alanine scanning, it is thought that some contact residues in TFPI-23 can be substituted with other residues without significantly affecting antigen binding. For example, Table 29A shows that some contact residues in TFPI-23 can be substituted with other residues, resulting in an effect of less than 0.5 kcal / mol on binding or stability ("less than 0.5 kcal / mol" means having a neutral effect on binding). In particular, as shown in Table 29A, column 4, three CDR positions and one framework position: H47, H58, L91, and L96 (numbered by Kabat) only allow one or two residues: (a) H47 is Trp or Tyr, (b) H58 is Tyr, (c) L91 is Tyr or Arg, and (d) L96 is Gly or Asn. Other CDR locations can be adapted to more substitutions, as summarized in Table 29A, column 4.

[0077] Therefore, in certain embodiments, the antibody or its antigen-binding fragment described herein is (a) H33 is Ala, Asn, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val, (b) H47 is Trp or Tyr, (c)H50 is Ala, Arg, Gly, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val, (d) H51 is Ile, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (e) H52 is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val, (f) H56 is Ser, Arg, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (g)H58 is Tyr, (h)H95 is Leu, Gln, Ile, Phe, or Tyr, (i) H96 is Gly, Ala, Arg, Asn, Asp, Gln, Ile, Lys, Met, Phe, Pro, Ser, Thr, or Val, (j)H97 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (k)H98 is Thr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (l)H99 is Ser, Ala, Gly, Phe, or Pro, (m)H100 is Leu, Arg, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr, or Val. (n)H100A is Ser, Ala, Arg, Asn, Asp, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, or Trp. (o)L29 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (p)L31 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val. (q) L91 is either Tyr or Arg, (r)L95A is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (s)L95B is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (t)L95C is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val Includes residues (numbered by Kabat), (u)L93 is Tyr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val, (v)L96 is Gly or Asn It may include things that are [like this]. Of these residues, H47 is a framework residue, and all the other residues are CDR residues.

[0078] When stricter substitution criteria are imposed (substitutions must result in an affinity of less than -0.5 kcal / mol, i.e., substitutions must have a positive (neutral / stabilizing) effect), the contact residues are as follows (numbered by Kabat) (Table 29A, column 5): (a) H33 is either Ala or Val, (b) H47 is a trump, (c)H50 is Ala, (d) H51 is Ile, (e) H52 is Ser, Arg, Lys, Phe, or Tyr, (f) H56 is Ser, Arg, or Lys, (g)H58 is Tyr, (h)H95 is Leu, (i) H96 is Gly, Ala, Arg, Asn, Lys, Pro, Ser, or Val, (j)H97 is Ala, (k)H98 is Thr, His, Ile, Leu, Met, Phe, or Tyr, (l) H99 is Ser, (m)H100 is Leu, Phe, Trp, or Tyr. (n)H100A is Ser, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, or Trp. (o)L29 is Ala, (p)L31 is Tyr, (q) L91 is Tyr, (r)L95A is Ser, Phe, Trp, or Tyr, (s)L95B is Gly, (t)L95C is Ser, Arg, Asn, Gln, Glu, Ile, Leu, Lys, Met, Phe, Trp, Tyr, or Val, (u)L93 may also be Ser, (v)L96 may be Gly.

[0079] Alternatively or additionally, the selection of acceptable substitutions can be based on column 6 of Table 29A, in which case the top three residues are selected based on their impact on affinity (i.e., the top three predicted positions that have the greatest stabilizing effect on affinity). Under this criterion, the contact residues are as follows (numbered by Kabat): (a) H33 is Ala, Val, His, or Phe, (b) H47 is Trp or Tyr, (c) H50 is Ala, Thr, Ser, or Phe, (d) H51 is Ile, Arg, Lys, or Pro, (e) H52 is Ser, Phe, Arg, or Tyr, (f) H56 is Ser, Lys, Tyr, or Phe, (g)H58 is Tyr, (h)H95 is Leu, Ile, Gln, or Phe, (i) H96 is Gly, Arg, Asn, or Lys, (j)H97 is Ala, Leu, Tyr, or Ile, (k)H98 is Thr, Tyr, Phe, or His. (l) H99 is Ser, Pro, Ala, or Phe, (m)H100 is Leu, Tyr, Trp, or Phe, (n)H100A is Ser, Arg, Leu, or Trp, (o)L29 is Ala, Glu, Asp, or Gln. (p)L31 is Tyr, Glu, Asp, or Trp. (q) L91 is either Tyr or Arg, (r)L95A is Ser, Phe, Tyr, or His, (s)L95B is Gly, Glu, Asp, or Pro. (t)L95C is Ser, Trp, Tyr, or Phe, (u)L93 may be Ser, Glu, Asp, or His. (v)L96 may be Gly or Asn.

[0080] The paratope residues of TFPI-23 (and its variants) may also refer to residues involved in hydrogen bonding with TFPI residues or with water molecules that are also hydrogen-bonded to TFPI, including: H58 Tyr, H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 Leu, L29 Ala, L31 Tyr, and L95B Gl (numbered by Kabat). See Table 29B.

[0081] Paratope residues of TFPI-23 (and its variants) may also refer to residues with non-zero changes in BSA due to interaction with TFPI, including (numbered by Kabat): H33 Ala, H58 Tyr, H95 Leu, H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 Leu, H100A Ser, L29 Ala, L31 Tyr, L91 Tyr, L93 Ser, L95A Ser, and L95B Gly. A cutoff (20 Å) is used to avoid including residues with minimal interaction. 2 The above, or BSAs (which involve electrostatic interactions), apply. See Table 29B.

[0082] If the BSA cutoff is not applied, the paratope residues include: H33 Ala, H34 Met, H47 Trp, H50 Ala, H51 Ile, H52 Ser, H56 Ser, H58 Tyr, H95 Leu, H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 Leu, H100A Ser, L28 Gly, L29 Ala, L31 Tyr, L91 Tyr, L93 Ser, L94 Ser, L95A Ser, L95B Gly, L95C Ser, and L96 Gly. See Table 29C.

[0083] The antibodies or antigen-binding fragments of the present invention may bind to the same epitopes or domains of TFPI as the antibodies specifically exemplified herein. For example, the antibodies or antigen-binding fragments can be identified by comparing their binding to TFPI with that of TFPI-23 or germline variants (e.g., TFPI-106 and TFPI-107), or by comparing the function of these antibodies with that of TFPI-23 and its variants. Analyses and assays that may be used for such identification purposes include assays to assess competition for TFPI binding, which are illustrated in the examples.

[0084] In one embodiment, the antibody or antigen-binding fragment of the present invention may bind to the same epitope or region as the antibodies described herein, such as TFPI-23 and its variants. This may include those in contact with the specific TFPI residues described above. For example, the antibody or antigen-binding fragment of the present invention may bind to TFPI in such a way that it is in contact (within 4 Å) with a residue selected from the group consisting of Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, Glu138 (as numbered in SEQ ID NO: 2), and any combination thereof. The antibody or antigen-binding fragment of the present invention may be able to bind to an epitope containing one or more residues selected from the group consisting of Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, Glu138 (as numbered in SEQ ID NO: 2), and any combination thereof.

[0085] The antibody or its antigen-binding fragment may contain at least one paratope residue (numbered by Kabat) within 4.0 Å of at least one epitope residue (numbered by Sequence ID No. 2) on the TFPI, for example: epitope residue 102 Asp is within 4.0 Å of paratope residue H58 Tyr; epitope residue 104 Gly is within 4.0 Å of paratope residue H58 Tyr; epitope residue 105 Ile is within 4.0 Å of paratope residues H33 Ala, H50 Ala, H51 Ile, H52 Ser, H56 Ser, H58 Tyr, H95 Leu; epitope residue 106 Cys is within 4.0 Å of paratope residues H100 Leu, H100A Ser; epitope residue 107 Arg is within 4.0 Å of paratope residues H96 Gly, H97 Ala, H98 Thr, H99 Ser is within 4.0 Å of H100 Leu; epitope residue 108 Gly is within 4.0 Å of paratope residue H100 Leu; epitope residue 112 Arg is within 4.0 Å of paratope residues L29 Ala and L31 Tyr; epitope residue 127 Tyr is within 4.0 Å of paratope residue L31 Tyr; epitope residue 129 Gly is within 4.0 Å of paratope residue L31 Tyr; epitope residue 130 Cys is within 4.0 Å of paratope residues L91 Tyr and L95B Gly; epitope residue 131 Leu is within 4.0 Å of paratope residues H47 Trp, H50 Ala, H58 Tyr, L95A Ser, L95B Gly, and L95C Ser; epitope residue 132 Gly is within 4.0 Å of paratope residue H58 Tyr, L95A Ser; epitope residue 134 Met is within 4.0 Å of paratope residue L95A Ser; and epitope residue 138 Glu is within 4.0 Å of paratope residue L29 Ala. See Tables 29A and 29B.

[0086] The antibody or its antigen-binding fragment may contain at least one paratope residue (numbered by Kabat) that can form hydrogen bonds with the epitope residue (numbered by Sequence ID No. 2) of TFPI, for example: epitope residue 102 Asp can form a hydrogen bond with paratope residue H58 Tyr; epitope residue 107 Arg can form a hydrogen bond with at least one paratope residue selected from the group consisting of H96 Gly, H97 Ala, H98 Thr, H99 Ser, and H100 Leu; epitope residue 112 Arg can form a hydrogen bond with paratope residue L29 Ala; epitope residue 127 Tyr can form a hydrogen bond with paratope residue L31 Tyr; epitope residue 131 Leu can form a hydrogen bond with paratope residue L95B Gly. See Table 29B.

[0087] The antibody or its antigen-binding fragment may also contain at least one paratope residue (numbered by Kabat) that results in a non-zero change in BSA due to interaction with an epitope residue (numbered by Sequence ID No. 2), for example: epitope residue 102 Asp interacts with paratope residue H58 Tyr; epitope residue 104 Gly interacts with paratope residue H58 Tyr; epitope residue 105 Ile interacts with at least one paratope residue selected from the group consisting of H33 Ala, H34 Met, H50 Ala, H51 Ile, H52 Ser, H56 Ser, H58 Tyr, and H95 Leu; epitope residue 106 Cys interacts with at least one paratope residue selected from the group consisting of H95 Leu, H100 Leu, H100A Ser, and L91 Tyr; epitope residue 107 Arg interacts with H96 Gly, H97 Epitope residue 108 Gly interacts with at least one paratope residue selected from the group consisting of Ala, H98 Thr, H99 Ser, and H100 Leu; epitope residue 108 Gly interacts with paratope residue H100 Leu; epitope residue 112 Arg interacts with at least one paratope residue selected from the group consisting of L29 Ala, L31 Tyr, and L93 Ser; epitope residue 127 Tyr interacts with at least one paratope residue selected from the group consisting of L31 Tyr and L95B Gly; epitope residue 129 Gly interacts with at least one paratope residue selected from the group consisting of H100A Ser, L31 Tyr, and L91 Tyr; epitope residue 130 Cys interacts with H95 Leu, H100A Ser, L31 Tyr, L91 Tyr, and L95B It interacts with at least one paratope residue selected from the group consisting of Gly; epitope residue 131 Leu interacts with at least one paratope residue selected from the group consisting of H47 Trp, H50 Ala, H58 Tyr, H95 Leu, L31 Tyr, L91 Tyr, L95A Ser, L95B Gly, L95C Ser, and L96 Gly;Epitope residue 132 Gly interacts with at least one paratope residue selected from the group consisting of H58 Tyr and L95A Ser; epitope residue 133 Asn interacts with paratope residue L95A Ser; epitope residue 134 Met interacts with at least one paratope residue selected from the group consisting of L93 Ser, L94 Ser, and L95A Ser; and epitope residue 138 Glu interacts with at least one paratope residue selected from the group consisting of L28 Gly, L29 Ala, and L93 Ser.

[0088] The antibody or antigen-binding fragment of the present invention may be any antibody or antigen-binding fragment comprising any of the above-mentioned paratope residues that interact with at least one of the epitope residues listed above.

[0089] In certain embodiments, the antibody or antigen-binding fragment described herein includes at least one, at least two, at least three, at least four, at least five, at least six, at least seven, at least eight, at least nine, at least ten, at least eleven, at least 12, at least 13, or all of the paratope residues described above, or any combination of the various categories of paratope residues described above. Furthermore, conservative substitutions may be introduced into these paratope residues. For example, the antibody or antigen-binding fragment may include one, two, three, four, five, six, seven, or eight conservative substitutions as shown in Table 34.

[0090] [Table 2]

[0091] The antibodies of the present invention may have the ability to compete with other antibodies for binding to TFPI as described herein. For example, the antibodies of the present invention may cross-compete with TFPI-23 and its variants as described herein for binding to TFPI, or to a suitable fragment or variant of TFPI bound by a TFPI-23 antibody. Such cross-competitive antibodies can be identified based on their ability to cross-compete with the antibodies of the present invention exemplified in standard binding assays. For example, cross-competition can be demonstrated using SPR (e.g., by using a Biacore® system), ELISA assay, or flow cytometry. Such cross-competition may suggest that the two antibodies bind to the same, overlapping, or similar epitopes.

[0092] Therefore, the antibodies of the present invention can be identified by a method comprising a binding assay that assesses whether a test antibody can compete for the binding site of a target molecule with an example antibody of the present invention (such as TFPI-23 as described herein, or any variant or fragment thereof).

[0093] In certain embodiments, the antibody or antigen-binding fragment described herein comprises the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 38, CDR-H2 containing SEQ ID NO: 39, CDR-H3 containing SEQ ID NO: 40, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 33, CDR-L2 containing SEQ ID NO: 34, and CDR-L3 containing SEQ ID NO: 35. In certain embodiments, the antibody or antigen-binding fragment described herein includes the following heavy chain CDR sequences: (i) CDR-H1 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 38, CDR-H2 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 39, and CDR-H3 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 40, and / or (ii) the following light chain CDR sequences: CDR-L1 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 33, CDR-L2 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 34, and CDR-L3 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 35. In certain embodiments, each CDR is subjected to 10 or fewer substitutions compared to sequence numbers 38, 39, 40, 33, 34, and 35, respectively, with 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions. In certain embodiments, the substitutions are conservative substitutions according to Table 34. In certain embodiments, the substitutions are according to Table 29A, column 4, column 5, or column 6.

[0094] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, the heavy chain framework sequence may be derived from the human VH3 germline, VH1 germline, VH5 germline, or VH4 germline. Preferred human germline heavy chain frameworks are those derived from the VH1, VH3, or VH5 germline. For example, the following VH frameworks derived from germlines can be used: IGHV3-23, IGHV3-7, or IGHV1-69 (germline names are based on the IMGT germline definition). Preferred human germline light chain frameworks are those derived from the VK or Vλ germline. For example, the following VL frameworks derived from germlines can be used: IGKV1-39 or IGKV3-20 (germline names are based on the IMGT germline definition). Alternatively or additionally, the framework sequence may be a framework of human germline consensus framework sequences, such as the human Vλ1 consensus sequence, VK1 consensus sequence, VK2 consensus sequence, VK3 consensus sequence, VH3 germline consensus sequence, VH1 germline consensus sequence, VH5 germline consensus sequence, or VH4 germline consensus sequence.

[0095] Human germline framework sequences are available from various public databases, such as V-base, IMGT, NCBI, or Abysis.

[0096] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a VH containing an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs. 41, 63, and 65, and / or (ii) a VL containing an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of SEQ ID NOs.

[0097] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 20, and / or (ii) a CL having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 26. Any combination of these CH and CL sequences is also encompassed by the present invention.

[0098] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0099] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a heavy chain having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 42, SEQ ID NO: 64, or SEQ ID NO: 66, and / or (ii) a light chain having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 37. Any combination of these heavy and light chain sequences is also encompassed by the present invention.

[0100] TFPI-24 and variants The co-crystal structure shows that TFPI-24 (and its variants) share several epitope residues with TFPI-23. Among these, Ile105, Arg107, and Leu131 (as numbered in human TFPI in SEQ ID NO: 2) are thought to be important for antibody-antigen interaction. Other shared epitope residues include Cys106, Gly108, C130, L131, and G132 (as numbered in SEQ ID NO: 2).

[0101] Epitope residues specific to TFPI-24 (and its variants) include Glu100, Glu101, Asp102, Gly104, and Tyr109. TFPI-23 and its variants do not bind to these residues. See Table 27. Accordingly, the present invention provides an isolated antibody or antigen-binding fragment thereof that specifically binds to an epitope in K2 of TFPI, wherein the epitope comprises (i) residues Ile105, Arg107, and Leu131, (ii) one or more residues selected from the group consisting of Cys106, Gly108, Cys130, Leu131, and Gly132, and (iii) one or more residues selected from the group consisting of Glu100, Glu101, Asp102, Gly104, and Tyr109 (as numbered in SEQ ID NO: 2).

[0102] In certain embodiments, the epitope does not contain one or more residues selected from the group consisting of P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, L140 (numbered by SEQ ID NO: 2), and any combination thereof. See Table 27. According to WO201007269 (Novo Nordisk), the reference antibody 4F36 recognizes epitopes including P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140.

[0103] In certain embodiments, the epitope does not contain one or more residues selected from the group consisting of D31, D32, P34, C35, K36, P103, K126, Y127, G128 (numbered by Sequence ID No. 2), and any combination thereof. See Table 27. According to Table 27, reference antibodies 2A8 and 2A8-200 recognize epitopes including D31, D32, P34, C35, K36, P103, K126, Y127, and G128.

[0104] Paratope residues derived from TFPI-24 (based on BSA) have also been characterized (see Table 24), including: H33 Ala, H35 Gln, H52 Ser, H53 Asn, H55 Arg, H56 Ser, H95 Phe, H96 Leu, H97 His, H99 Ser, H101 Asp, L31 Met, L32 Tyr, L34 His, L36 Tyr, L50 Arg, L91 Trp, and L96 Tyr. In certain embodiments, the antibody or antigen-binding fragment described herein includes at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, or all of these paratope residues. Furthermore, conservative substitutions may be introduced into these paratope residues. For example, the antibody or its antigen-binding fragment may contain 1, 2, 3, 4, 5, 6, 7, or 8 conservative substitutions as shown in Table 34.

[0105] In certain embodiments, the antibodies or antigen-binding fragments described herein include the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 48, CDR-H2 containing SEQ ID NO: 49, and CDR-H3 containing SEQ ID NO: 50, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 43, CDR-L2 containing SEQ ID NO: 44, and CDR-L3 containing SEQ ID NO: 45. In certain embodiments, the antibody or antigen-binding fragment described herein includes the following heavy chain CDR sequences: (i) CDR-H1, CDR-H2, CDR-H2, CDR-H3 In certain embodiments, each CDR is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions compared to SEQ ID NOs.48, 49, 50, 43, 44, and 45, respectively. In certain embodiments, the substitutions are conservative substitutions as shown in Table 34.

[0106] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, heavy chain framework sequences may be derived from the human VH3, VH1, VH5, or VH4 germline described above. Preferred human germline light chain frameworks are those derived from the VK or Vλ germline described above. Consensus human germline framework sequences can also be used as described above.

[0107] In certain embodiments, the antibody or antigen-binding fragment described herein is (i) an amino acid sequence selected from the group consisting of SEQ ID NOs. 67, 69, 51, and 79 that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to (i) an amino acid sequence selected from the group consisting of SEQ ID NOs. 67, 69, 51, and 79. The VL includes an amino acid sequence (VH) containing a amino acid sequence, and / or (ii) an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 71, 73, 75, and 77, which is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the VH sequence. Any combination of these VL and VH sequences is also included by the present invention.

[0108] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 20, and / or (ii) a CL having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 26. Any combination of these CH and CL sequences is also encompassed by the present invention.

[0109] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0110] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a heavy chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 52, SEQ ID NO: 68, SEQ ID NO: 70, or SEQ ID NO: 80, and / or (ii) a light chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 47, SEQ ID NO: 72, SEQ ID NO: 74, SEQ ID NO: 76, or SEQ ID NO: 78. Any combination of these heavy and light chain sequences is also included in the present invention.

[0111] 4D8 and variants The cocrystal structure also reveals epitope and paratope information for antibody 4D8 and its variants. The epitope residues of 4D8 and its variants, as numbered in SEQ ID NO: 2, are Glu101, Pro103, Tyr109, Thr111, Ser119, Gln121, Glu123, Arg124, Lys126, and Leu140.

[0112] In certain embodiments, the epitope does not contain one or more residues selected from the group consisting of E100, D102, R107, Y113, F114, N116, Q118, C122 (numbered by SEQ ID NO: 2), and any combination thereof. See Table 27. According to WO201007269 (Novo Nordisk), the reference antibody 4F36 recognizes epitopes including E100, D102, R107, Y113, F114, N116, Q118, and C122.

[0113] In certain embodiments, the epitope does not contain one or more residues selected from the group consisting of D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127, G128 (numbered by Sequence ID No. 2), and any combination thereof. See Table 27. According to Table 27, reference antibodies 2A8 and 2A8-200 recognize epitopes including D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127, and G128.

[0114] Paratope residues derived from 4D8 (based on BSA) have also been characterized (see Table 20), including: H50 Asp, H57 Thr, H58 Leu, H59 Tyr, H61 Gln, H98 Asp, H99 Tyr, H100 Asp, L30 His, L50 Trp, L92 Tyr, L93 Thr, L94 Thr, and L96 Tyr. In certain embodiments, the antibody or antigen-binding fragment described herein contains at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all of these paratope residues. Furthermore, conservative substitutions may introduce these paratope residues. For example, the antibody or antigen-binding fragment may contain 1, 2, 3, 4, 5, 6, 7, or 8 conservative substitutions according to Table 34.

[0115] In certain embodiments, the antibody or antigen-binding fragment described herein comprises the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 87, CDR-H2 containing SEQ ID NO: 88, and CDR-H3 containing SEQ ID NO: 89, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 81, CDR-L2 containing SEQ ID NO: 82, and CDR-L3 containing SEQ ID NO: 83. In certain embodiments, the antibody or antigen-binding fragment described herein includes the following heavy chain CDR sequences: (i) CDR-H1 identical to at least 85%, at least 90%, or at least 95% of SEQ ID NO: 87, CDR-H2 identical to at least 85%, at least 90%, or at least 95% of SEQ ID NO: 88, and CDR-H3 identical to at least 85%, at least 90%, or at least 95% of SEQ ID NO: 89, and / or (ii) the following light chain CDR sequences: CDR-L1 identical to at least 85%, at least 90%, or at least 95% of SEQ ID NO: 81, CDR-L2 identical to at least 85%, at least 90%, or at least 95% of SEQ ID NO: 82, and CDR-L3 identical to at least 85%, at least 90%, or at least 95% of SEQ ID NO: 83. In certain embodiments, each CDR is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions compared to SEQ ID NOs.87, 88, 89, 81, 82, and 83, respectively. In certain embodiments, the substitutions are conservative substitutions as shown in Table 34.

[0116] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, heavy chain framework sequences may be derived from the human VH3, VH1, VH5, or VH4 germline described above. Preferred human germline light chain frameworks are those derived from the VK or Vλ germline described above. Consensus human germline framework sequences can also be used as described above.

[0117] In a particular embodiment, the antibody or antigen-binding fragment described herein is (i) an amino acid sequence selected from the group consisting of SEQ ID NOs: 90, 95, 97, 99, 101, 103, 105, and 107, and at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, if (ii) a VH containing 100% identical amino acid sequences, and / or (ii) a VL containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences to an amino acid sequence selected from the group consisting of SEQ ID NOs: 84, 109, and 111. Any combination of these VL and VH sequences is also encompassed by the present invention.

[0118] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence of SEQ ID NO: 91 or SEQ ID NO: 85 by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence

[0119] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0120] In certain embodiments, the antibodies or antigen-binding fragments described herein are (i) at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, and less than (i) SEQ ID NOs: 92, 94, 96, 98, 100, 102, 104, 106, 108. (ii) a heavy chain containing 99% or 100% identical amino acid sequences, and / or (ii) a light chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequences to (ii) SEQ ID NO: 86, SEQ ID NO: 93, SEQ ID NO: 110, or SEQ ID NO: 112. Any combination of these heavy and light chain sequences is also included by the present invention.

[0121] TFPI-3 and variants TFPI-3 and its variants are also provided herein. Accordingly, antibodies based on TFPI-3 or their antigen-binding fragments include the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 16, CDR-H2 containing SEQ ID NO: 17, and CDR-H3 containing SEQ ID NO: 18, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 10, CDR-L2 containing SEQ ID NO: 11, and CDR-L3 containing SEQ ID NO: 12. In certain embodiments, the antibody or antigen-binding fragment described herein includes the following heavy chain CDR sequences: (i) CDR-H1 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 16, CDR-H2 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 17, and CDR-H3 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 18, and / or (ii) the following light chain CDR sequences: CDR-L1 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 10, CDR-L2 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 11, and CDR-L3 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 12. In certain embodiments, each CDR is subjected to 10 or fewer substitutions compared to sequence numbers 16, 17, 18, 10, 11, and 12, respectively, 9 or fewer, 8 or fewer, 7 or fewer, 6 or fewer, 5 or fewer, 4 or fewer, 3 or fewer, 2 or fewer, or 1 or fewer substitutions. In certain embodiments, the substitutions are conservative substitutions as shown in Table 34.

[0122] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, heavy chain framework sequences may be derived from the human VH3, VH1, VH5, or VH4 germline described above. Preferred human germline light chain frameworks are those derived from the VK or Vλ germline described above. Consensus human germline framework sequences can also be used as described above.

[0123] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a VH having an amino acid sequence identical to (i) SEQ ID NO: 19 by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, and / or (ii) a VL having an amino acid sequence identical to (ii) SEQ ID NO: 13 by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100%, respectively. Any combination of these VL and VH sequences is also encompassed by the present invention.

[0124] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence of SEQ ID NO: 91 or SEQ ID NO: 14 by at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the sequence the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence of the sequence

[0125] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0126] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a heavy chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 21, and / or (ii) a light chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 15. Any combination of these heavy and light chain sequences is also encompassed by the present invention.

[0127] TFPI-21 and variants TFPI-21 and its variants are also provided herein. Accordingly, antibodies based on TFPI-21 or their antigen-binding fragments include the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 28, CDR-H2 containing SEQ ID NO: 29, and CDR-H3 containing SEQ ID NO: 30, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 22, CDR-L2 containing SEQ ID NO: 23, and CDR-L3 containing SEQ ID NO: 24. In certain embodiments, the antibody or antigen-binding fragment described herein includes the following heavy chain CDR sequences: (i) CDR-H1 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 28, CDR-H2 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 29, and CDR-H3 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 30, and / or (ii) the following light chain CDR sequences: CDR-L1 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 22, CDR-L2 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 23, and CDR-L3 which is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 24. In certain embodiments, each CDR is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions compared to SEQ ID NOs.28, 29, 30, 22, 23, and 24, respectively. In certain embodiments, the substitutions are conservative substitutions as shown in Table 34.

[0128] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, heavy chain framework sequences may be derived from the human VH3, VH1, VH5, or VH4 germline described above. Preferred human germline light chain frameworks are those derived from the VK or Vλ germline described above. Consensus human germline framework sequences can also be used as described above.

[0129] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a VH having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 31, and / or (ii) a VL having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 25. Any combination of these VL and VH sequences is also encompassed by the present invention.

[0130] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 20, and / or (ii) a CL having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 91 or SEQ ID NO: 26. Any combination of these CH and CL sequences is also encompassed by the present invention.

[0131] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0132] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a heavy chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 32, and / or (ii) a light chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 27. Any combination of these heavy and light chain sequences is also encompassed by the present invention.

[0133] TFPI-26 and variants TFPI-26 and its variants are also provided herein. Accordingly, antibodies based on TFPI-26 or their antigen-binding fragments include the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 58, CDR-H2 containing SEQ ID NO: 59, and CDR-H3 containing SEQ ID NO: 60, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 53, CDR-L2 containing SEQ ID NO: 54, and CDR-L3 containing SEQ ID NO: 55. In certain embodiments, the antibody or antigen-binding fragment described herein includes the following heavy chain CDR sequences: (i) CDR-H1, CDR-H2, CDR-H2, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-L1, CDR-H1, CDR-H1, CDR-H1, CDR-H1, CDR-H1, CDR-H1, In certain embodiments, each CDR is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions compared to SEQ ID NOs.58, 59, 60, 53, 54, and 55, respectively. In certain embodiments, the substitutions are conservative substitutions as shown in Table 34.

[0134] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, heavy chain framework sequences may be derived from the human VH3, VH1, VH5, or VH4 germline described above. Preferred human germline light chain frameworks are those derived from the VK or Vλ germline described above. Consensus human germline framework sequences can also be used as described above.

[0135] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a VH containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 61, and / or (ii) a VL containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 56. Any combination of these VL and VH sequences is also encompassed by the present invention.

[0136] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 20, and / or (ii) a CL having an amino acid sequence identical to at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% of the SEQ ID NO: 26. Any combination of these CH and CL sequences is also encompassed by the present invention.

[0137] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0138] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a heavy chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 62, and / or (ii) a light chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 57. Any combination of these heavy and light chain sequences is also encompassed by the present invention.

[0139] 6B7.c5 and variant 6B7.c5 and its variants are also provided herein. Accordingly, antibodies based on 6B7.c5 or their antigen-binding fragments include the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 118, CDR-H2 containing SEQ ID NO: 119, and CDR-H3 containing SEQ ID NO: 120, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 113, CDR-L2 containing SEQ ID NO: 114, and CDR-L3 containing SEQ ID NO: 115. In certain embodiments, the antibody or antigen-binding fragment described herein includes the following heavy chain CDR sequences: (i) CDR-H1, CDR-H2, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-L3, CDR-H3, CDR-H3, CDR-L3, CDR-H3, CDR-L3, CDR-H3, CDR-H3, CDR-L3, CDR-H3, CDR-H3, CDR-L3, CDR-H3, CDR-H3, CDR-L2, CDR-H3, CDR-L3, CDR-H3, CDR-L2, CDR-H3, CDR-L2, CDR-H3, CDR-L3, CDR-H3, CDR-H1, CDR-H1, CDR-H1, CDR-H2, CDR-H3, CDR-L2, CDR-H3, CDR-H1 In certain embodiments, each CDR is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions compared to sequence numbers 118, 119, 120, 113, 114, and 115, respectively. In certain embodiments, the substitutions are conservative substitutions as shown in Table 34.

[0140] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, heavy chain framework sequences may be derived from the human VH3, VH1, VH5, or VH4 germline described above. Preferred human germline light chain frameworks are those derived from the VK or Vλ germline described above. Consensus human germline framework sequences can also be used as described above.

[0141] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a VH containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 121, and / or (ii) a VL containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 116. Any combination of these VL and VH sequences is also encompassed by the present invention.

[0142] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH containing an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 91, and / or (ii) a CL containing an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 91 or SEQ ID NO: 85. Any combination of these CH and CL sequences is also encompassed by the present invention.

[0143] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0144] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a heavy chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 122, and / or (ii) a light chain containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 117. Any combination of these heavy and light chain sequences is also encompassed by the present invention.

[0145] 7A4.D9 and variants 7A4.D9 and its variants are also provided herein. Accordingly, antibodies based on 7A4.D9 or their antigen-binding fragments include the following heavy chain CDR sequences: (i) CDR-H1 containing SEQ ID NO: 128, CDR-H2 containing SEQ ID NO: 129, and CDR-H3 containing SEQ ID NO: 130, and / or (ii) the following light chain CDR sequences: CDR-L1 containing SEQ ID NO: 123, CDR-L2 containing SEQ ID NO: 124, and CDR-L3 containing SEQ ID NO: 125. In certain embodiments, the antibodies or antigen-binding fragments described herein include the following heavy chain CDR sequences: (i) CDR-H1, CDR-H2, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-H3, CDR-L3, CDR-H3, CDR-L3, CDR-H3, CDR-L3, CDR-H3, CDR-L3, CDR-H3, CDR-L3, CDR-H3, CDR-L3, CDR-H3, CDR-L2, CDR-L2, CDR-L3, CDR-L2, CDR-L3, CDR-L2, CDR-L3, CDR-L2, CDR-L3, CDR-L2, CDR-L3, CDR-L2, CDR-L2, CDR-L3, CDR-L2, CDR-L3, CDR-L2 In certain embodiments, each CDR is subjected to 10 or fewer substitutions, 9 or fewer substitutions, 8 or fewer substitutions, 7 or fewer substitutions, 6 or fewer substitutions, 5 or fewer substitutions, 4 or fewer substitutions, 3 or fewer substitutions, 2 or fewer substitutions, or 1 or fewer substitutions compared to sequence numbers 128, 129, 130, 123, 124, and 125, respectively. In certain embodiments, the substitutions are conservative substitutions as shown in Table 34.

[0146] In certain embodiments, the antibodies or antigen-binding fragments described herein include human framework sequences. For example, heavy chain framework sequences may be derived from the human VH3, VH1, VH5, or VH4 germline described above. Preferred human germline light chain frameworks are those derived from the VK or Vλ germline described above. Consensus human germline framework sequences can also be used as described above.

[0147] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a VH containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 131, and / or (ii) a VL containing at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 126. Any combination of these VL and VH sequences is also encompassed by the present invention.

[0148] In certain embodiments, the antibody or antigen-binding fragment described herein includes (i) a CH containing an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 91, and / or (ii) a CL containing an amino acid sequence that is at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to an amino acid sequence of SEQ ID NO: 91 or SEQ ID NO: 85. Any combination of these CH and CL sequences is also encompassed by the present invention.

[0149] In certain embodiments, the antibody or antigen-binding fragment described herein includes an Fc domain. The Fc domain may be derived from IgA (e.g., IgA1 or IgA2), IgG, IgE, or IgG (e.g., IgG1, IgG2, IgG3, or IgG4).

[0150] In certain embodiments, the antibodies or antigen-binding fragments described herein include (i) a heavy chain having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 132, and / or (ii) a light chain having at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical amino acid sequence to SEQ ID NO: 127. Any combination of these heavy and light chain sequences is also encompassed by the present invention.

[0151] Also disclosed herein are antibodies or antigen-binding fragments that specifically bind to the K2 domain of TFPI and compete for binding to TFPI with any of the antibodies or antigen-binding fragments described herein, such as any of the antibodies (or antigen-binding fragments) listed in Table 3. For example, if the binding of an antibody or its antigen-binding moiety to TFPI interferes with subsequent binding to TFPI by TFPI-23 or TFPI-106, then this antibody or its antigen-binding moiety competes with TFPI-23 or TFPI-106 for TFPI binding.

[0152] Also disclosed herein are antibodies or antigen-binding fragments that specifically bind to the K2 domain of TFPI and bind to the same TFPI epitope as any of the antibodies or antigen-binding fragments described herein, for example, any one of the antibodies or antigen-binding fragments listed in Table 3.

[0153] An exemplary antibody competition assay (and overlapping epitope analysis) using SPR is provided in Example 6.

[0154] Antibodies and antigen-binding fragments disclosed herein include monoclonal antibodies, polyclonal antibodies, antibody fragments (e.g., Fab, Fab', F(ab')2, Fv, Fc, etc.), chimeric antibodies, bispecific antibodies, heteroconjugate antibodies, single-chain antibodies (ScFv), mutants thereof, fusion proteins containing antibody moieties, domain antibodies (dAb), humanized antibodies, and any other modified configurations of immunoglobulin molecules containing antigen recognition sites of the required specificity, including glycosylated variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies. Antibodies and antigen-binding fragments may be of mouse, rat, human, or any other origin (including chimeric or humanized antibodies). In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the antibody is a chimeric, humanized, or human antibody. In certain embodiments, the antibody is a human antibody. In certain embodiments, the antibody is a humanized antibody.

[0155] In certain embodiments, the antibody or antigen-binding fragment disclosed herein is approximately 1 × 10 -3 Less than M, for example, approximately 5 × 10 -4 M or less, approximately 4 x 10 -4 M or less, about 3 x 10 -4 M or less, approximately 2×10 -4 M or less, approximately 1×10 -4 M or less, approximately 9 x 10 -5 M or less, about 8 x 10 -5 M or less, about 7 x 10 -5 M or less, approximately 6×10 -5 M or less, about 5 x 10 -5 M or less, approximately 4 x 10-5 Below M, approximately 3×10 -5 Below M, approximately 2×10 -5 Below M, approximately 1×10 -5 Below M, approximately 9×10 -6 Below M, approximately 8×10 -6 Below M, approximately 7×10 -6 Below M, approximately 6×10 -6 Below M, approximately 5×10 -6 Below M, approximately 4×10 -6 Below M, approximately 3×10 -6 Below M, approximately 2×10 -6 Below M, approximately 1×10 -6 Below M, approximately 9×10 -7 Below M, approximately 8×10 -7 Below M, approximately 7×10 -7 Below M, approximately 6×10 -7 Below M, approximately 5×10 -7 Below M, approximately 4×10 -7 Below M, approximately 3×10 -7 Below M, approximately 2×10 -7 Below M, approximately 1×10 -7 Below M, approximately 9×10 -8 Below M, approximately 8×10 -8 Below M, approximately 7×10 -8 Below M, approximately 6×10 -8 Below M, approximately 5×10 -8 Below M, approximately 4×10 -8 Below M, approximately 3×10 -8 Below M, approximately 2×10 -8 Below M, approximately 1×10 -8 Below M, approximately 9×10 -9 Below M, approximately 8×10 -9 Below M, approximately 7×10 -9 Below M, approximately 6×10 -9 Below M, approximately 5×10 -9 Below M, approximately 4×10 -9 Below M, approximately 3×10 -9 Below M, approximately 2×10 -9 Below M, approximately 1×10 -9 Below M, approximately 1×10 -3 M ~ approximately 1×10 -13 M, 1×10 -4 M ~ approximately 1×10 -13 M, 1×10 -5 M ~ approximately 1×10 -13 M, approximately 1×10-6 M ~ approx. 1×10 -13 M, about 1 x 10 -7 M ~ approx. 1×10 -13 M, about 1 x 10 -8 M ~ approx. 1×10 -13 M, about 1 x 10 -9 M ~ approx. 1×10 -13 M, 1×10 -3 M ~ approx. 1×10 -12 M, 1×10 -4 M ~ approx. 1×10 -12 M, about 1 x 10 -5 M ~ approx. 1×10 -12 M, about 1 x 10 -6 M ~ approx. 1×10 -12 M, about 1 x 10 -7 M ~ approx. 1×10 -12 M, about 1 x 10 -8 M ~ approx. 1×10 -12 M, about 1 x 10 -9 M ~ approx. 1×10 -12 M, 1×10 -3 M ~ approx. 1×10 -11 M, 1×10 -4 M ~ approx. 1×10 -11 M, about 1 x 10 -5 M ~ approx. 1×10 -11 M, about 1 x 10 -6 M ~ approx. 1×10 -11 M, about 1 x 10 -7 M ~ approx. 1×10 -11 M, about 1 x 10 -8 M ~ approx. 1×10 -11 M, about 1 x 10 -9 M ~ approx. 1×10 -11 M, 1×10 -3 M ~ approx. 1×10 -10 M, 1×10 -4 M ~ approx. 1×10 -10 M, about 1 x 10 -5 M ~ approx. 1×10 -10 M, about 1 x 10 -6 M ~ approx. 1×10 -10 M, about 1 x 10 -7 M ~ approx. 1×10 -10 M, about 1 x 10 -8 M ~ approx. 1×10 -10 MM, or approximately 1 x 10 -9 M ~ approx. 1×10 -10It has affinity (Kd) values ​​such as M.

[0156] In certain embodiments, the dissociation constant is measured using surface plasmon resonance (SPR) (Biacore). Surface plasmon resonance refers to an optical phenomenon that enables real-time analysis of biospecific interactions by detecting changes in protein concentration within a biosensor matrix, for example, using a BIACORE® system. In certain embodiments, SPR measurements are performed using a Biacore T100 or T200 instrument.

[0157] For example, standard assay conditions for surface plasmon resonance (SPR) can be based on the immobilization of approximately 100 response units (RUs) of IgG ligand onto an SPR chip. The purified target protein is diluted in buffer to a series of final concentrations and injected at the required flow rate (e.g., 10–100 μl / min) to enable Ka calculation. Dissociation is allowed to proceed to establish the off-rate (Kd), followed by a 5-second pulse of 3M MgCl2 (or 20 mM NaOH) to regenerate the chip surface. The sensorgram is then analyzed using a kinetics evaluation software package.

[0158] In an exemplary embodiment, the SPR assay is performed under the conditions shown in Example 1, subtitled "Surface Plasmon Resonance (SPR)".

[0159] In certain embodiments, the dissociation constant is measured using a solution-based dynamic exclusion assay (KinExA®). In certain embodiments, the KinExA measurement is performed using a KinExA® 3200 instrument (Sapidyne). The dynamic exclusion assay (KinExA®) is a versatile immunoassay platform (essentially a flow fluorescence spectrometer) capable of measuring the equilibrium dissociation constant as well as the association and dissociation rate constants of antigen / antibody interactions. Since KinExA® is performed after equilibrium has been achieved, it is a favorable technique for measuring Kd of high-affinity interactions where the off-rate of the interaction may be very slow. The KinExA® method can generally be performed as described by Drake et al. (2004), Analytical Biochemistry, 328, 35-43.

[0160] Generally, TFPI antibodies need to bind to TFPI with high affinity to effectively block TFPI activity. However, since TFPI is also expressed on the cell surface, if the antibody's binding affinity is too high, the antibody can be rapidly internalized and degraded by the host cell. This can potentially lead to a short half-life and repeated injections. For example, the antibody TFPI-23 exhibits a lower binding affinity (Kd) compared to TFPI-24, which is more desirable under certain circumstances because it has a lower internalized rate and a longer half-life. Therefore, if a longer half-life is desired, 5 × 10⁻⁶ -7 M ~ approx. 5×10 -11 M, in particular, about 1 × 10 -8 M ~ approx. 1×10 -10 A binding affinity (Kd) of M (0.1 nM to 10 nM) is generally desirable. This range is thought to strike a balance between (i) the binding affinity required to effectively inhibit TFPI activity and (ii) a longer half-life and reduced internal transfer of the antibody.

[0161] In particular, maintaining weekly subcutaneous administration at 3 mg / kg requires approximately 1 × 10⁻⁶ -8 M ~ approx. 1×10 -10A Kd value of M (0.1nM to 10nM) is considered desirable.

[0162] Whether an antibody or its antigen-binding fragment reduces the activity of TFPI or reduces the binding of TFPI to its physiological substrate (e.g., FXa) can be determined by measuring the decrease in the binding affinity of TFPI to its physiological substrate, for example, by comparing (i) the binding affinity of TFPI to its substrate in the presence of an anti-TFPI antibody (or its antigen-binding fragment) with (ii) the binding affinity of TFPI to the same substrate in the absence of an anti-TFPI antibody. The reduction in the binding of TFPI to its physiological substrate (e.g., FXa) in the presence of an anti-TFPI antibody (or its antigen-binding fragment) may be at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%. The expected binding of TFPI to its physiological substrate in the absence of an antibody (or fragment) can be set to 100%.

[0163] The TFPI inhibitory activity of an anti-TFPI antibody or its antigen-binding fragment is also referred to herein as "reducing the activity of TFPI" and can be assessed in vivo models and / or in vitro, for example, using plasma systems. For example, the inhibitory activity of an antibody (or the level at which it reduces the activity of TFPI) can be assessed by (i) a decrease in clotting time as measured by a plasma-based diluted prothrombin time assay, (ii) a decrease in clotting time in whole blood as measured by thromboelastography, (iii) an increase in thrombin production, (iv) an increase in FXa in the presence of TFPI, (v) an enhancement of platelet accumulation in the presence of TFPI, (vi) an increase in fibrin production in the presence of TFPI, or (vii) any combination thereof. The inhibitory activity of an antibody or antigen-binding fragment may be dose-dependent (e.g., causing a dose-dependent decrease in clotting time as measured by a plasma-based diluted prothrombin time assay).

[0164] Several exemplary assays for assessing the TFPI inhibitory activity of antibodies are described in detail in the examples. For example, the plasma dilution prothrombin time (PT) assay is a modified PT assay that uses diluted thromboplastin or tissue factor to extend the coagulation time and dynamic range of the assay. Inhibitory / neutralizing anti-TFPI antibodies should reduce the dilution prothrombin time.

[0165] Another exemplary model system for determining TFPI inhibitory activity is the exogenous tenase assay, which tests the ability of an antibody or its antigen-binding fragment to restore exogenous complex-mediated FX activation in the presence of TFPI. Another model system for characterizing TFPI inhibitory activity is the FXa inhibitory assay, in which FXa activity is measured in the presence of TFPI (see Sprecher et al., Proc.Nat.Acad.Sci.USA, 91:3353~3357 (1994)).

[0166] The inhibitory activity of an antibody or its antigen-binding fragment can also be assessed in plasma-based assays. Thrombin formation can be induced in plasma substantially lacking FVIII or FIX activity (e.g., residual coagulation factor activity less than 1%) in the presence of an anti-TFPI antibody or its antigen-binding fragment. Thrombin formation can be detected using fluorescence-generating or chromogenic substrates. Prothrombin conversion can be measured, for example, using Thrombograph® (Thermo Scientific, Waltham, Mass.), and the resulting data can be compiled into a calibrated automated thrombogram generated by Thrombinoscope® software available from Thrombinoscope BV.

[0167] For example, an antibody or antigen-binding fragment may enhance TFPI-regulated thrombin production in the absence of FVIII (e.g., in FVIII-depleted plasma) to at least 1% of the level of TFPI-dependent thrombin production in normal plasma. Generally, normal (asymptomatic) plasma contains about 0.5 U / mL to about 2 U / mL of factor VIII. Therefore, in some cases, the antibody or antigen-binding fragment of the present invention will enhance thrombin formation in the absence of FVIII to at least 1% of what is observed in the presence of 0.5 U / mL to 2 U / mL of FVIII. In further embodiments, the antibody (or its antigen-binding fragment) enhances thrombin formation in the absence of factor VIII to at least about 2%, at least about 3%, at least about 5%, at least about 7%, or at least about 10% of the level of thrombin formation in normal plasma, i.e., in the presence of physiological levels of factor VIII.

[0168] Antibodies or antigen-binding fragments can also be administered to animal models of thrombin deficiency or hemophilia to characterize TFPI inhibitory activity in vivo. Such in vivo models are known in the art and include, for example, mice in which hemophilia A is induced by administration of anti-FVIII antibodies (Tranholm et al., Blood, 102, 3615-3620 (2003)); coagulation factor knockout models, for example, but not limited to FVIII knockout mice (Bi et al., Nat. Genet., 10(1), 119-121 (1995)) and FIX knockout mice (Wang et al., Proc. Nat. Acad. Sci. USA, 94(21):11563-11566 (1997)); induced hemophilia A in rabbits (Shen et al., Blood, 42(4):509-521 (1973)); and Chapel Hill There is also HA dogs (Lozier et al., Proc. Nat. Acad. Sci. USA, 99:12991~12996 (2002)).

[0169] In certain embodiments, the antibody (or antigen-binding fragment) disclosed herein enhances FXa activity in the presence of TFPI to a semi-maximal effective concentration (EC2). 50) is 1 × 10 -4 M or less, 1×10 -5 M or less, 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less, or 1 × 10 -12 It is less than or equal to M. Preferably, EC 50 It is approximately 5 x 10 -7 M~1×10 -11 M, for example, approximately 1 × 10 -7 M~5×10 -10 M, about 1 x 10 -7 M~1×10 -10 M, 1×10 -7 M~5×10 -9 M, 5×10 -7 M~5×10 -10 M, about 5 x 10 -7 M~1×10 -10 M, or approximately 5 x 10 -7 M~5×10 -9 Examples include M.

[0170] In certain embodiments, the antibody (or antigen-binding fragment) disclosed herein neutralizes TFPI inhibition of FVIIa / TF-mediated FX activation and reaches a semi-maximal effective concentration (EC2). 50 ) is 1 × 10 -4 M or less, 1×10 -5 M or less, 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less, or 1 × 10 -12 It is less than or equal to M. Preferably, EC 50 It is approximately 5 x 10 -7 M~1×10 -11 M, for example, approximately 1 × 10 -7 M~5×10 -10 M, about 1 x 10 -7 M~1×10 -10 M, 1×10 -7 M~5×10-9 M, 5×10 -7 M~5×10 -10 M, about 5 x 10 -7 M~1×10 -10 M, or approximately 5 x 10 -7 M~5×10 -9 Examples include M.

[0171] In certain embodiments, the antibodies (or antigen-binding fragments) disclosed herein reduce coagulation time when measured in a plasma-based diluted prothrombin time assay, and reach a semi-maximal effective concentration (EC2). 50 ) is 1 × 10 -4 M or less, 1×10 -5 M or less, 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8 M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less, or 1 × 10 -12 It is less than or equal to M. Preferably, EC 50 It is approximately 5 x 10 -7 M~1×10 -11 M, for example, approximately 1 × 10 -7 M~5×10 -10 M, about 1 x 10 -7 M~1×10 -10 M, 1×10 -7 M~5×10 -9 M, 5×10 -7 M~5×10 -10 M, about 5 x 10 -7 M~1×10 -10 M, or approximately 5 x 10 -7 M~5×10 -9 Examples include M.

[0172] In certain embodiments, the antibody (or antigen-binding fragment) disclosed herein increases the thrombin production rate index and reaches a semi-maximal effective concentration (EC2). 50 ) is 1 × 10 -4 M or less, 1×10 -5 M or less, 1×10 -6 M or less, 1×10 -7 M or less, 1×10 -8M or less, 1×10 -9 M or less, 1×10 -10 M or less, 1×10 -11 M or less, or 1 × 10 -12 It is less than or equal to M. Preferably, EC 50 It is approximately 5 x 10 -7 M~1×10 -11 M, for example, approximately 1 × 10 -7 M~5×10 -10 M, about 1 x 10 -7 M~1×10 -10 M, 1×10 -7 M~5×10 -9 M, 5×10 -7 M~5×10 -10 M, about 5 x 10 -7 M~1×10 -10 M, or approximately 5 x 10 -7 M~5×10 -9 Examples include M.

[0173] In certain embodiments, the antibodies and antibody fragments disclosed herein may be further assessed in other bioactivity assays to evaluate their efficacy, pharmacological activity, and potential potency, for example, as therapeutic agents. Such assays are known in the art and depend on the target antigen of the antibody and the intended use. Examples include, for example, tumor cell proliferation inhibition assays, antibody-dependent cell-mediated cytotoxicity (ADCC) and complementary-mediated cytotoxicity (CDC) assays, and agonist or antagonist activity assays.

[0174] C. Polynucleotides, vectors, and host cells The present invention also provides polynucleotides encoding any of the TFPI-binding antibodies of this disclosure, including antibody fragments and modified antibodies described herein, for example, antibodies with impaired Fc effector function. In another embodiment, the present invention provides a method for producing any of the polynucleotides described herein. Polynucleotides can be produced and expressed by procedures known in the art. Accordingly, the present invention provides compositions comprising polynucleotides encoding any of the TFPI antibodies of the present invention and their antigen-binding fragments, or pharmaceutical compositions comprising polynucleotides.

[0175] In one embodiment, the VH and VL domains, or their antigen-binding fragments, or the full-length HC or LC, are encoded by separate polynucleotides. Alternatively, both VH and VL, or their antigen-binding fragments, or the HC and LC, are encoded by a single polynucleotide.

[0176] The present invention provides a polynucleotide or a composition comprising a polynucleotide that encodes any of the TFPI antibodies of the present invention, including TFPI-23, TFPI-24, TFPI-106, TFPI-107, and 4D8, and their antigen-binding fragments, wherein the sequence of the polynucleotide includes the sequences of SEQ ID NO: 175 (encoding the TFPI-106 VH region), SEQ ID NO: 176 (encoding the TFPI-106 VL region), SEQ ID NO: 177 (encoding the TFPI-106 heavy chain), and SEQ ID NO: 178 (encoding the TFPI-106 light chain).

[0177] In another embodiment, the present invention provides an isolated nucleic acid that encodes the VH region of an antibody or its antigen-binding portion, comprising the nucleic acid sequence of an insert present in a plasmid deposited under ATCC accession number PTA-122329, which specifically binds TFPI.

[0178] In another embodiment, the present invention provides an isolated nucleic acid that encodes the VL region of an antibody or its antigen-binding portion, comprising the nucleic acid sequence of an insert present in a plasmid deposited under ATCC accession number PTA-122328, which specifically binds TFPI.

[0179] In another embodiment, the present invention provides polynucleotides and variants thereof encoding a TFPI antibody or a portion thereof, wherein such variant polynucleotides include nucleic acid sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any of the specific nucleic acid sequences disclosed herein. The degree of sequence identity over any length of a nucleotide sequence can be calculated using methods well known to those skilled in the art. In one embodiment, not limited to the present invention, the percentage sequence identity between two or more related nucleotide sequences can be determined using the Nucleotide BLAST server available from the National Library of Medicine (http: / / blast.ncbi.nlm.nih.gov / ). This software provides different settings that can be used by those skilled in the art to optimize sequence comparisons according to factors such as length, complexity, and other factors.

[0180] The present invention provides a nucleic acid molecule comprising any TFPI antibody of the present invention, including the amino acid sequence of the antibody or its antigen-binding fragment provided in Table 33 (for example, the amino acid sequences of SEQ ID NOs. 21 to 174), the amino acid sequence of the antigen-binding fragment of the present invention, and a nucleotide sequence encoding any antibody that binds to the same epitope as the antibody of the present invention and / or competes for TFPI binding.

[0181] In another aspect, the present invention provides polynucleotides and variants thereof encoding TFPI antibodies, wherein such variant polynucleotides encode amino acid sequences that share at least 70%, at least 75%, at least 80%, at least 85%, at least 87%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity with any TFPI antibody amino acid sequence disclosed herein.

[0182] In other embodiments, the degree of relevance between a nucleic acid comprising a variant polynucleotide sequence encoding a TFPI antibody or a portion thereof and any of the specific nucleotide sequences disclosed herein can be determined by testing whether the variant sequence (or its complement) can hybridize with the specific nucleotide sequence (or its complement) under moderate or highly stringent conditions in the form of a Northern blot or Southern blot assay. Exemplary and not limited to “moderately stringent conditions” include pre-washing in a solution of 5×SSC, 0.5% SDS, and 1.0 mM EDTA (pH 8.0); hybridization with 5×SSC overnight at 50°C to 65°C; and two subsequent washes of 2×, 0.5×, and 0.2×SSC, respectively, containing 0.1% SDS, at 65°C for 20 minutes each.

[0183] Exemplary and not limited to, “highly stringent conditions” or “high stringency conditions” include (1) using low ionic strength and high temperature for washing, e.g., 0.015 M sodium chloride / 0.0015 M sodium citrate / 0.1% sodium dodecyl sulfate at 50°C, and (2) using a denaturing agent during hybridization, e.g., formamide, e.g., 0.1% bovine serum albumin / 0.1% Ficol / 0.1% polyvinyl pylori at 42°C. This involves using 50% formamide containing 50 mM sodium phosphate buffer at pH 6.5, including Don / 750 mM sodium chloride and 75 mM sodium citrate, or (3) washing at 42°C in 0.2 × SSC (sodium chloride / sodium citrate), washing at 55°C in 50% formamide, followed by a high-stringency wash at 55°C using 0.1 × SSC containing EDTA, or using 50% formamide at 42°C, 5 × SSC (0.75 M NaCl, 0.075 M sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 × Denhardt's solution, sonicated salmon sperm DNA (50 μg / ml), 0.1% SDS, and 10% dextran sulfate. Those skilled in the art are expected to recognize how to adjust temperature, ionic strength, etc., as needed to accommodate factors such as probe length. Those skilled in the art are expected to be familiar with standard techniques for detecting the degree of association between variant nucleotide sequences and the specific nucleotide sequences of this disclosure by performing Northern blot or Southern blot assays.

[0184] Polynucleotides complementary to any such sequence are also included in the present invention. The polynucleotides may be single-stranded (coding or antisense) or double-stranded, and may be DNA (genomic, cDNA, or synthetic) molecules or RNA molecules. RNA molecules include hnRNA molecules containing introns and corresponding to DNA molecules in a one-to-one manner, and mRNA molecules that do not contain introns. Additional coding or non-coding sequences may, but are not required, be present within the polynucleotides of the present invention.

[0185] The variant may also be substantially homologous to the native gene or a portion of it or its complement. Such polynucleotide variants can hybridize to naturally occurring DNA sequences encoding native antibodies (or complementary sequences) under moderately stringent conditions.

[0186] Those skilled in the art will expect to recognize that, as a result of genetic coding degeneracy, there are many nucleotide sequences that can encode any or part of the TFPI antibodies disclosed herein. Some of these polynucleotides may encode the same amino acid sequence, although they may have a relatively low degree of sequence identity with any of the specific nucleotide sequences of the TFPI antibodies provided herein. Nevertheless, polynucleotides that vary due to differences in codon usage are also specifically anticipated by the present invention.

[0187] The polynucleotides of the present invention can be obtained by chemical synthesis, recombination, or PCR. Methods for chemical polynucleotide synthesis are well known in the art and do not need to be described in detail herein. Those skilled in the art can generate desired DNA sequences using the sequences provided herein and commercially available DNA synthesizers.

[0188] To prepare polynucleotides using recombinant methods, as will be further discussed herein, polynucleotides containing the desired sequence can be inserted into a suitable vector, and the vector can then be introduced into a suitable host cell for replication and amplification. Polynucleotides can be inserted into host cells by any means known in the art. Cells can be transformed by introducing exogenous polynucleotides by direct uptake, endocytosis, transfection, F-junction, or electroporation. After introduction, the exogenous polynucleotides can be maintained in the cell as a non-integrated vector (e.g., plasmid) or integrated into the host cell genome. The thus amplified polynucleotides can be isolated from host cells by methods well known in the art. See, for example, Sambrook et al., 1989.

[0189] Alternatively, PCR enables the replication of DNA sequences. PCR technology is well known in the art and is described in U.S. Patents 4,683,195, 4,800,159, 4,754,065, and 4,683,202, as well as in PCR: The Polymerase Chain Reaction, edited by Mullis et al., Birkauswer Press, Boston, 1994.

[0190] RNA can be obtained by using isolated DNA in a suitable vector and inserting it into a suitable host cell. Once the cell replicates and the DNA is transcribed into RNA, the RNA can be isolated using methods well known to those skilled in the art, for example, as shown above in Sambrook et al., 1989.

[0191] A suitable cloning vector may be constructed by standard techniques or selected from a large number of cloning vectors available in the art. The selection of a cloning vector may vary depending on the host cell intended for use, but a useful cloning vector will generally have the ability to self-replicate, may have a single target of a specific restriction endonuclease, and / or may carry a marker gene that can be used for selecting the clone containing the vector. Suitable examples include plasmids and bacterial viruses, e.g., pUC18, pUC19, Bluescript (e.g., pBS SK+) and its derivatives, mp18, mp19, pBR322, pMB9, ColE1, pCR1, RP4, phage DNA, and shuttle vectors, e.g., pSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers, e.g., BioRad, Stratagene, and Invitrogen.

[0192] Expression vectors are further provided. Expression vectors are generally replicable polynucleotide constructs containing polynucleotides according to the present invention. It is implied that expression vectors must be replicable in host cells as episomes or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, adenoviruses, adeno-associated viruses, retroviruses, cosmids, and plasmids and viral vectors, including the expression vector disclosed in PCT Publication WO87 / 04462. Vector components generally include, but are not limited to, a signal sequence, an origin of replication, one or more marker genes, and one or more appropriate transcriptional regulatory elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational regulatory elements, such as ribosome binding sites, translation initiation sites, and stop codons, are also usually required.

[0193] Vectors containing the target polynucleotide and / or the polynucleotide itself can be introduced into host cells by any of several suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; particulate guns; lipofection; and infection (for example, if the vector is an infectious agent such as vaccinia virus). The choice of introducing the vector or polynucleotide will often depend on the characteristics of the host cell.

[0194] The present invention also provides host cells containing any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used for the purpose of isolating genes encoding an antibody, polypeptide, or protein of interest. Non-limiting examples of mammalian host cells include, but are not limited to, monkey COS, human HeLa, human embryonic kidney (HEK) 293, Sp2.0, and Chinese hamster ovary (CHO) cells. See also PCT Publication WO87 / 04462. Suitable non-mammalian host cells include prokaryotes (such as Escherichia coli (E. coli) or B. subtillis) and yeasts (such as S. cerevisae, S. pombe, or K. lactis). Screening of cells expressing TFPI antibodies or their antigen-binding moieties can be detected using immunoconjugation assays, e.g., ELISA, FACS, or other assays well known to those skilled in the art.

[0195] Therefore, the antibody (or its antigen-binding fragment) of the present invention can be recombinantly produced using suitable host cells. The nucleic acid encoding the antibody or its antigen-binding fragment can be cloned into an expression vector, which can then be introduced into host cells that do not produce immunoglobulin proteins, such as Escherichia coli (E. coli) cells, yeast cells, insect cells, COS cells, CHO cells, or myeloma cells, to obtain a monoclonal antibody synthesis in recombinant host cells. Exemplary host cells include CHO cells, HEK293, and Sp2.0 cells.

[0196] Expression vectors can be used for the direct expression of TFPI antibodies. Those skilled in the art are well familiar with the administration of expression vectors for expressing foreign proteins in vivo. See, for example, U.S. Patents 6,436,908, 6,413,942, and 6,376,471. Administration of expression vectors includes injection, oral administration, particle gun or catheter intubation, and local or systemic administration, including local administration. According to certain, non-limited embodiments, expression vectors are administered directly to the liver, skeletal muscle, bone marrow, or other tissues.

[0197] Targeted delivery of therapeutic compositions containing expression vectors or subgenomic polynucleotides can also be used. Receptor-mediated DNA delivery techniques are described, for example, in Findeis et al., Trends Biotechnol., 1993, 11:202; Chiou et al., Gene Therapeutics: Methods And Applications Of Direct Gene Transfer, JAWolff (ed.), 1994; Wu et al., J. Biol. Chem., 1988, 263:621; Wu et al., J. Biol. Chem., 1994, 269:542; Zenke et al., Proc. Natl. Acad. Sci. USA, 1990, 87:3655; Wu et al., J. Biol. Chem., 1991, 266:338. Therapeutic compositions containing polynucleotides are administered locally in the range of approximately 100 ng to approximately 200 mg of DNA in gene therapy protocols. DNA in the concentration ranges of approximately 500 ng to 50 mg, 1 μg to 2 mg, 5 μg to 500 μg, and 20 μg to 100 μg can also be used in gene therapy protocols. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery media. Gene delivery media may be of viral or nonviral origin (see, in general, Jolly, Cancer Gene Therapy, 1994, 1:51; Kimura, Human Gene Therapy, 1994, 5:845; Connelly, Human Gene Therapy, 1995, 1:185; and Kaplitt, Nature Genetics, 1994, 6:148). Expression of such coding sequences can be induced using endogenous mammalian or xenogeneic promoters. Expression of coding sequences may be constitutive or regulated.

[0198] Viral vectors for the delivery of desired polynucleotides and their desired intracellular expression are well known in the art. Examples of viral media include, but are not limited to, recombinant retroviruses (see, for example, PCT Publications WO90 / 07936, WO94 / 03622, WO93 / 25698, WO93 / 25234, WO93 / 11230, WO93 / 10218, WO91 / 02805, U.S. Patents 5,219,740 and 4,777,127, GB Patent 2,200,651, and EP Patent 0345242), alphavirus vectors (see, for example, Sindbis virus vector, Semryqui Forest virus (ATCC VR-67, ATCC VR-1247), Ross River virus (ATCC VR-373, ATCC VR-1247), etc.) These include VR-1246), Venezuelan encephalitis virus (ATCC VR-923, ATCC VR-1250, ATCC VR 1249, ATCC VR-532), and adeno-associated virus (AAV) vectors (see, for example, PCT Publications WO94 / 12649, WO93 / 03769, WO93 / 19191, WO94 / 28938, WO95 / 11984, and WO95 / 00655). Administration of DNA linked to dead adenovirus, as described in Curiel, Hum. Gene Ther., 1992, 3:147, can also be used.

[0199] However, other methods of delivery may also be used, including polycation-condensed DNA linked to or unlinked to dead adenovirus alone (see, e.g., Curiel, Hum. Gene Ther., 1992, 3:147); ligand-linked DNA (see, e.g., Wu, J. Biol. Chem., 1989, 264:16985); eukaryotic cell delivery media (see, e.g., U.S. Patent No. 5,814,482, PCT Publications WO95 / 07994, WO96 / 17072, WO95 / 30763, and WO97 / 42338), as well as non-viral delivery media and methods including nuclear charge neutralization or fusion with the cell membrane. Naked DNA may also be used. Exemplary methods for delivering naked DNA are described in PCT Publication WO90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can act as gene delivery vehicles are described in U.S. Patent No. 5,422,120, PCT Publications WO95 / 13796, WO94 / 23697, WO91 / 14445, and EP0524968. Additional techniques are described in Philip, Mol. CellBiol., 1994, 14:2411 and Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.

[0200] The sequences of the desired antibody (or its antigen-binding fragment) and the nucleic acids encoding such an antibody (or its antigen-binding fragment) can be determined using standard sequencing techniques. The nucleic acid sequences encoding the desired antibody (or fragment) may be inserted into other vectors (such as cloning and expression vectors) for recombinant production and characterization. The heavy chain (or heavy chain fragment) and light chain (or light chain fragment) can be cloned into the same vector or different vectors.

[0201] Appropriate cloning and expression vectors may contain various components, such as promoters, enhancers, and other transcriptional regulatory sequences. Vectors may also be constructed to allow the transfer of antibody-variable domains between different vectors.

[0202] Antibody fragments can be produced by proteolysis or other degradation of antibodies, recombination, or chemical synthesis. Antibody polynucleotides, particularly shorter polypeptides of up to approximately 50 amino acids, are conveniently produced by chemical synthesis. Methods of chemical synthesis are known in the art and are commercially available.

[0203] The antibodies or antigen-binding fragments disclosed herein may be affinity-matured. For example, affinity-mature antibodies can be produced by procedures known in the art (Marks et al., 1992, Bio / Technology, 10:779-783; Barbas et al., 1994, Proc.Nat.Acad.Sci.USA, 91:3809~3813; Schier et al., 1995, Gene, 169:147~155; Yelton et al., 1995, J.Immunol., 155:1994~2004; Jackson et al., 1995, J.Immunol., 154(7):3310~3319; Hawkins et al., 1992, J.Mol.Biol., 226:889~896; and WO2004 / 058184).

[0204] 4. Formulation and Use The antibodies or antigen-binding fragments described herein can be formulated as pharmaceutical formulations. Pharmaceutical formulations may further include pharmaceutically acceptable carriers, excipients, or stabilizers in the form of lyophilized formulations or aqueous solutions (Remington: The Science and Practice of Pharmacy, 20th edition, 2000, edited by Lippincott Williams and Wilkins, KE. Hoover). Acceptable carriers, excipients, or stabilizers are non-toxic to the recipient at various doses and concentrations and include buffers, e.g., phosphoric acid, citrate, and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (octadecyldimethylbenzylammonium chloride; hexamethonium chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl, or benzyl alcohol; alkylparabens such as methyl or propylparaben; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol, etc.); low molecular weight (less than about 10 residues) polypeptides; proteins, e.g., serum albumin. This may include gelatin or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids, such as glycine, glutamine, asparagine, histidine, arginine, or lysine; monosaccharides, disaccharides, and other carbohydrates, including glucose, mannose, or dextran; chelating agents such as EDTA; sugars, such as sucrose, mannitol, trehalose, or sorbitol; salt-forming counterions such as sodium; metal complexes (e.g., Zn-protein complexes); and / or nonionic surfactants, such as TWEEN®, PLURONICS®, or polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described herein.

[0205] The antibodies or antigen-binding fragments described herein can be used for a variety of therapeutic or diagnostic purposes. For example, the antibodies or antigen-binding fragments can be used as affinity purifiers (e.g., for in vitro purification of TFPI) or as diagnostic agents (e.g., for detecting the expression of TFPI in specific cells, tissues, or serum).

[0206] Exemplary therapeutic uses of the antibodies and antibody fragments of the present invention include the treatment of thrombocytopenia, platelet disorders (impairment of platelet function or number), and hemorrhagic disorders (e.g., hemophilia A, hemophilia B, and hemophilia C). The antibodies and antibody fragments may also be used to treat uncontrolled bleeding in indications such as trauma and hemorrhagic stroke. The antibodies and antibody fragments may also be used in prophylactic treatment (e.g., preoperatively).

[0207] In particular, the antibodies or antigen-binding fragments described herein can be used to treat coagulation disorders or defects. For example, the antibodies or antigen-binding fragments described herein can be used to reduce or inhibit the interaction of TFPI with FXa, or to reduce the TFPI-dependent inhibition of TF / FVIIa / FXa activity.

[0208] For therapeutic purposes, the antibodies or antigen-binding fragments described herein can be administered to mammals, particularly humans, by conventional techniques, such as intravenously (as a bolus or by continuous infusion over a period of time), intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intraarticularly, intrabursally, subarachnoidally, or orally, topically, or by inhalation. Antibodies or antigen-binding fragments can also be appropriately administered by intratumoral, peritumoral, intralesional, or perilesional routes.

[0209] Accordingly, in one aspect, the present invention provides a method for reducing the activity of tissue factor pathway inhibitors (TFPIs), comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment described herein to a subject requiring such reduction. In another aspect, the present invention provides a method for shortening bleeding time, comprising administering a therapeutically effective amount of the antibody or antigen-binding fragment described herein to a subject requiring such reduction.

[0210] In certain embodiments, the subject is human.

[0211] In certain embodiments, subjects have or are susceptible to blood clotting disorders. Examples of blood clotting disorders include von Willebrand disease (vWD), hemophilia A, B, or C, and other platelet disorders (such as congenital platelet deficiency, congenital and acquired platelet pool deficiency, and prolonged bleeding time).

[0212] In certain embodiments, the antibody or antigen-binding fragment described herein is administered subcutaneously. In certain embodiments, the antibody or antigen-binding fragment described herein is administered intravenously.

[0213] The pharmaceutical composition may be administered to those in need at a frequency that may vary depending on the severity of the bleeding episode, or, in the case of prophylactic therapy, depending on the severity of the patient's coagulation disorder.

[0214] The composition may be administered to patients in need as a bolus or by continuous infusion. For example, a bolus administration of the antibody as a Fab fragment may be in amounts of 0.0025–100 mg / kg body weight, 0.025–0.25 mg / kg, 0.010–0.10 mg / kg, or 0.10–0.50 mg / kg. For continuous infusion, the antibody as a Fab fragment may be administered at a rate of 0.001–100 mg / kg body weight / min, 0.0125–1.25 mg / kg / min, 0.010–0.75 mg / kg / min, 0.010–1.0 mg / kg / min, or 0.10–0.50 mg / kg / min over periods of 1–24 hours, 1–12 hours, 2–12 hours, 6–12 hours, 2–8 hours, or 1–2 hours.

[0215] For the administration of antibodies that exist as full-length antibodies (entire constant region), the dosage is approximately 1 mg / kg to 10 mg / kg, 2 mg / kg to 10 mg / kg, 3 mg / kg to 10 mg / kg, 4 mg / kg to 10 mg / kg, 5 mg / kg to 10 mg / kg, 1 mg / kg to 20 mg / kg, 2 mg / kg to 20 mg / kg, 3 mg / kg to 20 mg / kg, 4 mg / kg to 20 mg / kg, 5 mg / kg to 20 mg / kg, and 1 The dosage may be mg / kg or more, approximately 2 mg / kg or more, approximately 3 mg / kg or more, approximately 4 mg / kg or more, approximately 5 mg / kg or more, approximately 6 mg / kg or more, approximately 7 mg / kg or more, approximately 8 mg / kg or more, approximately 9 mg / kg or more, approximately 10 mg / kg or more, approximately 11 mg / kg or more, approximately 12 mg / kg or more, approximately 13 mg / kg or more, approximately 14 mg / kg or more, approximately 15 mg / kg or more, approximately 16 mg / kg or more, approximately 17 mg / kg or more, approximately 19 mg / kg or more, or approximately 20 mg / kg or more. The frequency of administration depends on the severity of the condition. The frequency can range from 3 times a week to once every 2 weeks or 3 weeks.

[0216] Furthermore, the composition may be administered to the patient via subcutaneous injection. For example, a dose of 1 to 100 mg of anti-TFPI antibody may be administered to the patient via subcutaneous injection once daily, every two days, every three days, every four days, every five days, every six days, twice a week, weekly, every other week, or monthly.

[0217] In certain uses, the pharmaceutical composition is approximately 0.1 mg / kg to approximately 10 mg / kg, approximately 0.5 mg / kg to approximately 10 mg / kg, approximately 1 mg / kg to approximately 10 mg / kg, approximately 1.5 mg / kg to approximately 10 mg / kg, approximately 2 mg / kg to approximately 10 mg / kg, approximately 0.1 mg / kg to approximately 8 mg / kg, approximately 0.5 mg / kg to approximately 8 mg / kg, approximately 1 mg / kg to approximately 8 mg / kg, approximately 1.5 mg / kg to approximately 8 mg / kg, approximately 2 mg / kg to approximately 8 mg / kg, approximately 0.1 mg / kg to approximately 5 mg / kg, approximately 0.5 mg / kg to approximately 5 mg / kg, approximately 1 mg / kg to approximately 5 mg / kg, approximately 1.5 mg / It is administered subcutaneously once a week on a schedule in doses of approximately 5 mg / kg, 2 mg / kg to 5 mg / kg, 0.5 mg / kg, 1.0 mg / kg, 1.5 mg / kg, 2.0 mg / kg, 2.5 mg / kg, 3.0 mg / kg, 3.5 mg / kg, 4.0 mg / kg, 4.5 mg / kg, 5.0 mg / kg, 5.5 mg / kg, 6.0 mg / kg, 6.5 mg / kg, 7.0 mg / kg, 7.5 mg / kg, 8.0 mg / kg, 8.5 mg / kg, 9.0 mg / kg, 9.5 mg / kg, or 10.0 mg / kg.

[0218] In certain embodiments, the pharmaceutical composition is administered subcutaneously once a week at a dose of approximately 2.0 mg / kg. In certain embodiments, the pharmaceutical composition is administered subcutaneously once a week at a dose of approximately 3.0 mg / kg.

[0219] The antibodies and antibody fragments described herein can be used as monotherapy or in combination with other therapies to address hemostatic disorders. For example, co-administration of one or more antibodies (or antibody fragments) of the present invention with hemocoagulants such as factor VIIa, factor VIII, factor IX, or tranexamic acid may be useful in treating hemophilia.

[0220] In one embodiment, a method is provided for treating coagulation disorders or shortening bleeding time, comprising administering (a) a first amount of the antibody or antigen-binding fragment of the present invention and (b) a second amount of factor VIII or factor IX. Factor VII may not be co-administered. In another embodiment, a method is provided for treating coagulation disorders or shortening bleeding time, comprising administering (a) a first amount of the antibody or antigen-binding fragment of the present invention and (b) a second amount of factor VIII or factor IX. Factor VII may not be co-administered. Those skilled in the art will recognize that in the treatment of coagulation disorders, shortening bleeding time can also be called shortening coagulation time.

[0221] The present invention relates to a pharmaceutical composition comprising a therapeutically effective amount of the antibody (or antibody fragment) of the present invention in combination with factor VIII or factor IX, and also includes a pharmaceutical composition that does not contain factor VII. "Factor VII" includes factor VII and factor VIIa.

[0222] biological deposit Representative materials of the present invention were deposited on July 22, 2015, at the American Type Culture Collection, 10801 University Boulevard, Manassas, Va. 20110-2209, USA. Plasmid vector mAb-TFPI-106 VH, with ATCC accession number PTA-122329, contains a DNA insert encoding the heavy chain variable region of antibody TFPI-106, and plasmid vector mAb-TFPI-106 VL, with ATCC accession number PTA-122328, contains a DNA insert encoding the light chain variable region of antibody TFPI-106. The deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and Regulations thereunder (Budapest Treaty). This guarantees the maintenance of a viable culture of the deposited material for 30 years from the date of deposit. The deposited material will be made available to the public by the ATCC in accordance with the provisions of the Budapest Convention and the agreement between Pfizer Inc. and the ATCC, which guarantees the perpetual and unrestricted availability of the offspring of the culture of the deposited material to the public at the earlier of the issuance of the relevant U.S. patent or the public disclosure of any U.S. or foreign patent application, and guarantees the availability of the offspring to persons whom the Commissioner of the U.S. Patent and Trademark Office determines to be qualified in accordance with Section 122 of the U.S. Patent Act and the Commissioner's Rules thereunder (including Section 1.14 of Title 37 of the Code of Rules, with particular reference to 886OG638).

[0223] The assignee of this application agrees to immediately replace, upon notice, the deposited material if the culture of the material dies, is lost, or is destroyed when cultured under appropriate conditions. The availability of the deposited material should not be construed as a license to practice the invention in violation of any right granted under the patent law of any government. [Examples]

[0224] The present invention will be further described in detail with reference to the following experimental examples. These examples are provided for illustrative purposes only and are not intended to be limiting unless otherwise specified. Therefore, the present invention should not be construed as being limited to the following examples, but rather as encompassing any and all variations that become apparent as a result of the teachings provided herein.

[0225] (Example 1) Materials and methods for the experiment 1. TFPI protein reagent Table 1 lists the protein reagents used for immunization, phage display selection, and characterization of anti-TFPI antibodies, and Table 2 lists their sequence numbers.

[0226] The TFPI construct (pSMED2 vector) was transiently expressed in HEK293F cells, and the conditioned medium was collected 120 hours after transfection. The target protein was captured from the conditioned medium using nickel Sepharose HP and further purified by size exclusion chromatography. Factor Xa and Factor X were obtained from Haematologic Technologies, Inc. The chromogenic substrate for the amid degradation assay of Factor Xa, Spectrozyme® FXa, was obtained from Sekisui Diagnostics.

[0227] [Table 3]

[0228] [Table 4]

[0229] 2. Antibody reagents Table 3 lists the monoclonal antibodies used for comparative purposes (reference antibodies 2A8, 2A8-200, 3F18, hz4F36). The antibody descriptions, sources, and sequences are listed in Table 4 (Figure 5). Light and heavy chain sequences were cloned into appropriate vectors, monoactively expressed in human embryonic kidney-293 (HEK-293) cells, and purified by protein A Sepharose and size exclusion chromatography. Mab 2974 was obtained from R&D Systems (catalog #MAB2974).

[0230] [Table 5]

[0231] 3.TFPI binding ELISA Recombinant humTFPI K1K2, murTFPI K1K2, cynTFPI K1K2, ratTFPI K1K2, rabTFPI K1K2, or TFPI2 was biotinylated using the AviTag® system and mixed in 1 × 10⁶ of ELISA assay buffer. -8 The antibody was captured at concentration M on a Greiner streptavidin-coated 96-well plate. The purified anti-TFPI antibody was diluted to 1 μg / ml in ELISA assay buffer, and then serially diluted 3-fold to produce an 8-dilution series. The diluted antibody was added to each well at a volume of 100 μL. The plate was incubated at room temperature for 2 hours. After washing the plate with PBS / 0.05% Tween20, the plate was incubated with goat anti-mouse IgG-Fc polyclonal antibody conjugated with horseradish peroxidase (Pierce) at a dilution of 1:10,000. After 1 hour of incubation, the conjugated antibody was detected by adding TMB substrate solution. Absorbance was read at 450 nm, and the data was analyzed using GraphPad Prism software.

[0232] 4. Surface plasmon resonance (SPR) Anti-human Fc sensor chips were prepared by amine coupling anti-human IgG antibody (catalog number BR-1008-39, GE Healthcare) to all four flow cells of a carboxymethylated dextran coated sensor chip (CM5) (catalog number BR100530, GE Healthcare). The flow cells were activated by injecting a 1:1 mixture of 400 mM 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS) at a flow rate of 10 μl / min for 7 minutes. The anti-human IgG antibody was diluted to 25 μg / ml in 10 mM sodium acetate pH 5.0 and injected into all flow cells at a flow rate of 10 μl / min for 7 minutes. All flow cells were blocked with 1 M ethanolamine-HCl (ETH) at a flow rate of 10 μl / min for 7 minutes. The final immobilization level of the captured antibody was approximately 10,000 resonance units (RU). The running buffer for immobilization and kinetics was 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% (v / v) Tween-20 (HBS-EP+). To characterize the binding of anti-TFPI antibody to human TFPI, the antibody was diluted to 0.5 μg / mL in HBS-EP+ and immobilized with anti-human IgG on flow cells 2, 3, and 4, and captured at a flow rate of 5 μL / min for 30 seconds to 1 minute to achieve capture levels of 70 to 300 RU. Flow cell 1 was used as a reference surface. After antibody capture, the flow rate was increased to 50 μL / min, and human TFPI in the buffer or HBS-EP+ at a concentration range of 0.2 nM to 200 nM was injected onto all flow cells for 1.0 minute of association, followed by dissociation for 10 to 15 minutes. The buffer cycles used to collect each capture antibody were used for dual reference (Myszka, DG, J.Mol.Recognit., 12, 279-284 (1999)). At the end of each cycle, the entire anti-IgG surface was regenerated with a 60-second pulse of 3M MgCl2. The kinetic assay was performed at 25°C with a BIAcore T200 instrument (GE Healthcare) at a collection rate of 10 Hz. The rate constant and affinity were determined by fitting the data to a 1:1 model using BIAcore T200 Evaluation software version 1.0 (GE).

[0233] 5. Factor Xa TFPI Inhibition Removal Assay The ability of purified anti-TFPI antibodies to restore factor Xa activity in the presence of inhibitory TFPI concentrations was assessed in vitro. Anti-TFPI antibodies diluted in PBS at concentrations ranging from 1 nM to 500 nM were pre-incubated at 37°C for 30 minutes with 10 nM recombinant human TFPI K1K2 or 10 nM rabbit TFPI K1K2 protein in activation buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 5 mM CaCl2, 0.5 mg / mL BSA). 2 nM human plasma-derived factor Xa was added, and the reaction was incubated at 37°C for 30 minutes. The chromogenic substrate Spectrozyme Xa was added to a final concentration of 500 μM per 100 μL of final reaction volume. Control reaction materials included those without factor Xa to control assay background, those without TFPI, those allowing maximum FXa production (100% activity), or those without anti-TFPI antibody (PBS alone). The absorbance of the reactants was immediately read at a wavelength of 405 nm at 2-minute intervals over a 60-minute period using a SpectraMax M5e multimode plate reader. 50 This was calculated using Prism Graph Pad software.

[0234] 6.2-Step TF-FVIIa-FX Inhibition Removal Assay The ability of purified anti-TFPI antibodies to restore factor VIIa-tissue factor activity in the presence of inhibitory TFPI concentrations was assessed in vitro. Anti-TFPI antibodies in the concentration range of 1 nM to 500 nM were pre-incubated with 10 nM recombinant TFPI protein in active buffer at 37°C for 30 minutes. Approximately 1 pM of lipidized tissue factor and 1 nM recombinant factor VIIa (NovoSeven) were added to the reaction mixture and incubated at 37°C for 5 minutes. 150 nM human factor X was introduced into the reaction mixture. The chromogenic substrate Spectrozyme Xa was added to each well for a final reaction volume of 100 μL to a final concentration of 500 μM. Control reaction mixtures included those without factor VIIa, tissue factor, factor X, TFPI, or anti-TFPI antibody (PBS alone). The absorbance of the reactants was immediately read at a wavelength of 405 nm at 2-minute intervals over a 60-minute period using a SpectraMax M5e multimode plate reader. 50 This was calculated using Prism Graph Pad software.

[0235] 7. Thrombin production assay (TGA) The ability of purified anti-TFPI antibodies to restore thrombin production in plasma with attenuated factor VIII activity was assessed in a thrombin production assay using a calibrated automated thrombogram (CAT) system. Anti-TFPI antibodies diluted in PBS at concentrations ranging from 1 nM to 500 nM were introduced into human factor VIII-deficient plasma and a reaction mixture containing PPP-Low reagent with 4 μM phospholipid and 1 pM tissue factor. A control reaction mixture using antibody-free PBS was used. The reaction was induced by adding a fluorescent thrombin substrate and Fluca buffer containing CaCl2. Fluorescence from each reaction was immediately read at 20-second intervals over 60 minutes using a Fluoroskan Ascent plate reader with Thrombinoscope software. Each reaction was compared to a calibrator control well containing PBS, thrombin calibrator, FVIII-deficient plasma, and Fluca buffer. Thrombinoscope thrombin production curves (nM thrombin vs. time) were analyzed using Thrombinoscope software (Thrombinoscope BV version) to extract delay time, peak height, time to peak, and area under the curve or endogenous thrombin potential (ETP). The rate exponent (peak thrombin concentration / time to peak - delay time) was calculated using the data.

[0236] The ability of purified anti-TFPI antibodies to restore thrombin production in rabbit plasma with attenuated FVIIIa activity was also assessed. Normal New Zealand white rabbit plasma was treated with either 100 μg / mL of anti-FVIII antibody (GM-8015) or 100 μg / mL of control mouse anti-human IgG2a at 37°C for 60 minutes. Immediately before adding to the reaction wells, the rabbit plasma was diluted 1:3 in buffer (20 mM HEPES, 140 mM NaCl). The thrombin production assay using FVIII-neutralized rabbit plasma was performed as described above.

[0237] 8. Generation of TFPI K2 domains in cynomolgus monkeys for structural studies Cynomolgus monkey (cyno) TFPI K1K2 (Table 1) was expressed in HEK293 cells, and the conditioned medium was collected 120 hours after transfection. Purified cynomolgus monkey TFPI K1K2 was incubated with human neutrophil elastase (HNE) at RT for 120 minutes at a molar ratio of 1:70 (HNE:TFPI) to induce cleavage. Cynomolgus monkey K1 was separated from cynomolgus monkey K2 using anion exchange chromatography on HQ50 (Poros). Size exclusion chromatography using Superdex75 was performed as the final purification step. Residual AviTag was cleaved from the C-terminus of the cynomolgus monkey K2 domain using endoproteinase AspN.

[0238] 9. Generation of antibody Fab / cynomolgus monkey TFPI K2 complex for structural study Anti-TFPI antibodies 4D8.b1, TFPI-23, TFPI-24, 2A8-200 (Table 3), and Mab2974 (R&D systems) were digested with immobilized papain according to the manufacturer's protocol (Thermo / Pierce). Fab was purified from the digest using MabSelect SuRe and then used for complex formation with cynomolgus monkey TFPI K2. The Fab / cynomolgus monkey TFPI K2 complex was then concentrated to approximately 16 mg / ml. The concentrated complex was then screened for protein crystallization conditions.

[0239] (Example 2) Generation of mouse anti-TFPI antibodies 1. Mouse immunization and hybridoma generation A cohort of five BALB / c mice was subcutaneously immunized with a mixture of 5 μg of humTFPI K1K2 and 5 μg of murTFPI K1K2 protein emulsified in complete Freund's adjuvant. The mice were then immunized twice per week alternately with the protein mixture emulsified in incomplete Freund's adjuvant or diluted in PBS. Blood samples were collected on days 17 and 27 (after the 5th and 7th immunizations, respectively), and serum was tested for the presence of circulating anti-TFPI antibodies by ELISA. By day 27, each mouse received a final booster immunization of the protein mixture (10 μg) intraperitoneally. Four days later, aspiration lymph nodes (axillary, inguinal, and popliteal) were collected, and pooled lymph node cells were mixed with P3X63.Ag8.653 cells in a 1:1 ratio and subjected to electrocellular fusion. The fusion cells were treated with FBS (25%), NCTC-109 (12.5%), Glutamax (1%), Penicillin-Streptomycin (1%), Hybridoma Cloning Supplement (5%), and HAT (1 × 10⁻¹⁴). -4 M-hypoxanthine, 4 x 10 -7 M aminopterin, and 1.6 × 10 -5 The hybridomas were seeded in RPMI1640 medium supplemented with M-thymidine. Fourteen days after fusion, the hybridoma culture supernatant was tested for binding to humTFPI K1K2 by ELISA. Based on their binding activity and potency in functional assays, antibodies derived from three hybridomas, 4D8, 6B7, and 7A4, were selected and further characterized.

[0240] 2. Cloning and sequencing of hybridoma-derived anti-TFPI antibodies RNAs were prepared from hybridomas 4D8, 6B7, and 7A4, and variable region DNA sequences derived from the expressed antibodies were obtained by RT-PCR cloning. The PCR products were cloned into a TOPO-TA cloning vector and then sequenced using conventional methods. One heavy / light cDNA pair was detected from hybridomas 6B7 and 7A4. Two heavy / light cDNA pairs were detected from 4D8.

[0241] (Example 3) Characterization of mouse hybridoma anti-TFPI antibodies Monoclonal hybridoma cell lines were obtained by subcloning parent hybridomas 4D8, 6B7, and 7A4 using limiting dilution. Positive subclones were identified and expanded by ELISA screening for reactivity with humTFPI K1K2. Purified antibodies derived from one subclone of each hybridoma were further characterized.

[0242] 1.TFPI binding Purified anti-TFPI antibodies 4D8.B1, 6B7.C5, and 7A4.D9 were tested for binding to recombinant human and rabbit TFPI proteins by protein-binding ELISA. The EC50 values ​​for each antibody, both humTFPI K1K2-aviHis10 and rabTFPI K1K2, are shown in Table 5.

[0243] [Table 6]

[0244] Surface plasmon resonance (SPL) experiments were performed to assess the affinity of purified mouse anti-TFPI antibodies to human and rabbit TFPI K1K2 proteins. The k-level of each antibody binding to human and rabbit TFPI K1K2 was also assessed. a , k d , and K D The values ​​are shown in Table 6.

[0245] [Table 7]

[0246] 2. In vitro activity assay Anti-TFPI mouse monoclonal antibodies were tested for activity in FXa and TF-FXa-FVIIa inhibition deactivation assays and thrombin production assays (TGA). The most potent antibody, 4D8.B1, was selected for further study.

[0247] [Table 8]

[0248] (Example 4) Generation of chimeric and humanized antibodies from clone 4D8 1. Generation of mouse-human chimeric antibody 4D8 Variable region cDNA derived from hybridoma 4D8 was subcloned into a mammalian expression vector. The mouse heavy chain variable region was fused in-frame to human IgG1 3M (SEQ20, Table 4), and the mouse light chain variable region was fused in-frame to human Ig kappa constant region (SEQ62, Table 4) to generate chimeric antibodies. The chimeric constructs were transiently transfected into HEK293 cells. A total of four transient transfections were performed using all possible heavy and light chain combinations to identify the correct heavy / light chain pair from hybridoma 4D8. The antibody generated from one of the transfections was referred to as the hu-mu 4D8 chimera (Tables 3 and 4).

[0249] 2. Characterization of mouse-human chimeric antibody 4D8 (mu-hu 4D8) The Mu-hu 4D8 chimera was tested for its ability to bind both human and rabbit TFPI K1K2 proteins using protein-binding ELISA (Table 8) and SPR (Table 9). KD and EC50 values ​​closely matched those measured for purified mouse MAb 4D8.B1, demonstrating that transplantation of the mouse variable region into a human IgG1 background preserved binding activity.

[0250] [Table 9]

[0251] [Table 10]

[0252] 3. Humanization of hu-mu 4D8 Chimera The hu-mu 4D8 chimeric sequence was humanized by CDR transplantation into a human acceptor framework sequence. The DP54 framework and DPK9 framework were selected. Combinations of heavy and light chain constructs (see Table 3) were then expressed. Antibodies were tested for human and rabbit TFPI binding in ELISA assays (Table 10) and human TFPI binding in SPR assays (Table 11).

[0253] [Table 11]

[0254] [Table 12]

[0255] Based on these data, 4D8 Vk1.1×VH1.4 was selected for further characterization and designated as hz4D8 (Table 3). The humanized anti-TFPI antibody (hz4D8) was compared to mouse 4D8.B1 for activity in the FXa inhibition deactivation assay, the two-step TF-FVIIa-FX inhibition assay, and the thrombin production assay. The data in Table 12 show that the humanized antibody showed improved activity in all three assays compared to the mouse antibody, indicating that TFPI binding activity was fully retained within the humanized antibody.

[0256] [Table 13]

[0257] (Example 5) Generation of additional anti-TFPI antibodies by phage display 1. Selection of anti-TFPI antibodies by phage display Recombinant human and mouse TFPI K1K2-binding single-chain fragment variable (scFv) antibodies were identified according to a four-round selection using a phage display library of scFv antibody fragments derived from non-immunized human donors. Phage selection was performed in solution using streptavidin beads. Binding phages were eluted by incubation with 140 mM triethanolamine (TEA) pH 11.5 or 50 mM MES pH 5.5 for 10 minutes at room temperature in a rotary shaker, and neutralized with 1 M Tris-HCl, pH 7.5.

[0258] Using the eluted phage pool, the medium-term logarithmic phase (approximately 0.5 OD) 600 The phages were inoculated into 10 mL of E. coli (ER2738) culture medium that had grown to the corresponding size. The bacteria were infected with the phages at 37°C for 30 minutes without shaking, concentrated by centrifugation, seeded, and then grown overnight at 30°C. For the next round of selection, the phages were inoculated with 25 mL of 2 × TYAG / tetracycline to an OD of approximately 0.1. 600 Cells were rescued and grown at 37°C until their OD600 reached 0.3–0.5. The cells were co-infected with MK13K07 helper phage at a cell / helper phage ratio of 1:20 and incubated at 37°C for 30 minutes without shaking, then for 60 minutes with shaking at 150 rpm. The cells were then centrifuged, and the pellet was resuspended in kanamycin / non-glucose medium. This culture was grown overnight at 25°C. The phages were collected in the supernatant after centrifugation and used for selection in the next round.

[0259] 2. Preparation of crude periplasmic material for use in ELISA assays. The scFv antibody fragment can be expressed on the surface of phage particles or in solution within the interstitial space of bacterial periplasm, depending on the growth conditions used. To induce the release of the scFv antibody fragment into the periplasm, thawed glycerol stock was inoculated into a 96-deep-well plate containing 2×TY medium with 0.1% glucose / 100 μg / ampicillin 1 mL and grown at 37°C for approximately 4 hours. The contents of the bacterial periplasm (periprep) were released by osmotic shock. The plate was centrifuged and the scFv-containing supernatant was collected.

[0260] 3. ELISA to measure the binding of scFv expressed in the periplasm to human and mouse TFPI K1K2. A total of 1984 clones were randomly selected from rounds 2, 3, and 4 of all branching selections. TFPI scFv binders were identified by periplasm preparation (periprep) conjugated ELISA. Biotinylated human and mouse TFPI K1K2 were coated in 384-well Nunc Maxisorp streptavidin plates at a concentration of 1 μg / mL in PBS. The TFPI K1K2 solution was removed, and the plates were blocked in 0.05% Tween20 / 1% BSA / PBS at room temperature for 1 hour. Periprep was prepared and blocked in equal volumes of 6% milk / 1% BSA at room temperature for 1 hour. 20 μl / well of blocked periplasm scFv and control antibody were transferred to appropriate plates and incubated at room temperature for 1 hour. scFV or anti-TFPI control antibody conjugated to 1:2,000 dilution of anti-myc horseradish peroxidase (HRP) or 1:10,000 dilution of goat anti-human HRP secondary antibody was detected. The signal was chromogenically developed using 3,3',5,5'-tetramethylbenzidine, and the absorbance was read at 450 nm using an Envision plate reader (Perkin Elmer). A total of 883 scFV clones were identified as TFPI binders. The 883 TFPI-conjugated scFVs were sequenced to identify unique clones. 288 unique clones were selected and tested for competitive TFPI / FXa binding.

[0261] 4. ELISA to identify scFvs that compete with FXa for binding to human and mouse TFPI K1K2. A total of 288 unique clones were tested in FXa / TFPI competitive binding ELISA. Human FXa was coated overnight in 384-well Nunc Maxisorp plates at a concentration of 1 μg / mL in PBS. The FXa solution was removed, and the plate surface was blocked in 0.05% Tween 20 / 1% BSA / PBS at room temperature for 1 hour. Periprep was prepared and blocked in an equal volume of 6% milk / 1% BSA at room temperature for 1 hour. 20 μl / well of blocked periplasm scFv and control antibody were mixed with biotinylated humTFPI K1K2 and incubated at room temperature for 1 hour. The mixture was transferred to FXa-coated plates and incubated at room temperature for 1 hour. 1:2000 dilution of streptavidin-horseradish peroxidase was added to detect bound TFPI. The signal was chromogenically developed using 3,3',5,5'-tetramethylbenzidine, and absorbance was read at 450 nm with an Envision plate reader. A total of 48 scFV antibodies were classified as competitive inhibitors of TFPI / FXa binding.

[0262] 5. Conversion of ScFv to human IgG A total of 48 ScFv antibodies with unique sequences demonstrating binding to and inhibition of TFPI in TFPI / FXa competitive ELISA were selected and subcloned into human IgG-3M cloning vectors. Briefly, the fragments were amplified by standard PCR. VH or VL fragments were gel-purified and ligated into mammalian expression vectors containing human IgG1-3M(VH) or kappa or lambda constant regions (VK / VL). The VH and VK / VL pair expression vectors were then used for transient mammalian expression and purification in HEK293 cells.

[0263] 6. Characterization of human IgG-3M anti-TFPI antibodies Forty-eight anti-TFPI antibodies were ranked using various assays, including FXa and TF / FVIIa / FXa deactivation assays. TFPI-3, TFPI-21, TFPI-23, TFPI-24, and TFPI-26 possessed desirable properties, such as low or no heterobinding of TFPI to humTFPI2 K1K2K3 (Table 13). FXa and TF / FVIIa / FXa deactivation assay data for these same five antibodies are shown in Table 14, and SPR binding data are shown in Table 15.

[0264] [Table 14]

[0265] [Table 15]

[0266] [Table 16]

[0267] (Example 6) Epitope mapping of anti-TFPI antibodies by SPR The epitopes of the anti-TFPI antibodies discovered and disclosed in this document (TFPI-21, TFPI-23, TFPI-24, 4D8, 6B7.c5, and 7A4.D9) were mapped using a sandwich SPR assay. Other known reference antibodies (hz4F36, 2A8-200, and Mab2974) were also included in the epitope mapping experiments. Antibody 1 was immobilized on a CM5 biacore chip using NHS chemistry. Human TFPI (humTFPI K1K2) was initially injected onto the chip until binding was near apparent equilibrium. Immediately after stopping the human TFPI injection, antibody 2 was injected onto the chip. If antibody 2 binds to the complex of antibody 1 and human TFPI on the surface of the CM5 chip, antibody 2 has a distinct, non-overlapping binding epitope for antibody 1 (scored as +). If antibody 2 does not show binding, it is scored as a significantly overlapping epitope (negative (-)) with antibody 1. If antibody 2 shows weak binding, antibodies 1 and 2 are considered to have some partial overlap in the TFPI epitope (scored as + / -). As shown in Table 16, TFPI-21 and TFPI-23 have similar epitopes, and the data also show that TFPI-21 and TFPI-23 have completely distinct epitopes with mab2974 and hz4F36.

[0268] [Table 17]

[0269] (Example 7) TFPI-23 antibody germline-derived human framework Two variants of TFPI-23 were created to increase the content of human framework germline residues. TFPI-106 contained mutations from H1Q to E and H5V to L (Kabat numbered). TFPI-107 (Tables 3 and 4) contained mutations from H1Q to E, H5V to L, and H94I to K (Kabat numbered). TFPI-106, TFPI-107, and TFPI-23 were expressed, purified, and tested for binding to humTFPI K1K2 by SPR. The data in Table 17 show that the TFPI-106 germline variant maintained complete binding affinity compared to the TFPI-23 parental antibody.

[0270] [Table 18]

[0271] (Example 8) TFPI-24 antibody germline-derived human framework Four TFPI-24 VL variants were created (TFPI-110, TFPI-111, TFPI-112, TFPI-113) and paired with the TFPI-24 VH sequence. Three TFPI-24 VH variants (TFPI-108, TFPI-109, TFPI-114) were created and paired with the TFPI-24 VL sequence. Based on this data, the best VL variant, TFPI-113, was paired with the best VH variant, TFPI-108, to generate the antibody TFPI-118. When TFPI-118 and TFPI-24 were tested for binding to human TFPI by SPR, the results in Table 18 show comparable binding kinetics.

[0272] [Table 19]

[0273] (Example 9) SPR binding dynamics of anti-TFPI antibodies to TFPIs from various species Anti-TFPI antibodies (TFPI-106, TFPI-118, and hz4F36) were analyzed by SPR to determine their binding kinetics to TFPI derived from different animal species (human (huTFPI K1K2), cynomolgus monkey (cynTFPI K1K2), rabbit (rabTFPI K1K2), mouse (murTFPI K1K2), and rat (ratTFPI K1K2); (Table 1)). Three comparator antibodies (hz4F36, 2A8, and 2A8-200) were also included in this experiment.

[0274] [Table 20]

[0275] (Example 10) Anti-TFPI antibody / TFPI complex structure 1.4D8.b1 Fab / Cynomolgus TFPI K2 complex structure A complex was formed by mixing 4D8.b1 Fab and cynomolgus monkey TFPI K2 in a 1:1 molar ratio. Final purification was performed using a Superdex200 column. The complex was concentrated to 12.6 mg / ml for structural study. Crystals of the TFPI K2+4D8 Fab complex were obtained in 100 mM Tris-HCl pH 8.5, 20% PEG10000. It yielded rod-shaped crystals diffracted down to 2.9 Å. The crystals were transiently cryoprotected, and synchrotron data acquisition was performed remotely at an Advanced Photon Source. Image frames were processed using the software AutoPROC (Global Phasing Ltd). The data belong to space group P212121, with unit cells as follows: a=62.102 Å, b=82.284 Å, c=103.628 Å, α=β=γ=90°, and one complex per asymmetric unit. Compelling answers for each component were obtained through molecular substitution searches using homology models of 4D8 Fab and publicly available structures of the TFPI K2 domain (RSCB Protein Databank, PDB codes 1TFX and 4DTG). Refinement using the software autoBUSTER (Global Phasing Ltd) revealed final R / Rfree factors of 0.1707 and 0.2424, respectively, with a binding RMSD of 0.010 Å and an angle RMSD of 1.26°. The epitopes and paratopes of 4D8 Fab were determined based on the buried surface area (BSA) and percentage BSA (%BSA) of residues at the Fab / TFPI K2 interface. The following residues in the K2 domain of TFPI are involved in direct contact with 4D8 Fab (BSA-mediated epitopes): E101, P103, Y109, I110, T111, Y113, F114, S119, Q121, C122, E123, R124, F125, K126, and L140. The following residues in the heavy chain of 4D8 Fab contain heavy chain paratopes: D50, T57, L58, Y59, Q61, K64, D98, Y99, and D100. The following residues in the light chain of 4D8 Fab contain light chain paratopes: H30, W50, H91, Y92, T93, T94, P95, and Y96.Table 20 shows the BSA and %BSA values ​​for epitope and paratope residues.

[0276] [Table 21]

[0277] 2.2A8&2A8-200 Fab / Cynomolgus K1K2 complex structure A complex was formed by mixing 2A8 Fab and cynomolgus monkey TFPI K1K2 in a 1:1 molar ratio. Final purification was performed using a Superdex200 column. The complex was concentrated to 10.8 mg / ml for structural study. Crystals of the complex containing 2A8 Fab and TFPI K1K2 were obtained under the following two conditions: (1) needle-shaped crystals diffracted to 3.0 Å in 100 mM HEPES pH 7.5, 12.5% ​​PEG8000; (2) block-shaped crystals diffracted to 3.3 Å in 100 mM HEPES pH 7.5, 1600 mM ammonium sulfate, 2% PEG1000. The crystals were transiently cryoprotected, and synchrotron data acquisition was performed remotely at an Advanced Photon Source. Image frames were processed using the software AutoPROC (Global Phasing Ltd). The data belong to space group P3221, with unit cells as follows: a=b=196.146 Å, c=41.262 Å, α=β=90°, γ=120°, and one complex per asymmetric unit. Compelling answers for each component were obtained by molecular substitution search using the homology model of 2A8 Fab and publicly available structures of the TFPI K2 domain (RSCB Protein Databank, PDB codes 1TFX and 4DTG). Refinement using the software PHENIX yielded final R / Rfree factors of 0.1667 and 0.2088 at 3.0 Å, respectively, with a binding RMSD of 0.011 Å and an angle RMSD of 1.474°. The epitopes and paratopes of the complexes were determined based on the buried surface area (BSA) and percentage BSA (%BSA) of residues at the Fab TFPI K1K2 interface. The following residues in the TFPI K1K2 domain of TFPI are involved in direct contact with 2A8 Fab (BSA-mediated epitopes): D31, D32, G33, P34, C35, K36, E100, E101, P103, G104, I105, C106, R107, G108, Y109, E123, K126, Y127, and G128.The following residues in the heavy chain of 4D8 Fab contain the heavy chain paratopes: G26, T28, S31, Y32, Y96, R97, Y98, W99, and D101 (Kabat numbered). The following residues in the light chain of 2A8 Fab contain the light chain paratopes: L28, R29, N30, Y31, Y32, Y49, Y50, D51, and N66 (Kabat numbered). The BSA and %BSA values ​​of the epitope and paratope residues are shown in Table 21. The very closely related antibody, 2A8-200, was also elucidated in complex with TFPI K1K2 using essentially the same method. The epitopes and paratopes of this antibody were identical to those of 2A8.

[0278] [Table 22]

[0279] 3.Mab 2974 Fab / TFPI K2 complex structure A complex was formed by mixing Mab 2974 (R&D Systems) Fab and cynomolgus monkey TFPI K1K2 in a molar ratio of 1:1.2. Final purification was performed using a Superdex200 column. The complex was concentrated to 17.5 mg / ml for structural study. Crystals of the complex containing Mab 2974 Fab and TFPI K2 were obtained in 100 mM sodium citrate pH 5.6, 20% isopropanol, and 20% PEG4000. It produced blocky crystals diffracted to 2.15 Å. The crystals were transiently cryoprotected, and synchrotron data acquisition was performed remotely at an Advanced Photon Source. Image frames were processed using the software AutoPROC (Global Phasing Ltd). The complex data belong to space group P212121, with the unit cell dimensions being: a=82.075 Å, b=117.829 Å, c=170.945 Å, α=β=γ=90°, and there are three complexes per asymmetric unit. Since the sequence of Mab 2947 Fab was unavailable, a high-resolution dataset of Fab alone (1.63 Å) was collected in conjunction with bioinformatics analysis, and the protein sequence was deciphered. Compelling answers for each component were obtained by molecular substitution searches using the structure of Mab 2974 Fab and publicly available structures of the TFPI K2 domain (RSCB Protein Databank, PDB codes 1TFX and 4DTG). Refinement was performed using the software autoBUSTER, and the final R / Rfree factors at 2.15 Å were 0.1702 and 0.2161, respectively, with a binding RMSD of 0.010 Å and an angle RMSD of 1.13°. Epitopes of the complex were determined based on the buried surface area (BSA) and percent BSA (%BSA) of residues at the Fab TFPI K2 interface. The following residues in the TFPI K2 domain of TFPI are involved in direct contact with Mab 2974 Fab (epitopes by BSA): E100, E101, P103, R107, Y109, T111, N116, Q118, S119, Q121, E123, R124, F125, and K126. The BSA and %BSA values ​​of the epitope residues are shown in Table 22.

[0280] [Table 23]

[0281] 4. Structure of the TFPI-23 Fab / cynomolgus monkey TFPI K2 complex TFPI-23 Fab and cynomolgus monkey TFPI K2 were mixed in a 1:2 molar ratio to form a complex. Final purification was performed using a Superdex200 column. The complex was concentrated to 12.4 mg / ml for structural studies. Crystals of the TFPI K2+4D8 Fab complex were obtained in 100 mM Bis-Tris pH 6.5, 20% PEGMME5000. It produced fibrous crystals diffracted down to 2.9 Å. The crystals were transiently cryoprotected, and synchrotron data acquisition was performed remotely at an Advanced Photon Source. Image frames were processed using the software AutoPROC (Global Phasing Ltd). The data belong to space group P1, with unit cells as follows: a=74.669 Å, b=101.372 Å, c=119.275 Å, α=101.83°, β=92.27°, γ=96.78°, and 6 complex copies per asymmetric unit. Compelling answers for each component were obtained by molecular substitution search using the homology model of TFPI-23 Fab and publicly available structures of the TFPI K2 domain (RSCB Protein Databank, PDB codes 1TFX and 4DTG). Refinement was performed using the software autoBUSTER, and the final R / Rfree factors at 2.9 Å were 0.1961 and 0.2344, respectively, with a binding RMSD of 0.010 Å and an angle RMSD of 1.22°. Epitope and paratope of the complex were determined based on the buried surface area (BSA) and percentage BSA (%BSA) of residues at the Fab TFPI K2 interface. The following residues in the K2 domain of TFPI are involved in direct contact with TFPI-23 Fab (epitopes by BSA): D102, I105, C106, R107, G108, R112, Y127, G129, C130, L131, G132, M134, and E138. The following residues in the heavy chain of 4D8 Fab contain heavy chain paratopes: A33, W47, A50, I51, S52, S56, Y58, L95, G96, A97, T98, S99, L100, and S100A. The following residues in the light chain of 4D8 Fab contain the light chain paratope: A29, Y31, Y91, S95A, G95B, and S95C.Table 23 shows the BSA and %BSA values ​​for epitope and paratope residues.

[0282] [Table 24]

[0283] 5. Structure of the TFPI-24 Fab / cynomolgus monkey TFPI K2 complex TFPI-24 Fab and cynomolgus monkey TFPI K2 were mixed in a 1:2 molar ratio to form a complex. Final purification was performed using a Superdex200 column. The complex was concentrated to 12.2 mg / ml for structural study. Crystals of the TFPI K2 / TFPI-24 Fab complex were obtained in 20% PEG3350, 200 mM ammonium nitrate. This yielded crystals diffracted down to 1.75 Å. The crystals were transiently cryoprotected, and synchrotron data acquisition was performed remotely at an Advanced Photon Source. Image frames were processed using the software AutoPROC. The data belong to space group P212121, with unit cells as follows: a=42.817 Å, b=71.362 Å, c=148.729 Å, α=β=γ=90°, and one complex per asymmetric unit. Compelling answers for each component were obtained through molecular substitution searches using homology models of the TFPI-24 Fab and publicly available structures of the TFPI K2 domain (RSCB Protein Databank, PDB codes 1TFX and 4DTG). Refinement using the software autoBUSTER revealed final R / Rfree factors of 0.1900 and 0.2269 at 1.75 Å, respectively, with a binding RMSD of 0.010 Å and an angle RMSD of 1.18°. The epitopes and paratopes of the complex were determined based on the buried surface area (BSA) and percentage BSA (%BSA) of residues at the Fab / TFPI K2 interface. The following residues in the K2 domain of TFPI are involved in direct contact with TFPI-24 Fab (BSA-mediated epitopes): E100, E101, D102, G104, I105, C106, R107, G108, Y109, I110, G129, C130, L131, and G132. The following residues in the heavy chain of TFPI-24 Fab contain heavy chain paratopes: A33, Q35, W47, G50, I51, S52, N53, R55, S56, I57, G58, F95, L96, H97, S99, and D101. The following residues in the light chain of TFPI-24 Fab contain light chain paratopes: M31, Y32, H34, Y36, L46, R50, W91, and Y96.Table 24 shows the BSA and %BSA values ​​for epitope and paratope residues.

[0284] [Table 25]

[0285] Epitope analysis of 6.hz4F36 The structure of hz4F36 fab in complex with the human TFPI K2 domain is available in the Protein Databank (PDB access code 4DTG). Based on the BSA and percent BSA (%BSA) of interface residues in the hz4F36 / TFPI K2 complex structure, the epitope residues were defined as shown in Table 25.

[0286] [Table 26]

[0287] 7. Comparison of anti-TFPI antibody epitopes The anti-TFPI antibody epitopes shown in Tables 20-25 are compared in Tables 26 and 27. Table 26 shows the antibody epitopes that are specific to the TFPI K2 domain. Table 27 includes two additional antibodies (2A8 and 2A8-200) that bind to both the K1 and K2 domains.

[0288] [Table 27]

[0289] [Table 28]

[0290] [Table 29]

[0291] [Table 30-1]

[0292] [Table 30-2]

[0293] [Table 31]

[0294] [Table 32]

[0295] (Example 11) Diluted prothrombin time (dPT) The ability of anti-TFPI antibodies to inhibit endogenous TFPI in human FVIII-deficient plasma (hemophilia A) was studied using a diluted prothrombin time (PT) assay. Diluted PT is a modified PT assay that uses diluted tissue factor (innovin) to prolong clotting time.

[0296] For dPT analysis in human FVIII-deficient plasma (George King Biomedical), Innovin® reagent was diluted 1:3000 in dilution buffer (50 mM imidazole, 0.1 M sodium chloride, 1 mg / mL BSA, 8.34 mM calcium chloride, pH 7.4) and pre-incubated at 37°C. Plasma was thawed in a 37°C water bath for 5 minutes and immediately assayed. Dilution of anti-TFPI antibody was prepared in PBS and added to the plasma, and the plasma was incubated at room temperature for 20 minutes. After this incubation, 50 μL of plasma was incubated at 37°C for 1 minute, and the coagulation reaction was immediately started by adding 50 μL of a 1:3000 dilution of Innovin® reagent warmed to 37°C. The time to coagulation was measured at 37°C using a STart® 4 Coagulation Analyzer. Data points were collected in two ways, entered into Microsoft Excel, and the effective concentration (EC50) at 50% was estimated using GraphPad Prism®. The results are shown in Table 30.

[0297] TFPI downmodulates the exogenous FVIIa / TF / FXa coagulation pathway, reducing the production of FXa and ultimately thrombin. The effect on the exogenous coagulation pathway is measured by dPT. The data show that the addition of anti-TFPI antibody to hemophilia A plasma resulted in a dose-dependent reduction in coagulation time. A 300 nM control IgG had no effect on coagulation time.

[0298] [Table 33]

[0299] (Example 12) Tromboelastography (TEG) Thromboelastography (TEG) is a comprehensive hemostatic assay that measures the dynamics of blood clot formation in whole blood. Whole blood was isolated from healthy human donors by drawing it into plastic blood collection tubes containing 3.2% sodium citrate, and the first tube into which the blood was drawn was discarded to minimize the introduction of coagulation activators such as tissue factor. Citrated whole blood was treated for 1 hour with control mouse anti-human IgG2 (100 mcg / mL) or inhibitory FVIII antibody (GM1805 (Green Mountain), 100 mcg / mL) to inhibit endogenous FVIII and induce a hemophilia A-like phenotype. Whole blood (320 μL) administered with anti-TFPI antibody or IgG1 control antibody was added to a TEG® reaction cup containing 20 μL of 0.2 M calcium chloride, 20 μL of lipid-derived tissue factor (Innovin®) diluted in 20 mM HEPES, 150 mM sodium chloride, and pH 7.4, yielding a final lipid-derived tissue factor dilution of 1:200,000 in each reaction. The reaction was run in two ways, starting immediately after adding whole blood to the TEG cup. Analysis was performed on a TEG® 5000 Hemostasis analyzer using TEG® software according to the manufacturer's instructions, after calibration with Level I and Level II controls (Haemonetics). The reaction was carried out at 37°C for 60 minutes. See Table 31.

[0300] [Table 34]

[0301] Table 31 shows that treatment of whole blood with FVIII antibody significantly prolonged the TEG-R value up to 41.5 minutes. In the presence of whole blood, anti-TFPI106 showed a favorable profile compared to 2A8-200 and 4F36. Addition of TFPI-106, 2A8-200, or 4F36 (300 nM) shortened the TEG-R value by 17.85 minutes, 20.4 minutes, and 24.05 minutes, respectively. Anti-TFPI106 promoted coagulation in hemophilic blood, as indicated by the decrease in TEG-R value and the observed increase in the TEG-alpha angle.

[0302] (Example 13) Neutralization of TFPI and thrombin production Neutralization of TFPI by TFPI antibodies was measured using two chromogenic assays, a direct factor Xa activity assay, and a two-step FVIIa / TF / FXa assay based on TF-FVIIa-mediated inhibition of FXa production by TF. In the first assay, TFPI-106, TFPI-118, hz4D8, and two reference antibodies, 4F36 or 2A8-200, were pre-incubated with fixed concentrations of human recombinant TFPI K1K2 and FXa at various concentrations (0-500 nM) to form complexes. FXa activity was evaluated using a chromogenic FXa substrate. Addition of the TFPI antibody of the present invention resulted in a dose-dependent increase in FXa activity in this assay (Table 32, Xa inhibition assay, EC). 50 (See value).

[0303] In vivo, the two dominant forms of TFPI are TFPI-alpha (K1K2K3) and TFPI-beta (K1K2). The ability of TFPI antibodies to inhibit recombinant TFPI K1K2 or TFPI K1K2K3 was assessed using a two-step FVIIa / TF / FXa assay. This assay measures the combined effect of neutralizing both FXa and FVIIa / TF / FXa TFPI inhibition. Antibodies were incubated with TFPI at increasing concentrations (0–500 nM) and added to the assay with FVIIa / TF / FXa, and FXa activity was measured using an FXa-chromogenic substrate. The TFPI antibodies of the present invention neutralized TFPI K1K2 inhibition of FVIIa / TF-mediated FX activation (Table 32, FVIIa / TF / Xa EC). 50 (See value). The exemplary antibodies of the present invention were also effective in inhibiting TFPI K1K2K3 (EC of TFPI-106). 50 (The neutralization of FVIIa / TF / FXa inhibition by TFPI K1K2K3 is 8.47 nM). The data demonstrate inhibition of full-length and truncated TFPI.

[0304] The clinical severity of hemophilia is related to the residual levels of coagulation factor activity. Factor activity of less than 1% is associated with a severe phenotype, moderate hemophilia is associated with factor activity of 2–5%, and mild hemophilia is associated with factor activity of greater than 5% but less than 40%. Defects in the endogenous coagulation pathway in hemophilia result in insufficient thrombin production. The inhibition of endogenous TFPI in platelet-poor hemophilia plasma was examined using a thrombin production assay (TGA). The TGA assay measures the initiation, activation, and inactivation phases of thrombin production. The ability of TFPI antibodies to restore thrombin production in platelet-poor hemophilia plasma was tested using a calibrated automated thrombin (CAT) production assay. TFPI-106, TFPI-118, hz4D8, and two reference antibodies, 4F36 or 2A8-200 (0–500 nM), were incubated in hemophilia plasma to neutralize TFPI before being added to the assay. Compared to normal human pooled plasma, thrombin production was significantly reduced in human hemophilia plasma. A dose-dependent response was observed when FACT, standardized with a normal control and 1 U / mL FVIII, was spiked in hemophilia A plasma. Similarly, the addition of 200 ng / mL (1 U / mL) of B-domain deletion FVIII restored thrombin production to 100 nM. Minimal thrombin production was observed in hemophilia plasma over the course of a 60-minute assay. Incubation of plasma with the antibody of the present invention resulted in dose-dependent increases in peak thrombin, endogenous thrombin potential, and rate index. Reference antibodies 2A8-200 and 4F36 were also assayed for comparison (Table 32, TGA rate EC). 50 value).

[0305] (Example 14) In vivo efficacy in a hemophilia mouse model The efficacy of certain anti-TFPI antibodies as coagulation promoters was tested using an acute tail amputation assay in hemophilic mice. This assay involved amputation of the distal portion of the tail, resulting in substantial blood loss. This blood loss can be reduced if hemostatic agents are administered before or immediately after transsection. Hemophilic mice received a single intravenous (IV) dose of either anti-TFPI antibody (6 mg / kg), a nonspecific IgG control (6 mg / kg), or 4 ml / kg via the tail vein in saline. The effect of the antibodies on bleeding was assessed at different time points after administration, as follows:

[0306] Mice were anesthetized intraperitoneally with a ketamine / xylazine cocktail. Their tails were immersed in 50 mL of pre-warmed phosphate-buffered solution (PBS) at 37°C for 2 minutes. A 3 mm tail section was removed, and blood was collected in PBS over a 10-minute period. The volume of blood loss was then quantified by measuring the hemoglobin content of the PBS using the following technique: The tubes were centrifuged to collect red blood cells, which were resuspended in 5 mL of lysis buffer (8.3 g / l ammonium chloride, 1.0 g / l potassium bicarbonate, and 0.037 g / l EDTA). The absorbance of the sample at 575 nm was measured using a spectrophotometer. The absorbance values ​​were converted to total blood loss (μL) using a calibration curve. Statistical significance of differences between means was assessed by analysis of variance (ANOVA) using GraphPad® Prism software, followed by Dunnett's multiple comparison test. Results are expressed as mean ± standard error of the mean (SEM). In the figures below, statistical significance is defined as a P-value < 0.05 and is indicated by an asterisk above the data.

[0307] Figure 3A shows the effect of administering the 2A8-200 antibody at different times prior to tail amputation on blood loss in hemophilia A mice (indicated as FVIII- / -) after tail amputation, compared to a medium control (saline). Figure 3B shows the effect of administering the 2A8 antibody at different times prior to tail amputation on blood loss in hemophilia A mice (indicated as FVIII- / -) after tail amputation, compared to a medium control (saline) and nonspecific human IgG1 (indicated as hIgG1). Figure 3B also shows the effect of administering the 2A8 antibody at two different times prior to tail amputation on blood loss in normal mice (indicated as FVIII+ / +) after tail amputation. Figure 3C shows the effect of administering antibodies 4D8, 21, 23, and 24 at different times prior to tail amputation on blood loss in hemophilia A mice after tail amputation, compared to a medium control (saline). Figure 3D shows the effect of administering antibody 106 at different times before tail amputation compared to a medium control (physiological saline) on blood loss in hemophilia A mice after tail amputation. Figure 3E shows the effect of administering antibody 118 at different times before tail amputation compared to a medium control (physiological saline) on blood loss in hemophilia A mice after tail amputation. Figure 4 shows the effect of administering antibody 106 at...

Claims

1. An isolated antibody or its antigen-binding fragment that specifically binds to an epitope in the Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), wherein the antibody comprises a heavy chain variable region (VH) complementarity-determining region 1 (CDR-H1) containing the amino acid sequence of SEQ ID NO: 48, a CDR-H2 containing the amino acid sequence of SEQ ID NO: 49, and a CDR-H3 containing the amino acid sequence of SEQ ID NO: 50, and a light chain variable region (VL) complementarity-determining region 1 (CDR-L1) containing the amino acid sequence of SEQ ID NO: 43, a CDR-L2 containing the amino acid sequence of SEQ ID NO: 44, and a CDR-L3 containing the amino acid sequence of SEQ ID NO:

45.

2. An isolated antibody or antigen-binding fragment that specifically binds to K2 of TFPI, comprising (i) the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 present in the amino acid sequence of SEQ ID NO: 51, and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 present in the amino acid sequence of SEQ ID NO: 46, or (ii) the isolated antibody or antigen-binding fragment comprising the amino acid sequences of CDR-H1, CDR-H2, and CDR-H3 present in the amino acid sequence of SEQ ID NO: 67, and the amino acid sequences of CDR-L1, CDR-L2, and CDR-L3 present in the amino acid sequence of SEQ ID NO:

77.

3. An isolated antibody or its antigen-binding fragment that specifically binds to K2 of TFPI, comprising a VH containing the amino acid sequence of SEQ ID NO: 51, 67, 69, or 79, and a VL containing the amino acid sequence of SEQ ID NO: 46, 71, 73, 75, or 77.

4. An isolated antibody or its antigen-binding fragment that specifically binds to K2 of TFPI, comprising (i) a VH containing the amino acid sequence of SEQ ID NO: 51 and a VL containing the amino acid sequence of SEQ ID NO: 46, or (ii) a VH containing the amino acid sequence of SEQ ID NO: 67 and a VL containing the amino acid sequence of SEQ ID NO:

77.

5. An isolated antibody or its antigen-binding fragment that specifically binds to K2 of TFPI, comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 52 and a light chain consisting of the amino acid sequence of SEQ ID NO:

47.

6. An isolated antibody or its antigen-binding fragment that specifically binds to K2 of TFPI, comprising a heavy chain consisting of the amino acid sequence of SEQ ID NO: 68 and a light chain consisting of the amino acid sequence of SEQ ID NO:

78.

7. 1 x 10 －８ M~1 x 10 －１０ An antibody or antigen-binding fragment according to any one of claims 1 to 6, which binds to TFPI at a binding affinity (Kd) value of M.

8. An antibody or antigen-binding fragment according to any one of claims 1 to 7, which (i) reduces clotting time when measured by a plasma-based diluted prothrombin time assay, (ii) reduces clotting time in whole blood when measured by thromboelastography, (iii) increases thrombin production, (iv) increases FXa activity in the presence of TFPI, (v) enhances platelet accumulation in the presence of TFPI, (vi) increases fibrin production in the presence of TFPI, or (vii) any combination thereof.

9. The antibody or antigen-binding fragment according to claim 8, wherein the plasma or whole blood is deficient in factor VIII or factor IX.

10. An isolated nucleic acid molecule comprising a nucleotide sequence encoding an antibody or an antigen-binding fragment according to any one of claims 1 to 9.

11. The nucleic acid molecule according to claim 10, wherein the nucleic acid encodes (i) VH comprising CDR-H1 comprising the amino acid sequence of SEQ ID NO: 48, CDR-H2 comprising the amino acid sequence of SEQ ID NO: 49, and CDR-H3 comprising the amino acid sequence of SEQ ID NO: 50, and VL comprising CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43, CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44, and CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45, (ii) VH comprising the amino acid sequence of SEQ ID NO: 51 and VL comprising the amino acid sequence of SEQ ID NO: 46, or (iii) VH comprising the amino acid sequence of SEQ ID NO: 67 and VL comprising the amino acid sequence of SEQ ID NO:

77.

12. A pharmaceutical composition comprising an antibody or its antigen-binding fragment according to claims 1 to 8 and a pharmaceutically acceptable carrier or excipient.

13. The pharmaceutical composition according to claim 12 for treating uncontrolled bleeding in a subject.

14. The pharmaceutical composition according to claim 12 for shortening bleeding time in subjects requiring it.

15. The pharmaceutical composition according to claim 13 or 14, wherein the subject is suffering from or susceptible to hemophilia A, hemophilia B, von Willebrand disease (vWD), or platelet disorder.

16. The pharmaceutical composition according to claims 12 to 15, further comprising a coagulant or for use in combination with a coagulant.

17. The pharmaceutical composition according to claim 16, wherein the coagulant is selected from the group consisting of factor VIIa, factor VIII, factor IX, and tranexamic acid.

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