Tissue factor pathway inhibitor antibody and its uses

Abstract

The present invention relates to antibodies and antigen-binding fragments thereof that specifically bind to TFPI and inhibit its activity. These antibodies and fragments can be used to treat hemorrhagic disorders and shorten the clotting time.

Description

[0001] This application is a divisional application of Chinese Patent Application No. 201680048417.2, titled "Tissue Factor Pathway Inhibitor Antibodies and Their Uses", filed on August 10, 2016. Technical Field

[0002] The present invention relates to antibodies that bind to tissue factor pathway inhibitor (TFPI). Background Art

[0003] Hemophilia A and B are X-linked genetic diseases caused by functional deficiencies of the plasma proteins factor VIII (FVIII) or factor IX (FIX), respectively. The clinical severity of hemophilia is related to the residual level of the clotting factor activity. Factor activity of <1% is associated with the severe phenotype, intermediate hemophilia is associated with 2% to 5% factor activity, and mild form is associated with 5% to 40% factor activity.

[0004] The standard of care for these diseases is to replace the missing clotting factor by intravenous infusion. The replacement factor is usually a recombinant protein, such as Xyntha (factor VIII) or BeneFIX (FIX), but plasma-derived products of various purities are still used. Treatment with the replacement factor can be either episodic (treating bleeding as needed when it occurs) or prophylactic (preventing bleeding by maintaining factor levels within a protective range). There is clear evidence that prophylactic treatment can prevent bleeding and related joint damage, which is the major morbidity in hemophilia patients. Effective prophylactic treatment requires intravenous injection of the factor 3 to 4 times a week, which poses difficulties in compliance and reduces the quality of life. Due to the complexity of manufacturing clotting factors, the treatment cost is also expensive. In addition, a large number of patients (up to 32% of severe hemophilia A patients) develop neutralizing antibodies against the administered factor, which is regarded as a foreign protein by patients with mutations in these genes. These patients require alternative treatment modalities, such as bypassing factors, factor VIIa (NovoSeven).

[0005] An alternative approach to treatment is to bypass the need for replacement factors by augmenting the intact extrinsic pathway. Hemophilia patients have some ability to stop bleeding through their intact extrinsic pathway; however, this is not sufficient to stop major bleeds or prevent spontaneous bleeding. The extrinsic pathway is not sufficient to provide protection because it is rapidly shut down by tissue factor pathway inhibitor (TFPI).

[0006] Although WO 2010 / 017196 (Bayer), WO 2011 / 109452 (Bayer), WO 2014 / 144577 (Bayer), WO 2010 / 072687 (Novo Nordisk), WO 2012 / 001087 (Novo Nordisk), WO 2014 / 140240 (Novo Nordisk), and WO 2015 / 007880 (Novo Nordisk) disclose antibodies that bind human TFPI, they do not provide the antibodies of the present invention, which have the characteristics of being potential therapeutic agents for novel hemophilia.

[0007] A product that can provide prophylactic protection while reducing the frequency of coagulation factor administration, reducing the amount of factor used, allowing alternative delivery routes (such as subcutaneous), and having a lower risk of generating neutralizing antibodies would meet a large number of unmet needs of hemophilia patients. Summary of the Invention

[0008] The present invention discloses and exemplifies antibodies (and their antigen-binding fragments) that bind tissue factor pathway inhibitor (TFPI).

[0009] Those skilled in the art will recognize or be able to determine many equivalents of the specific embodiments of the present invention described herein using only routine experimentation. Such equivalents are intended to be included in the following embodiments (E).

[0010] E1. An isolated antibody or its antigen-binding fragment that specifically binds to an epitope of the Kunitz domain 2 (K2) of tissue factor pathway inhibitor (TFPI), wherein the epitope comprises residues Ile105, Arg107, and Leu131 (numbered according to SEQ ID NO: 2).

[0011] E2. The antibody or its antigen-binding fragment of embodiment 1, wherein the antibody or its antigen-binding fragment does not bind to the Kunitz domain 1 (K1) of TFPI.

[0012] E3. The antibody or its antigen-binding fragment of 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 (numbered according to SEQ ID NO: 2).

[0013] E4. The antibody or its antigen-binding fragment of any one of embodiments 1 to 3, wherein the epitope further comprises residues Cys106, Gly108, Cys130, Leu131, and Gly132 (numbered according to SEQ ID NO: 2).

[0014] The antibody or antigen-binding fragment thereof 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 (numbered according to SEQ ID NO: 2).

[0015] E6. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 5, wherein the epitope further comprises Asp102, Arg112, Tyr127, Gly129, Met134, and Glu138 (numbered according to SEQ ID NO: 2).

[0016] E7. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the epitope does not comprise 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 (numbered according to SEQ ID NO: 2).

[0017] E8. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 7, wherein the epitope does not comprise: E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140 (numbered according to SEQ ID NO: 2).

[0018] E9. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6, wherein the epitope does not comprise one or more residues selected from the group consisting of D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128 (numbered according to SEQ ID NO: 2).

[0019] E10. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 6 and 9, wherein the epitope does not comprise: D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, and G128 (numbered according to SEQ ID NO: 2).

[0020] The antibody or antigen-binding fragment thereof 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 (numbered according to SEQ ID NO: 2), and wherein the epitope residues have a non-zero change in the buried surface area (BSA) due to interaction with the antibody or antigen-binding fragment thereof.

[0021] The antibody or antigen-binding fragment thereof according to embodiment 11, wherein the epitope comprises: Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Asn133, Met134, and Glu138 (numbered according to SEQ ID NO: 2).

[0022] The antibody or antigen-binding fragment thereof 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 (numbered according to SEQ ID NO: 2), and wherein the epitope residues participate in hydrogen bonds with residues from the antibody or antigen-binding fragment thereof.

[0023] The antibody or antigen-binding fragment thereof according to embodiment 13, wherein the epitope comprises: Asp102, Arg107, Arg112, Tyr127, and Leu131 (numbered according to SEQ ID NO: 2).

[0024] The antibody or antigen-binding fragment thereof 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 (numbered according to SEQ ID NO: 2).

[0025] An antibody or antigen-binding fragment thereof according to embodiment 15, wherein the epitope comprises: Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, and Glu138 (numbered according to SEQ ID NO: 2).

[0026] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 16, which comprises the following heavy (H)-chain and light (L)-chain paratope residues that have a non-zero change in BSA due to interaction with TFPI (according to Kabat numbering): 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.

[0027] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 17, which comprises the following contacting residues (according to Kabat numbering): (1) H47 is Trp or Tyr; (b) H58 is Tyr; and (c) L91 is Tyr or Arg; and optionally comprises: (d) L96 is Gly or Asn.

[0028] An antibody or antigen-binding fragment thereof according to any one of embodiments E19, comprising the following contacting residues (according to Kabat numbering): (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 or Trp, Tyr, 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 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; and (t) L95C is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and optionally comprises the following residues: (u) L93 is Tyr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and (v) L96 is Gly or Asn.;

[0029] E20. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 18, which comprises the following contacting residues (according to Kabat numbering): (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; (1) 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; and (t) L95C is Ser, Arg, Asn, Gln, Glu, Ile, Leu, Lys, Met, Phe, Trp, Tyr or Val; and optionally comprises the following residues: (u) L93 is Ser; and (v) L96 is Gly.

[0030] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 18, which comprises the following contacting residues (according to Kabat numbering): (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 Tyr or Arg; (r) L95A is Ser, Phe, Tyr or His; L95B is Gly, Glu, Asp or Pro; and (t) L95C is Ser, Trp, Tyr or Phe; and optionally comprises the following residues: (u) L93 is Ser, Glu, Asp or His; and (v) L96 is Gly or Asn.

[0031] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 18, which comprises the following contacting residues (according to Kabat numbering): 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 optionally comprises the following residues: L93 Ser and L96 Gly.

[0032] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 22, which comprises a heavy chain variable region (VH), said VH comprising:

[0033] (a) a VH complementarity determining region 1 (CDR-H1) comprising the amino acid sequence of SEQ ID NO: 38;

[0034] (b) A VH complementarity-determining region 2 (CDR-H2) comprising the amino acid sequence of SEQ ID NO: 39; and

[0035] (c) A VH complementarity-determining region 3 (CDR-H3) comprising the amino acid sequence of SEQ ID NO: 40.

[0036] E24. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 22, which comprises CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO: 41.

[0037] E25. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 24, which comprises a human VH3 framework sequence.

[0038] E26. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 24, which comprises a human VH1 framework sequence.

[0039] E27. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 24, which comprises a human VH5 framework sequence.

[0040] E28. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 24, which comprises a VH framework sequence of human germline IGHV3-23 or IGHV1-69.

[0041] E29. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 24, which comprises a VH framework sequence of human germline IGHV3-7.

[0042] E30. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 24, which comprises a human VH germline consensus framework sequence.

[0043] E31. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 30, which comprises a VH that contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 41, 63, and 65.

[0044] E32. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 31, which comprises a VH that contains an amino acid sequence selected from the group consisting of SEQ ID NO: 41, 63, and 65.

[0045] E33. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 32, which comprises a VH that contains the amino acid sequence of SEQ ID NO: 41.

[0046] E34. An antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 32, which comprises a VH that contains the amino acid sequence of SEQ ID NO: 63.

[0047] E35. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 32, which comprises a VH that contains the amino acid sequence of SEQ ID NO: 65.

[0048] E36. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 35, which comprises a variable light chain (VL), the VL comprising:

[0049] (a) a VL complementarity determining region 1 (CDR-L1) that contains the amino acid sequence of SEQ ID NO: 33;

[0050] (b) a VL complementarity determining region 2 (CDR-L2) that contains the amino acid sequence of SEQ ID NO: 34; and

[0051] (c) a VL complementarity determining region 3 (CDR-L3) that contains the amino acid sequence of SEQ ID NO: 35.

[0052] E37. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 35, which comprises CDR-L1, CDR-L2 and CDR-L3 sequences of SEQ ID NO: 36.

[0053] E38. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, which comprises a human V K framework sequence.

[0054] E39. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, which comprises a human V λ framework sequence.

[0055] E40. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, which comprises a VL framework sequence of human germline IGKV3-20.

[0056] E41. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, which comprises a VL framework sequence of human germline IGKV1-39.

[0057] E42. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 37, which comprises a human VL germline consensus framework sequence.

[0058] E43. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 42, which comprises a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 36.

[0059] E44. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 43, which comprises a VL that contains the amino acid sequence of SEQ ID NO: 36.

[0060] E45. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 44, comprising a heavy chain constant region (CH), said CH having an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

[0061] E46. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 45, comprising a CH, said CH having the amino acid sequence of SEQ ID NO: 20.

[0062] E47. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 46, comprising a light chain constant region (CL), said CL having an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0063] E48. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 47, comprising a CL, said CL having the amino acid sequence of SEQ ID NO: 26.

[0064] E49. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 48, comprising an Fc domain.

[0065] E50. The antibody or antigen-binding fragment thereof according to embodiment 49, wherein the Fc domain is the Fc domain of IgA.

[0066] E51. The antibody or antigen-binding fragment thereof according to embodiment 50, wherein the IgA is IgA 1 or IgA 2 .

[0067] E52. The antibody or antigen-binding fragment thereof according to embodiment 49, wherein the Fc domain is the Fc domain of IgD.

[0068] E53. The antibody or antigen-binding fragment thereof according to embodiment 49, wherein the Fc domain is the Fc domain of IgE.

[0069] E54. The antibody or antigen-binding fragment thereof according to embodiment 49, wherein the Fc domain is the Fc domain of IgM.

[0070] E55. The antibody or antigen-binding fragment thereof according to embodiment 49, wherein the Fc domain is the Fc domain of IgG.

[0071] E56. The antibody or antigen-binding fragment thereof according to embodiment 55, wherein the IgG is IgG 1 , IgG 2 , IgG 3 or IgG 4 .

[0072] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 56, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 42.

[0073] E58. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 56, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 64.

[0074] E59. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 56, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 66.

[0075] E60. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 59, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 37.

[0076] E61. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 60, comprising a VH sequence encoded by an insert present in a plasmid deposited with ATCC accession number PTA-122329.

[0077] E62. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 61, comprising a VL sequence encoded by an insert present in a plasmid deposited with ATCC accession number PTA-122328.

[0078] E63. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, wherein the epitope further comprises one or more residues selected from the group consisting of Glu 100, Glu 101, Asp 102, Gly 104, and Tyr 109 (numbered according to SEQ ID NO: 2).

[0079] E64. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63, wherein the epitope further comprises Glu 100, Glu 101, Asp 102, Gly 104, and Tyr 109 (numbered according to SEQ ID NO: 2).

[0080] E65. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 64, wherein the epitope does not comprise 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).

[0081] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 65, wherein the epitope does not comprise: P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126 and L140 (numbered according to SEQ ID NO: 2).

[0082] E67. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 64, wherein the epitope does not comprise 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).

[0083] E68. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4, 63 to 64 and 67, wherein the epitope does not comprise: D31, D32, P34, C35, K36, P103, K126, Y127, G128 (numbered according to SEQ ID NO: 2).

[0084] E69. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 68, which comprises the following residues (according to Kabat numbering): 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.

[0085] E70. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 69, which comprises a VH that contains:

[0086] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 48;

[0087] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 49; and

[0088] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 50.

[0089] E71. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 69, which comprises the CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO: 51.

[0090] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 71, which comprises a human VH3, VH1 or VH5 framework sequence.

[0091] E73. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 72, which comprises a VH framework sequence of human germline IGHV3-23 or IGHV1-69.

[0092] E74. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 72, which comprises a VH framework sequence of human germline IGHV3-7.

[0093] E75. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 71, which comprises a human VH germline consensus framework sequence.

[0094] E76. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 75, which comprises a VH that contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 67, 69, 51, and 79.

[0095] E77. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 76, which comprises a VH that contains an amino acid sequence selected from the group consisting of SEQ ID NO: 67, 69, 51, and 79.

[0096] E78. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 77, which comprises a VH that contains the amino acid sequence of SEQ ID NO: 67.

[0097] E79. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 77, which comprises a VH that contains the amino acid sequence of SEQ ID NO: 69.

[0098] E80. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 77, which comprises a VH that contains the amino acid sequence of SEQ ID NO: 51.

[0099] E81. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 77, which comprises a heavy chain variable region (VH) that contains the amino acid sequence of SEQ ID NO: 79.

[0100] E82. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 81, which comprises a VL that contains:

[0101] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 43;

[0102] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 44; and

[0103] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 45.

[0104] E83. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 81, which comprises CDR-L1, CDR-L2 and CDR-L3 sequences of SEQ ID NO: 46.

[0105] E84. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 83, which comprises a human V K or V λ framework sequence.

[0106] E85. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 84, which comprises a VL framework sequence of human germline IGKV3-20 or IGKV1-39.

[0107] E86. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 83, which comprises a human VL germline consensus framework sequence.

[0108] E87. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 86, which comprises a VL that contains an amino acid sequence that is at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 46, 71, 73, 75 and 77.

[0109] E88. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 87, which comprises a VL that contains an amino acid sequence selected from the group consisting of SEQ ID NOs: 46, 71, 73, 75 and 77.

[0110] E89. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 88, which comprises a VL that contains the amino acid sequence of SEQ ID NO: 46.

[0111] E90. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 88, which comprises a VL that contains the amino acid sequence of SEQ ID NO: 71.

[0112] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 88, which comprises a VL that contains the amino acid sequence of SEQ ID NO: 73.

[0113] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 88, which comprises a VL that contains the amino acid sequence of SEQ ID NO: 75.

[0114] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 88, which comprises a VL that contains the amino acid sequence of SEQ ID NO: 77.

[0115] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 93, which comprises a CH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

[0116] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 94, which comprises a CH that contains the amino acid sequence of SEQ ID NO: 20.

[0117] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 95, which comprises a CL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0118] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 96, which comprises a CL that contains the amino acid sequence of SEQ ID NO: 26.

[0119] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and any one of 63 to 97, which comprises an Fc domain.

[0120] An antibody or antigen-binding fragment thereof according to embodiment 98, wherein the Fc domain is the Fc domain of IgA.

[0121] An antibody or antigen-binding fragment thereof according to embodiment 99, wherein the IgA is IgA 1 or IgA 2 。

[0122] An antibody or antigen-binding fragment thereof according to embodiment 98, wherein the Fc domain is the Fc domain of IgD, IgE or IgM.

[0123] An antibody or antigen-binding fragment thereof according to embodiment 98, wherein the Fc domain is the Fc domain of IgG.

[0124] E103. An antibody or antigen-binding fragment thereof according to embodiment 102, wherein the IgG is IgG 1 , IgG 2 , IgG 3 or IgG 4 .

[0125] E104. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 103, which comprises a heavy chain having the amino acid sequence of SEQ ID NO: 52.

[0126] E105. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 103, which comprises a heavy chain having the amino acid sequence of SEQ ID NO: 68.

[0127] E106. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 103, which comprises a heavy chain having the amino acid sequence of SEQ ID NO: 70.

[0128] E107. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 103, which comprises a heavy chain having the amino acid sequence of SEQ ID NO: 80.

[0129] E108. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 107, which comprises a light chain having the amino acid sequence of SEQ ID NO: 47.

[0130] E109. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 107, which comprises a light chain having the amino acid sequence of SEQ ID NO: 72.

[0131] E110. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 107, which comprises a light chain having the amino acid sequence of SEQ ID NO: 74.

[0132] E111. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 107, which comprises a light chain having the amino acid sequence of SEQ ID NO: 76.

[0133] E112. An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 4 and 63 to 107, which comprises a light chain having the amino acid sequence of SEQ ID NO: 78.

[0134] E113. 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), wherein the epitope comprises the residues Glu101, Pro103, Tyr109, Thr111, Ser119, Gln121, Glu123, Arg124, Lys126, and Leu140 (numbered according to SEQ ID NO: 2).

[0135] E114. The antibody or antigen-binding fragment thereof of embodiment 113, wherein the antibody or antigen-binding fragment thereof does not bind to Kunitz domain 1 (K1) of TFPI.

[0136] E115. The antibody or antigen-binding fragment thereof of embodiment 113 or 114, wherein the epitope does not comprise 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).

[0137] E116. The antibody or antigen-binding fragment thereof of any one of embodiments 113 to 115, wherein the epitope does not comprise: E100, D102, R107, Y113, F114, N116, Q118, and C122 (numbered according to SEQ ID NO: 2).

[0138] E117. The antibody or antigen-binding fragment thereof of embodiment 113 or 114, wherein the epitope does not comprise 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).

[0139] E118. The antibody or antigen-binding fragment thereof of any one of embodiments 113 to 114 and 117, wherein the epitope does not comprise: D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127, and G128 (numbered according to SEQ ID NO: 2).

[0140] E119. The antibody or antigen-binding fragment thereof of embodiments 113 to 118, which comprises the following residues (according to Kabat numbering): H50 Asp, H57 Thr, H58 Leu, H59 Tyr, H61 Gln, H98 Asp, H99 Tyr, H100 Asp, L30 His, L50Trp, L92Tyr, L93 Thr, L94 Thr, and L96 Tyr.

[0141] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 119, comprising a VH, said VH comprising:

[0142] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 87;

[0143] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 88; and

[0144] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 89.

[0145] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 119, comprising CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO: 90.

[0146] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 121, comprising a human VH3, VH1 or VH5 framework sequence.

[0147] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 122, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69 or IGHV3-7.

[0148] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 121, comprising a human VH germline consensus framework sequence.

[0149] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 124, comprising a VH, said VH comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 90, 95, 97, 99, 101, 103, 105 and 107.

[0150] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 125, comprising a VH, said VH comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 90, 95, 97, 99, 101, 103, 105 and 107.

[0151] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH, said VH comprising the amino acid sequence of SEQ ID NO: 90.

[0152] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH, said VH comprising the amino acid sequence of SEQ ID NO: 95.

[0153] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH that contains the amino acid sequence of SEQ ID NO: 97.

[0154] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH that contains the amino acid sequence of SEQ ID NO: 99.

[0155] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH that contains the amino acid sequence of SEQ ID NO: 101.

[0156] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH that contains the amino acid sequence of SEQ ID NO: 103.

[0157] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH that contains the amino acid sequence of SEQ ID NO: 105.

[0158] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 126, comprising a VH that contains the amino acid sequence of SEQ ID NO: 107.

[0159] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 134, comprising a VL that contains:

[0160] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 81;

[0161] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 82; and

[0162] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 83.

[0163] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 134, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 84.

[0164] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 136, comprising a human V K or V λ framework sequence.

[0165] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 137, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39.

[0166] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 136, comprising a human VL germline consensus framework sequence.

[0167] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 139, comprising a VL that contains an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 84, 109, and 111.

[0168] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 140, comprising a VL that contains an amino acid sequence selected from the group consisting of SEQ ID NO: 84, 109, and 111.

[0169] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 141, comprising a VL that contains the amino acid sequence of SEQ ID NO: 84.

[0170] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 141, comprising a VL that contains the amino acid sequence of SEQ ID NO: 109.

[0171] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 141, comprising a VL that contains the amino acid sequence of SEQ ID NO: 111.

[0172] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 144, comprising a CH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

[0173] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 145, comprising a CH that contains the amino acid sequence of SEQ ID NO: 20.

[0174] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 144, comprising a CH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 91.

[0175] An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 144 and 147, comprising a CH that contains the amino acid sequence of SEQ ID NO: 91.

[0176] An antibody or an antigen-binding fragment thereof according to any one of embodiments 113 to 148, which comprises a CL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 14.

[0177] An antibody or an antigen-binding fragment thereof according to any one of embodiments 113 to 149, which comprises a CL that contains the amino acid sequence of SEQ ID NO: 14.

[0178] An antibody or an antigen-binding fragment thereof according to any one of embodiments 113 to 148, which comprises a CL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 85.

[0179] An antibody or an antigen-binding fragment thereof according to any one of embodiments 113 to 148 and 151, which comprises a CL that contains the amino acid sequence of SEQ ID NO: 85.

[0180] An antibody or an antigen-binding fragment thereof according to any one of embodiments 113 to 152, which comprises an Fc domain.

[0181] An antibody or an antigen-binding fragment thereof according to embodiment 153, wherein the Fc domain is the Fc domain of IgA (such as IgA 1 or IgA 2 ).

[0182] An antibody or an antigen-binding fragment thereof according to embodiment 153, wherein the Fc domain is the Fc domain of IgD, IgE or IgM.

[0183] An antibody or an antigen-binding fragment thereof according to embodiment 153, wherein the Fc domain is the Fc domain of IgG.

[0184] An antibody or an antigen-binding fragment thereof according to any one of embodiments 156, wherein the IgG is IgG 1 , IgG 2 , IgG 3 or IgG 4 .

[0185] An antibody or an antigen-binding fragment thereof according to any one of embodiments 113 to 157, which comprises a heavy chain that contains the amino acid sequence of SEQ ID NO: 92.

[0186] An antibody or an antigen-binding fragment thereof according to any one of embodiments 113 to 157, which comprises a heavy chain that contains the amino acid sequence of SEQ ID NO: 94.

[0187] E160. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 157, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 96.

[0188] E161. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 157, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 98.

[0189] E162. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 157, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 100.

[0190] E163. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 157, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 102.

[0191] E164. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 157, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 104.

[0192] E165. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 157, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 106.

[0193] E166. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 157, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 108.

[0194] E167. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 166, comprising a light chain having the amino acid sequence of SEQ ID NO: 86.

[0195] E168. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 166, comprising a light chain having the amino acid sequence of SEQ ID NO: 93.

[0196] E169. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 166, comprising a light chain having the amino acid sequence of SEQ ID NO: 110.

[0197] E170. An antibody or antigen-binding fragment thereof according to any one of embodiments 113 to 166, comprising a light chain having the amino acid sequence of SEQ ID NO: 112.

[0198] E171. An isolated antibody or antigen-binding fragment thereof that specifically binds to the Kunitz domain 2 (K2) of TFPI, comprising a VH, wherein the VH contains:

[0199] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16;

[0200] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; and

[0201] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18.

[0202] E172. 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: 19.

[0203] E173. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising a VL, wherein the VL contains:

[0204] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10;

[0205] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and

[0206] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0207] E174. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 13.

[0208] El75. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising:

[0209] (i) VH, which contains:

[0210] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 16;

[0211] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 17; and

[0212] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 18;

[0213] and (ii) VL, which contains:

[0214] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 10;

[0215] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 11; and

[0216] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 12.

[0217] E176. 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: 19 and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 13.

[0218] E177. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 176, comprising a human VH3, VH1, or VH5 framework sequence.

[0219] E178. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 177, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7.

[0220] E179. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 176, comprising a human VH germline consensus framework sequence.

[0221] E180. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 179, comprising a VH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 19.

[0222] E181. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 180, comprising a VH that contains the amino acid sequence of SEQ ID NO: 19.

[0223] E182. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 181, comprising a human V K or V λ framework sequence.

[0224] E183. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 182, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39.

[0225] E184. The antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 181, comprising a human VL germline consensus framework sequence.

[0226] E185. An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 184, comprising a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 13.

[0227] E186. An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 185, comprising a VL that contains the amino acid sequence of SEQ ID NO: 13.

[0228] E187. An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 186, comprising a CH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

[0229] E188. An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 187, comprising a CH that contains the amino acid sequence of SEQ ID NO: 20.

[0230] E189. An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 188, comprising a CL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 14.

[0231] E190. An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 189, comprising a CL that contains the amino acid sequence of SEQ ID NO: 14.

[0232] E191. An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 190, comprising an Fc domain.

[0233] E192. The antibody or antigen-binding fragment thereof according to embodiment 191, wherein the Fc domain is the Fc domain of IgA (e.g., IgA 1 or IgA 2 ), IgD, IgE or IgM.

[0234] E193. The antibody or antigen-binding fragment thereof according to embodiment 191, wherein the Fc domain is the Fc domain of IgG.

[0235] E194. The antibody or antigen-binding fragment thereof according to embodiment 191, wherein the IgG is IgG 1 , IgG 2 , IgG 3 or IgG 4 .

[0236] An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 194, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 21.

[0237] An antibody or antigen-binding fragment thereof according to any one of embodiments 171 to 195, comprising a light chain having the amino acid sequence of SEQ ID NO: 15.

[0238] An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising a VH that contains:

[0239] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28;

[0240] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29; and

[0241] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30.

[0242] 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.

[0243] An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising a VL that:

[0244] (a) comprises CDR-L1 having the amino acid sequence of SEQ ID NO: 22;

[0245] (b) comprises CDR-L2 having the amino acid sequence of SEQ ID NO: 23; and

[0246] (c) comprises CDR-L3 having the amino acid sequence of SEQ ID NO: 24.

[0247] An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 25.

[0248] An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising:

[0249] (i) VH, which contains:

[0250] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 28;

[0251] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 29; and

[0252] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 30;

[0253] and (ii) VL, which comprises:

[0254] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 22;

[0255] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 23; and

[0256] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 24.

[0257] 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.

[0258] 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.

[0259] 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.

[0260] 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.

[0261] E206. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 205, comprising a VH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 31.

[0262] E207. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 206, comprising a VH that contains the amino acid sequence of SEQ ID NO: 31.

[0263] E208. The antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 207, comprising a human V Kor V λ framework sequence.

[0264] E209. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 208, comprising a VL framework sequence of human germline IGKV3-20 or IGKV1-39.

[0265] E210. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 207, comprising a human VL germline consensus framework sequence.

[0266] E211. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 210, comprising a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 25.

[0267] E212. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 211, comprising a VL that contains the amino acid sequence of SEQ ID NO: 25.

[0268] E213. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 212, comprising a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

[0269] E214. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 213, comprising a VL that contains the amino acid sequence of SEQ ID NO: 20.

[0270] E215. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 214, comprising a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0271] E216. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 215, comprising a VL that contains the amino acid sequence of SEQ ID NO: 26.

[0272] E217. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 216, comprising an Fc domain.

[0273] E218. The antibody or antigen-binding fragment thereof according to embodiment 217, wherein the Fc domain is an Fc domain of IgA (e.g., IgA 1 or IgA 2 ), IgD, IgE or IgM.

[0274] E219. The antibody or antigen-binding fragment thereof according to embodiment 217, wherein the Fc domain is an IgG (e.g., IgG 1, IgG 2 , IgG 3 or IgG 4 ) Fc domain.

[0275] E220. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 219, comprising a heavy chain having the amino acid sequence of SEQ ID NO: 32.

[0276] E221. An antibody or antigen-binding fragment thereof according to any one of embodiments 197 to 220, comprising a light chain having the amino acid sequence of SEQ ID NO: 27.

[0277] E222. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising a heavy chain variable region (VH) that contains:

[0278] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58;

[0279] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 59; and

[0280] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60.

[0281] E223. 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: 61.

[0282] E224. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising a VL that contains:

[0283] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 53;

[0284] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 54; and

[0285] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 55.

[0286] E225. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 56.

[0287] E226. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising:

[0288] (i) VH, which comprises:

[0289] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 58;

[0290] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 59; and

[0291] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 60;

[0292] and (ii) VL, which comprises:

[0293] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 53;

[0294] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 54; and

[0295] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 55.

[0296] E227. 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: 61 and the CDR-L1, CDR L3, and CDR-L3 sequences of SEQ ID NO: 56.

[0297] E228. The antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 227, comprising a human VH3, VH1, or VH5 framework sequence.

[0298] E229. The antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 228, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7.

[0299] E230. The antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 227, comprising a human VH germline consensus framework sequence.

[0300] E231. The antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 230, comprising a VH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 61.

[0301] An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 231, comprising a VH that contains the amino acid sequence of SEQ ID NO: 61.

[0302] E233. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 232, comprising a human V K or V λ framework sequence.

[0303] E234. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 233, comprising a human germline IGKV3-20 or IGKV1-39 framework sequence.

[0304] E235. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 232, comprising a human VL germline consensus framework sequence.

[0305] E236. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 235, comprising a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 56.

[0306] E237. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 236, comprising a VL that contains the amino acid sequence of SEQ ID NO: 56.

[0307] E238. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 237, comprising a CH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 20.

[0308] E239. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 238, comprising a CH that contains the amino acid sequence of SEQ ID NO: 20.

[0309] E240. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 239, comprising a CL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 26.

[0310] E241. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 240, comprising a CL that contains the amino acid sequence of SEQ ID NO: 26.

[0311] E242. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 241, comprising an Fc domain.

[0312] An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 242, wherein the Fc domain is the Fc domain of IgA (e.g., IgA 1 or IgA 2 ), IgD, IgE or IgM.

[0313] E244. An antibody or antigen-binding fragment thereof according to embodiment 242, wherein the Fc domain is the Fc domain of IgG (e.g., gG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0314] E245. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 244, which comprises an amino acid sequence containing SEQ ID NO: 62.

[0315] E246. An antibody or antigen-binding fragment thereof according to any one of embodiments 222 to 245, which comprises an amino acid sequence containing SEQ ID NO: 57.

[0316] E247. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, which comprises a VH that contains:

[0317] (a) CDR-H1 containing an amino acid sequence of SEQ ID NO: 118;

[0318] (b) CDR-H2 containing an amino acid sequence of SEQ ID NO: 119; and

[0319] (c) CDR-H3 containing an amino acid sequence of SEQ ID NO: 120.

[0320] E248. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, which comprises CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO: 121.

[0321] E249. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, which comprises a VL that contains:

[0322] (a) CDR-L1 containing an amino acid sequence of SEQ ID NO: 113;

[0323] (b) CDR-L2 containing an amino acid sequence of SEQ ID NO: 114; and

[0324] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0325] E250. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 116.

[0326] E251. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising:

[0327] (i) VH, which comprises:

[0328] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 118;

[0329] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 119; and

[0330] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 120;

[0331] and (ii) VL, which comprises:

[0332] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 113;

[0333] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 114; and

[0334] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 115.

[0335] E252. 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: 121, and the CDR-L1, CDR-L2, and CDR-L3 sequences of SEQ ID NO: 116.

[0336] E253. The antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 253, comprising a human VH3, VH1, or VH5 framework sequence.

[0337] E254. The antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 253, comprising a VH framework sequence of human germline IGHV3-23, IGHV1-69, or IGHV3-7.

[0338] An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 252, which comprises a human VH germline consensus framework sequence.

[0339] E256. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 255, which comprises a VH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 121.

[0340] E257. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 256, which comprises a VH that contains the amino acid sequence of SEQ ID NO: 121.

[0341] E258. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 257, which comprises a human V K or V λ framework sequence.

[0342] E259. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 258, which comprises a human germline IGKV3-20 or IGKV1-39 framework sequence.

[0343] E260. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 257, which comprises a human VL germline consensus framework sequence.

[0344] E261. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 260, which comprises a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 116.

[0345] E262. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 261, which comprises a VL that contains the amino acid sequence of SEQ ID NO: 116.

[0346] E263. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 262, which comprises a CH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 91.

[0347] E264. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 263, which comprises a CH that contains the amino acid sequence of SEQ ID NO: 91.

[0348] E265. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 264, which comprises a CL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 85.

[0349] An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 265, which comprises a CL, and the CL contains the amino acid sequence of SEQ ID NO: 85.

[0350] E267. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 266, which comprises an Fc domain.

[0351] E268. The antibody or antigen-binding fragment thereof according to embodiment 267, wherein the Fc domain is an Fc domain of IgA (such as IgA 1 or IgA 2 ), IgD, IgE or IgM.

[0352] E269. The antibody or antigen-binding fragment thereof according to embodiment 267, wherein the Fc domain is an Fc domain of IgG (such as gG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0353] E270. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 269, which comprises a heavy chain, and the heavy chain contains the amino acid sequence of SEQ ID NO: 122.

[0354] E271. An antibody or antigen-binding fragment thereof according to any one of embodiments 247 to 270, which comprises a light chain, and the light chain contains the amino acid sequence of SEQ ID NO: 117.

[0355] E272. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, which comprises a VH, and the VH contains:

[0356] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 128;

[0357] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 129; and

[0358] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 130.

[0359] E273. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, which comprises the CDR-H1, CDR-H2 and CDR-H3 sequences of SEQ ID NO: 131.

[0360] E274. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, which comprises a VL, and the VL contains the following groups:

[0361] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123;

[0362] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and

[0363] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

[0364] E275. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising CDR-L1, CDR L3, and CDR-L3 sequences of SEQ ID NO: 126.

[0365] E276. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising:

[0366] (i) VH, which comprises:

[0367] (a) CDR-H1 comprising the amino acid sequence of SEQ ID NO: 128;

[0368] (b) CDR-H2 comprising the amino acid sequence of SEQ ID NO: 129; and

[0369] (c) CDR-H3 comprising the amino acid sequence of SEQ ID NO: 130;

[0370] and (ii) VL, which comprises:

[0371] (a) CDR-L1 comprising the amino acid sequence of SEQ ID NO: 123;

[0372] (b) CDR-L2 comprising the amino acid sequence of SEQ ID NO: 124; and

[0373] (c) CDR-L3 comprising the amino acid sequence of SEQ ID NO: 125.

[0374] E277. An isolated antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, comprising CDR-H1, CDR-H2, and CDR-H3 sequences of SEQ ID NO: 131 and CDR-L1, CDR L3, and CDR-L3 sequences of SEQ ID NO: 126. E278. The antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 277, comprising a human VH3, VH1, or VH5 framework sequence.

[0375] An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 278, comprising a VH framework sequence of human IGHV3-23, IGHV1-69 or IGHV3-7.

[0376] E280. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 277, comprising a human VH germline consensus framework sequence.

[0377] E281. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 280, comprising a VH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 131.

[0378] E282. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 281, comprising a VH that contains the amino acid sequence of SEQ ID NO: 131.

[0379] E283. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 282, comprising a human V K or V λ framework sequence.

[0380] E284. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 283, comprising a human germline IGKV3-20 or IGKV1-39 framework sequence.

[0381] E285. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 282, comprising a human VL germline consensus framework sequence.

[0382] E286. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 285, comprising a VL that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 126.

[0383] E287. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 286, comprising a VL that contains the amino acid sequence of SEQ ID NO: 126.

[0384] E288. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 287, comprising a CH that contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 91.

[0385] E289. An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 288, comprising a CH that contains the amino acid sequence of SEQ ID NO: 91.

[0386] An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 289, which comprises a CL, and the CL contains an amino acid sequence that is at least 90% identical to SEQ ID NO: 85.

[0387] An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 290, which comprises a CL, and the CL contains the amino acid sequence of SEQ ID NO: 85.

[0388] An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 291, which comprises an Fc domain.

[0389] An antibody or antigen-binding fragment thereof according to embodiment 292, wherein the Fc domain is the Fc domain of IgA (such as IgA 1 or IgA 2 ), IgD, IgE or IgM.

[0390] An antibody or antigen-binding fragment thereof according to embodiment 292, wherein the Fc domain is the Fc domain of IgG (such as IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0391] An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 294, which comprises a heavy chain, and the heavy chain contains the amino acid sequence of SEQ ID NO: 132.

[0392] An antibody or antigen-binding fragment thereof according to any one of embodiments 272 to 295, which comprises a light chain, and the light chain contains the amino acid sequence of SEQ ID NO: 127.

[0393] An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment competes with the antibody or antigen-binding fragment according to any one of embodiments 1 to 296 for binding to TFPI.

[0394] E298. An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment thereof competes with antibodies selected from the following group for binding to TFPI: 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 chimeric, 4D8-Vk1.0 x VH1.0, 4D8-Vk1.0 x VH1.1, 4D8-Vk1.0 x VH1.2, 4D8-Vk1.0 x VH1.3, 4D8-Vk1.0 x VH1.4, 4D8-Vk1.0 x VH1.5, 4D8-Vk1.0 x VH1.6, 4D8-Vk1.1 x VH1.0, 4D8-Vk1.1 x VH1.1, 4D8-Vk1.1 x VH1.2, 4D8-Vk1.1x VH1.3, 4D8-Vk1.1x VH1.4, 4D8-Vk1.1 x VH1.5, 4D8-Vk1.1 x VH1.6, hz4D8, 6B7.c5, and 7A4.D9.

[0395] E299. An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment thereof competes with antibodies selected from the following group for binding to TFPI: TFPI-23, TFPI-24, TFPI-106, and TFPI-118.

[0396] E300. The antibody or antigen-binding fragment thereof according to embodiment 299, wherein the antibody or antigen-binding fragment thereof competes with TFPI-23 or TFPI-106 for binding to TFPI.

[0397] E301. The antibody or antigen-binding fragment thereof according to embodiment 299, wherein the antibody or antigen-binding fragment thereof competes with TFPI-24 or TFPI-118 for binding to TFPI.

[0398] E302. An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment thereof competes with the antibody 4D8 for binding to TFPI.

[0399] E303. An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment thereof binds to the same TFPI epitope as the antibody or antigen-binding fragment thereof of any one of Embodiments 1 to 296.

[0400] E304. An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment thereof binds to the same TFPI epitope as the antibodies selected from the group consisting of: 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 chimeric, 4D8-Vk1.0 x VH1.0, 4D8-Vk1.0 x VH1.1, 4D8-Vk1.0 x VH1.2, 4D8-Vk1.0 x VH1.3, 4D8-Vk1.0 x VH1.4, 4D8-Vk1.0 x VH1.5, 4D8-Vk1.0 x VH1.6, 4D8-Vk1.1 x VH1.0, 4D8-Vk1.1 x VH1.1, 4D8-Vk1.1 x VH1.2, 4D8-Vk1.1 x VH1.3, 4D8-Vk1.1 x VH1.4, 4D8-Vk1.1 x VH1.5, 4D8-Vk1.1 x VH1.6, hz4D8, 6B7.c5 and 7A4.D9.

[0401] E305. An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment thereof binds to the same TFPI epitope as the antibodies selected from the group consisting of: TFPI-23, TFPI-24, TFPI-106 and TFPI-118.

[0402] E306. The antibody or antigen-binding fragment thereof of Embodiment 305, wherein the antibody or antigen-binding fragment thereof binds to the same TFPI epitope as TFPI-23 or TFPI-106.

[0403] E307. The antibody or antigen-binding fragment thereof of Embodiment 305, wherein the antibody or antigen-binding fragment thereof binds to the same TFPI epitope as TFPI-24 or TFPI-118.

[0404] E308. An antibody or antigen-binding fragment thereof that specifically binds to the K2 domain of TFPI, wherein the antibody or antigen-binding fragment thereof binds to the same TFPI epitope as antibody 4D8.

[0405] E309. The antibody or antigen-binding fragment thereof according to any one of embodiments 297 to 308, wherein the antibody or antigen-binding fragment thereof does not bind to the K1 domain of TFPI.

[0406] E310. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 309, wherein the antibody or antigen-binding fragment thereof is an Fc fusion protein, a monobody, a maxibody, a bispecific antibody, an scFab, an scFv, a peptibody, or any antigen-binding fragment of the foregoing.

[0407] E311. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 310, wherein the antibody or antigen-binding fragment thereof binds to TFPI with a binding affinity (Kd) value of about 1×10 -7 M to about 1×10 -12 M.

[0408] E312. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 311, wherein the antibody or antigen-binding fragment thereof binds to TFPI with a binding affinity (Kd) value of about 5×10 -7 M to about 5×10 -11 M.

[0409] E313. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 312, wherein the antibody or antigen-binding fragment thereof binds to TFPI with a binding affinity (Kd) value of about 1×10 -8 M to about 1×10 -10 M.

[0410] E314. The antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 313, wherein the antibody or antigen-binding fragment thereof: (i) reduces the clotting time measured in a plasma-based diluted prothrombin time (dPT) assay; (ii) reduces the whole blood clotting time measured by thromboelastrography or thromboelastometry; (iii) increases thrombin generation; (iv) increases FXa activity in the presence of TFPI; or (v) any combination thereof.

[0411] E315. The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the antibody or antigen-binding fragment thereof reduces the clotting time measured in a plasma-based diluted prothrombin time assay.

[0412] An antibody or antigen-binding fragment thereof according to embodiment 315, wherein the reduction in clotting time measured in a plasma-based diluted prothrombin time assay is dose-dependent.

[0413] An antibody or antigen-binding fragment thereof according to embodiment 314, wherein the antibody or antigen-binding fragment thereof reduces whole blood clotting time measured by thromboelastography or rotational thromboelastometry.

[0414] An antibody or antigen-binding fragment thereof according to embodiment 317, wherein the reduction in whole blood clotting time measured by thromboelastography or rotational thromboelastometry is dose-dependent.

[0415] An antibody or antigen-binding fragment thereof according to embodiment 314, wherein the antibody or antigen-binding fragment increases thrombin generation.

[0416] An antibody or antigen-binding fragment thereof according to embodiment 319, wherein the increase in thrombin generation is dose-dependent.

[0417] An antibody or antigen-binding fragment thereof according to embodiment 314, wherein the antibody or antigen-binding fragment increases FXa activity in the presence of TFPI.

[0418] An antibody or antigen-binding fragment thereof according to embodiment 321, wherein the increase in FXa activity in the presence of TFPI is dose-dependent.

[0419] An antibody or antigen-binding fragment thereof according to embodiment 322, wherein the antibody enhances platelet accumulation in the presence of TFPI.

[0420] An antibody or antigen-binding fragment thereof according to embodiment 323, wherein the enhancement of platelet accumulation in the presence of TFPI is dose-dependent.

[0421] An antibody or antigen-binding fragment thereof according to embodiment 324, wherein the antibody increases fibrin formation in the presence of TFPI.

[0422] An antibody or antigen-binding fragment thereof according to embodiment 325, wherein the increase in fibrin formation in the presence of TFPI is dose-dependent.

[0423] An antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in whole blood clotting time is measured using whole blood obtained from a human patient with severe hemophilia A.

[0424] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in whole blood clotting time is measured using whole blood obtained from a human patient with severe hemophilia A and an inhibitory antibody against human factor VIII.

[0425] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in whole blood clotting time is measured using whole blood obtained from a human patient with moderate hemophilia A.

[0426] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in whole blood clotting time is measured using whole blood obtained from a human patient with severe hemophilia B.

[0427] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in whole blood clotting time is measured using whole blood obtained from a human patient with severe hemophilia B and an inhibitory antibody against human factor IX.

[0428] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in whole blood clotting time is measured using whole blood obtained from a human patient with moderate hemophilia B.

[0429] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in clotting time measured in the dPT assay is measured using plasma obtained from a human patient with severe hemophilia A.

[0430] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in clotting time measured in the dPT assay is measured using plasma obtained from a human patient with severe hemophilia A and an inhibitory antibody against human factor VIII.

[0431] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in clotting time measured in the dPT assay is measured using plasma obtained from a human patient with moderate hemophilia A.

[0432] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in clotting time measured in the dPT assay is measured using plasma obtained from a human patient with severe hemophilia B.

[0433] The antibody or antigen-binding fragment thereof according to embodiment 314, wherein the reduction in clotting time measured in the dPT assay is measured using plasma obtained from a human patient with severe hemophilia B and an inhibitory antibody against human factor IX.

[0434] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E338, wherein the reduction in clotting time measured in the dPT assay is determined using plasma obtained from a human patient with moderate hemophilia B.

[0435] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E339, wherein the increase in thrombin generation is determined using plasma obtained from a human patient with severe hemophilia A.

[0436] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E340, wherein the increase in thrombin generation is determined using plasma obtained from a human patient with severe hemophilia A and an inhibitory antibody against human factor VIII.

[0437] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E341, wherein the increase in thrombin generation is determined using plasma obtained from a human patient with moderate hemophilia A.

[0438] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E342, wherein the increase in thrombin generation is determined using plasma obtained from a human patient with severe hemophilia B.

[0439] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E343, wherein the increase in thrombin generation is determined using plasma obtained from a human patient with severe hemophilia B and an inhibitory antibody against human factor IX.

[0440] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E344, wherein the increase in thrombin generation is determined using plasma obtained from a human patient with moderate hemophilia.

[0441] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E345, wherein 100 nM of the antibody or antigen-binding fragment thereof is at least as effective as the amount of recombinant human factor VIII sufficient to achieve 5% normal clotting activity in reducing the clotting time of whole blood obtained from a human patient with severe hemophilia A.

[0442] The antibody or antigen-binding fragment thereof according to item 314 of Embodiment E346, wherein 100 nM of the antibody or antigen-binding fragment thereof is at least as effective as the amount of recombinant human factor VIII sufficient to achieve 5% normal clotting activity in increasing the peak thrombin generation of platelet-rich plasma obtained from a human patient with severe hemophilia A.

[0443] The antibody or antigen-binding fragment thereof according to any one of Embodiments 1 to 346, wherein the TFPI is human TFPI.

[0444] An antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 347, wherein the TFPI comprises residues 91 to 147 of SEQ ID NO: 2.

[0445] An isolated nucleic acid molecule or class of nucleic acid molecules comprising one or more nucleotide sequences encoding an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 348.

[0446] An isolated nucleic acid molecule encoding an antibody or antigen-binding fragment thereof that specifically binds TFPI, wherein the nucleic acid comprises a nucleic acid sequence selected from the group consisting of: the nucleic acid sequence of SEQ ID NO: 175, the nucleic acid sequence of SEQ ID NO: 176, the nucleic acid sequence of SEQ ID NO: 177, the nucleic acid sequence of SEQ ID NO: 178, the insert nucleic acid sequence of the mAb-TFPI-106VL vector deposited under ATCC accession number PTA-122328, and the insert nucleic acid sequence of the mAb-TFPI-106VH vector deposited under ATCC accession number PTA-122329.

[0447] A vector comprising the nucleic acid molecules of embodiments 349 and 350.

[0448] A host cell comprising the nucleic acid molecule of embodiment 349 or 350 or the vector of embodiment 351.

[0449] The host cell of embodiment 352, wherein the cell is a mammalian cell.

[0450] The host cell of embodiment 353, wherein the host cell is a CHO cell, a HEK-293 cell, or an Sp2.0 cell.

[0451] A method for preparing an antibody or 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 thereof is expressed by the host cell.

[0452] The method of embodiment 355, further comprising isolating the antibody or antigen-binding fragment thereof.

[0453] An antibody or antigen-binding fragment thereof obtained by the method of embodiment 355 or 356.

[0454] A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of embodiments 1 to 347 and 357 and a pharmaceutically acceptable carrier or excipient.

[0455] E359. A method of reducing the activity of tissue factor pathway inhibitor (TFPI), comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 347 and 357, or a pharmaceutical composition according to embodiment 358.

[0456] E360. A method of shortening bleeding time, comprising administering to a subject in need thereof a therapeutically effective amount of an antibody or an antigen-binding fragment thereof according to any one of embodiments 1 to 347 and 357, or a pharmaceutical composition according to embodiment 358.

[0457] E361. The method according to embodiment 358 or 360, wherein the subject is human.

[0458] E362. The method according to any one of embodiments 359 to 361, wherein the subject has or is susceptible to a blood coagulation deficiency.

[0459] E363. The method according to any one of embodiments 359 to 361, wherein the subject has or is susceptible to platelet abnormalities.

[0460] 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.

[0461] E365. The method according to any one of embodiments 359 to 361, wherein the subject has or is susceptible to hemophilia A or B.

[0462] E366. The method according to any one of embodiments 359 to 361, wherein the subject has or is susceptible to von Willebrand disease (vWD).

[0463] E367. The method according to embodiment 360, further comprising administering a therapeutically effective amount of FVII.

[0464] E368. The method according to embodiment 367, wherein the method increases thrombin generation in the presence of TFPI.

[0465] E369. The method according to any one of embodiments 359 to 368, comprising administering the antibody or an antigen-binding fragment thereof or the pharmaceutical composition intravenously.

[0466] E370. The method according to any one of embodiments 359 to 368, comprising administering the antibody or an antigen-binding fragment thereof or the pharmaceutical composition subcutaneously.

[0467] The method of any one of embodiments 359 to 368, wherein the antibody or its antigen-binding fragment or the 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.

[0468] The antibody or its antigen-binding fragment of any one of embodiments 1 to 347 and 357 or the pharmaceutical composition of embodiment 358, for use as a medicament.

[0469] The antibody or its antigen-binding fragment of any one of embodiments 1 to 347 and 357 or the pharmaceutical composition of embodiment 358 is for reducing the TFPI activity in a subject.

[0470] The antibody or its antigen-binding fragment of any one of embodiments 1 to 347 and 357 or the pharmaceutical composition of embodiment 358 is for shortening the bleeding time in a subject.

[0471] The antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiments 372 to 374, wherein the subject is human.

[0472] The antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiments 372 to 375, wherein the subject has or is susceptible to a blood coagulation deficiency.

[0473] The antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiments 372 to 375, wherein the subject has or is susceptible to hemophilia A, B or C.

[0474] The antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiments 372 to 375, wherein the subject has or is susceptible to hemophilia A or B.

[0475] The antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiments 372 to 375, wherein the subject has or is susceptible to von Willebrand disease (vWD).

[0476] The antibody or antigen-binding fragment or pharmaceutical composition of any one of embodiments 372 to 375, wherein the subject has or is susceptible to platelet abnormalities.

[0477] Use of an antibody or its antigen-binding fragment of any one of embodiments 1 to 348 and 357 or a pharmaceutical composition of embodiment 358 for reducing the TFPI activity in a subject.

[0478] Use of an antibody according to any one of embodiments 1 to 348 and 357, or an antigen-binding fragment thereof, or a pharmaceutical composition according to embodiment 358, in the preparation of a medicament for reducing TFPI activity in a subject.

[0479] E383. Use of an antibody according to any one of embodiments 1 to 348 and 357, or an antigen-binding fragment thereof, or a pharmaceutical composition according to embodiment 358, in shortening the bleeding time in a subject.

[0480] E384. Use of an antibody according to any one of embodiments 1 to 348 and 357, or an antigen-binding fragment thereof, or a pharmaceutical composition according to embodiment 358, in the preparation of a medicament for shortening the bleeding time in a subject.

[0481] E385. Use according to any one of embodiments 381 to 384, wherein the subject is human.

[0482] E386. Use according to any one of embodiments 381 to 384, wherein the subject has or is prone to having a blood coagulation deficiency.

[0483] E387. Use according to any one of embodiments 381 to 384, wherein the subject has or is prone to having hemophilia A, B or C.

[0484] E388. Use according to any one of embodiments 381 to 384, wherein the subject has or is prone to having hemophilia A or B.

[0485] E389. Use according to any one of embodiments 381 to 384, wherein the subject has or is prone to having von Willebrand disease (vWD).

[0486] E390. Use according to any one of embodiments 381 to 384, wherein the subject has or is prone to having platelet abnormalities. BRIEF DESCRIPTION OF THE DRAWINGS

[0487] Figures 1A to 1F The figure shows the cocrystal structures of various anti-TFPI antibodies and the K2 domain of TFPI. Specifically, as Figure 1F shown, compared with other reference antibodies, the exemplary antibodies TFPI-23, TFPI-24 and 4D8 disclosed in the present invention all bind to non-overlapping epitopes of the K2 domain. 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 Mab 2974 from R&D Systems. The Novo2021 antibody is also referred to as "hz4F36". "Clone 23" refers to TFPI-23; "clone 24" refers to TFPI-24.

[0488] The diagrams of FIGS. 2A-2E show the interactions between the epitope residues within the K2 domain of TFPI and the antigenic complementarity determining region residues from various anti-TFPI antibodies. "R&D" or "R&D Fab" refers to antibody Mab 2974 from R&D Systems. "Clone 23" refers to TFPI-23; "Clone 24" refers to TFPI-24.

[0489] FIGS. 3A-3E show the in vivo potencies of various anti-TFPI antibodies in a murine injury model. FIGS. 3A and 3B show the duration of reduced bleeding in factor VIII-deficient (FVIII- / -) hemophilia A mice when administered 2A8-200 and 2A8 antibodies (used as reference antibodies in this study). FIG. 3C shows the duration of the antibody effects in hemophilia A mice when administered TFPI 4D8 (control), TFPI-21, TFPI-23, and TFPI-24 antibodies. FIGS. 3D and 3E show the duration in hemophilia A mice when administered TFPI-106 and TFPI-118 antibodies. At the indicated time points (hours (h)) prior to injury, antibodies were administered intravenously at 6 mg / kg to hemophilia A mice (FVIII- / -). Then, after tail transection, the total volume of blood loss (μL) was measured. Vehicle (saline)-treated hemophilia A mice served as controls. All measurements are presented as mean ± SEM. * = P < 0.05. FVIII+ / + (wild-type) mice received saline. n = 5 / group.

[0490] Figure 4 The figure shows the bleeding duration in hemophilia B mice after tail transection when administered TFPI-106 antibody. At the indicated time points (hours (h)) prior to injury, antibodies were administered intravenously at 6 mg / kg to hemophilia B mice. Then, after tail transection, the total volume of blood loss (μL) was measured. Vehicle (saline)-treated hemophilia A mice served as controls. All measurements are presented as mean ± SEM. * = P < 0.05. n = 4-5 / group.

[0491] Figure 5 contains Group A and Group B, each containing six sets (Figure 5A1 to 6 and Figure 5B1 to 6), showing micrographs of intravital microscopy (IVM), demonstrating that TFPI was detected in platelet thrombi at the site of vascular injury and along the endothelium in wild-type mice. Figure 5A shows an increase in platelet thrombi at the injury site detected using CD42c labeled with Dylight 649 that binds to GP1bβ on platelets. The presence of platelets was demonstrated by the fluorescence signals detected in Group 1 (0 seconds); Group 2 (15 seconds); Group 3 (30 seconds); Group 4 (60 seconds); Group 5 (90 seconds); and Group 6 (120 seconds). Alexa 488-labeled negative control IgG was also administered and no fluorescence was detected. Figure 5B containing Groups 1 to 6 shows micrographs of IVM, which demonstrate the presence of platelet thrombi and TFPI along the endothelium in wild-type mice after laser-induced vascular injury. Alexa 488-labeled TFPI (green signal shown in gray) was not detected at 0 seconds (Figure 5B, Group 1), while a faint signal could be seen at 15 seconds (Figure 5B, Group 2). At 30 seconds (Figure 5B, Group 3), the green fluorescence signal had increased and a faint red signal (Dylight 649-labeled CD42c) could be seen, indicating platelet accumulation detected at approximately the same site where TFPI was detected. Figure 5B Group 4 (60 seconds) shows strong green and red fluorescence signals (both light gray, where the red fluorescence can be seen on the left side of the vascular injury site, while the green signal is mainly detected on the right side towards the injury site), demonstrating platelet accumulation and TFPI detected at the injury site. Figure 5B Group 5 shows red (platelet) and green (TFPI) fluorescence signals at the injury site at 60 seconds, where both signals are stronger than those at 30 seconds. Figure 5B Group 6 shows a decrease in the red (platelet) signal and green (TFPI) signal, and both can still be detected at the injury site at 120 seconds.

[0492] The figure of Figure 6A shows the hemostatic effect of TFPI-106 after laser-induced vascular injury in hemophilia A mice evaluated using IVM, where the amount of platelet thrombi is represented by the area under the curve (AUC) (* = P < 0.005 is indicated). Figure 6A shows platelet accumulation at the injury site in wild-type mice (WT) at 0.5 hours after injury compared to a lack of platelet accumulation in hemophilia A mice administered saline at 0.5 hours. Platelet accumulation was detected in hemophilia A mice administered recombinant factor VIII (rFVIII) or TFPI-106 at 0.5 hours. Platelet thrombus accumulation could still be detected in hemophilia A mice administered TFPI-106 at 168 hours.

[0493] The figure in Figure 6B shows the hemostatic effect of TFPI-106 after laser-induced vascular injury in hemophilia A mice using IVM, where the fibrin generation amount is expressed as the area under the curve (AUC) (* = P < 0.005 was indicated). Figure 6B shows that there was no detectable fibrin generation in hemophilia A mice administered saline at 0.5 hours, and fibrin generation at the injury site in wild-type mice (WT) receiving only saline control at 0.5 hours after injury. Among them, fibrin generation was detectable in hemophilia A mice administered recombinant factor VIII (rFVIII) or TFPI-106 at 0.5 hours. Fibrin generation was still detectable in hemophilia A mice administered TFPI-106 at 168 hours.

[0494] Figure 7 The depicted figure shows the effect of TFPI-106 and recombinant factor VIIa (rFVIIa) on the thrombin generation assay (TGA) in severe hemophilia A plasma in the presence of 1 pM tissue factor and 4 μM phospholipids. The figure shows the thrombin generation plots of hemophilia A plasma with TFPI-106 alone (16 μg / ml) and in combination with rFVIIa (20 μg / ml; 2 μg / ml; or 0.2 μg / ml). The thrombin generation plots of hemophilia A plasma without either TFPI-106 or rFVIIa and non-hemophilia plasma are also shown in the figure.

[0495] Figure 8A The depicted thrombin generation plot shows the effect of TFPI-106 with or without rFVIIa, or rFVIIa alone on thrombin generation in hemophilia A plasma in the presence of 1 pM tissue factor and 4 μM phospholipids. Non-hemophilia plasma is included as a control.

[0496] Figure 8B The depicted thrombin generation plot shows the effect of TFPI-106 with or without rFVIIa, or rFVIIa alone on thrombin generation in citrated platelet-poor hemophilia A plasma in the presence of three Bethesda units (3BU) of inhibitor. Non-hemophilia plasma is included as a control. The figure also includes a non-hemophilia plasma control with TFPI-106 (16 μg / ml) added.

[0497] Figure 8CThe depicted thrombin generation graphs show the effect of TFPI-106 with or without rFVIIa, or rFVIIa alone, on thrombin generation in citrated platelet-poor hemophilia B plasma in the presence of an inhibitor at three Bethesda units (3BU). Non-hemophilic plasma was included as a control. The figure also includes a non-hemophilic plasma control to which TFPI-106 (16 μg / ml) was added.

[0498] Figure 9A shows the effect of administering antibody TFPI-106 (6 mg / kg) and separate recombinant factor VIII (200 units / kg) to hemophilia A mice immediately after tail transection on blood loss, compared to the control. Figure 9B shows the effect of administering antibody TFPI-106 (6 mg / kg) and separate recombinant factor VIII (200 units / kg) to hemophilia A mice 2 minutes after tail transection on blood loss, compared to the control group.

[0499] Figure 10A shows the effect of different concentrations of TFPI 106 on the clotting time of whole blood from human patients with severe hemophilia A, compared to recombinant factor VIII. Figure 10B shows the effect of different concentrations of TFPI 106 on the peak thrombin generation of platelet-rich plasma from human patients with severe hemophilia A, compared to recombinant factor VIII.

[0500] Figure 11A shows the effect of different concentrations of TFPI 106 on the clotting time of whole blood from human patients with severe hemophilia A and an inhibitor against FVIII. Figure 11B shows the effect of different concentrations of TFPI 106 on the peak thrombin generation of platelet-rich plasma from human patients with severe hemophilia A and an inhibitor against FVIII. Figure 11C Shows the effect of different concentrations of TFPI 106 on the clotting time of platelet-poor plasma from human patients with severe hemophilia A and an inhibitor against FVIII.

[0501] Figure 12A shows the effect of different concentrations of TFPI 106 on the clotting time of whole blood from human patients with moderate hemophilia A, compared to recombinant factor VIII. Figure 12B shows the effect of different concentrations of TFPI 106 on the peak thrombin generation of platelet-rich plasma from human patients with moderate hemophilia A, compared to recombinant factor VIII. Figure 12C Shows the effect of different concentrations of TFPI 106 on the clotting time of platelet-poor plasma from human patients with moderate hemophilia A.

[0502] Figure 13A shows the effect of different concentrations of TFPI 106 on the whole blood clotting time from human patients with moderate hemophilia B compared to recombinant factor IX. Figure 13B shows the effect of different concentrations of TFPI 106 on the peak thrombin generation in platelet-rich plasma from human patients with moderate hemophilia B compared to recombinant factor IX. Figure 13C Shows the effect of different concentrations of TFPI 106 on the clotting time of platelet-poor plasma from human patients with moderate hemophilia B.

[0503] Figure 14A shows the effect of different concentrations of TFPI 106 on the whole blood clotting time from multiple human patients with hemophilia A compared to recombinant factor VIII. Figure 14B shows the effect of different concentrations of TFPI 106 on the peak thrombin generation in platelet-rich plasma from multiple human patients with hemophilia A compared to recombinant factor VIII. Figure 14C Shows the effect of different concentrations of TFPI 106 on the clotting time of platelet-poor plasma from multiple human patients with hemophilia B. DETAILED DESCRIPTION OF THE INVENTION

[0504] 1. GENERAL OVERVIEW

[0505] As indicated above, hemophilia patients have some hemostatic ability through their intact extrinsic pathway; however, this extrinsic pathway is not sufficient to provide protection as it is rapidly shut down by tissue factor pathway inhibitor (TFPI). Blocking / neutralizing TFPI inhibition in these patients can compensate for insufficient FXa generation and normalize the bleeding diathesis. Accordingly, the present invention discloses and exemplifies antibodies and antigen-binding fragments thereof that specifically bind TFPI and inhibit its activity.

[0506] 2. DEFINITIONS

[0507] "Reducing the activity of TFPI" means that the antibody or its antigen-binding fragment can: (i) reduce the clotting time as measured, for example, by a plasma-based diluted prothrombin time assay, compared to the clotting time in the absence of the antibody; (ii) reduce the whole blood clotting time as detected, for example, by thromboelastography or rotational thromboelastometry, compared to the clotting time in the absence of the antibody; (iii) increase thrombin generation; (iv) increase FXa activity in the presence of TFPI; (v) enhance platelet accumulation in the presence of TFPI; (vi) increase fibrin generation in the presence of TFPI; or (vii) any combination thereof. The inhibitory activity of the antibody or antigen-binding fragment can, but need not necessarily be, dose-dependent (e.g., causing a dose-dependent reduction in clotting time as measured in a plasma-based diluted prothrombin time assay).

[0508] In addition, as disclosed and exemplified herein, a co-crystal structure of an anti-TFPI antibody and the Kunitz domain 2 (K2 domain) of TFPI was obtained. Structural analysis showed that the exemplary antibodies of the present invention recognize a unique epitope of TFPI compared to other disclosed TFPI antibodies (which were used as reference antibodies in the examples). For example, as Figures 1A to 1F shown in FIGS. 2A-2E, compared to several reference TFPI antibodies (R&D (Mab2479) Fab, Novo2021 (also known as hz4F36) Fab, 2A8 Fab), the TFPI-23, TFPI-24, and 4D8 antibodies bind to non-overlapping sites in the K2 domain of TFPI.

[0509] Accordingly, 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 this co-crystal structure and computational alanine scanning, this epitope contains the following three residues important for antibody-antigen interaction: Ile105, Arg107, and Leu131 (numbered according to human TFPI as shown in SEQ ID NO: 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) both recognize this epitope.

[0510] In certain embodiments, recognition of the key epitope residues disclosed herein allows the antibody (and its antigen-binding fragments) to reduce the activity of TFPI. In particular, the crystal structure shows that the K2 domain of TFPI adopts a conical structure, and the tip of the cone (especially Arg107) binds to FXa. Both TFPI-23 and TFPI-24 recognize the tip of this conical region and block the binding of TFPI to FXa. The antibody 4D8 recognizes a different epitope in the K2 domain. Although it does not directly interact with the residues at the tip of the cone, 4D8 blocks the binding of TFPI to FXa. Table 15 summarizes the non-overlapping epitope residues recognized by the exemplary antibodies of the present invention when compared to other well-known TFPI antibodies.

[0511] In addition, in certain embodiments, the antibodies and antigen-binding fragments disclosed herein have shown desirable pharmacological activities and pharmacokinetic properties for treating coagulation defects (e.g., hemophilia) and for shortening bleeding time.

[0512] Antibodies that "preferably bind" or "specifically bind" (used interchangeably in the present invention) to an epitope are terms well known in the art, and methods for determining such specific or preferred binding are also well known in the art. A molecule is said to exhibit "specific binding" or "preferred binding" if it reacts or binds more frequently and more rapidly to a particular cell or substance with a longer duration and / or greater affinity as compared to reacting or binding to alternative cells or substances. An antibody "specifically binds" or "preferably binds" to a target if it binds to the target with greater affinity, avidity, more readily, and / or for a longer duration as compared to its binding to other substances. In addition, an antibody "specifically binds" or "preferably binds" to a target in a sample if it binds to the target in the sample with greater affinity, avidity, more readily, and / or for a longer duration as compared to its binding to other substances present in the sample. For example, an antibody that specifically binds or preferably binds to a TFPI epitope is an antibody that binds to that epitope with greater affinity, avidity, more readily, and / or for a longer duration as compared to its binding to other TFPI epitopes or non-TFPI epitopes. It can also be understood from this definition that, for example, an antibody (or portion or epitope) that "specifically binds" or "preferably binds" to a first target may or may not specifically bind or preferably bind to a second target. Thus, "specific binding" or "preferred binding" does not necessarily require (although it can include) exclusive binding. Generally, but not necessarily, reference to binding means preferred binding. "Specific binding" or "preferred binding" includes compounds such as proteins, nucleic acids, antibodies, etc. that can recognize and bind to a specific molecule in a sample but that essentially do not recognize or bind other molecules. For example, an antibody or peptide receptor that recognizes and binds to a cognate ligand or binding partner in a sample (such as an anti-TFPI antibody that binds TFPI) but that essentially does not recognize or bind other molecules in the sample specifically binds to that cognate ligand or binding partner. Thus, under specified assay conditions, the particular binding moiety (such as an antibody or its antigen-binding portion or a receptor or its ligand-binding portion) preferably binds to a specific target molecule and does not bind to other components present in the test sample in significant amounts.

[0513] A variety of assay formats can be used to select antibodies or peptides that specifically bind to a molecule of interest. For example, solid-phase ELISA immunoassays, immunoprecipitation, Biacore TM (GE Healthcare, Piscataway, NJ), KinExA, fluorescence-activated cell sorting (FACS), Octet TM(FortéBio, Inc, Menlo Park, CA) and Western blot analysis are among the various assays that can be used to identify antibodies that specifically react with an antigen or a receptor or a ligand-binding portion thereof that specifically binds to a cognate ligand or binding partner. Typically, the specific or selective reaction will be at least twice the background signal or noise, more typically more than 10-fold above background, more typically more than 50-fold above background, more typically more than 100-fold above background, more typically more than 500-fold above background, and even more typically more than 1000-fold above background, and even more typically more than 10000-fold above background. In addition, when the equilibrium dissociation constant (K D ) ≤ 7 nM, the antibody is said to “specifically bind” the antigen.

[0514] In the present invention, the term “binding affinity” is used as a measure of the strength of the non-covalent interaction between two molecules, such as an antibody or a fragment thereof and an antigen. The term “binding affinity” is used to describe a monovalent interaction (intrinsic activity).

[0515] By virtue of the monovalent interaction, the binding affinity between two molecules, such as an antibody or a fragment thereof and an antigen, can be quantified by determining the dissociation constant (K D ). In turn, K D can be determined by measuring the kinetics of complex formation and dissociation, for example, using surface plasmon resonance (SPR) methods (Biacore). The rate constants corresponding to the binding and dissociation of the monovalent complex are referred to as the association rate constant k a (or k on ) and the dissociation rate constant k d (or k off ), respectively. K D is related to k D and k d by the equation K a = k a / k d . The value of the dissociation constant can be directly determined by known methods and can even be calculated for a complex mixture by methods such as those shown in Caceci et al. (1984, Byte 9: 340-362). For example, K DIt can be established using assays such as the dual-filter nitrocellulose filter binding assay 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 against target antigens) are known in the art, including, for example, ELISA, Western blotting, RIA, and flow cytometry analysis, and the assays exemplified elsewhere in the present invention. The binding kinetics and binding affinity of antibodies can also be evaluated by standard assays known in the art, such as surface plasmon resonance (SPR) (e.g., using a Biacore TM system or KinExA).

[0516] Competitive binding assays can be performed, in which the binding of an antibody to an antigen is compared to the binding of a target to another ligand of that target (such as another antibody or a soluble receptor that binds the target in another way). The concentration at which 50% inhibition occurs is called K i . Under ideal conditions, this K i is equal to K D . This K i value will never be less than K D , so the measurement of K i can be conveniently replaced by the provided upper limit of K D .

[0517] According to the above definition, the binding affinity associated with interactions with different molecules (e.g., comparing the binding affinities of different antibodies for a specified antigen) can be compared by comparing the K D values of individual antibody / antigen complexes. The K D values of antibodies or other binding partners can be determined using well-established methods in the art. One method for determining the K D value is to use surface plasmon resonance (usually using a biosensor system such as system).

[0518] Similarly, the specificity of an interaction can be evaluated by determining the K D value of the interaction of interest (e.g., the specific interaction between an antibody and an antigen) and comparing it to the K D value of an interaction not of interest (e.g., a control antibody known not to bind TFPI).

[0519] Antibodies that specifically bind their targets can bind their targets with high affinity (i.e., exhibit a low K D as discussed above) and bind other non-target molecules with lower affinity. For example, an antibody can bind at 1×10 -6 M or higher (preferably 1×10 -5 M or higher, more preferably 1×10-4 M or higher, more preferably 1×10 -3 M or higher, more preferably 1×10 -2 M or higher) of K D value binds to non-target molecules. Preferably, the antibodies of the present invention can bind to their targets with an affinity that is at least 2-fold, 10-fold, 50-fold, 100-fold, 200-fold, 500-fold, 1,000-fold or 10,000-fold or higher than their binding affinity to another non-TFPI molecule.

[0520] Generally, TFPI antibodies need to bind to TFPI with high affinity to effectively reduce the activity of TFPI. However, when the binding affinity of the antibody is too high, the antibody can be quickly internalized and degraded by host cells. This can potentially lead to a short half-life and repeated injections. For example, antibody TFPI-23 shows a lower binding affinity (Kd) than TFPI-24, and in some cases, it seems more favorable for clinical use because it has a lower internalization rate and a longer half-life. Therefore, a binding affinity (Kd) in the range of 5×10 -7 to about 5×10 -11 M, especially about 1×10 -8 to about 1×10 -10 M is generally desirable, especially for treating chronic diseases (such as hemophilia) that require repeated injections. Without wishing to be bound by any particular theory, this affinity range is considered to achieve a balance between (i) the binding affinity required to effectively inhibit the activity of TFPI and (ii) a longer half-life and reduced antibody internalization.

[0521] The specific amino acid residue positions in TFPI are numbered according to SEQ ID NO: 2 (human TFPIα K1K2K3). However, the present invention is not limited to SEQ ID NO: 2. The corresponding residues from other TFPI homologs, isotypes, variants or fragments can be identified based on sequence alignments or structural alignments known in the art. For example, the alignment can be done manually or using well-known sequence alignment programs such as ClustalW2 or "BLAST 2 Sequences" with default parameters. For example, Arg107 of SEQ ID NO: 2 corresponds to Arg104 of mouse TFPI K1K2 (SEQ ID NO: 4).

[0522] An "antigen-binding fragment" of an antibody refers to a fragment of a full-length antibody that retains the ability to specifically bind an antigen (preferably having 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') 2Fragments, including: (i) a divalent fragment containing two Fab fragments bridged by a disulfide bond in the hinge region; (iii) an Fd fragment consisting of the VH and CH1 domains; (iv) an Fv fragment consisting of the VL and VH domains of a single arm of an antibody; (v) a dAb fragment consisting of a VH domain (Ward et al., (1989) Nature 341:544-546); and (vi) isolated complementarity determining regions (CDRs), disulfide-linked Fvs (dsFvs), and anti-idiotypic (anti-Id) antibodies and intrabody. In addition, although the two domains of the Fv fragment, VL and VH, are encoded by separate genes, they can be joined using recombinant methods by a synthetic linker to make them into a single protein chain (where the VL and VH regions pair to form a monovalent molecule (called a single-chain Fv (scFv)); see, e.g., Bird et al. Science 242:423-426 (1988) and Huston et al. Proc. Natl. Acad. Sci. USA 85:5879-5883 (1988). Also included are other forms of single-chain antibodies, such as diabodies. Diabodies are bivalent, bispecific antibodies in which the VH and VL domains are expressed on a single polypeptide chain, but the linker used is too short to allow pairing between the two domains on the same chain, thus forcing the domains to pair with the complementary domains on the other chain and creating two antigen-binding sites (see, e.g., Holliger et al. Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993); Poljak et al., 1994, Structure 2:1121-1123).

[0523] The "variable domain" of an antibody refers to the variable region of the antibody light chain (VL) or the variable region of the antibody heavy chain (VH), either alone or in combination. As is 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 participate in forming the antigen-binding site of the antibody.

[0524] Residues in the variable domain are numbered according to Kabat, a numbering system used to compile the heavy chain variable domain or the light chain variable domain of an antibody. See, Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991). Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to deletions or insertions in the FR or CDR of the variable domain. For example, the heavy chain variable domain may include a single amino acid insertion after residue 52 of H2 (residue 52a (according to Kabat)) and residues inserted after heavy chain FR residue 82 (e.g., residues 82a, 82b, and 82c (according to Kabat)). The Kabat numbering of the residues of a specified antibody can be determined by aligning the antibody homology region with a "standard" Kabat numbered sequence. Various algorithms for assigning Kabat numbers are available. Unless otherwise specified, the present invention uses the algorithm published by Abysis (www.abysis.org) in 2012 to assign Kabat numbers to the variable regions.

[0525] The specific amino acid residue positions in an antibody (e.g., the antigen complementarity determining region residues disclosed in the present invention) are also numbered according to Kabat.

[0526] "Complementary determining regions" (CDRs) can be identified according to the Kabat, Chothia definitions, the cumulative of both Kabat and Chothia, AbM, contact, and / or conformational definitions or any method for determining CDRs well known in the art. See, e.g., Kabat et al., 1991, Sequences of Proteins of Immunological Interest, 5th ed. (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 and uses Oxford Molecular's AbM antibody modeling software The "contact" definition of CDR is based on the observed antigen contact (described in MacCallum et al., 1996, J. Mol. Biol., 262:732 - 745). The "conformation" definition of CDR is based on the residues that contribute enthalpy to antigen binding (see, e.g., Makabe et al., 2008, Journal of Biological Chemistry, 283:1156 - 1166). There are other CDR boundary definitions that may not strictly follow one of the above methods, but will still overlap with at least a portion of the Kabat CDRs, and may be shorter or longer in view of predictions or experimental findings that specific residues or groups of residues or even entire CDRs do not significantly affect antigen binding. As used herein, CDR may refer to CDRs defined by any method known in the art (including combinations of methods).

[0527] In the examples (see Tables 3 and 4), the CDRs are defined as follows (according to Kabat numbering; H: heavy chain; L: light chain):

[0528] CDR-H1: H26 - H35B; CDR-H2: H50 - H65; CDR-H3: H95 - H102

[0529] CDR-L1: L24 - L34; CDR-L2: L50 - L56; CDR-L3: L89 - L97

[0530] "Framework" (FR) residues are the antibody variable domain residues other than the CDR residues. The VH or VL domain framework contains four framework sub-regions: FR1, FR2, FR3, and FR4, interspersed with CDRs of the following structure: FR1 - CDR1 - FR2 - CDR2 - FR3 - CDR3 - FR4.

[0531] In the examples (see Tables 3 and 4), the FR residues include the following (according to Kabat numbering; H: heavy chain; L: light chain):

[0532]

[0533] "Epitope" refers to the antigen (Ag) region or area to which an antibody specifically binds, such as a region or area containing residues that interact with the antibody (Ab). Epitopes can be linear or conformational. In a linear epitope, all points of interaction between the protein and the interacting molecule (such as an antibody) are linearly present along the primary amino acid sequence of the protein. A "non-linear epitope" or "conformational epitope" contains non-contiguous polypeptides (or amino acids) within the antigenic protein, and an antibody specific for the epitope binds to the antigenic protein. The term "epitope" as used in the present invention is defined as a portion of an antigen that can specifically bind to an antibody as determined by any method well known in the art (such as by conventional immunoassays). Alternatively, during the discovery process, the generation and characterization of antibodies can elucidate information regarding the desired epitope. Using this information, antibodies that bind to the same epitope can then be competitively screened. A method to achieve this is to conduct competition and cross-competition studies to identify antibodies that compete or cross-compete with each other for binding to TFPI. That is, antibodies compete for binding to the antigen, such that the antibody competes for binding to the antigen-binding site of the anti-TFPI antibody of the present disclosure.

[0534] The term "paratope" is derived by reversing the perspective of the above definition of "epitope" and refers to the region or area of the antibody molecule that participates in antigen binding, such as a region or area containing residues that interact with the antigen. Paratopes can be linear or conformational (such as discontinuous residues in the CDRs).

[0535] The epitope / paratope of a designated 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 methods of competitive binding. Since each method relies on unique principles, the description of the epitope is closely linked to the method used to determine it. Thus, the epitope / paratope of a designated antibody / antigen pair will be defined differently depending on the mapping method employed.

[0536] At the most detailed level, the epitope / paratope for the interaction between antibodies and antibodies can be defined by specifying the spatial coordinates of the atomic contacts present in the Ag-Ab interaction and information regarding their relative contributions to the binding thermodynamics. At one level, the epitope / paratope residues are characterized by specifying the spatial coordinates of the atomic contacts between the Ag and the Ab. In one aspect, the epitope / paratope residues can be defined by specific criteria, such as the distance between atoms in the Ab and the Ag (such as the distance between the heavy atoms of a homologous antibody and the heavy atoms of the antigen being equal to or less than (“Contact” residues)). On the other hand, epitope / paratope residues are characterized by participating in hydrogen bonding interactions with the cognate antibody / antigen, or interactions with water molecules such that the water molecule also hydrogen bonds with the cognate antibody / antigen (water-mediated hydrogen bonding). On the other hand, epitope / paratope residues are characterized by forming salt bridges with residues of the cognate antibody / antigen. On the other hand, epitope / paratope residues are characterized by having non-zero changes in the buried surface area (BSA) due to interaction with the cognate antibody / antigen. At a less detailed level, an epitope / paratope can be characterized by function (e.g., competing with other Abs for binding). The epitope / paratope can also be more generally defined as including amino acid residues that, when substituted by another amino acid, will change the nature of the interaction between the Ab and the Ag (e.g., alanine scanning).

[0537] In the context of an X-ray-derived crystal structure defined by the spatial coordinates of a complex between an antibody (e.g., a Fab fragment or two Fab fragments) and its antigen, unless otherwise stated, an epitope residue refers to a TFPI residue as described below (i) having a heavy atom (i.e., a non-hydrogen atom) that is within of the heavy atom of the cognate antibody (also referred to as a “contact” residue), (ii) participating in hydrogen bonding with a residue of the cognate antibody or with a water molecule such that the water molecule also hydrogen bonds with the cognate antibody (water-mediated hydrogen bonding), (iii) participating in a salt bridge with a residue of the cognate antibody, and / or (iv) having a non-zero change in the buried surface area (BSA) due to interaction with the cognate antibody. Generally, a cut-off is applied to the BSA to avoid including residues with minimal interactions. Thus, unless otherwise stated, if the BSA of the epitope residue is or greater or participates in electrostatic interactions when the antibody binds to TFPI, an epitope residue of category (iv) is selected. Similarly, unless otherwise stated or inconsistent with the context, in the context of an X-ray-derived crystal structure, a paratope residue refers to an antibody residue as described below (i) having a heavy atom (i.e., a non-hydrogen atom) that is within of the heavy atom of TFPI (also referred to as a “contact” residue), (ii) participating in hydrogen bonding with a TFPI residue or with a water molecule such that the water molecule also hydrogen bonds with TFPI (water-mediated hydrogen bonding), (iii) participating in forming a salt bridge with a residue of TFPI, and / or (iv) having a non-zero change in the buried surface area (BSA) due to interaction with TFPI. Furthermore, unless otherwise stated, if the BSA of the paratope residue is or greater or participates in electrostatic interactions when the antibody binds to TFPI, a paratope residue of category (iv) is selected.

[0538] Based on the epitope mapping methods used and the fact that epitope descriptions and definitions are obtained at different levels of detail, the comparison of epitopes of different Abs on the same Ag can be carried out in a similar manner at different levels of elaboration. For example, if they contain the same set of amino acid residues, epitopes described at the amino acid level (e.g., from X-ray structure determination) are said to be the same. If the epitopes do not share amino acid residues, these epitopes are considered to be distinct (unique). Epitopes characterized by competitive binding are said to be overlapping if binding to one Ab precludes binding to other Abs simultaneously or sequentially; while if the antigen can accommodate binding to two corresponding Abs simultaneously, the epitope is said to be distinct (unique).

[0539] The epitopes and antigenic determinants of a specified Ab / Ag pair can be identified by conventional methods. For example, the approximate location of an epitope can be determined by assessing the ability of an antibody to bind to different fragments or variant TFPI polypeptides (which is more fully described elsewhere hereinabove). Specific residues within TFPI that contact specific residues within the antibody can also be determined using conventional methods (such as those described in the Examples). For example, the Ab / Ag complex can be crystallized. The crystal structure can be determined and used to identify specific sites of interaction between the antibody and the antigen.

[0540] As used herein, the term "competition" with respect to an antibody means that the binding of a first antibody or its antigen-binding portion to an antigen reduces the subsequent binding of a second antibody or its antigen-binding portion to the same antigen. Generally, binding of the first antibody creates steric hindrance, conformational changes, or binding to a common epitope (or portion thereof), thereby reducing the 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 assay suitable for antibody competition involves the use of Biacore technology, which can utilize surface plasmon resonance (SPR) technology (usually using a biosensor system (e.g., The system measures the degree of interaction. 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 is an ELISA-based method. In addition, International Patent Application No. WO2003 / 48731 describes a high-throughput method for "binning" antibodies based on antibody competition. If one antibody (or fragment) reduces the binding of another antibody (or fragment) to TFPI, there is competition. For example, a sequential binding competition assay can be used by sequentially adding different antibodies. The addition of the first antibody may achieve near-saturating binding. Then, the second antibody is added. If no binding of the second antibody to TFPI is detected, or if the binding of the second antibody to TFPI is significantly reduced (e.g., reduced by 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%) compared to a parallel assay in the absence of the first antibody (the value of which can be set to 100%), the two antibodies are considered to compete with each other. An exemplary antibody competition assay (and overlapping epitope analysis) by SPR is provided in Example 6.

[0541] The anti-TFPI antibodies of the present disclosure may have the ability to compete or cross-compete with another antibody of the present disclosure for binding to TFPI as described in the present invention. For example, the antibodies of the present disclosure may compete or cross-compete with the antibodies described in the present invention for binding to TFPI, or for binding to a suitable TFPI fragment or variant bound by the antibodies disclosed in the present invention.

[0542] That is, if a first anti-TFPI antibody competes with a second antibody for binding to TFPI, but it does not compete when the second antibody binds to TFPI first, this first antibody is considered to "compete" with the second antibody (also referred to as one-way competition). When one antibody competes with another antibody, regardless of which antibody binds to TFPI first, then 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 a known antibody of the present invention in a standard binding assay. For example, SPR (e.g., by using a Biacore TM system), ELISA assays, or flow cytometry can be used to demonstrate competition / cross-competition. Such competition / cross-competition may imply that the two antibodies bind to the same, overlapping, or similar epitopes.

[0543] Thus, the anti-TFPI antibodies of the present disclosure can be identified by methods that include a binding assay that evaluates whether a test antibody is able to compete / cross-compete for binding to the binding site on the target molecule with a reference antibody of the present 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).

[0544] An “Fc fusion” protein is a protein in which one or more polypeptides are operably linked to an Fc polypeptide. The Fc fusion protein incorporates the Fc region of an immunoglobulin as a fusion partner.

[0545] The binding affinity of an antibody can be expressed as a Kd value, which refers to the dissociation rate of a specific antigen-antibody interaction. Kd is the ratio of the rate of dissociation (also referred to as the “off-rate (Koff)”) to the rate of binding (or “on-rate (kon)”). Thus, the Kd value is equal to Koff / kon and is expressed in molar concentration (M), and the smaller the Kd value, the stronger the binding affinity. The Kd value of an antibody can be determined using well-established methods in the art. An exemplary method for measuring Kd is surface plasmon resonance (SPR), typically using a biosensor system, such as system. BIAcore kinetic analysis involves analyzing the binding and dissociation of an antigen to a chip having immobilized molecules on its surface (e.g., molecules containing an epitope-binding domain). Another method for determining the Kd of an antibody is to use Bio-Layer Interferometry, typically using technology (Octet QKe system, ForteBio). Additionally or alternatively, it can also be determined using (kinetic exclusion assay) available from Sapidyne Instruments (Boise, Id).

[0546] The term “therapeutically effective amount” means an amount of an anti-TFPI antibody or fragment thereof, or a combination comprising said antibody or fragment thereof, sufficient to achieve a desired purpose (e.g., increasing blood clotting, or in the case of hemophilia, shortening the clotting time, or otherwise causing a measurable benefit in a subject in need). The exact amount will depend on many factors, including but not limited to the components and physical properties of the therapeutic composition, the intended patient population, individual patient considerations, etc., and can be determined by a person skilled in the art.

[0547] The term "co-therapeutically effective amount" means an amount of an anti-TFPI antibody or an antigen-binding fragment thereof that, when provided together with a second therapeutic agent (e.g., factor VIIa (FVIIa)), provides a measurable benefit (e.g., reduced clotting time, reduced bleeding time, increased fibrin generation, enhanced platelet accumulation, etc.) that is higher than the additive measurable benefit of each therapeutic agent or antibody administered alone.

[0548] The term "treatment" includes prophylactic and / or therapeutic treatment. If the treatment is administered prior to the clinical manifestation of a disorder, it is considered prophylactic. Therapeutic treatment includes, for example, increasing or decreasing the severity of a disease or shortening the length of a disease.

[0549] As used herein, the term "about" means + / - 10% of a numerical value.

[0550] 3. Anti-TFPI Antibody

[0551] Disclosed and exemplified herein are antibodies (and antigen-binding fragments thereof) that bind tissue factor pathway inhibitor (TFPI). The antibody and antibody fragment bind to a unique epitope of TFPI. In certain embodiments, recognition of certain epitope residues in TFPI enables the antibody (and antigen-binding fragment thereof) to reduce the activity of TFPI. In addition, in certain embodiments, the antibodies (and antigen-binding fragments thereof) disclosed herein have demonstrated desirable pharmaceutical activities and pharmacokinetic properties for the treatment of coagulation defects and reduction of bleeding time.

[0552] A. Tissue Factor Pathway Inhibitor (TFPI)

[0553] TFPI is a multivalent Kunitz domain-containing protease inhibitor. Exemplary sequences of human, mouse, cynomolgus monkey, rabbit, and rat TFPI are provided in Table 2.

[0554] Human TFPI is an extracellular glycoprotein that has two major forms, TFPI-α and TFPI-β. TFPIα (which is a glycosylated protein with 276 amino acids (MW 43 kD)) is the largest form of TFPI and consists of three Kunitz-like domains and a basic carboxyl-terminal region. Alternative splicing generates TFPI-β, which contains Kunitz domain 1 (K1) and Kunitz domain 2 (K2), but contains an alternative C-terminal portion that lacks Kunitz domain 3 (K3) and the basic region. TFPI-β is anchored to the cell membrane as a glycosylphosphatidylinositol (GPI)-anchored protein by post-translational modification.

[0555] The main targets of TFPI are protease factor Xa (FXa) and factor VIIa (FVIIa), which are key factors in the initial stage of the blood coagulation cascade. Biochemical analysis has revealed that K2 is an inhibitor of FXa, while K1 inhibits the FVIIa-tissue factor complex. The role of K3 is unclear, as it does not appear to have direct protease inhibitory activity, but can serve as a recognition site for the cofactor protein S. The C-terminal domain unique to TFPI-α may be involved in the recognition of prothrombinase on the platelet surface.

[0556] Kunitz domain 1 (K1) corresponds to amino acid residues 26-76 of SEQ ID NO: 2, while Kunitz domain 2 (K2) corresponds to residues 91 to 147 of SEQ ID NO: 2. The K1 and K2 domains from other TFPI homologs, isotypes, variants, or fragments can be identified by sequence alignment or structural alignment against the sequence of SEQ ID NO: 2.

[0557] The TFPI of the present disclosure includes any naturally occurring form of TFPI, which can be derived from any suitable organism. For example, TFPI can be mammalian TFPI, such as human, mouse, rat, non-human primate, bovine, ovine, canine, feline, or porcine TFPI. In certain embodiments, TFPI is human TFPI. TFPI can be the mature form of TFPI (i.e., the TFPI protein that has undergone post-translational processing in a suitable cell). For example, such a mature TFPI protein can be glycosylated.

[0558] The TFPI of the present disclosure includes any functional fragment or variant derived from naturally occurring TFPI. A functional fragment of TFPI can be any TFPI constituent or portion that retains the activity of TFPI (e.g., the activity of inhibiting factor Xa (FXa), inhibiting the FVIIa-tissue factor complex, and / or the ability to act as a negative regulator of blood coagulation or hemostasis). For example, a functional fragment can contain a Kunitz domain, such as the K1 domain, K2 domain, or both K1 and K2 domains of TFPI.

[0559] Compared to naturally occurring TFPI, the functional variants can contain one or more mutations and still retain the activity of naturally occurring TFPI, such as the ability to inhibit factor Xa (FXa) or the ability to inhibit the activity of the FVIIa-tissue factor complex. For example, the variants can have different degrees of sequence identity with SEQ ID NO: 1, 2, 3, 4, 5, 6, or 7, such as at least 60%, 70%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% identical to the sequences listed in SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7.

[0560] The TFPI fragments, variants, isotypes, and homologs of the present invention should retain important epitope residues (e.g., Ile105, Arg107, and Leu131 if using TFPI-23 and TFPI-24 antibodies). In addition, TFPI can contain 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more surface-accessible residues of the K2 domain of TFPI. Surface-accessible residues are residues with a relative accessibility higher than 40%.

[0561] For example, for the K2 domain of TFPI (see, e.g., SEQ ID NO: 2), the following amino acid residues have a relative accessibility higher than 40%: 94-95, 98, 100-110, 118-121, 123-124, 131, 134, 138-142, and 144-145. TFPI can contain 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more of these residues, such as TFPI fragments that include 5 or more, 8 or more, 10 or more, 12 or more, or 15 or more of these residues.

[0562] B. Anti-TFPI Antibodies

[0563] The antibodies or antigen-binding fragments thereof of the present invention specifically bind to the K2 domain of TFPI and can inhibit its interaction with FXa and / or reduce the activity of TFPI.

[0564] TFPI-23 and Variants

[0565] In one aspect, the present invention includes the preparation of antibodies TFPI-23 and variants of TFPI-23 with increased content of human framework germline residues (“germlining”). For example, TFPI-106 contains mutations from H1Q to E and H5V to L (Kabat numbering), while TFPI-107 contains mutations from H1Q to E, H5V to L, and H94I to K (Kabat numbering). For the purposes of the present invention, the TFPI-23 parental antibody and the TFPI-106 germline variant are interchangeable in their epitope residue and paratope residue interactions.

[0566] In one aspect, the present invention provides an isolated antibody or an 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 (numbering according to SEQ ID NO: 2). In certain embodiments, the antibody or its antigen-binding fragment does not bind to the Kunitz domain 1 (K1) of TFPI.

[0567] As disclosed and exemplified in the present invention, a unique epitope in the K2 domain of TFPI has been discovered based on the co-crystal structure and computational alanine scanning. In particular, the crystal structure shows that the K2 domain of TFPI adopts a conical structure, and the tip of the cone (especially Arg107) binds to FXa. TFPI-23, TFPI-24, and their variants all recognize residues near the tip of this conical region and block the binding of TFPI to FXa. Therefore, recognizing epitope residues located near the tip of the cone is particularly useful for inhibiting TFPI activity.

[0568] In certain embodiments, the present invention discloses a TFPI epitope comprising three residues important for antibody-antigen interaction: Ile105, Arg107, and Leu131 (numbering according to human TFPI as shown in SEQ ID NO: 2). Mutating these three residues to alanine results in the loss of binding of TFPI-23, TFPI-24, and their variants. See Table 28, which summarizes the results of alanine scanning.

[0569] Additional TFPI residues involved in antibody binding have also been identified, 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 (numbering according to SEQ ID NO: 2) and any combination thereof. These epitope residues can be recognized by TFPI-23, TFPI-24, and their variants. See Table 27, which shows the common epitope residues shared by TFPI-23 and TFPI-24.

[0570] In certain embodiments, the epitope further comprises one or more residues selected from the group consisting of Asp102, Arg112, Tyr127, Gly129, Met134, Glu138 (numbered according to SEQ ID NO: 2) and any combination thereof. These epitope residues can be recognized by TFPI-23 and its variants (such as TFPI-106, TFPI-107), but not by TFPI-24 (and its variants). See Table 27.

[0571] In certain embodiments, the epitope does not comprise 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 according to SEQ ID NO: 2) and any combination thereof. See Table 27. According to WO201007269 (Novo Nordisk A / S), the reference antibody 4F36 recognizes an epitope comprising: E100, E101, P103, Y109, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126 and L140.

[0572] In certain embodiments, the epitope does not comprise one or more residues selected from the group consisting of D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126, G128 (numbered according to SEQ ID NO: 2) and any combination thereof. See Table 27. According to Table 27, the reference antibodies 2A8 and 2A8-200 recognize an epitope comprising: D31, D32, P34, C35, K36, E100, E101, P103, Y109, K126 and G128.

[0573] In certain embodiments, the epitope may refer to one or more TFPI "contact" residues (with a heavy atom within a distance of a heavy atom of the homologous antibody i.e., non-hydrogen atom), and comprises one or more residues selected from the group consisting of Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, Glu138 (numbered according to SEQ ID NO: 2) and any combination thereof. See Table 29B.

[0574] In certain embodiments, the epitope can refer to one or more TFPI residues that participate in hydrogen bonding with antibody residues or with water molecules (which also hydrogen bond with the cognate antibody, water-mediated hydrogen bonding), and includes one or more residues selected from the group consisting of Asp102, Arg107, Arg112, Tyr127, and Leu131 (numbered according to SEQ ID NO: 2) and any combination thereof. These epitope residues participate in hydrogen bonding with the cognate antibody. See Table 29B.

[0575] In certain embodiments, the epitope can refer to residues that contain non-zero changes in the buried surface area (BSA) due to interaction with the cognate antibody, and includes 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 (numbered according to SEQ ID NO: 2) and any combination thereof. These See Table 29B.

[0576] Any combination of these different classes of epitope residues is also included within the present invention.

[0577] In certain embodiments, the epitope includes at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all of the above epitope residues, or any combination of the various classes of epitope residues described above.

[0578] The paratope residues of TFPI-23 (and variants) can refer to the following contacting residues (located within the TFPI epitope residues): 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 optionally, L93 Ser and L96 Gly (according to Kabat numbering). L93 Ser and L96 Gly are optional because the distance slightly exceeds but is close enough to be rounded to

[0579] It should be noted that the above contact residues are the original residues from the TFPI-23 antibody. However, based on structural analysis and alanine scanning, it is believed that many of the contact residues in TFPI-23 can be replaced by another residue without significantly affecting antigen binding. For example, Table 29A shows that many of the contact residues in TFPI-23 can be replaced by other residues with an impact on binding or stability of <0.5 kcal / mol ("<0.5 kcal / mol" means that the substitution has a neutral effect on binding). In particular, as shown in column 4 of Table 29A, 3 CDR positions and 1 framework position: H47, H58, L91, and L96 (according to Kabat numbering) can only tolerate 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. As summarized in column 4 of Table 29A, other CDR positions can accommodate more substitutions.

[0580] Accordingly, in certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise the following residues (according to Kabat numbering):

[0581] (a) H33 is Ala, Asn, Gly, His, Lys, Met, Phe, Pro, Ser, Thr, Trp, or Val;

[0582] (b) H47 is Trp or Tyr;

[0583] (c) H50 is Ala, Arg, Gly, Lys, Met, Phe, Pro, Ser, Thr, Tyr, or Val;

[0584] (d) H51 is Ile, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0585] (e) H52 is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Trp, Tyr, or Val;

[0586] (f) H56 is Ser, Arg, Gly, His, Ile, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr, or Val;

[0587] (g) H58 is Tyr;

[0588] (h) H95 is Leu, Gln, Ile, Phe, or Tyr;

[0589] (i) H96 is Gly, Ala, Arg, Asn, Asp, Gln, Ile, Lys, Met, Phe, Pro, Ser, Thr or Val;

[0590] (j) H97 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0591] (k) H98 is Thr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0592] (1) H99 is Ser, Ala, Gly, Phe or Pro;

[0593] (m) H100 is Leu, Arg, His, Ile, Leu, Lys, Phe, Pro, Trp, Tyr or Val;

[0594] (n) H100A is Ser, Ala, Arg, Asn, Asp, Gln, Glu, His, Leu, Lys, Met, Phe, Pro, Ser, Thr or Trp;

[0595] (o) L29 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0596] (p) L31 is Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0597] (q) L91 is Tyr or Arg;

[0598] (r) L95A is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0599] (s) L95B is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and

[0600] (t) L95 is Ser, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val;

[0601] and optionally contains (u) L93 is Tyr, Ala, Arg, Asn, Asp, Gln, Glu, Gly, His, Leu, Lys, Met, Phe, Pro, Ser, Thr, Trp, Tyr or Val; and

[0602] (v) L96 is Gly or Asn.

[0603] Among these residues, H47 is a framework residue; all others are CDR residues.

[0604] When more stringent substitution criteria are applied - the substitution must result in an affinity of < -0.5 kcal / mol, which means the substitution must have a positive (neutral / stable) effect - the contacting residues are as follows (according to Kabat numbering) (column 5 of Table 29A):

[0605] (a) H33 is Ala or Val;

[0606] (b) H47 is Trp;

[0607] (c) H50 is Ala;

[0608] (d) H51 is Ile;

[0609] (e) H52 is Ser, Arg, Lys, Phe or Tyr;

[0610] (f) H56 is Ser, Arg or Lys;

[0611] (g) H58 is Tyr;

[0612] (h) H95 is Leu;

[0613] (i) H96 is Gly, Ala, Arg, Asn, Lys, Pro, Ser or Val;

[0614] (j) H97 is Ala;

[0615] (k) H98 is Thr, His, Ile, Leu, Met, Phe or Tyr;

[0616] (1) H99 is Ser;

[0617] (m)H100 is Leu, Phe, Trp or Tyr;

[0618] (n)H100A is Ser, Arg, Asn, Gln, Glu, His, Leu, Lys, Met, Phe, Pro or Trp;

[0619] (o)L29 is Ala;

[0620] (p)L31 is Tyr;

[0621] (q)L91 is Tyr;

[0622] (r)L95A is Ser, Phe, Trp or Tyr;

[0623] (s)L95B is Gly; and

[0624] (t)L95C is Ser, Arg, Asn, Gln, Glu, Ile, Leu, Lys, Met, Phe, Trp, Tyr or Val;

[0625] And optionally: (u) L93 is Ser; and

[0626] (v) L96 is Gly.

[0627] In addition or alternatively, the selection of acceptable substitutions can be based on column 6 of Table 29A, where the first 3 residues are selected according to their effect on affinity (i.e., the top 3 predicted sites that are most stabilizing to affinity). According to this criterion, the contacting residues are as follows (according to Kabat numbering):

[0628] (a) H33 is Ala, Val, His or Phe;

[0629] (b) H47 is Trp or Tyr;

[0630] (c) H50 is Ala, Thr, Ser or Phe;

[0631] (d) H51 is Ile, Arg, Lys or Pro;

[0632] (e) H52 is Ser, Phe, Arg or Tyr;

[0633] (f) H56 is Ser, Lys, Tyr or Phe;

[0634] (g) H58 is Tyr;

[0635] (h) H95 is Leu, Ile, Gln or Phe;

[0636] (i) H96 is Gly, Arg, Asn or Lys;

[0637] (j) H97 is Ala, Leu, Tyr or Ile;

[0638] (k) H98 is Thr, Tyr, Phe or His;

[0639] (1) H99 is Ser, Pro, Ala or Phe;

[0640] (m) H100 is Leu, Tyr, Trp or Phe;

[0641] (n) H100A is Ser, Arg, Leu or Trp;

[0642] (o) L29 is Ala, Glu, Asp or Gln;

[0643] (p) L31 is Tyr, Glu, Asp or Trp;

[0644] (q) L91 is Ty or Arg;

[0645] (r) L95A is Ser, Phe, Tyr or His;

[0646] (s) L95B is Gly, Glu, Asp or Pro; and

[0647] (t) L95C is Ser, Trp, Tyr or Phe;

[0648] And optionally (u) L93 is Ser, Glu, Asp or His;

[0649] (v) L96 is Gly or Asn.

[0650] The antigenic complementarity determining residues of TFPI-23 (and variants) can also refer to residues involved in hydrogen bonding with residues of TFPI or with water molecules (which also hydrogen bond with TFPI), and include the following: H58 Tyr, H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 Leu, L29 Ala, L31 Tyr and L95B Gly (according to Kabat numbering). See Table 29B.

[0651] The antigenic complementarity-determining residues of TFPI-23 (and variants) can also refer to residues that have non-zero changes in BSA due to interaction with TFPI and include the following: (according to Kabat numbering): 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. Truncation is applied (BSA is or greater, or involved in electrostatic interactions) to avoid including residues with minimal interaction. See Table 29B.

[0652] If truncation of BSA is not applied, the antigenic complementarity-determining residues include the following: 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.

[0653] The antibodies or antigen-binding fragments of the present invention can bind to the same TFPI epitope or domain as the antibodies specifically exemplified in the present invention. For example, the antibodies or antigen-binding fragments can be identified by comparing their binding to TFPI with the binding of TFPI-23 or germline variants (such as TFPI-106 and TFPI-107) to TFPI; or by comparing the functions of these antibodies with TFPI-23 and its variants. Assays and determinations that can be used for these identification purposes include assays that evaluate competitive binding to TFPI and will be exemplified in the Examples.

[0654] In one embodiment, the antibodies or antigen-binding fragments of the present invention can bind to the same epitope or region as the antibodies described in the present invention (such as TFPI-23 and its variants). This can include their contact with specific TFPI residues as described above. For example, the antibodies or antigen-binding fragments of the present invention can contact residues selected from the following group (in bind to TFPI in a manner within: Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, Glu138 (numbered according to SEQ ID NO: 2) and any combination thereof. The antibody or antigen-binding fragment thereof of the present invention may be capable of binding to an epitope comprising one or more residues selected from the group consisting of: Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Arg112, Tyr127, Gly129, Cys130, Leu131, Gly132, Met134, Glu138 (numbered according to SEQ ID NO: 2) and any combination thereof.

[0655] The antibody and its antigen-binding fragment may comprise at least one epitope residue located on TFPI (numbered according to SEQ ID NO: 2) and the antigen-complementary site residues (numbered according to Kabat) within, as follows: The epitope residue 102Asp is within the antigen-complementary site residue H58 ; The epitope residue 104Gly is within the antigen-complementary site residue H58 ; The epitope residue 105Ile is within the antigen-complementary site residues H33 Ala, H50 Ala, H51 Ile, H52 Ser, H56 Ser, H58 Tyr, H95 ; The epitope residue 106Cys is within the antigen-complementary site residues H100 Leu and H100A ; The epitope residue 107Arg is within the antigen-complementary site residues H96 Gly, H97 Ala, H98 Thr, H99 Ser, H100 ; The epitope residue 108Gly is within the antigen-complementary site residue H100 ; The epitope residue 112Arg is within the antigen-complementary site residues L29Ala, L31 ; The epitope residue 127Tyr is within the antigen-complementary site residue L31 ; The epitope residue 129Gly is within the antigen-complementary site residue L31 ; The epitope residue 130Cys is within the antigen-complementary site residues L91 Tyr, L95B ; The epitope residue 131Leu is within the antigen-complementary site residues H47 Trp, H50 Ala, H58 Tyr, L95A Ser, L95B Gly, L95C within; the epitope residue 132 Gly is located within the antigen - complementary residues H58 Tyr, L95A Ser, H58 Tyr, L95A within; the epitope residue 134 Met is located within the antigen - complementary residue L95A within; and the epitope residue 138 Glu is located within the antigen - complementary residue L29 See Tables 29A and 29B.

[0656] The antibody or its antigen - binding fragment may also comprise at least one antigen - complementary residue (numbered according to Kabat) that can form a hydrogen bond with an epitope residue (numbered according to SEQ ID NO: 2) of TFIP, as follows: the epitope residue 102 Asp can form a hydrogen bond with the antigen - complementary residue H58 Tyr; the epitope residue 107 Arg can form a hydrogen bond with at least one antigen - complementary residue selected from the group consisting of H96 Gly, H97 Ala, H98 Thr, H99 Ser, and H100 Leu; the epitope residue 112 Arg can form a hydrogen bond with the antigen - complementary residue L29 Ala; the epitope residue 127 Tyr can form a hydrogen bond with the antigen - complementary residue L31 Tyr; and the epitope residue 131 Leu can form a hydrogen bond with the antigen - complementary residue L95B Gly. See Table 29B.

[0657] The antibody or its antigen-binding fragment may also comprise at least one antigen-complementary residue (numbered according to Kabat) having a non-zero change in BSA due to interaction with an epitope residue (numbered according to SEQ ID NO: 2), as follows: epitope residue 102Asp interacts with antigen-complementary residue H58 Tyr; epitope residue 104Gly interacts with antigen-complementary residue H58 Tyr; epitope residue 105Ile interacts with at least one antigen-complementary 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 106Cys interacts with at least one antigen-complementary residue selected from the group consisting of: H95 Leu, H100 Leu, H100A Ser and L91 Tyr; epitope residue 107Arg interacts with at least one antigen-complementary residue selected from the group consisting of: H96 Gly, H97 Ala, H98 Thr, H99 Ser and H100 Leu; epitope residue 108 Gly interacts with antigen-complementary residue H100 Leu; epitope residue 112Arg interacts with at least one antigen-complementary residue selected from the group consisting of: L29 Ala, L31 Tyr and L93 Ser; epitope residue 127Tyr interacts with at least one antigen-complementary residue selected from the group consisting of: L31 Tyr and L95B Gly; epitope residue 129Gly interacts with at least one antigen-complementary residue selected from the group consisting of: H100A Ser, L31 Tyr and L91 Tyr; epitope residue 130Cys interacts with at least one antigen-complementary residue selected from the group consisting of: H95 Leu, H100A Ser, L31 Tyr, L91 Tyr and L95B Gly; epitope residue 131Leu interacts with at least one antigen-complementary 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 132Gly interacts with at least one antigen-complementary residue selected from the group consisting of: H58 Tyr and L95A Ser; epitope residue 133 Asn interacts with antigen-complementary residue L95A Ser; epitope residue 134Met interacts with at least one antigen-complementary residue selected from the group consisting of: L93 Ser, L94 Ser and L95A Ser; and epitope residue 138Glu interacts with at least one antigen-complementary residue selected from the group consisting of: L28 Gly, L29 Ala and L93 Ser.

[0658] The antibody or antigen-binding fragment thereof of the present invention can be any antibody or antigen-binding fragment that contains any of the above antigen-combining site residues that interact with at least one of the above-listed epitope residues.

[0659] In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention contains at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all of the above antigen-combining site residues, or any combination of the above various classes of antigen-combining site residues. In addition, conservative substitutions can be introduced among these antigen-combining site residues. For example, the antibody or antigen-binding fragment thereof can contain 1, 2, 3, 4, 5, 6, 7, or 8 conservative substitutions according to Table 34.

[0660] Table 34 Conservative Substitutions

[0661]

[0662]

[0663] The antibody of the present invention, as described in the present invention, can be capable of competing with another antibody for binding to the TFPI of the present invention. For example, the antibody of the present invention can cross-compete with TFPI-23 and its variants described in the present invention for binding to TFPI, or bind to a suitable fragment or variant of TFPI that is bound by the TFPI-23 antibody. Such cross-competing antibodies can be identified based on their ability to cross-compete with the exemplary antibodies of the present invention in a standard binding assay. For example, SPR (e.g., by using a Biacore TM system), ELISA assay, or flow cytometry can be used to demonstrate cross-competition. Such cross-competition may imply that the two antibodies bind to the same, overlapping, or similar epitopes.

[0664] Therefore, the antibody of the present invention can be identified by a method comprising a binding assay that evaluates whether a test antibody can compete with an exemplary antibody of the present invention (e.g., TFPI-23 or any variant or fragment thereof described in the present invention) for binding to a binding site on a target molecule.

[0665] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise the following heavy chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 38, CDR-H2 comprising SEQ ID NO: 39, and CDR-H3 comprising SEQ ID NO: 40; and / or (ii) the following light chain CDR sequences: CDR-L1 comprising SEQ ID NO: 33, CDR-L2 comprising SEQ ID NO: 34, and CDR-L3 comprising SEQ ID NO: 35. In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise the following heavy chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 38, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 39, and CDR-H3 that 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 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 33, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 34, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 35. In certain embodiments, relative to SEQ ID NOs: 38, 39, 40, 33, 34, and 35, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34. In certain embodiments, the substitution is according to Column 4, Column 5, or Column 6 of Table 29A.

[0666] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise human framework sequences. For example, the heavy chain framework sequence can be derived from human VH3 germline, VH1 germline, VH5 germline or VH4 germline. Preferably, the human germline heavy chain framework is a framework derived from VH1, VH3 or VH5 germline. For example, VH frameworks from the following germlines can be used: IGHV3-23, IGHV3-7 or IGHV1-69 (germline names are based on IMGT germline definitions). Preferably, the human germline light chain framework is a framework derived from VK or Vλ germline. For example, VL frameworks from the following germlines can be used: IGKV1-39 or IGKV3-20 (germline names are based on IMGT germline definitions). Additionally or alternatively, the framework sequence can be a human germline consensus framework sequence, such as a framework of 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.

[0667] Sequences of human germline frameworks can be obtained from various publicly available databases, such as V-base, IMGT, NCBI or Abysis.

[0668] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a VH that comprises 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 that comprises 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: 36. Any combination of these VL and VH sequences is also included within the present invention.

[0669] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a CH that comprises 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: 20; and / or (ii) a CL that comprises 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: 26. Any combination of these CH and CL sequences is also included within the present invention.

[0670] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0671] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a heavy chain that comprises 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: 42, SEQ ID NO: 64 or SEQ ID NO: 66; and / or (ii) a light chain that comprises 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: 37. Any combination of these heavy chain and light chain sequences is also included within the present invention.

[0672] TFPI-24 and variants

[0673] The eutectic structure shows that TFPI-24 (and its variants) share many epitope residues with TFPI-23. Among them, Ile105, Arg107, and Leu131 (numbered according to human TFPI as shown in SEQ ID NO: 2) are considered important for antibody-antigen interaction. Other shared epitope residues include: Cys106, Gly108, C130, L131, and G132 (numbered according to SEQ ID NO: 2).

[0674] 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 an antigen-binding fragment thereof that specifically binds to an epitope in K2 of TFPI, wherein the epitope (i) comprises the residues Ile105, Arg107, and Leu131; (ii) optionally comprises one or more residues selected from the group consisting of Cys106, Gly108, Cys130, Leu131, and Gly132; and (iii) further optionally comprises one or more residues selected from the group consisting of Glu100, Glu101, Asp102, Gly104, and Tyr109 (numbered according to SEQ ID NO: 2).

[0675] In certain embodiments, the epitope does not comprise one or more residues selected from the group consisting of P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, L140 (numbered according to SEQ ID NO: 2) and any combination thereof. See Table 27. According to WO201007269 (Novo Nordisk A / S), the reference antibody 4F36 recognizes an epitope comprising: P103, T111, Y113, F114, N116, Q118, Q121, C122, E123, R124, F125, K126, and L140.

[0676] In certain embodiments, the epitope does not comprise 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) and any combination thereof. See Table 27. According to Table 27, the reference antibodies 2A8 and 2A8-200 recognize an epitope comprising: D31, D32, P34, C35, K36, P103, K126, Y127, and G128.

[0677] Also characterize the antigen complementarity determining region residues from TFPI-24 (based on BSA) (see Table 24) and include the following: H33Ala, 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 thereof described in the present invention comprises 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 antigen complementarity determining region residues. Additionally, conservative substitutions can be introduced among these antigen complementarity determining region residues. For example, the antibody or antigen-binding fragment thereof can comprise 1, 2, 3, 4, 5, 6, 7, or 8 conservative substitutions according to Table 34.

[0678] In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention comprises the following heavy chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 48, CDR-H2 comprising SEQ ID NO: 49, and CDR-H3 comprising SEQ ID NO: 50; and / or (ii) the following light chain CDR sequences: CDR-L1 comprising SEQ ID NO: 43, CDR-L2 comprising SEQ ID NO: 44, and CDR-L3 comprising SEQ ID NO: 45. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention comprises the following heavy chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 48, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 49, and CDR-H3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 50; and / or (ii) the following light chain CDR sequences: CDR-L1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 43, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 44, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 45. In certain embodiments, relative to SEQ ID NO: 48, 49, 50, 43, 44, and 45, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34.

[0679] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise human framework sequences. For example, the heavy chain framework sequence can be derived from human VH3 germline, VH1 germline, VH5 germline, or VH4 germline as described above. Preferably, the human germline light chain framework is a framework derived from VK or Vλ germline as described above. Consensus human germline framework sequences as described above can also be used.

[0680] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise (i) a VH that comprises 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: 67, 69, 51, and 79; and / or (ii) a VL that comprises 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: 46, 71, 73, 75, and 77. Any combination of these VL and VH sequences is also included within the present invention.

[0681] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a CH that comprises 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: 20; and / or (ii) a CL that comprises 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: 26. Any combination of these CH and CL sequences is also included within the present invention.

[0682] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2)、IgG, IgE or IgG (such as IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0683] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a heavy chain comprising 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: 52, SEQ ID NO: 68, SEQ ID NO: 70 or SEQ ID NO: 80; and / or (ii) a light chain comprising 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: 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 within the present disclosure.

[0684] 4D8 and variants

[0685] The co-crystal structure also discloses epitope and paratope information of antibody 4D8 and its variants. The epitope residues of 4D8 and its variants include: Glu101, Pro103, Tyr109, Thr111, Ser119, Gln121, Glu123, Arg124, Lys126 and Leu140 (numbered according to SEQ ID NO: 2).

[0686] 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 according to SEQ ID NO: 2) and any combination thereof. See Table 27. According to WO201007269 (Novo Nordisk A / S), the reference antibody 4F36 recognizes an epitope comprising E100, D102, R107, Y113, F114, N116, Q118 and C122.

[0687] In certain embodiments, the epitope does not include one or more residues selected from the group consisting of D31, D32, P34, C35, K36, E100, I105, R107, G108, Y127, G128 (numbered according to SEQ ID NO: 2) and any combination thereof. See Table 27. According to Table 27, the reference antibodies 2A8 and 2A8-200 recognize an epitope that includes D31, D32, P34, C35, K36, P103, K126, Y127, and G128.

[0688] Also characterized are the antigen complementarity determining residues from 4D8 (based on BSA) (see Table 24) and include the following: 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 antibodies or antigen-binding fragments thereof described in the present invention include at least 8, at least 9, at least 10, at least 11, at least 12, at least 13, or all of these antigen complementarity determining residues. In addition, conservative substitutions may be introduced into these antigen complementarity determining residues. For example, the antibody or antigen-binding fragment thereof may include 1, 2, 3, 4, 5, 6, 7, or 8 conservative substitutions according to Table 34.

[0689] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise the following heavy-chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 87, CDR-H2 comprising SEQ ID NO: 88, and CDR-H3 comprising SEQ ID NO: 89; and / or (ii) the following light-chain CDR sequences: CDR-L1 comprising SEQ ID NO: 81, CDR-L2 comprising SEQ ID NO: 82, and CDR-L3 comprising SEQ ID NO: 83. In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise the following heavy-chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 87, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 88, and CDR-H3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 89; and / or (ii) the following light-chain CDR sequences: CDR-L1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 81, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 82, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 83. In certain embodiments, relative to SEQ ID NOs: 87, 88, 89, 81, 82, and 83, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34.

[0690] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise human framework sequences. For example, the heavy-chain framework sequence can be from the human VH3 germline, VH1 germline, VH5 germline, or VH4 germline as described above. Preferably, the human germline light-chain framework is a framework derived from the VK or Vλ germline as described above. The consensus human germline framework sequence as described above can also be used.

[0691] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a VH that comprises 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: 90, 95, 97, 99, 101, 103, 105, and 107; and / or (ii) a VL that comprises 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: 84, 109, and 111. Any combination of these VL and VH sequences is also included within the present invention.

[0692] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a CH that comprises 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: 20; and / or (ii) a CL that comprises 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 or SEQ ID NO: 85. Any combination of these CH and CL sequences is also included within the present invention.

[0693] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE, or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0694] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a heavy chain comprising 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: 92, SEQ ID NO: 94, SEQ ID NO: 96, SEQ ID NO: 98, SEQ ID NO: 100, SEQ ID NO: 102, SEQ ID NO: 104, SEQ ID NO: 106, SEQ ID NO: 108; and / or (ii) a light chain comprising 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: 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 within the present invention.

[0695] TFPI-3 and variants

[0696] The present invention also provides TFPI-3 and its variants. Accordingly, an antibody based on TFPI-3 or an antigen-binding fragment thereof comprises the following heavy-chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 16, CDR-H2 comprising SEQ ID NO: 17, and CDR-H3 comprising SEQ ID NO: 18; and / or (ii) the following light-chain CDR sequences: CDR-L1 comprising SEQ ID NO: 10, CDR-L2 comprising SEQ ID NO: 11, and CDR-L3 comprising SEQ ID NO: 12. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention comprises the following heavy-chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 16, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 17, and CDR-H3 that 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 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 10, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 11, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 12. In certain embodiments, relative to SEQ ID NOs: 16, 17, 18, 10, 11, and 12, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34.

[0697] In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention comprises a human framework sequence. For example, the heavy-chain framework sequence can be from a human VH3 germline, VH1 germline, VH5 germline, or VH4 germline as described above. The preferred human germline light-chain framework is a framework derived from a VK or Vλ germline as described above. A consensus human germline framework sequence as described above can also be used.

[0698] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a VH that comprises 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: 19, and / or (ii) a VL that comprises 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: 13. Any combination of these VL and VH sequences is also included within the present invention.

[0699] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a CH that comprises 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: 20; and / or (ii) a CL that comprises 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 or SEQ ID NO: 14. Any combination of these CH and CL sequences is also included within the present invention.

[0700] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0701] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a heavy chain comprising 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: 21; and / or (ii) a light chain comprising 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: 15. Any combination of these heavy and light chain sequences is also included within the present invention.

[0702] TFPI-2 1 and variants

[0703] The present invention also provides TFPI-21 and its variants. Accordingly, an antibody or an antigen-binding fragment thereof based on TFPI-21 comprises the following heavy-chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 28, CDR-H2 comprising SEQ ID NO: 29, and CDR-H3 comprising SEQ ID NO: 30; and / or (ii) the following light-chain CDR sequences: CDR-L1 comprising SEQ ID NO: 22, CDR-L2 comprising SEQ ID NO: 23, and CDR-L3 comprising SEQ ID NO: 24. In certain embodiments, the antibody or an antigen-binding fragment thereof described in the present invention comprises the following heavy-chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 28, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 29, and CDR-H3 that 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 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 22, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 23, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 24. In certain embodiments, relative to SEQ ID NO: 28, 29, 30, 22, 23, and 24, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34.

[0704] In certain embodiments, the antibody or an antigen-binding fragment thereof described in the present invention comprises human framework sequences. For example, the heavy-chain framework sequence can be from a human VH3 germline, VH1 germline, VH5 germline, or VH4 germline as described above. The preferred human germline light-chain framework is a framework derived from a VK or Vλ germline as described above. The consensus human germline framework sequences described above can also be used.

[0705] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a VH that comprises 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: 31, and / or (ii) a VL that comprises 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: 25. Any combination of these VL and VH sequences is also included within the present invention.

[0706] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a CH that comprises 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: 20; and / or (ii) a CL that comprises 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 or SEQ ID NO: 26. Any combination of these CH and CL sequences is also included within the present invention.

[0707] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0708] In certain embodiments, the antibodies or antigen-binding fragments thereof described herein comprise: (i) a heavy chain comprising 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: 32; and / or (ii) a light chain comprising 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: 27. Any combination of these heavy and light chain sequences is also included within the present invention.

[0709] TFPI-26 and variants

[0710] The present invention also provides TFPI-26 and its variants. Accordingly, an antibody or an antigen-binding fragment thereof based on TFPI-26 comprises the following heavy chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 58, CDR-H2 comprising SEQ ID NO: 59, and CDR-H3 comprising SEQ ID NO: 60; and / or (ii) the following light chain CDR sequences: CDR-L1 comprising SEQ ID NO: 53, CDR-L2 comprising SEQ ID NO: 54, and CDR-L3 comprising SEQ ID NO: 55. In certain embodiments, the antibody or an antigen-binding fragment thereof described in the present invention comprises the following heavy chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 58, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 59, and CDR-H3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 60; and / or (ii) the following light chain CDR sequences: CDR-L1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 53, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 54, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 55. In certain embodiments, relative to SEQ ID NOs: 58, 59, 60, 53, 54, and 55, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34.

[0711] In certain embodiments, the antibody or an antigen-binding fragment thereof described in the present invention comprises a human framework sequence. For example, the heavy chain framework sequence can be from a human VH3 germline, VH1 germline, VH5 germline, or VH4 germline as described above. The preferred human germline light chain framework is a framework derived from a VK or Vλ germline as described above. A consensus human germline framework sequence as described above can also be used.

[0712] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a VH that comprises 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: 61, and / or (ii) a VL that comprises 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: 56. Any combination of these VL and VH sequences is also included within the present invention.

[0713] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a CH that comprises 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: 20; and / or (ii) a CL that comprises 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: 26. Any combination of these CH and CL sequences is also included within the present invention.

[0714] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0715] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a heavy chain comprising 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: 62; and / or (ii) a light chain comprising 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: 57. Any combination of these heavy and light chain sequences is also included within the present invention.

[0716] 6B7.c5 and variants

[0717] The present invention also provides 6B7.c5 and its variants. Accordingly, an antibody based on 6B7.c5 or an antigen-binding fragment thereof comprises the following heavy-chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 118, CDR-H2 comprising SEQ ID NO: 119, and CDR-H3 comprising SEQ ID NO: 120; and / or (ii) the following light-chain CDR sequences: CDR-L1 comprising SEQ ID NO: 113, CDR-L2 comprising SEQ ID NO: 114, and CDR-L3 comprising SEQ ID NO: 115. In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention comprises the following heavy-chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 118, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 119, and CDR-H3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 120; and / or (ii) the following light-chain CDR sequences: CDR-L1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 113, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 114, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 115. In certain embodiments, relative to SEQ ID NOs: 118, 119, 120, 113, 114, and 115, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34.

[0718] In certain embodiments, the antibody or antigen-binding fragment thereof described in the present invention comprises a human framework sequence. For example, the heavy-chain framework sequence can be from a human VH3 germline, VH1 germline, VH5 germline, or VH4 germline as described above. The preferred human germline light-chain framework is a framework derived from a VK or Vλ germline as described above. A consensus human germline framework sequence as described above can also be used.

[0719] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a VH that comprises 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: 121, and / or (ii) a VL that comprises 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: 116. Any combination of these VL and VH sequences is also included within the present invention.

[0720] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a CH that comprises 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 that comprises 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 or SEQ ID NO: 85. Any combination of these CH and CL sequences is also included within the present invention.

[0721] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0722] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a heavy chain comprising 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: 122; and / or (ii) a light chain comprising 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: 117. Any combination of these heavy and light chain sequences is also included within the present invention.

[0723] 7A4.D9 and variants

[0724] The present invention also provides 7A4.D9 and its variants. Accordingly, an antibody or an antigen-binding fragment thereof based on 7A4.D9 comprises the following heavy-chain CDR sequences: (i) CDR-H1 comprising SEQ ID NO: 128, CDR-H2 comprising SEQ ID NO: 129, and CDR-H3 comprising SEQ ID NO: 130; and / or (ii) the following light-chain CDR sequences: CDR-L1 comprising SEQ ID NO: 123, CDR-L2 comprising SEQ ID NO: 124, and CDR-L3 comprising SEQ ID NO: 125. In certain embodiments, the antibody or an antigen-binding fragment thereof described in the present invention comprises the following heavy-chain CDR sequences: (i) CDR-H1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 128, CDR-H2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 129, and CDR-H3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 130; and / or (ii) the following light-chain CDR sequences: CDR-L1 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 123, CDR-L2 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 124, and CDR-L3 that is at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 125. In certain embodiments, relative to SEQ ID NOs: 128, 129, 130, 123, 124, and 125, no more than 10, no more than 9, no more than 8, no more than 7, no more than 6, no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 substitution is made in each CDR. In certain embodiments, the substitution is a conservative substitution according to Table 34.

[0725] In certain embodiments, the antibody or an antigen-binding fragment thereof described in the present invention comprises a human framework sequence. For example, the heavy-chain framework sequence can be from a human VH3 germline, VH1 germline, VH5 germline, or VH4 germline as described above. The preferred human germline light-chain framework is a framework derived from a VK or Vλ germline as described above. A consensus human germline framework sequence as described above can also be used.

[0726] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a VH that comprises 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: 131, and / or (ii) a VL that comprises 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: 126. Any combination of these VL and VH sequences is also included within the present invention.

[0727] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a CH that comprises 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 that comprises 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 or SEQ ID NO: 85. Any combination of these CH and CL sequences is also included within the present invention.

[0728] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise an Fc domain. The Fc domain can be derived from IgA (e.g., IgA 1 or IgA 2 ), IgG, IgE or IgG (e.g., IgG 1 , IgG 2 , IgG 3 or IgG 4 ).

[0729] In certain embodiments, the antibodies or antigen-binding fragments thereof described in the present invention comprise: (i) a heavy chain comprising 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: 132; and / or (ii) a light chain comprising 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: 127. Any combination of these heavy and light chain sequences is also included within the present invention.

[0730] Also disclosed are antibodies or antigen-binding fragments thereof that specifically bind to the K2 domain of TFPI and compete for binding to TFPI with any of the antibodies or antigen-binding fragments thereof described in the present invention (e.g., any of the antibodies (or antigen-binding fragments thereof) listed in Table 3). For example, an antibody or antigen-binding portion thereof competes for binding to TFPI with TFPI-23 or TFPI-106 if its binding to TFPI inhibits subsequent binding of TFPI-23 or TFPI-106 to TFPI.

[0731] Also disclosed are antibodies or antigen-binding fragments thereof 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 thereof described in the present invention (e.g., any of the antibodies (or antigen-binding fragments thereof) listed in Table 3).

[0732] Exemplary antibody competition assays (and overlapping epitope analyses) by SPR are provided in Example 6.

[0733] The antibodies and antigen-binding fragments disclosed in the present invention 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 (ScFv), mutants thereof, fusion proteins containing antibody portions, domain antibodies (dAb), humanized antibodies, and any other modified configurations of immunoglobulin molecules containing the desired specific antigen recognition site (including glycosylation variants of antibodies, amino acid sequence variants of antibodies, and covalently modified antibodies). The antibody and antigen-binding fragment can 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.

[0734] In certain embodiments, the affinity (Kd) value of the antibodies and antigen-binding fragments disclosed in the present invention does not exceed about 1×10 -3 M, for example, does not exceed about 5×10 -4 M, does not exceed about 4×10 -4 M, does not exceed about 3×10 -4 M, does not exceed about 2×10 -4 M, does not exceed about 1×10 -4 M, does not exceed about 9×10 -5 M, does not exceed about 8×10 -5 M, does not exceed about 7×10 -5 M, does not exceed about 6×10 -5 M, does not exceed about 5×10 -5 M, does not exceed about 4×10 -5 M, does not exceed about 3×10 -5 M, does not exceed about 2×10 -5 M, does not exceed about 1×10 -5 M, does not exceed about 9×10 -6 M, does not exceed about 8×10 -6 M, does not exceed about 7×10 -6 M, does not exceed about 6×10 -6 M, does not exceed about 5×10 -6 M, does not exceed about 4×10 -6 M, does not exceed about 3×10 -6 M, does not exceed about 2×10 -6 M, does not exceed about 1×10 -6 M, does not exceed about 9×10 -7 M, does not exceed about 8×10 -7 M, does not exceed about 7×10 -7 M, does not exceed about 6×10 -7 M, does not exceed about 5×10-7 M, not exceeding about 4×10 -7 M, not exceeding about 3×10 -7 M, not exceeding about 2×10 -7 M, not exceeding about 1×10 -7 M, not exceeding about 9×10 -8 M, not exceeding about 8×10 -8 M, not exceeding about 7×10 -8 M, not exceeding about 6×10 -8 M, not exceeding about 5×10 -8 M, not exceeding about 4×10 -8 M, not exceeding about 3×10 -8 M, not exceeding about 2×10 -8 M, not exceeding about 1×10 -8 M, not exceeding about 9×10 -9 M, not exceeding about 8×10 -9 M, not exceeding about 7×10 -9 M, not exceeding about 6×10 -9 M, not exceeding about 5×10 -9 M, not exceeding about 4×10 -9 M, not exceeding about 3×10 -9 M, not exceeding about 2×10 -9 M, not exceeding about 1×10 -9 M, about 1×10 -3 to about 1×10 -13 M, 1×10 -4 to about 1×10 -13 M, 1×10 -5 to about 1×10 -13 M, about 1×10 -6 to about 1×10 -13 M, about 1×10 -7 to about 1×10 -13 M, about 1×10 -8 to about 1×10 -13 M, about 1×10 -9 to about 1×10 -13 M, 1×10 -3 to about 1×10 -12 M, 1×10 -4 to about 1×10 -12 M, about 1×10 -5 to about 1×100 -12 M, about 1×10 -6 to about 1×10 -12 M, about 1×10 -7 to about 1×10 -12 M, about 1×10 -8to about 1×10 -12 M, about 1×10 -9 to about 1×10 -12 M, 1×10 -3 to about 1×10 -11 M, 1×10 -4 to about 1×10 -11 M, about 1×10 -5 to about 1×10 -11 M, about 1×10 -6 to about 1×10 -11 M, about 1×10 -7 to about 1×10 -11 M, about 1×10 -8 to about 1×10 -11 M, about 1×10 -9 to about 1×10 -11 M, 1×10 -3 to about 1×10 -10 M, 1×10 -4 to about 1×10 -10 M, about 1×10 -5 to about 1×10 -10 M, about 1×10 -6 to about 1×10 -10 M, about 1×10 -7 to about 1×10 -10 M, about 1×10 -8 to about 1×10 -10 M or about 1×10 -9 to about 1×10 -10 M.

[0735] In certain embodiments, the dissociation constant is measured using surface plasmon resonance (SPR) method (Biacore). Surface plasmon resonance refers to an optical phenomenon which is, for example, by using BIACORE TM system to detect changes in protein concentration within a biosensor matrix to analyze biorecognition interactions in real time. In certain embodiments, the SPR measurement is performed using a Biacore T100 or T200 instrument.

[0736] For example, standard assay conditions for surface plasmon resonance can be based on immobilizing about 100 IgG reaction units (RU) of ligand on the SPR chip. The purified target protein is diluted to the final concentration range with buffer and injected at the required flow rate (e.g., 10 - 100 μl / min) to calculate Ka. Dissociation is allowed to proceed to establish the dissociation rate (Kd), and then 5 seconds of 3M MgCl 2(or 20 mM NaOH) pulses to regenerate the chip surface. Then, the sensorgram is analyzed using a kinetic evaluation software package.

[0737] In an exemplary embodiment, the SPR assay is performed according to the conditions listed under the subheading "Surface Plasmon Resonance (SPR)" in Example 1.

[0738] In certain embodiments, the dissociation constant is measured using solution-based kinetic exclusion assay (KinExA TM ). In a particular embodiment, the KinExA measurement is performed using a KinExA TM 3200 instrument (Sapidyne). The kinetic exclusion assay (KinExA TM ) is a general immunoassay platform (essentially a flow spectrofluorometer) capable of measuring the equilibrium dissociation constant and the association and dissociation rate constants of antigen / antibody interactions. Since KinExA TM is performed after equilibrium has been achieved, it is a favorable technique for measuring the Kd of high-affinity interactions where the dissociation rate of the interaction may be slow. The KinExA TM method can be generally performed as described in Drake et al (2004) Analytical Biochemistry 328, 35 - 43.

[0739] Generally, TFPI antibodies need to bind to TFPI with high affinity to effectively block the activity of TFPI. However, since TFPI is also expressed on the cell surface, when the binding affinity of the antibody is too high, the antibody can be rapidly internalized and degraded by host cells. This can potentially result in a short half-life and repeated injections. For example, compared to TFPI-24, the antibody TFPI-23 shows a lower binding affinity (Kd) and is in some cases more desirable because it has a lower internalization rate and a longer half-life. Thus, if a longer half-life is desired, a binding affinity (Kd) in the range of 5×10 -7 M to about 5×10 - 11 M, especially about 1×10 -8 to about 1×10 -10 M (0.1 nM to 10 nM) is generally desired. This range is considered to achieve a balance between (i) the binding affinity required to effectively inhibit the activity of TFPI and (ii) a longer half-life and reduced antibody internalization.

[0740] In particular, it is considered that in order to maintain a subcutaneous administration of 3 mg / kg per week, the Kd value is about 1×10 -8M to about 1×10 -10 M (0.1 nM to 10 nM) is desired.

[0741] Whether an antibody or an antigen-binding fragment thereof reduces the activity of TFPI or decreases the binding of TFPI to a physiological substrate (e.g., FXa) can be determined by measuring a decrease in the binding affinity of TFPI for the physiological substrate, e.g., by comparing (i) the binding affinity of TFPI for its substrate in the presence of an anti-TFPI antibody (or an antigen-binding fragment thereof) with (ii) the binding affinity of TFPI for the same substrate in the absence of the anti-TFPI antibody. In the presence of an anti-TFPI antibody (or an antigen-binding fragment thereof), the binding of TFPI to a physiological substrate (e.g., FXa) can be reduced by 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%. When the antibody (or fragment) is absent, the expected binding of TFPI to its physiological substrate can be set at 100%.

[0742] The TFPI inhibitory activity of an anti-TFPI antibody or an antigen-binding fragment thereof (also referred to herein as "reducing the activity of TFPI") can also be evaluated in an in vivo model and / or in vitro using, for example, a plasma system. For example, the inhibitory activity of the antibody (or the level of reducing the activity of TFPI) can be evaluated by: (i) reducing the clotting time measured in a plasma-based diluted prothrombin time assay; (ii) reducing the whole blood clotting time measured by thromboelastography; (iii) increasing thrombin generation; (iv) increasing FXa activity in the presence of TFPI; (v) enhanced platelet accumulation in the presence of TFPI; (vi) increased fibrin generation in the presence of TFPI; or (vii) any combination thereof. The inhibitory activity of the antibody or antigen-binding fragment can be dose-dependent (e.g., causing a dose-dependent decrease in the clotting time measured in a plasma-based diluted prothrombin time assay).

[0743] Several exemplary assays for evaluating the TFPI inhibitory activity of an antibody are described in detail in the Examples. For example, the plasma-diluted prothrombin time (PT) assay is a modified PT assay that uses diluted thromboplastin or tissue factor to extend the clotting time and dynamic range of the assay. An inhibitory / neutralizing anti-TFPI antibody should reduce the diluted prothrombin time.

[0744] Another exemplary model system for determining TFPI inhibitory activity is the extrinsic tenase assay, which tests the ability of an antibody or an antigen-binding fragment thereof to restore FX activation mediated by the extrinsic complex in the presence of TFPI. Another model system for characterizing TFPI inhibitory activity is the FXa inhibition 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)).

[0745] The inhibitory activity of an antibody or an antigen-binding fragment thereof can also be evaluated in a plasma-based assay. In the presence of an anti-TFPI antibody or an antigen-binding fragment thereof, thrombin formation can be triggered in plasma that is substantially devoid of FVIII or FIX activity (e.g., residual clotting factor activity is less than 1%). Thrombin formation can be detected using a fluorogenic substrate or a chromogenic substrate. Prothrombin conversion can be measured using, for example, a Thrombograph TM (Thermo Scientific, Waltham, Mass.), and the resulting data can be compiled by Thrombinoscope software obtained from Thrombinoscope BV to generate a calibrated automatic thrombogram. TM Software to generate a calibrated automatic thrombogram.

[0746] For example, an antibody or antigen-binding fragment can increase TFPI-regulated thrombin generation in the absence of FVIII (e.g., in FVIII-depleted plasma) to at least 1% of the TFPI-dependent thrombin generation level in normal plasma. Generally, normal (unperturbed) plasma contains approximately 0.5 U / mL to approximately 2 U / mL of factor VIII. Thus, in some cases, the antibodies or antigen-binding fragments of the present invention will enhance thrombin formation in the absence of FVIII to at least about 1% of that observed in the presence of 0.5 U / mL to 2 U / mL of FVIII. In a further embodiment, the antibody (or an antigen-binding fragment thereof) enhances thrombin formation in the absence of FVIII to at least about 2%, at least about 3%, at least about 5%, at least about 7%, or at least about 10% of the thrombin formation level in normal plasma (i.e., in the presence of physiological levels of factor VIII).

[0747] The antibody or antigen-binding fragment can also be administered to animal models of thrombin deficiency or hemophilia to characterize the in vivo inhibitory activity of TFPI. Such in vivo models are known in the art and include, for example, mice in which hemophilia A is induced by administration of an anti-FVIII antibody (Tranholm et al., Blood, 102, 3615-3620 (2003)); coagulation factor knockout models such as, 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)); rabbit-induced hemophilia A (Shen et al., Blood, 42(4):509-521 (1973)); and Chapel Hill HA dogs (Lozier et al., Proc. Nat. Acad. Sci. USA 99:12991-12996 (2002)).

[0748] In certain embodiments, the antibody (or antigen-binding fragment) disclosed herein enhances FXa activity in the presence of TFPI, with a half-maximal effective concentration (EC 50 ) not exceeding 1×10 -4 M, not exceeding 1×10 -5 M, not exceeding 1×10 -6 M, not exceeding 1×10 -7 M, not exceeding 1×10 -8 M, not exceeding 1×10 -9 M, not exceeding 1×10 -10 M, not exceeding 1×10 -11 M or not exceeding 1×10 -12 M. Preferably, the EC 50 is from about 5×10 -7 M to 1×10 -11 M, such as from about 1×10 -7 M to 5×10 -10 M, from about 1×10 -7 M to 1×10 -10 M, 1×10 -7 M to 5×10 -9 M, 5×10 -7 M to 5×10 -10 M, from about 5×10 -7 M to 1×10 -10 M or from about 5×10 -7 M to 5×10 -9 M.

[0749] In certain embodiments, the antibodies (or antigen-binding fragments) of the invention neutralize the inhibition of FVIIa / TF-mediated FX activation by TFPI, and the half maximal effective concentration (EC 50 ) is not more than 1×10 -4 M, not more than 1×10 -5 M, not more than 1×10 -6 M, not more than 1×10 -7 M, not more than 1×10 -8 M, not more than 1×10 -9 M, not more than 1×10 -10 M, not more than 1×10 -11 M or not more than 1×10 -12 M. Preferably, the EC 50 is about 5×10 -7 M to 1×10 -11 M, such as about 1×10 -7 M to 5×10 -10 M, about 1×10 -7 M to 1×10 -10 M, 1×10 -7 M to 5×10 -9 M, 5×10 -7 M to 5×10 -10 M, about 5×10 -7 M to 1×10 -10 M or about 5×10 -7 M to 5×10 -9 M.

[0750] In certain embodiments, as measured in a plasma-based diluted prothrombin time assay, the antibodies (or antigen-binding fragments) disclosed by the invention reduce clotting time, and the half maximal effective concentration (EC 50 ) is not more than 1×10 -4 M, not more than 1×10 -5 M, not more than 1×10 -6 M, not more than 1×10 -7 M, not more than 1×10 -8 M, not more than 1×10 -9 M, not more than 1×10 -10 M, not more than 1×10 -11 M or not more than 1×10 -12 M. Preferably, the EC 50 is about 5×10 -7 M to 1×10 -11 M, such as about 1×10 -7 M to 5×10 -10 M, about 1×10 -7 M to 1×10-10 M, 1 × 10 -7 M to 5 × 10 -9 M, 5 × 10 -7 M to 5 × 10 -10 M, approximately 5 × 10 -7 M to 1 × 10 -10 M or approximately 5 × 10 -7 M to 5 × 10 -9 M.

[0751] In certain embodiments, the antibodies (or antigen-binding fragments) disclosed herein increase the thrombin generation rate index, and the half-maximal effective concentration (EC 50 ) does not exceed 1 × 10 -4 M, does not exceed 1 × 10 -5 M, does not exceed 1 × 10 -6 M, does not exceed 1 × 10 -7 M, does not exceed 1 × 10 -8 M, does not exceed 1 × 10 -9 M, does not exceed 1 × 10 -10 M, does not exceed 1 × 10 -11 M or does not exceed 1 × 10 -12 M. Preferably, the EC 50 is approximately 5 × 10 -7 M to 1 × 10 -11 M, such as approximately 1 × 10 -7 M to 5 × 10 -10 M, approximately 1 × 10 -7 M to 1 × 10 -10 M, 1 × 10 -7 M to 5 × 10 -9 M, 5 × 10 -7 M to 5 × 10 -10 M, approximately 5 × 10 -7 M to 1 × 10 -10 M or approximately 5 × 10 -7 M to 5 × 10 -9 M.

[0752] In certain embodiments, the antibodies and antibody fragments disclosed herein can also be further evaluated by other bioactivity assays, e.g., to assess their potency, pharmaceutical activity, and potential efficacy as therapeutic agents. These assays are known in the art and depend on the target antigen and the intended use of the antibody. Examples include, for example, tumor cell growth inhibition assays; antibody-dependent cell cytotoxicity (ADCC) and complement-mediated cytotoxicity (CDC) assays; agonist activity or antagonist activity assays.

[0753] C. Polynucleotides, Vectors, and Host Cells

[0754] The present invention also provides a polynucleotide encoding any TFPI-binding antibody of the present disclosure, including the antibody fragments and modified antibodies described in the present invention, e.g., an antibody having attenuated Fc effector function. In another aspect, the present invention provides a method of preparing any polynucleotide described in the present invention. The polynucleotide can be prepared and expressed by methods known in the art. Accordingly, the present invention provides a polynucleotide or a composition (including a pharmaceutical composition comprising a polynucleotide encoding any TFPI antibody of the present invention and its antigen-binding fragment).

[0755] In one embodiment, the VH and VL domains, or their antigen-binding fragments, or the full-length HC or LC are encoded by different polynucleotides. Alternatively, both VH and VL, or their antigen-binding fragments, or HC and LC are encoded by a single polynucleotide.

[0756] The present invention provides a polynucleotide or a composition comprising a polynucleotide encoding any TFPI antibody of the present invention and its antigen-binding fragment (including, but not limited to, TFPI-23, TFPI-24, TFPI-106, TFPI-107, and 4D8), wherein the sequence of the polynucleotide comprises 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).

[0757] In another aspect, the present invention provides an isolated nucleic acid encoding the VH region or an antigen-binding portion thereof of an antibody that specifically binds TFPI, which comprises the nucleic acid sequence of the insert present in the plasmid deposited with ATCC accession number PTA-122329.

[0758] In another aspect, the present invention provides an isolated nucleic acid encoding the VL region or an antigen-binding portion thereof of an antibody that specifically binds TFPI, which comprises the nucleic acid sequence of the insert present in the plasmid deposited with ATCC accession number PTA-122328.

[0759] In another aspect, the present invention provides polynucleotides encoding TFPI antibodies or portions thereof and variants thereof, wherein these variant polynucleotides comprise nucleic acid sequences having 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 to any of the specific nucleic acid sequences disclosed in the present invention. The degree of sequence identity over any length of nucleotide sequence can be calculated using methods well known to those of ordinary skill in the art. In a non-limiting embodiment, the percentage of sequence identity between two or more related nucleotide sequences can be determined using the nucleotide BLAST server obtained from the National Library of Medicine (http: / / blast.ncbi.nlm.nih.gov / ). The software provides different settings such that those of ordinary skill in the art can optimize sequence comparisons based on factors such as length, complexity, and other factors.

[0760] The present invention provides nucleic acid molecules comprising nucleotide sequences encoding the amino acid sequences of any of the TFPI antibodies of the present invention and antigen-binding fragments thereof (including but not limited to the amino acid sequences of the antibodies or antigen-binding fragments thereof provided in Table 4, such as the amino acid sequences of SEQ ID NO: 21-174), and the amino acid sequences of any antibodies that bind to the same epitope and / or compete with the antibodies of the present invention for binding to TFPI.

[0761] In another aspect, the present invention provides polynucleotides encoding TFPI antibodies and variants thereof, wherein these variant polynucleotides encode amino acid sequences having 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 to any of the TFPI antibody amino acid sequences disclosed in the present invention.

[0762] In other embodiments, the degree of relatedness between a nucleic acid comprising a variant polynucleotide sequence encoding a TFPI antibody or a portion thereof and any particular nucleotide sequence disclosed herein can be determined by testing whether the variant sequence (or its complement) can hybridize to the particular nucleotide sequence (or its complement) in a Northern or Southern blot assay format under moderately or highly stringent conditions. Exemplary and non-limiting "moderately stringent conditions" include prewashing in a solution of 5×SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0); hybridizing at 50°C to 65°C in 5×SSC overnight; followed by washing twice at 65°C for 20 minutes each with 2×, 0.5×, and 0.2× SSC containing 0.1% SDS.

[0763] Exemplary and non-limiting "highly stringent conditions" or "high stringency conditions" refer to the following: (1) using low ionic strength and high temperature for washing, such as at 50°C, 0.015 sodium chloride / 0.0015 sodium citrate / 0.1% sodium dodecyl sulfate; (2) using a denaturing agent during hybridization at 42°C, such as formamide (e.g., 50% (v / v) formamide) with 0.1% bovine serum albumin / 0.1% Ficoll / 0.1% polyvinylpyrrolidone / 50 mM sodium phosphate buffer, pH 6.5, plus 750 mM sodium chloride, 75 mM sodium citrate; or (3) using 50% formamide, 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 at 42°C, and washing at 42°C with 0.2×SSC (sodium chloride / sodium citrate) and at 55°C with 50% formamide, followed by washing at 55°C with a high stringency wash consisting of 0.1×SSC containing EDTA. Those skilled in the art will recognize how to adjust temperature, ionic strength, etc. as needed to accommodate factors such as probe length. Those of ordinary skill in the art will also be familiar with standard techniques for performing Northern or Southern blot assays to detect the degree of relatedness between a nucleotide sequence variant and a particular nucleotide sequence of the present disclosure.

[0764] The invention also encompasses polynucleotides that are complementary to any of these sequences. The polynucleotides can be single-stranded (coding or antisense) or double-stranded, and can be DNA (genomic, cDNA, or synthetic) or RNA molecules. RNA molecules include hnRNA molecules (which contain introns and correspond to DNA molecules in a one-to-one manner) and mRNA molecules that do not contain introns. Additional coding or non-coding sequences can, but need not, be present in the polynucleotides of the invention.

[0765] Alternatively, the variant may also be substantially homologous to a native gene, or a portion or complement thereof. These polynucleotide variants are capable of hybridizing to the native DNA sequence (or complementary sequence) encoding the native antibody under moderately stringent conditions.

[0766] One of ordinary skill in the art will appreciate that due to the degeneracy of the genetic code, there are many nucleotide sequences that can encode any of the TFPI antibodies or portions thereof disclosed herein. Some of these polynucleotides may have a relatively low sequence identity with any particular nucleotide sequence of the TFPI antibodies provided herein, but encode the same amino acid sequence. However, polynucleotides that vary due to differences in codon usage are specifically contemplated by the present invention.

[0767] The polynucleotides of the present invention can be obtained using chemical synthesis, recombinant methods, or PCR. Chemical polynucleotide synthesis methods are well known in the art and need not be described in detail herein. One of ordinary skill in the art can use the sequences provided herein and commercially available DNA synthesizers to generate the desired DNA sequences.

[0768] In the case of preparing polynucleotides using recombinant methods, the polynucleotide containing the desired sequence can be inserted into a suitable vector, and then the vector can be introduced into a suitable host cell for replication and amplification (as further discussed herein). The polynucleotide can be inserted into the host cell by any method known in the art. The cell can be transformed by direct uptake, endocytosis, transfection, F-mating, or electroporation to introduce the exogenous polynucleotide. Once introduced, the exogenous polynucleotide can be maintained within the cell in the form of a non-integrating vector (e.g., plasmid) or integrated into the host cell genome. The polynucleotide so amplified can be isolated from the host cell by methods well known in the art. See, e.g., Sambrook et al., 1989.

[0769] Alternatively, PCR allows the replication of DNA sequences. PCR techniques are well known in the art and are described in U.S. Patent Nos. 4,683,195, 4,800,159, 4,754,065, and 4,683,202, and in PCR: The Polymerase Chain Reaction, Mullis et al. eds., Birkauswer Press, Boston, 1994.

[0770] RNA can be obtained by using the isolated DNA in a suitable vector and inserting it into a suitable host cell. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those of ordinary skill in the art (e.g., as listed in Sambrook et al., 1989 (supra)).

[0771] Suitable cloning vectors can be constructed according to standard techniques or selected from a large number of cloning vectors available in the art. Although the selected cloning vector can vary depending on the host cell to be used, useful cloning vectors generally have the ability to self-replicate, may possess a single target for a particular restriction endonuclease, and / or may carry a gene that can be used as a marker for selection of clones containing the vector. Suitable examples include plasmids and bacteriophages, such as pUC18, pUC19, Bluescript (such as pBS SK+), and their derivatives, mp18, mp19, pBR322, pMB9, ColE1, PCR1, RP4, phage DNA, and shuttle vectors, such as PSA3 and pAT28. These and many other cloning vectors are available from commercial suppliers such as BioRad, Stratagene, and Invitrogen.

[0772] Expression vectors are further provided. An expression vector is generally a replicable polynucleotide construct containing a polynucleotide according to the invention. This means that the expression vector must be able to replicate in a host cell, either as an episome or as an integral part of chromosomal DNA. Suitable expression vectors include, but are not limited to, plasmids, viral vectors (including adenovirus, adeno-associated virus, retrovirus, cosmid), and the expression vectors disclosed in PCT Publication No. WO 87 / 04462. Vector components generally may include, but are not limited to, one or more of the following: a signal sequence; an origin of replication; one or more marker genes; suitable transcriptional control elements (such as promoters, enhancers, and terminators). For expression (i.e., translation), one or more translational control elements, such as ribosome binding sites, translation initiation sites, and termination codons, are generally also required.

[0773] The vector and / or the polynucleotide itself containing the polynucleotide of interest can be introduced into a host cell by any of a variety of suitable means, including electroporation, transfection using calcium chloride, rubidium chloride, calcium phosphate, DEAE-dextran, or other substances; microprojectile bombardment; liposome transfection; and infection (such as where the vector is an infective agent, such as vaccinia virus). The choice of introducing the vector or polynucleotide generally depends on the characteristics of the host cell.

[0774] The present invention also provides host cells comprising any of the polynucleotides described herein. Any host cell capable of overexpressing heterologous DNA can be used to isolate genes encoding antibodies, polypeptides, or proteins of interest. Non-limiting examples of mammalian host cells include, but are not limited to: simian COS, human HeLa, human embryonic kidney (HEK) 293, Sp2.0, and Chinese hamster ovary (CHO) cells. See also PCT Publication No. WO 87 / 04462. Suitable non-mammalian host cells include prokaryotes (e.g., Escherichia coli (E. coli) or Bacillus subtilis (B. subtillis)) and yeast (e.g., Saccharomyces cerevisiae (S. cerevisae), Schizosaccharomyces pombe (S. pombe); or Kluyveromyces lactis (K. lactis)). Screening of host cells expressing TFPI antibodies or antigen-binding portions thereof can be detected using immuno-binding assays, such as ELISA, FACS, or other assays familiar to those of ordinary skill in the art.

[0775] Accordingly, the antibodies (or antigen-binding fragments thereof) of the present invention can be recombinantly produced using suitable host cells. Nucleic acids encoding the antibody or antigen-binding fragment thereof can be cloned into an expression vector, which can then be introduced into host cells that do not otherwise produce immunoglobulins (e.g., E. coli cells, yeast cells, insect cells, COS cells, CHO cells, or myeloma cells) to obtain monoclonal antibodies synthesized in the recombinant host cells. Exemplary host cells include CHO cells, HEK 293, and Sp2.0 cells.

[0776] Expression vectors can be used to directly express TFPI antibodies. Those skilled in the art are familiar with how to administer expression vectors to obtain in vivo expression of foreign proteins. See, for example, U.S. Patent Nos. 6,436,908; 6,413,942; and 6,376,471. Administration of the expression vector includes local or systemic administration, including injection, oral administration, particle gun or catheter insertion administration, and local application. According to certain non-limiting embodiments, the expression vector is directly administered to the liver, skeletal muscle, bone marrow, or other tissues.

[0777] Therapeutic compositions containing expression vectors or subgenomic polynucleotides can also be used for targeted delivery. 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, J.A. Wolff, 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. In gene therapy protocols, therapeutic compositions containing polynucleotides for local administration are administered in the range of about 100 ng to about 200 mg of DNA. Concentrations in the range of about 500 ng to about 50 mg, about 1 μg to about 2 mg, about 5 μg to about 500 μg, about 20 μg to about 100 μg of DNA can also be used during gene therapy protocols. Therapeutic polynucleotides and polypeptides can be delivered using gene delivery vehicles. The gene delivery vehicle can be of viral or non-viral origin (see generally 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 these coding sequences can be induced using endogenous mammalian promoters or heterologous promoters. The expression of the coding sequences can be constitutive or regulated.

[0778] Viral-based vectors for delivering a desired polynucleotide and expressing it in a desired cell are well known in the art. Exemplary viral-based vectors include, but are not limited to, recombinant retroviruses (see, e.g., PCT Publication Nos. WO 90 / 07936; WO 94 / 03622; WO 93 / 25698; WO 93 / 25234; WO 93 / 11230; WO 93 / 10218; WO 91 / 02805; U.S. Patent Nos. 5,219,740 and 4,777,127; GB Patent No. 2,200,651; and EP Patent No. 0345 242), alphavirus-based vectors (e.g., Sindbis virus vectors, Semliki forest virus (ATCC VR-67; ATCC VR-1247), Ross River virus (ATCC VR-373; ATCC VR-1246), and Venezuelan equine encephalitis virus (ATCC VR-923; ATCC VR-1250, ATCC VR 1249; ATCC VR-532)), and adeno-associated virus (AAV) vectors (see, e.g., PCT Publication Nos. WO 94 / 12649, WO 93 / 03769; WO 93 / 19191; WO 94 / 28938; WO 95 / 11984, and WO 95 / 00655). DNA conjugated to inactivated adenovirus as described in Curiel, Hum. Gene Ther., 1992, 3:147 can also be used.

[0779] Non-viral delivery vehicles and methods can also be employed, including but not limited to polycationic condensed DNA conjugated or unconjugated to a separate inactivated adenovirus (see, e.g., Curiel, Hum. Gene Ther., 1992, 3:147); ligand-conjugated DNA (see, e.g., Wu, J. Biol. Chem., 1989, 264:16985); eukaryotic cell delivery vehicle cells (see, e.g., U.S. Patent No. 5,814,482; PCT Publication Nos. WO 95 / 07994; WO 96 / 17072; WO 95 / 30763; and WO 97 / 42338), and nucleic acid charge neutralization or fusion with cell membranes. Naked DNA can also be employed. Exemplary methods for introducing naked DNA are described in PCT Publication No. WO 90 / 11092 and U.S. Patent No. 5,580,859. Liposomes that can serve as gene delivery vehicles are described in U.S. Patent No. 5,422,120; PCT Publication Nos. WO 95 / 13796; WO 94 / 23697; WO 91 / 14445; and EP 0524968. Additional methods are described in Philip, Mol. Cell Biol., 1994, 14:2411 and Woffendin, Proc. Natl. Acad. Sci., 1994, 91:1581.

[0780] The sequences of the desired antibodies (or antigen-binding fragments thereof) and the nucleic acids encoding these antibodies (or antigen-binding fragments thereof) can be determined using standard sequencing techniques. The nucleic acid sequences encoding the desired antibodies (or fragments) can be inserted into other vectors (e.g., cloning and expression vectors) for recombinant production and characterization. The heavy chain (or a fragment of the heavy chain) and the light chain (or a fragment of the light chain) can be cloned in the same vector or in different vectors.

[0781] Suitable cloning and expression vectors can include various components, such as promoters, enhancers, and other transcriptional regulatory sequences. The vector can also be constructed to allow the movement of antibody variable domains between different vectors.

[0782] Antibody fragments can be produced by proteolysis or other antibody degradation methods, by recombinant methods, or by chemical synthesis. The polypeptides of antibodies, especially shorter polypeptides of up to about 50 amino acids, can be conveniently prepared by chemical synthesis. Chemical synthesis methods are known in the art and are commercially available.

[0783] The antibody or antigen-binding fragment thereof disclosed by the present invention may be affinity matured. For example, affinity matured antibodies can be made by methods 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).

[0784] 4. Dosage Forms and Uses

[0785] The antibody or antigen-binding fragment described in the present invention can be formulated into a pharmaceutical dosage form. The pharmaceutical dosage form in the form of a freeze-dried dosage form or an aqueous solution may further comprise a pharmaceutically acceptable delivery agent, excipient or stabilizer (Remington: The Science and practice of Pharmacy 20th Ed., 2000, Lippincott Williams and Wilkins, Ed. K.E. Hoover). The acceptable delivery agent, excipient or stabilizer is non-toxic to the recipient at the dosage and concentration, and may include buffering agents, such as phosphates, citrates and other organic acids; antioxidants, including ascorbic acid and methionine; preservatives (such as octadecyl dimethyl benzyl ammonium chloride; hexamethylene diamine chloride; benzalkonium chloride, benzethonium chloride; phenol, butyl or benzyl alcohol; alkyl p-hydroxybenzoates, such as methyl or propyl p-hydroxybenzoate; catechol; resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin or immunoglobulin; 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; counterions forming salts, such as sodium; metal complexes (such as Zn-protein complexes); and / or nonionic surfactants, such as TWEEN TM , PLURONICS TMor polyethylene glycol (PEG). Pharmaceutically acceptable excipients are further described in the present invention.

[0786] The antibodies or antigen-binding fragments described in the present invention can be used for various therapeutic or diagnostic purposes. For example, the antibody or its antigen-binding fragment can be used as an affinity purifying agent (e.g., for in vitro purification of TFPI), as a diagnostic agent (e.g., for detecting TFPI expression in specific cells, tissues or sera).

[0787] Exemplary therapeutic uses of the antibodies and antibody fragments of the present invention include treating thrombocytopenia, platelet abnormalities (disorders of platelet function or number), and bleeding disorders (such as hemophilia A, hemophilia B, and hemophilia C). The antibodies and antibody fragments can also be used to treat uncontrolled bleeding in indications such as trauma and hemorrhagic stroke. The antibodies and antibody fragments can also be used for prophylactic treatment (e.g., before surgery).

[0788] In particular, the antibodies or antigen-binding fragments described in the present invention can be used to treat coagulation deficiencies or defects. For example, the antibodies or antigen-binding fragments described in the present invention can be used to reduce or inhibit the interaction of TFPI with FXa, or to reduce TFPI-dependent inhibition of TF / FVIIa / FXa activity.

[0789] In terms of therapeutic applications, the antibodies or antigen-binding fragments described in the present invention can be administered to mammals, especially humans, by conventional techniques, such as intravenously (as a bolus or by continuous infusion over a period of time), intramuscularly, intraperitoneally, intracerebrospinally, subcutaneously, intra-articularly, intrasynovially, intrathecally, orally, topically, or by inhalation. The antibodies or antigen-binding fragments are also suitable for administration by intratumoral, peritumoral, intralesional, or perilesional routes.

[0790] Thus, in one aspect, the present invention provides a method for reducing the activity of tissue factor pathway inhibitor (TFPI), which comprises administering a therapeutically effective amount of the antibody or antigen-binding fragment of the present invention to a subject in need thereof. In another aspect, the present invention provides a method for shortening bleeding time, which comprises administering a therapeutically effective amount of the antibody or antigen-binding fragment described in the present invention to a subject in need thereof.

[0791] In certain embodiments, the subject is human.

[0792] In certain embodiments, the subject has or is prone to coagulation defects. Coagulation defects include, for example, von Willebrand disease (VWD), hemophilia A, B or C, and other platelet abnormalities (such as congenital platelet defects, congenital and acquired storage pool deficiencies, prolonged bleeding time).

[0793] In certain embodiments, the antibodies or antigen-binding fragments described herein are administered subcutaneously. In certain embodiments, the antibodies or antigen-binding fragments described herein are administered intravenously.

[0794] The pharmaceutical composition can be administered to a subject in need thereof at different frequencies, which can vary with the severity of the bleeding event and, in the case of prophylactic treatment, may vary with the severity of the patient's coagulation defect.

[0795] The composition can be administered to a patient in need thereof as a bolus or by continuous infusion. For example, for a bolus administration of an antibody presented as a Fab fragment, the amount used can be 0.0025 to 100 mg / kg (body weight), 0.025 to 0.25 mg / kg, 0.010 to 0.10 mg / kg or 0.10 to 0.50 mg / kg. For continuous infusion, the amount of an antibody presented as a Fab fragment administered can be 0.001 to 100 mg / kg (body weight) / minute, 0.0125 to 1.25 mg / kg / minute, 0.010 to 0.75 mg / kg / minute, 0.010 to 1.0 mg / kg / minute or 0.10 to 0.50 mg / kg / minute delivered over a period of 1 to 24 hours, 1 to 12 hours, 2 to 12 hours, 6 to 12 hours, 2 to 8 hours or 1 to 2 hours.

[0796] In the case of administering an antibody in the form of a full-length antibody (with an intact constant region), the dose can be about 1 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 3 mg / kg to about 10 mg / kg, about 4 mg / kg to about 10 mg / kg, about 5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 20 mg / kg, about 2 mg / kg to about 20 mg / kg, about 3 mg / kg to about 20 mg / kg, about 4 mg / kg to about 20 mg / kg, about 5 mg / kg to about 20 mg / kg, about 1 mg / kg or more, about 2 mg / kg or more, about 3 mg / kg or more, about 4 mg / kg or more, about 5 mg / kg or more, more than about 6 mg / kg, about 7 mg / kg or more, about 8 mg / kg or more, about 9 mg / kg or more, about 10 mg / kg or more, about 11 mg / kg or more, about 12 mg / kg or more, about 13 mg / kg or more, about 14 mg / kg or more, about 15 mg / kg or more, about 16 mg / kg or more, about 17 mg / kg or more, about 19 mg / kg or more or about 20 mg / kg or more. The frequency of administration will depend on the severity of the condition. The frequency range can be three times a week to once every two or three weeks.

[0797] In addition, the composition can be administered to a patient by subcutaneous injection. For example, an anti-TFPI antibody in a dose of 1 to 100 mg can be administered to the patient once a day, once every 2 days, once every 3 days, once every 4 days, once every 5 days, once every 6 days, twice a week, once a week, once every two weeks, or once a month by subcutaneous injection.

[0798] In certain embodiments, the pharmaceutical composition is administered subcutaneously on a weekly schedule at a dose of about 0.1 mg / kg to about 10 mg / kg, about 0.5 mg / kg to about 10 mg / kg, about 1 mg / kg to about 10 mg / kg, about 1.5 mg / kg to about 10 mg / kg, about 2 mg / kg to about 10 mg / kg, about 0.1 mg / kg to about 8 mg / kg, about 0.5 mg / kg to about 8 mg / kg, about 1 mg / kg to about 8 mg / kg, about 1.5 mg / kg to about 8 mg / kg, about 2 mg / kg to about 8 mg / kg, about 0.1 mg / kg to about 5 mg / kg, about 0.5 mg / kg to about 5 mg / kg, about 1 mg / kg to about 5 mg / kg, about 1.5 mg / kg to about 5 mg / kg, about 2 mg / kg to about 5 mg / kg, about 0.5 mg / kg, about 1.0 mg / kg, about 1.5 mg / kg, about 2.0 mg / kg, about 2.5 mg / kg, about 3.0 mg / kg, about 3.5 mg / kg, about 4.0 mg / kg, about 4.5 mg / kg, about 5.0 mg / kg, about 5.5 mg / kg, about 6.0 mg / kg, about 6.5 mg / kg, about 7.0 mg / kg, about 7.5 mg / kg, about 8.0 mg / kg, about 8.5 mg / kg, about 9.0 mg / kg, about 9.5 mg / kg, or about 10.0 mg / kg.

[0799] In certain embodiments, the pharmaceutical composition is administered subcutaneously on a weekly schedule at a dose of about 2.0 mg / kg. In certain embodiments, the pharmaceutical composition is administered subcutaneously on a weekly schedule at a dose of about 3.0 mg / kg.

[0800] The antibodies and antibody fragments described in the present invention can be used alone or in combination with other therapies to address hemostatic abnormalities. For example, co-administering one or more antibodies (or antibody fragments) of the present invention with a coagulant (such as factor VIIa, factor VIII, factor IX, or tranexamic acid) is useful for treating hemophilia.

[0801] In one embodiment, a method for treating a coagulation defect or shortening bleeding time is provided, which comprises administering (a) a first amount of an antibody or antigen-binding fragment of the present invention, and (b) a second amount of factor VIII or factor IX. Optionally, factor VII is not co-administered. In another embodiment, a method for treating a coagulation defect or shortening bleeding time is provided, which comprises administering (a) a first amount of an antibody or antigen-binding fragment of the present invention, and (b) a second amount of factor VIII or factor IX. Optionally, factor VII is not co-administered. Those skilled in the art will understand that when treating a coagulation defect, shortening bleeding time may also be referred to as shortening clotting time.

[0802] The present invention also includes a pharmaceutical composition comprising a therapeutically effective amount of an antibody (or antibody fragment) of the present invention and factor VIII or factor IX, wherein the composition does not contain factor VII. "Factor VII" includes factor VII and factor VIIa.

[0803] Biological Deposits

[0804] Representative materials of the present invention were deposited on July 22, 2015 with the American Type Culture Collection (10801 University Boulevard, Manassas, Va. 20110-2209, USA). The plasmid vector mAb-TFPI-106 VH with ATCC accession number PTA-122329 contains a DNA insert encoding the heavy chain variable region of the antibody TFPI-106, and the plasmid vector mAb-TFPI-106 VL with ATCC accession number PTA-122328 contains a DNA insert encoding the light chain variable region of the antibody TFPI-106. This deposit was made in accordance with the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purposes of Patent Procedure (Budapest Treaty). This ensures that the deposited cultures will remain viable for 30 years from the date of deposit. The deposited materials will be made available by the ATCC under the terms of the Budapest Treaty and will be subject to an agreement between Pfizer Inc. and the ATCC, which ensures the permanent and unrestricted availability of the progeny of the deposited cultures upon the issuance of the relevant U.S. patent or upon the opening to the public of any U.S. or foreign patent application (whichever is earlier), and ensures the availability of the progeny as determined by the U.S. Commissioner of Patents and Trademarks in accordance with 35 U.S.C. Section 122 and the Commissioner's rules (including 37 C.F.R. Section 1.14, with particular reference to 886 OG 638).

[0805] The assignee of the present application has agreed to replace another identical substance in a timely manner upon notification if the deposited culture dies, is lost or damaged when cultured under appropriate conditions. The availability of the deposited substance should not be construed as permission to practice the invention contrary to any license granted by any government under its patent laws. Examples

[0806] The present invention is further described in detail by reference to the following experimental examples. Unless otherwise specified, these examples are for illustrative purposes only and are not intended to be limiting. Accordingly, the present invention should not be construed in any way as limited to the following examples, but should be construed as encompassing any and all variations made apparent by the teachings provided herein.

[0807] Example 1: Experimental Materials and Methods

[0808] 1. TFPI Protein Reagents

[0809] The protein reagents used for immunization, phage display selection, and characterization of anti-TFPI antibodies are listed in Table 1, and their sequence IDs are listed in Table 2.

[0810] The TFPI construct (pSMED2 vector) was transiently expressed in HEK293F cells and the conditioned medium was harvested 120 hours after transfection. The protein of interest 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 amidolytic assay of Factor Xa was obtained from Sekisui Diagnostics

[0811] Table 1

[0812] Protein Reagents for Immunization, Phage Display Selection, and Identification of Anti-TFPI Antibodies

[0813]

[0814] Table 1

[0815] TFPI Reagent Sequence Identification Number, Description, and Sequence

[0816]

[0817]

[0818] 2. Antibody Reagents

[0819] Monoclonal antibodies for comparison purposes (reference antibodies 2A8, 2A8-200, 3F18, hz4F36) are listed in Table 3. Antibody descriptions, sources, and sequences are listed in Table 4 (Figure 5). The light and heavy chain sequences were cloned into appropriate vectors and transiently expressed in human embryonic kidney-293 (HEK-293) cells, and then purified by Protein A Sepharose and size exclusion chromatography. Mab2974 was obtained from R&D Systems (Catalog #MAB2974).

[0820] Table 3

[0821] Light chain (LC) CDR1, 2, 3, variable light (VL), constant light (CL), light chain (LC), heavy chain (HC) CDR1, 2, 3, variable heavy (VH), constant heavy (CH), and heavy chain (HC) regions. The sequence compositions of SeqID numbers are in Table 4.

[0822]

[0823]

[0824] 3. TFPI Binding ELISA

[0825] Using the AviTag TM system, recombinant humTFPI K1K2, murTFPI K1K2, cynTFPI K1K2, ratTFPIK1K2, rabTFPI K1K2, or TFPI2 was biotinylated and captured onto a Greiner streptavidin-coated 96-well plate at a concentration of 1×10 -8 M in ELISA assay buffer. The purified anti-TFPI antibody was diluted to 1 μg / ml in ELISA assay buffer and serially diluted 3-fold to generate an 8-point dilution series. Diluted antibody with a volume of 100 μL was added to each well. The plate was incubated at room temperature for 2 hours. After washing the plate with PBS / 0.05% Tween20, the plate was incubated with a goat anti-mouse IgG-Fc polyclonal antibody conjugated to horseradish peroxidase (Pierce) diluted 1:10,000. After 1 hour of incubation, bound antibody was detected by adding the TMB substrate solution. Absorbance at 450 nm was read and the data were analyzed by GraphPad Prism software.

[0826] 4. Surface Plasmon Resonance (SPR)

[0827] An anti-human Fc sensor chip was 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) for 7 minutes at a flow rate of 10 μl / minute. The anti-human IgG antibody was diluted to 25 μg / ml in 10 mM sodium acetate (pH 5.0) and injected onto all flow cells at a flow rate of 10 μl / minute for 7 minutes. All flow cells were blocked with 1 M ethanolamine HCl (ETH) for 7 minutes at a flow rate of 10 μl / minute. The final immobilization level of the capture 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, 0.05% (v / v) Tween-20 (HBS-EP+). To characterize the binding of the anti-TFPI antibody to human TFPI, the antibody was diluted to 0.5 μg / ml in HBS-EP+ and captured by the anti-human IgG immobilized in flow cells 2, 3, and 4 at a flow rate of 5 μL / minute for 30 seconds to 1 minute to achieve a capture level of 70 to 300 RU. Flow cell 1 was used as a reference surface. After capturing the antibody, the flow rate was increased to 50 μL / minute and human TFPI in the concentration range of 0.2 nM to 200 nM in buffer or in HBS-EP+ was injected onto all flow cells for 1.0 minute to bind, and then allowed to dissociate for 10 to 15 minutes. The buffer cycle of each captured antibody was collected for dual reference (Myszka, D.G. J. Mol. Recognit. 12, 279-284 (1999)). At the end of each cycle, the entire anti-IgG surface was regenerated by a 60-second pulse of 3M MgCl 2 At 25 °C, kinetic measurements were performed on a BIAcore T200 instrument (GE Healthcare) at a collection rate of 10 Hz. The rate constants and affinities were determined by fitting the data to a 1:1 model of the BIAcore T200 evaluation software version 1.0 (GE).

[0828] 5. Factor Xa TFPI Inhibition Recovery Assay

[0829] In vitro evaluation of the ability of purified anti-TFPI antibodies to restore factor Xa activity in the presence of inhibitory concentrations of TFPI. Anti-TFPI antibodies diluted in PBS in the concentration range of 1 nM to 500 nM were pre-incubated with 10 nM recombinant human TFPI K1K2 or 10 nM rabbit TFPI K1K2 protein in the active buffer (20 mM HEPES, pH 8.0, 150 mM NaCl, 5 mM CaCl 2 , 0.5 mg / mL BSA) at 37 °C for 30 minutes. 2 nM factor Xa derived from human plasma was added and the reaction was incubated at 37 °C for 30 minutes. The chromogenic substrate Spectrozyme Xa was added thereto to a final concentration of 500 μM in a final reaction volume of 100 μl. Control reactions included reactions without factor Xa to control the assay background, without TFPI to allow maximum generation of FXa (100% activity), or without anti-TFPI antibody (PBS alone). Immediately, the absorbance of the reaction was read at 405 nm every 2 minutes over a 60-minute period on a SpectraMax M5e multimode plate reader. The EC 50 .

[0830] 6. Two-stage TF-FVIIa-FX inhibition recovery assay

[0831] In vitro evaluation of the ability of purified anti-TFPI antibodies to restore factor VIIa-tissue factor activity in the presence of inhibitory concentrations of TFPI. Anti-TFPI antibodies in the concentration range of 1 nM to 500 nM were pre-incubated with 10 nM recombinant TFPIK1K2 protein in the active buffer at 37 °C for 30 minutes. Approximately 1 pM lipidated tissue factor and 1 nM recombinant factor VIIa (NovoSeven) were added to the reaction and incubated at 37 °C for 5 minutes. 150 nM human factor X was introduced into the reaction. The chromogenic substrate Spectrozyme Xa was added to each well to a final concentration of 500 μM in a final reaction volume of 100 μl. Control reactions included reactions without factor VIIa, without tissue factor, without factor X, without TFPI or without anti-TFPI antibody (PBS alone). Immediately, the absorbance of the reaction was read at 405 nm every 2 minutes over a 60-minute period on a SpectraMax M5e multimode plate reader. The EC 50 .

[0832] 7. Thrombin generation assay (TGA)

[0833] The ability of a purified anti-TFPI antibody to restore thrombin generation in factor VIII-depleted plasma was evaluated in a thrombin generation assay using a calibrated automated thrombin generation (CAT) system. Anti-TFPI antibodies at concentrations from 1 nM to 500 nM diluted in PBS were introduced into a reaction mixture containing human factor VIII-deficient plasma and PPP-Low reagent, with 4 μM phospholipids and 1 pM tissue factor. The control reaction mixture used PBS without antibody. A Fluca buffer containing a fluorescent thrombin substrate and CaCl 2 was added to trigger the reaction. Fluorescence of each reaction mixture was read every 20 seconds for 60 minutes using a Fluoroskan Ascent plate reader with Thrombinoscope software. Each reaction mixture was compared to a calibrator control well containing PBS, a thrombin calibrator, factor VIII-deficient plasma, and FLUCA buffer. Thrombinoscope thrombin generation curves (nM thrombin vs. time) were analyzed using Thrombinoscope software (Thrombinoscope BV version) to deduce lag time, peak height, time to peak, and area under the curve or endogenous thrombin potential (ETP). These data were used to calculate a velocity index (peak thrombin concentration / time to peak - lag time).

[0834] The ability of a purified anti-TFPI antibody to restore thrombin generation in factor VIII-depleted rabbit plasma was also evaluated. Normal New Zealand white rabbit plasma was treated with anti-FVIII antibody (GM-8015) at a final concentration of 100 μg / mL or control mouse anti-human IgG2a at a concentration of 100 μg / mL for 60 minutes at 37°C. Shortly before addition to the reaction wells, the rabbit plasma was diluted 1:3 in buffer (20 mM HEPES, 140 mM NaCl). The thrombin generation assay was performed with FVIII-neutralized rabbit plasma as described above.

[0835] 8. Generation of cynomolgus TFPI K2 domain for structural studies

[0836] Cynomolgus (cyno) TFPI K1K2 (Table 1) was expressed in HEK293 cells and the conditioned medium was harvested 120 hours after transfection. Purified Cyno TFPI K1K2120 was incubated with human neutrophil elastase (HNE) at a 1:70 (molar ratio HNE:TFPI) for 120 minutes at room temperature for cleavage. Cyno K1 was separated from cyno K2 using anion exchange chromatography on HQ50 (Poros). Size exclusion chromatography using Superdex 75 was performed as the final purification step. The AviTag residue was trimmed off the C-terminus of the cyno K2 domain using endoprotease AspN.

[0837] 9. Generation of antibody Fab / cyno TFPI K2 complex for structural studies

[0838] According to the manufacturer's (Thermo / Pierce) protocol, anti-TFPI antibodies 4D8.b1, TFPI-23, TFPI-24, 2A8-200 (Table 3), and Mab 2974 (R&D Systems) were digested with immobilized papain. Fab was purified from the digestion using MabSelect SuRe and then complexed with cyno TFPI K2. The Fab / cyno TFPI K2 complex was then concentrated to approximately 16 mg / ml. The concentrate was then used to screen for protein crystallization conditions.

[0839] Example 2. Generation of mouse anti-TFPI antibodies

[0840] 1. Mouse immunization and generation of hybridomas

[0841] A cohort of 5 BALB / c mice were each immunized subcutaneously with a 5 μg mixture of humTFPIK1K2 and 5 μg murTFPI K1K2 proteins emulsified in complete Freund's adjuvant. Subsequently, the mice were immunized 2 times per week with the protein mixture emulsified in incomplete Freund's adjuvant or diluted in PBS. Blood samples were taken on days 17 and 27 (after the fifth and seventh immunizations, respectively) and tested by ELISA for the presence of circulating anti-TFPI antibodies in the serum. On day 27, each mouse received a booster injection of the final protein mixture (10 μg) by intraperitoneal injection. Four days later, the draining lymph nodes (axillary, inguinal, and popliteal) were harvested and the pooled lymph node cells were mixed with P3X63.Ag8.653 cells at a 1:1 ratio and subjected to electro-cell fusion. The fused cells were plated in medium supplemented with FBS (25%), NCTC-109 (12.5%), Glutaman (1%), penicillin-streptomycin (1%), hybridoma cloning supplement (5%), and HAT (1×10 -4M hypoxanthine, 4×10 -7 M aminopterin, and 1.6×10 -5 M thymidine) in RPMI 1640 medium. After 14 days, the binding of the fusion hybridoma culture supernatant to humTFPI K1K2 was tested by ELISA. Antibodies from three hybridomas 4D8, 6B7, and 7A4 were selected for further characterization based on their binding activity and potency in functional assays.

[0842] 2. Cloning and sequencing of anti-TFPI antibodies derived from hybridomas

[0843] RNA was prepared from hybridomas 4D8, 6B7, and 7A4 and the variable region DNA sequences of the antibodies from this expression were cloned by RT-PCR. The PCR products were cloned into TOPO-TA cloning vectors and then sequenced by conventional methods. One pair of heavy-chain and light-chain cDNAs was detected from hybridomas 6B7 and 7A4. Two pairs of heavy-chain and light-chain cDNAs were detected from 4D8.

[0844] Example 3. Characterization of murine hybridoma anti-TFPI antibodies

[0845] The parental hybridomas 4D8, 6B7, and 7A4 were subcloned by limiting dilution to obtain monoclonal hybridoma cell lines. Reactivity with humTFPI K1K2 was screened by ELISA to identify positive subclones and amplify them. The purified antibodies from one subclone of each hybridoma were further characterized.

[0846] 1. TFPI binding

[0847] The binding of the purified anti-TFPI antibodies 4D8.B1, 6B7.C5, and 7A4.D9 to recombinant human and rabbit TFPI proteins was tested by protein-binding ELISA. The EC50 values of each antibody for humTFPI K1K2-aviHis10 and rabTFPI K1K2 are shown in Table 5.

[0848] Table 5

[0849] EC50 (nM) values of murine anti-TFPI monoclonal antibodies for human and rabbit TFPI

[0850] antibody EC50 (nM) humTFPI K1K2 EC50 (nM) rabTFPI K1K2 4D8.B1 0.0959 0.0976 6B7.C5 0.1209 0.1289 7A4.D9 0.0887 0.0887

[0851] Surface plasmon resonance experiments were performed to evaluate the affinity of the purified murine anti-TFPI antibodies for human and rabbit TFPI K1K2 proteins. The k a 、k d and K D values of the binding of each antibody to human and rabbit TFPI K1K2 are shown in Table 6.

[0852] Table 6

[0853] Kinetic measurements of anti-TFPI murine hybridoma clones binding to human and rabbit TFPI

[0854]

[0855]

[0856] 2. In vitro activity assay

[0857] The activity of anti-TFPI murine monoclonal antibodies was tested in the FXa and TF-FXa-FVIIa inhibition recovery assays and the thrombin generation assay (TGA). The most potent antibody, 4D8.B1, was selected for further study.

[0858] Table 7

[0859] Activity of anti-TFPI murine monoclonal antibodies in the FXa and TF-FXa-FVIIa inhibition recovery assays and the thrombin generation assay (TGA)

[0860] antibody FXa EC50 (nM) FXa - FVIIa EC50 (nM) Rate Index of TGA at 20 nM 4D8.B1 5.9 6.67 26.3 6B7.C5 13.9 12.13 23.5 7A4.D9 22.3 9.35 23.3

[0861] Example 4. Generation of chimeric and humanized antibodies from clone 4D8

[0862] 1. Generation of murine-human chimeric antibody 4D8

[0863] The variable region cDNAs derived from hybridoma 4D8 were subcloned into a mammalian expression vector to generate a chimeric antibody in which the murine heavy chain variable region was fused in-frame with human IgG1 3M (SEQ 20, Table 4), and the murine light chain variable region was fused in-frame with the human Igκ constant region (SEQ 62, Table 4). The chimeric construct was transiently transfected into HEK293 cells. Four transient transfections were performed with all possible heavy and light chain combinations to identify the correct heavy and light chain pairs from hybridoma 4D8. The antibody produced from one of the transfections was designated the hu-mu 4D8 chimera (Tables 3 and 4).

[0864] 2. Characterization of murine-human chimeric antibody 4D8 (hu-mu 4D8)

[0865] The ability of the Mu-hu 4D8 chimera to bind to human and rabbit TFPIK1K2 proteins was tested by protein-binding ELISA (Table 8) and SPR (Table 9). The KD and EC50 values were very comparable to those measured for purified murine Mab 4D8.B1, demonstrating that the transplantation of murine variable regions into the human IgG1 background retained binding activity.

[0866] Table 8

[0867] EC50 (nM) values of mu - hu 4D8 chimeras for human and rabbit TFPI

[0868] antibody EC50 (nM) humTFPI K1K2 EC50 (nM) rabTFPI K1K2 mu - hu 4D8 chimera 0.0577 0.0680

[0869] Table 9

[0870] Kinetic measurements of mu - hu 4D8 chimeras for human and rabbit TFPI

[0871] analyte ligand <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (nM)]]> humTFPI K1K2 mu - hu 4D8 chimera <![CDATA[9.58x10 5 > <![CDATA[1.14x10 -3 > 1.19 rabTFPI K1K2 mu - hu 4D8 chimera <![CDATA[2.01x10 6 > <![CDATA[1.26x10 -3 > 0.63

[0872] 3. Humanized hu - mu 4D8 chimeras

[0873] The hu - mu 4D8 chimera sequences were humanized by CDR grafting onto human receptor framework sequences. The DP54 framework and DPK9 framework were selected. Then combinations of heavy and light chain constructs were expressed (see Table 3). The antibodies were tested for binding to human and rabbit TFPI in ELISA binding assays (Table 10) and for binding to human TFPI in SPR binding assays (Table 11).

[0874] Table 10

[0875] EC50 (nM) values of humanized 4D8 antibodies binding to human and rabbit TFPI K1K2 proteins

[0876]

[0877]

[0878] Table 11

[0879] SPR analysis of humanized 4D8 antibodies binding to humTFPI K1K2 protein

[0880] antibody <![CDATA[k a (1 / Ms)]]> <![CDATA[K d (1 / s)]]> <![CDATA[K D , nM]]> <![CDATA[4D8 V k 1.0 x V H 1.0]]> <![CDATA[6.77x10 4 > <![CDATA[4.70x10 -4 > <![CDATA[6.94x10 -9 > <![CDATA[4D8 V k 1.0 x V H 1.1]]> <![CDATA[6.06x10 4 > <![CDATA[1.54x10 -3 > <![CDATA[2.54x10 -8 > <![CDATA[4D8 V k 1.0 x V H 1.2]]> <![CDATA[2.38x10 5 > <![CDATA[1.65x10 -4 > <![CDATA[6.95x10 -10 > <![CDATA[4D8 V k 1.0 x V H 1.3]]> <![CDATA[7.95x10 4 > <![CDATA[5.71x10 -4 > <![CDATA[7.19x10 -9 > <![CDATA[4D8 V k 1.0 x V H 1.4]]> <![CDATA[7.55x10 4 > <![CDATA[8.35x10 -4 > <![CDATA[1.11x10 -8 > <![CDATA[4D8 V k 1.1 x V H 1.0]]> <![CDATA[1.25x10 5 > <![CDATA[8.35x10 -4 > <![CDATA[5.50x10 -10 > <![CDATA[4D8 V k 1.1 x V H 1.1]]> <![CDATA[1.93x10 5 > <![CDATA[1.61x10 -4 > <![CDATA[8.32x10 -10 > <![CDATA[4D8 V k 1.1 x V H 1.2]]> <![CDATA[1.51x10 5 > <![CDATA[9.49x10 -5 > <![CDATA[6.27x10 -10 > <![CDATA[4D8 V k 1.1 x V H 1.3]]> <![CDATA[1.59x10 5 > <![CDATA[1.31x10 -4 > <![CDATA[8.23x10 -10 <!-- 69 -->]]> <![CDATA[4D8 V k 1.1 x V H 1.4]]> <![CDATA[2.21x10 5 > <![CDATA[5.28x10 -5 > <![CDATA[2.93x10 -10 >

[0881] Based on these data, 4D8 Vk 1.1 xVH 1.4 was selected for further characterization and named hz4D8 (Table 3). The activities of the humanized anti - TFPI antibody (hz4D8) and murine 4D8.B1FXa were compared in inhibition recovery assays, two - stage TF - FVIIa - FX inhibition assays, and thrombin generation assays. The data in Table 12 show that the humanized antibody has improved activity in all three assays compared to the murine antibody, indicating that TFPI - binding activity is fully retained in the humanized antibody.

[0882] Table 12

[0883] Comparison of the activities of 4D8.B1 and hz4D8 antibodies in FXa and TF-FXa-FVIIa inhibition recovery assays and thrombin generation assay (TGA)

[0884] antibody FXa EC50 (nM) FXa - FVIIa EC50 (nM) Rate Index of TGA at 20 nM 4D8.B1 4.15 3.5 13.5 Hz4D8 1.87 1.57 15.7

[0885] Example 5. Generation of additional anti-TFPI antibodies by phage display technology

[0886] 1. Selection of anti-TFPI antibodies by phage display

[0887] Single-chain fragment variable (scFv) antibodies that bind recombinant human and mouse TFPI K1K2 were identified after 4 rounds of 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. Bound phages were eluted by incubation with 140 mM triethanolamine (TEA) pH 11.5 or 50 mM MES pH 5.5 for 10 minutes on a rotary shaker at room temperature and neutralized with 1 M Tris-HCl pH 7.5.

[0888] The eluted phage library was used to infect 10 mL of Escherichia coli ER2738 culture grown to mid-log phase (equivalent to an OD 600 of approximately 0.5). The bacteria were infected with phages for 30 minutes at 37°C without shaking, concentrated by centrifugation and plated, and then grown overnight at 30°C. In the next round of selection, the phages were rescued by inoculating into 25 mL of 2×TYAG / tetracycline to an OD 600 of approximately 0.1 and then growing it to an OD 600 of 0.3 to 0.5 at 37°C. The cells were super-infected with MK13K07 helper phage at a ratio of 1:20 cells / helper phage and incubated for 30 minutes at 37°C without shaking, then shaken at 150 rpm for 60 minutes. The cells were then centrifuged and the pellet was resuspended in medium containing kanamycin / glucose-free. The culture was grown overnight at 25°C. The phages in the supernatant were harvested after centrifugation and used for the next round of selection.

[0889] 2. Preparation of crude periplasmic material for ELISA assay

[0890] Depending on the growth conditions used, the ScFv antibody fragment can be expressed on the surface of phage particles or in solution in the bacterial periplasmic space. To induce the release of the scFv antibody fragment into the periplasm, inoculate from a thawed glycerol stock into a 96-deep well plate containing 2X TY medium with 0.1% glucose / 100 μg / mL ampicillin and grow at 37 °C for about 4 hours. Release the contents of the bacterial periplasm (peripreps) by osmotic shock. Centrifuge the plate and harvest the supernatant containing the scFv.

[0891] 3. ELISA measurement of the binding of scFv expressed in the periplasm to human and mouse TFPI K1K2

[0892] A total of 1984 clones were picked from the 2nd, 3rd, and 4th rounds randomly selected from all branches. TFPI scFv binders were identified by periplasm preparation (periprep) binding ELISA. Biotinylated human and mouse TFPI K1K2 at a concentration of 1 μg / mL in PBS were coated onto a 384-well Nunc Maxisorp streptavidin plate. The TFPI K1K2 solution was removed and the plate was blocked with 0.05% Tween 20 / 1% BSA / PBS at room temperature for 1 hour. Prepare the periplasm preparation and block it with an equal volume of 6% milk / 1% BSA at room temperature for 1 hour. Transfer 20 μl / well of the blocked periplasmic scFv and control antibodies to the appropriate plate and incubate at room temperature for 1 hour. Add anti-myc horseradish peroxidase (HRP) diluted 1:2,000 or goat anti-human HRP secondary antibody diluted 1:10,000 to detect the bound scFv or anti-TFPI control antibody. Develop the signal using 3,3′,5,5′-tetramethylbenzidine and read the absorbance at 450 nm on an Envision plate reader (Perkin Elmer). A total of 883 scFV clones were identified as TFPI binders. The 883 TFPI-binding scFvs were sequenced to identify unique clones. Two hundred and eighty-eight unique clones were selected to test TFPI / FXa competitive binding.

[0893] 4. ELISA identification of scFvs that competitively bind human and mouse TFPI K1K2 with FXa

[0894] A total of 288 unique clones were tested in the FXa / TFPI competitive binding ELISA. Human FXa at a concentration of 1 μg / mL in PBS was coated onto 384-well Nunc Maxisorp plates overnight. The FXa solution was removed and the plates were blocked with 0.05% Tween 20 / 1% BSA / PBS for 1 hour at room temperature. The periplasmic preparations were prepared and blocked with 6% milk / 1% BSA for 1 hour at room temperature. 20 μl / well of the blocked periplasmic scFv and control antibodies were mixed with biotinylated humTFPI K1K2 and incubated for 1 hour at room temperature. The mixture was transferred to the FXa-coated plates and incubated for 1 hour at room temperature. Streptavidin horseradish peroxidase diluted 1:2000 was added to detect the bound TFPI. The signal was developed using 3,3′,5,5′-tetramethylbenzidine and the absorbance at 450 nm was read on an Envision plate reader (Perkin Elmer). A total of 48 scFV antibodies were classified as competitive inhibitors of TFPI / FXa binding.

[0895] 5. ScFv conversion to human IgG

[0896] A total of 48 scFV antibodies with unique sequences that showed binding to TFPI and inhibition in the TFPI / FXa competitive ELISA were selected for subcloning into the human IgG-3M cloning vector. Briefly, the fragments were amplified by standard PCR. The VH or VL fragments were gel purified and ligated into mammalian expression vectors containing the human IgG1-3M (VH) or κ or λ constant regions (VK / VL). Then, the expression vectors paired with VH and VK / VL were used for transient mammalian expression and purification in HEK293 cells.

[0897] 6. Characterization of human IgG-3M anti-TFPI antibodies

[0898] The 48 anti-TFPI antibodies were ranked in various assays including FXa and TF / FVII / FXa inhibition recovery assays. TFPI-3, TFPI-21, TFPI-23, TFPI-24, and TFPI-26 had the desired properties, such as cross-species TFPI and low or no binding to humTFPI2 K1K2K3 (Table 13). The FXa and TF / FVIIa / FXa inhibition recovery assay data for these same 5 antibodies are shown in Table 14, while the SPR binding data are shown in Table 15.

[0899] Table 13

[0900] TFPI antibodies showed binding to multiple TFPI species (human, monkey, mouse, rabbit, and rat) and weak or no binding to human TFPI2

[0901] Table 14

[0902] Activity of TFPI antibodies in FXa, TF / FVIIa / FXa inhibition recovery assays and thrombin generation assays

[0903] antibody FXa EC50 (nM) FXa - FVIIa EC50 (nM) TGA Velocity Index at 20 nM TFPI - 3 207.9 37.4 NT TFPI - 21 80.64 54.1 24.71 TFPI - 23 46 32.2 23.28 TFPI - 24 23 9.3 23.21 TFPI - 26 12.8 9.6 18.65

[0904] Table 15

[0905] SPR binding kinetics of TFPI antibodies to human and murine TFPI

[0906] analyte ligand <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (nM)]]> humTFPI K1K2 TFPI - 3 <![CDATA[6.25x10 4 > <![CDATA[1.7x10 -3 > 27.05 humTFPI K1K2 TFPI - 21 <![CDATA[7.85x10 5 > <![CDATA[3.47x10 -2 > 43.95 humTFPI K1K2 TFPI - 23 <![CDATA[9.18x10 5 > <![CDATA[9.71x10 -3 > 9.89 humTFPI K1K2 TFPI - 24 <![CDATA[1.59x10 5 > <![CDATA[8.62x10 -4 > 5.46 humTFPI K1K2 TFPI - 26 <![CDATA[2.05x10 5 > <![CDATA[1.18x10 -3 > 5.79 murTFPI K1K2 TFPI - 3 <![CDATA[2.15x10 5 > <![CDATA[8.24x10 -4 > 3.81 murTFPI K1K2 TFPI - 21 <![CDATA[1.73x10 6 > <![CDATA[7.71x10 -3 > 4.54 murTFPI K1K2 TFPI - 23 <![CDATA[1.69x10 6 > <![CDATA[2.48x10 -3 > 1.46 murTFPI K1K2 TFPI - 24 <![CDATA[2.48x10 5 > <![CDATA[5.96x10 -3 > 24.1 murTFPI K1K2 TFPI - 26 <![CDATA[1.29x10 6 > <![CDATA[4.76x10 -3 > 3.68

[0907] Example 6. Epitope mapping of anti-TFPI antibodies by SPR

[0908] Use a sandwich SPR assay to map the epitopes of anti-TFPI antibodies (TFPI-21, TFPI-23, TFPI-24, 4D8, 6B7.c5, and 7A4.D9) discovered and disclosed herein. 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 first injected onto the chip until binding approached apparent equilibrium. Immediately after stopping the injection of human TFPI, 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, then antibody 2 has a different and non-overlapping TFPI binding epitope relative to antibody 1 (denoted as +). If antibody 2 shows no binding, it is denoted as having a significantly overlapping epitope relative to antibody 1 (negative (-)). If antibody 2 shows weak binding, antibodies 1 and 2 are considered to have partially overlapping TFPI epitopes (denoted as + / -). As shown in Table 16, TFPI-21 and TFPI-23 have similar epitopes and the data also shows that TFPI-21 and TFPI-23 have completely different epitopes from mab2974 and hz4F36.

[0909] Table 16

[0910] Epitope mapping of anti-TFPI antibodies using an SPR sandwich assay

[0911]

[0912] Antibody 1 was immobilized on the surface of a CM5 chip. Then human TFPI (humTFPI K1K2) was injected onto the surface until the measurement approached apparent equilibrium. Immediately after stopping the TFPI injection, antibody 2 was injected to measure the binding to the antibody 1 / TFPI complex. Antibody 1 and 2 pairs with completely different epitopes were given a "+" notation. Antibody pairs with strongly overlapping epitopes were given a "-" notation. If antibody 2 showed weak binding, antibody 1 and 2 were considered to have some partial overlap in the TFPI epitope (denoted as + / -).

[0913] Example 7. Human framework of germline TFPI-23 antibody

[0914] Two variants of TFPI-23 were prepared to increase the content of human framework germline residues. TFPI-106 contains mutations from H1Q to E and H5V to L (Kabat numbering), while TFPI-107 (Tables 3 and 4) contains mutations from H1Q to E, H5V to L, and H94I to K (Kabat numbering). TFPI-106, TFPI-107, and TFPI-23 were expressed and their binding to humTFPI K1K2 was tested by SPR after purification. The data in Table 17 show that the TFPI-106 germline variant retains all binding affinity when compared to the TFPI-23 parental antibody.

[0915] Table 17

[0916] SPR binding kinetics of TFPI-23 human framework germline variants to human TFPI

[0917] analyte ligand <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (nM)]]> humTFPI K1K2 TFPI - 23 <![CDATA[9.18x10 5 > <![CDATA[9.71x10 -3 > 9.89 humTFPI K1K2 TFPI - 106 <![CDATA[2.72x10 6 > <![CDATA[9.74x10 -3 > 3.7 humTFPI K1K2 TFPI - 107 - - No binding

[0918] When compared to the parental TFPI-23 antibody, TFPI-106 showed a moderately increased binding.

[0919] Example 8. Human framework of germline TFPI-24 antibody

[0920] Four TFPI-24 VL variants (TFPI-110, TFPI-111, TFPI-112, TFPI-113) were prepared and paired with TFPI-24 VH sequences. Three TFPI-24 VH variants (TFPI-108, TFPI-109, TFPI-114) were prepared and paired with TFPI-24 VL sequences. Based on these data, the best VL variant TFPI-113 was paired with the best VH variant TFPI-108 to generate antibody TFPI-118. The binding of TFPI-118 and TFPI-24 to human TFPI was tested by SPR, and the results in Table 18 show comparable binding kinetics.

[0921] Table 18

[0922] Using SPR to compare the binding kinetics of TFPI-24 and human framework variant TFPI-118 with human TFPI

[0923] analyte ligand <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (nM)]]> humTFPI K1K2 TFPI - 24 <![CDATA[9.18x10 5 > <![CDATA[9.71x10 -3 > 3.68 humTFPI K1K2 TFPI - 118 <![CDATA[1.25x10 5 > <![CDATA[1.19x10 -3 > 9.61

[0924] TFPI-118 showed binding kinetics comparable to the parental TFPI-23 antibody.

[0925] Example 9. SPR Binding Kinetics of Anti-TFPI Antibodies to TFPI from Different Species

[0926] Anti-TFPI antibodies (TFPI-106, TFPI-118, and hz4F36) were analyzed by SPR to determine the binding kinetics to TFPI from different animal species (human (huTFPI K1K2), cynomolgus monkey (cynTFPI K1K2), rabbit (rabTFPI K1K2), mouse (murTFPI K1K2), and rat (ratTFPI K1K2); Table 1). Three comparitor antibodies (hz4F36, 2A8, and 2A8-200) were also included in this experiment.

[0927] Table 19

[0928] SPR Binding Kinetics of Anti-TFPI Antibodies to Human, Cynomolgus Monkey, Rabbit, Rat, and Mouse TFPI (K d values are the average of two experiments)

[0929] analyte ligand <![CDATA[k a (1 / Ms)]]> <![CDATA[k d (1 / s)]]> <![CDATA[K D (nM)]]> humTFPI K1K2 TFPI - 106 <![CDATA[2.72x10 6 > <![CDATA[9.74x10 -3 > 3.7 humTFPI K1K2 TFPI - 118 <![CDATA[1.25x10 5 > <![CDATA[1.19x10 -3 > 9.61 humTFPI K1K2 hz4D8 <![CDATA[3.16x10 6 > <![CDATA[1.32x10 -3 > 0.42 humTFPI K1K2 hz4F36 <![CDATA[1.89x10 6 > <![CDATA[9.30x10 -4 > 0.49 humTFPI K1K2 2A8 <![CDATA[3.55x10 5 > <![CDATA[3.77x10 -3 > 10.6 humTFPI K1K2 2A8-200 <![CDATA[1.16x10 6 > <![CDATA[3.81x10 -3 > 0.327 cynTFPI K1K2 TFPI - 106 <![CDATA[6.55x10 6 > <![CDATA[8.01x10 -3 > 1.22 cynTFPI K1K2 TFPI - 118 <![CDATA[7.21x10 5 > <![CDATA[1.27x10 -3 > 1.8 cynTFPI K1K2 hz4D8 <![CDATA[2.93x10 7 > <![CDATA[1.95x10 -3 > 0.067 cynTFPI K1K2 hz4F36 <![CDATA[6.86x10 6 > <![CDATA[2.88x10 -3 > 0.425 cynTFPI K1K2 2A8 <![CDATA[2.37x10 5 > <![CDATA[3.06x10 -2 > 13.25 cynTFPI K1K2 2A8-200 <![CDATA[1.25x10 6 > <![CDATA[8.09x10 -4 > 0.637 rabTFPI K1K2 TFPI - 106 <![CDATA[3.66x10 6 > <![CDATA[1.55x10 -2 > 4.25 rabTFPI K1K2 TFPI - 118 <![CDATA[2.01x10 5 > <![CDATA[1.55x10 -2 > 5.79 rabTFPI K1K2 hz4D8 <![CDATA[3.16x10 6 > <![CDATA[2.9x10 -3 > 0.502 rabTFPI K1K2 hz4F36 <![CDATA[4.2x10 6 > <![CDATA[7.6x10 -3 > 1.81 rabTFPI K1K2 2A8 <![CDATA[7.3x10 5 > <![CDATA[1.23x10 -3 > 1.69 rabTFPI K1K2 2A8-200 <![CDATA[1.92x10 6 > <![CDATA[2.77x10 -4 > 0.145 murTFPI K1K2 TFPI-106 <![CDATA[4.05x10 6 > <![CDATA[2.32x10 -3 > 0.575 murTFPI K1K2 TFPI-118 <![CDATA[2.19x10 5 > <![CDATA[9.82x10 -3 > 45.6 murTFPI K1K2 hz4D8 - - No binding murTFPI K1K2 hz4F36 - - No binding murTFPI K1K2 2A8 <![CDATA[1.91x10 5 > <![CDATA[6.32x10 -3 > 33.65 murTFPI K1K2 2A8-200 <![CDATA[2.54x10 6 > <![CDATA[1.17x10 -3 > 0.455 ratTFPI K1K2 TFPI-106 <![CDATA[3.01x10 6 > <![CDATA[4.71x10 -3 > 1.57 ratTFPI K1K2 TFPI-118 <![CDATA[4.83x10 5 > <![CDATA[1.75x10 -3 > 3.65 ratTFPI K1K2 hz4D8 - - No binding ratTFPI K1K2 hz4F36 - - No binding ratTFPI K1K2 2A8 - - Not tested ratTFPI K1K2 2A8-200 - - Not tested

[0930] Example 10. Anti-TFPI Antibody / TFPI Complex Structure

[0931] 1.4D8.b1 Fab / cyno TFPI K2 Complex Structure

[0932] 4D8.b1 Fab and cyno TFPI K2 were mixed at a 1:1 molar ratio to form a complex. Final purification was performed using a Superdex200 column. The complex was concentrated to 12.6 mg / ml for structural studies. Crystals of the TFPI K2 + 4D8 Fab complex were obtained in 100 mM Tris-HCl pH 8.5 - 20% PEG 10000. They produced diffraction up to Rod-shaped crystals. The crystals were cryoprotected instantaneously and synchrotron data collection was performed remotely at the Advanced Photon Source. The image frames were processed using the software AutoPROC (Global Phasing Ltd). The data belong to the space group P212121, and the unit cell is as follows: α = β = γ = 90°, and each asymmetric unit has one complex. A molecular replacement search was performed using a homology model of 4D8 Fab and the structures of the publicly available TFPI K2 domain (RSCB Protein Data Bank; PDB codes 1TFX and 4DTG) to generate a convincing solution for each component. Refinement was carried out using the software autoBUSTER (Global Phasing Ltd), and at the final R / Rfree factors were 0.1707 and 0.2424, respectively, and the RMSD of the bonds was The RMSD of the angles was 1.26°. The epitope and paratope of 4D8 Fab were determined based on the buried surface area (BSA) and BSA percentage (%BSA) of the residues at the Fab / TFPI K2 interface. The following residues in the K2 domain of TFPI are involved in direct contact with 4D8 Fab (epitope according to BSA): 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 the heavy chain paratope: D50, T57, L58, Y59, Q61, K64, D98, Y99, and D100. The following residues in the light chain of 4D8 Fab contain the light chain paratope: H30, W50, H91, Y92, T93, T94, P95, and Y96. The BSA and %BSA values of these epitope and paratope residues are shown in Table 20.

[0933] Table 20

[0934] Anti-TFPI antibody 4D8.b1 epitope and paratope residues (antibody light chain (LC) and heavy chain (HC) residues using Kabat-defined numbering) as defined by the buried surface area (BSA) and BSA percentage (%BSA) of the interface residues in the 4D8.b1 Fab / cyno TFPI K2 complex structure. Truncation ( or larger BSA, or involved in electrostatic interactions) was applied to the BSA analysis.

[0935]

[0936]

[0937] 2.2A8 and 2A8-200 Fab / cyno K1K2 complex structure

[0938] Mix 2A8 Fab and cyno TFPI K1K2 in a 1:1 molar ratio to form a complex. Perform final purification using a Superdex 200 column. Concentrate the complex to 10.8 mg / ml for structural studies. Crystals of the complex containing 2A8 Fab and TFPI K1K2 were obtained under the following two conditions: (1) 100 mM HEPES pH 7.5, 12.5% PEG 8000, which produced needle-shaped crystals that diffracted to ; (2) 100 mM HEPES pH 7.5, 1600 mM ammonium sulfate, 2% PEG 1000, which produced block-shaped crystals that diffracted to . Flash-freeze the crystals and perform synchrotron data collection remotely at the Advanced Photon Source. Process the image frames using the software AutoPROC (Global Phasing Ltd). The data belong to the space group P3221, and the unit cell is as follows: α = β = 90°, γ = 120°, and each asymmetric unit has one complex. Perform a molecular replacement search using a homology model of 2A8 Fab and the structures of the publicly available TFPI K2 domain (RSCB Protein Data Bank; PDB codes 1TFX and 4DTG) to generate a convincing solution for each component. Refine using the software PHENIX. The final R / Rfree factors at are 0.1667 and 0.2088, respectively, and the RMSD of the bonds is The RMSD of the angle is 1.474°. The epitope and paratope of the complex were determined based on the buried surface area (BSA) and BSA percentage (%BSA) of the 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 (epitope according to BSA): 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 2A8 Fab contain the heavy chain paratope: G26, T28, S31, Y32, Y96, R97, Y98, W99, and D101 (Kabat numbering). The following residues in the light chain of 2A8 Fab contain the light chain paratope: L28, R29, N30, Y31, Y32, Y49, Y50, D51, and N66 (Kabat numbering). The BSA and %BSA values of these epitope and paratope residues are shown in Table 21. An antibody 2A8-200 closely related to 2A8 in the complex with TFPI K1K2 was also resolved using essentially the same method. The epitope and paratope of this antibody are identical to those of 2A8.

[0939] Table 21

[0940] Anti-TFPI antibody 2A8 epitope and paratope residues as defined by the buried surface area (BSA) and BSA percentage (%BSA) of interface residues in the 2A8 Fab / cyno TFPI K2 complex structure (antibody ligh...

Claims

1. 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), wherein the antibody comprises: (a) a VH comprising heavy chain variable region (VH) complementarity determining region 1 (CDR-H1), CDR-H2, and CDR-H3 and a VL comprising light chain variable region (VL) complementarity determining region 1 (CDR-L1), CDR-L2, and CDR-L3, wherein CDR-H1 consists of the amino acid sequence of SEQ ID NO: 48, CDR-H2 consists of the amino acid sequence of SEQ ID NO: 49, CDR-H3 consists of the amino acid sequence of SEQ ID NO: 50, CDR-L1 consists of the amino acid sequence of SEQ ID NO: 43, CDR-L2 consists of the amino acid sequence of SEQ ID NO: 44, and CDR-L3 consists of the amino acid sequence of SEQ ID NO: 45; (b) a VH consisting of the amino acid sequence of SEQ ID NO: 51 and a VL consisting of the amino acid sequence of SEQ ID NO: 46; (c) a VH consisting of the amino acid sequence of SEQ ID NO: 67 and a VL consisting of the amino acid sequence of SEQ ID NO: 77; (d) 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; or (e) 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.

2. The antibody or antigen-binding fragment thereof of claim 1, wherein the epitope comprises the residues numbered according to SEQ ID NO: 2: Glu100, Glu101, Asp102, Gly104, Ile105, Cys106, Arg107, Gly108, Tyr109, Cys130, Leu131, and Gly132.

3. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment thereof binds to TFPI with a binding affinity (Kd) value of from about 1×10 -8 M to about 1×10 -10 M.

4. The antibody or antigen-binding fragment thereof of claim 1, wherein the antibody or antigen-binding fragment: (i) reduces clotting time as measured by a plasma-based diluted prothrombin time assay; (ii) reduces whole blood clotting time as measured by thromboelastography; (iii) increases thrombin generation; (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.

5. The antibody or antigen-binding fragment thereof of claim 4, wherein the plasma or whole blood is deficient in factor VIII or factor IX.

6. One or more isolated nucleic acid molecules comprising a nucleotide sequence encoding the antibody or antigen-binding fragment thereof of claim 1.

7. One or more isolated nucleic acid molecules of claim 6, wherein the nucleotide sequence encodes VH and VL, the VH comprises CDR-H1, CDR-H2 and CDR-H3 and the VL comprises CDR-L1, CDR-L2 and CDR-L3, the CDR-H1 consists of the amino acid sequence of SEQ ID NO: 48, the CDR-H2 consists of the amino acid sequence of SEQ ID NO: 49 and the CDR-H3 consists of the amino acid sequence of SEQ ID NO: 50, and the CDR-L1 consists of the amino acid sequence of SEQ ID NO: 43, the CDR-L2 consists of the amino acid sequence of SEQ ID NO: 44 and the CDR-L3 consists of the amino acid sequence of SEQ ID NO:

45.

8. One or more isolated nucleic acid molecules of claim 7, wherein the nucleotide sequence encodes a VH consisting of the amino acid sequence of SEQ ID NO: 51 and a VL consisting of the amino acid sequence of SEQ ID NO:

46.

9. One or more isolated nucleic acid molecules of claim 7, wherein the nucleotide sequence encodes a VH consisting of the amino acid sequence of SEQ ID NO: 67 and a VL consisting of the amino acid sequence of SEQ ID NO:

77.

10. A pharmaceutical composition comprising the antibody of claim 1 or an antigen-binding fragment thereof and a pharmaceutically acceptable delivery vehicle or excipient.

11. Use of the antibody of claim 1 or an antigen-binding fragment thereof in the preparation of a medicament for treating hemophilia A, hemophilia B, von Willebrand disease (vWD) or platelet abnormalities in a subject in need thereof.

12. Use according to claim 11, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain consists of the amino acid sequence of SEQ ID NO: 68 and the light chain consists of the amino acid sequence of SEQ ID NO:

78.

13. Use of the antibody of claim 1 or an antigen-binding fragment thereof in the preparation of a medicament for treating bleeding disorders in a subject in need thereof.

14. Use according to claim 13, wherein the antibody or an antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain consists of the amino acid sequence of SEQ ID NO: 68 and the light chain consists of the amino acid sequence of SEQ ID NO:

78.

15. Use according to claim 11, wherein the subject has or is susceptible to hemophilia A, hemophilia B, von Willebrand disease (vWD) or platelet abnormalities.

16. Use according to claim 13, wherein the subject has or is susceptible to hemophilia A, hemophilia B, von Willebrand disease (vWD) or platelet abnormalities.

17. Use according to claim 11, wherein the medicament further comprises FVIIa.

18. Use according to claim 12, wherein the medicament further comprises FVIIa.

19. Use according to claim 13, wherein the medicament further comprises a coagulant.

20. Use according to claim 19, wherein the subject has or is prone to having hemophilia A or hemophilia B, and the coagulant is selected from the following: factor VIIa, factor VIII, factor IX, and tranexamic acid.

21. Use according to claim 20, wherein the antibody or antigen-binding fragment thereof comprises a heavy chain and a light chain, the heavy chain consists of the amino acid sequence of SEQ ID NO: 68, and the light chain consists of the amino acid sequence of SEQ ID NO:

78.

22. A vector comprising one or more nucleic acid molecules according to any one of claims 6-9.

23. A host cell comprising one or more nucleic acid molecules according to any one of claims 6-9 or the vector according to claim 22.

24. The host cell according to claim 23, wherein the cell is a mammalian cell.

25. The host cell according to claim 24, wherein the host cell is a CHO cell, a HEK-293 cell, or an Sp2.0 cell.

26. A method for preparing an antibody or an antigen-binding fragment thereof, comprising culturing a host cell according to any one of claims 23-25 under conditions for expressing the antibody or antigen-binding fragment in the host cell.

27. The method according to claim 26, further comprising isolating the antibody or antigen-binding fragment thereof.

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