Human antibodies against tissue factor

Abstract

Isolated human monoclonal antibodies and related antibody-based compositions and molecules that bind to human TF are disclosed. Also disclosed are pharmaceutical compositions comprising the antibodies, as well as therapeutic and diagnostic methods using the antibodies.

Description

[0001] This application is a divisional application based on a patent application with an application date of December 9, 2009, a priority date of December 9, 2008, an application number of 201610367892.8, and an invention name of "Human Antibodies Against Tissue Factor". Field of the Invention

[0002] The present invention relates to antibodies against tissue factor, particularly human tissue factor, and uses of such antibodies, particularly their use in the treatment of cancer, inflammation and vascular diseases. Background of the Invention

[0004] Tissue factor (TF), also known as thromboplastin, factor III or CD142, is a protein present in subendothelial tissue, platelets and leukocytes, which is necessary to initiate the formation of thrombin from the zymogen prothrombin. Thrombin formation ultimately leads to blood coagulation. Tissue factor enables cells to trigger the coagulation cascade and function as a high-affinity receptor for factor VII. The resulting complex provides a catalytic event responsible for initiating the coagulation protease cascade by specific limited proteolysis. Unlike other cofactors in these protease cascades that circulate as non-functional precursors, this factor is a powerful initiator that is fully functional when expressed on the cell surface.

[0005] Tissue factor is the cell surface receptor of serine protease factor VIIa (FVIIa). The combination of FVIIa and tissue factor has been found to start a signaling process in cells, and the signaling function plays a role in angiogenesis. Although angiogenesis is a normal process in growth and development and wound healing, it is also a basic step in the process of tumor transformation from dormancy to malignant state: when cancer cells obtain the ability to produce proteins that participate in angiogenesis, so-called angiogenesis growth factors, these proteins are released by the tumor into nearby tissues, and stimulate existing healthy blood vessels to sprout new blood vessels toward the tumor and enter the tumor interior. Once the new blood vessels enter the tumor, it can quickly increase its size and invade local tissues and organs. Through these new blood vessels, cancer cells can further escape into the circulation and inhabit new tumors (metastasis) in other organs.

[0006] In addition, TF plays a role in inflammation. The action of TF is believed to be mediated by blood coagulation (AJ Chu: "Tissue factor mediates inflammation" in Archives of biochemistry and biophysics, 2005, vol. 440, No. 2, pp. 123-132). Therefore, inhibition of TF by, for example, monoclonal anti-TF antibodies is of great significance in interfering with the coagulation-inflammation cycle, which is not only helpful for anti-inflammatory but also for anti-vascular disease.

[0007] TF expression has been observed in many types of cancer and is associated with more aggressive disease. Moreover, human TF also exists in an alternatively spliced ​​form, asHTF. It was recently found that asHTF promotes tumor growth (Hobbs et al. 2007 Thrombosis Res. 120 (2) S13-S21).

[0008] Antibodies that bind to TF have been disclosed in the prior art:

[0009] WO98 / 40408 discloses antibodies that can bind to native human TF, either alone or in a TF:VIIa complex, and are effective in preventing factor X from binding to TF or the complex, thereby reducing coagulation. The document discloses that the antibodies can be used to reduce thrombosis after invasive medical procedures, such as arterial or cardiac surgery, or to eliminate coagulation caused by the use of medical measures. Other antibodies are disclosed for use in in vivo diagnostic methods, including in vivo diagnostic imaging of native human TF.

[0010] WO04 / 094475 provides antibodies capable of binding to human tissue factor which do not inhibit factor-mediated coagulation compared to a normal plasma control. No human antibodies are described. The antibodies are said to be useful in treating cancer.

[0011] WO03 / 093422 relates to antibodies that bind to the TF:VIIa complex with greater affinity than to TF itself and proposes the use of these antibodies as anticoagulants in the treatment of certain diseases, such as sepsis, disseminated intravascular coagulation, ischemic stroke, thrombosis, acute coronary syndrome and coagulopathy in advanced cancer.

[0012] WO01 / 27079 discloses compositions and methods for inhibiting abnormal cell proliferation, particularly endothelial cell proliferation, such as cancer, abnormal embryonic development, malfunctioning immune responses, and angiogenesis associated with neovascularization and tumor growth. Many active substances are provided, including antibodies, but no specific antibodies are disclosed.

[0013] WO03 / 037361 relates to the use of TF agonists or antagonists for apoptosis-related treatments.

[0014] WO03 / 029295 relates to isolated human antibodies which immunoreact with human TF and inhibit the binding of Factor VIIa. However, this application does not disclose any examples of antibodies having these properties.

[0015] Several monoclonal antibody therapies have been approved for the treatment of different types of tumors, including bevacizumab Cetuximab Panitumumab (Vectibix TM ) and trastuzumab SUMMARY OF THE INVENTION

[0016] Despite the progress that has been made, there remains a need for improved therapeutic antibody-based approaches for treating serious diseases, such as improved cancer treatments.

[0017] One object of the present invention is to provide new highly specific, effective human anti-TF antibodies for medical use. The antibodies of the present invention show TF binding properties that are different from those of antibodies already described in the art. In a preferred embodiment, the antibodies of the present invention have a high affinity for human tissue factor, mediate antibody-dependent cellular cytotoxicity (ADCC), inhibit FVIIa binding to TF, inhibit FVIIa-induced ERK phosphorylation and IL-8 release, and do not inhibit or only weakly inhibit coagulation.

[0018] The present invention includes the following items:

[0019] 1. A human antibody that binds to human tissue factor.

[0020] 2. The antibody of claim 1, wherein the antibody has an apparent affinity (EC) of 3 nM or less, such as 0.50 nM or less, such as 0.35 nM or less, such as 0.20 nM or less, such as 0.1 nM or less when measured using the assay method described in Example 13. 50 ) binds to the extracellular domain of tissue factor.

[0021] 3. The antibody of any of the preceding items, wherein the antibody binds to mammalian cells expressing tissue factor, such as A431 cells transfected with a construct encoding tissue factor, preferably with an apparent affinity (EC) of 0.1% when measured using the assay described in Example 14. 50 ) is 10 nM or lower, such as 8 nM or lower, such as 5 nM or lower, such as 2 nM or lower, such as 1 nM or lower, such as 0.5 nM or lower, such as 0.3 nM or lower.

[0022] 4. The antibody of any of the preceding items, wherein the antibody is capable of inducing antibody-dependent cellular cytotoxicity in A431 cells, preferably, when measured using the assay method described in Example 20, its EC 50 The value is 2 nM or less, such as 1 nM or less, such as 0.7 nM or less, or 0.3 nM or less, such as 0.20 nM or less, or 0.1 nM or less, or 0.05 nM or less.

[0023] 5. The antibody of any of the preceding items, wherein the antibody is effective in inhibiting the growth of established MDA-MB-231 tumors when measured by the method described in Example 24, and / or is effective in inhibiting the growth of established BxPC3 tumors when measured by the method described in Example 26.

[0024] 6. The antibody of any of the preceding items, wherein the antibody inhibits tissue factor-induced coagulation, preferably with a median inhibitory concentration of less than 10 nM, such as less than 5 nM, such as less than 2 nM, such as less than 1 nM, when determined using the assay method described in Example 19.

[0025] 7. The antibody of any of the preceding items, wherein the antibody inhibits the binding of FVIIa to tissue factor, preferably with a maximum inhibition value greater than 80%, such as greater than 90%, when measured using the assay method described in Example 15.

[0026] 8. The antibody of any of the preceding items, wherein the antibody inhibits FVIIa-induced IL-8 release from MDA-MB-231 cells, preferably, when measured using the assay method described in Example 17, the maximum inhibition value of the inhibition is greater than 40%, such as greater than 50%, such as greater than 60%.

[0027] 9. The antibody of any of the preceding items, wherein the antibody inhibits the conversion of FX to FXa by the TF / FVIIa complex, preferably by less than 50%, such as less than 40%, such as in the range of 1-30%, when determined using the assay method described in Example 18.

[0028] 10. The antibody of any of the preceding items, wherein the antibody competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65.

[0029] 11. The antibody of any of the preceding items, wherein binding to tissue factor does not involve any of the following amino acids of tissue factor: W at position 45, K at position 46, or Y at position 94.

[0030] 12. The antibody of any of the preceding items, wherein the antibody competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 37 and a VL region comprising the sequence of SEQ ID NO: 93.

[0031] 13. The antibody of any of the preceding items, wherein the antibody inhibits FVIIa-induced ERK phosphorylation, preferably with a median inhibitory concentration of less than 10 nM, such as less than 5 nM, such as less than 2 nM, when assayed using the assay method described in Example 16.

[0032] 14. An antibody according to any one of items 1-7 and 9-13, wherein the antibody inhibits ERK phosphorylation, preferably with a median inhibitory concentration of less than 10 nM, for example less than 5 nM, for example less than 2 nM, when measured using the assay method described in Example 16, and the inhibition of FVII-induced IL-8 release as described in the assay method in Example 17 does not exceed a maximum of 10%.

[0033] 15. The antibody of any of the preceding items, wherein the antibody is capable of inducing C3c and C4c deposition, preferably, the antibody is capable of inducing C3c and C4c deposition as determined as described in Example 21.

[0034] 16. The antibody of any of the preceding items, wherein the antibody Fab fragment binds to the extracellular domain of tissue factor as described in Example 28, and the EC50 value measured by ELISA is less than 0.1 μg / mL, such as less than 0.05 μg / mL, such as less than 0.04 μg / mL.

[0035] 17. The antibody of any one of items 1-15, wherein the antibody Fab fragment binds to the extracellular domain of tissue factor as described in Example 28, and the EC50 value measured by ELISA is higher than 1.0 μg / mL.

[0036] 18. The antibody of any one of items 1-15, wherein the antibody Fab fragment binds to the extracellular domain of tissue factor as described in Example 28, with an EC50 value of less than 10 μg / mL, for example, less than 1 μg / mL, for example, less than 0.5 μg / mL, or less than 0.2 μg / mL.

[0037] 19. An antibody according to any of the above, wherein the antibody binds to human tissue factor but not to mouse tissue factor, and shows reduced binding to the shuffled construct 42-84mm compared to human TF, wherein the shuffled construct comprises the human TF sequence except that amino acids 42-84 are replaced by the mouse sequence, as described in Example 27.

[0038] 20. An antibody of items 1-18, wherein the antibody binds to human tissue factor but not to mouse tissue factor, and shows reduced binding to the shuffled construct 85-122mm compared to human TF, wherein the shuffled construct comprises the human TF sequence except that amino acids 85-122 are replaced by the mouse sequence, as described in Example 27.

[0039] 21. An antibody of items 1-18, wherein the antibody binds to human tissue factor but not to mouse tissue factor, and shows reduced binding to the shuffled construct 123-137mm compared to human TF, wherein the shuffled construct comprises the human TF sequence except that amino acids 123-137 are replaced by the mouse sequence, as described in Example 27.

[0040] 22. An antibody of items 1-18, wherein the antibody binds to human tissue factor but not to mouse tissue factor, and shows reduced binding to the shuffled construct 185-225mm compared to human TF, wherein the shuffled construct comprises the human TF sequence except that amino acids 185-225 are replaced by the mouse sequence, as described in Example 27.

[0041] 23. An antibody of items 1-18, wherein the antibody binds to human tissue factor but not to mouse tissue factor, and shows reduced binding to the shuffled construct 226-250mm compared to human TF, wherein the shuffled construct comprises the human TF sequence except that amino acids 226-250 are replaced by the mouse sequence, as described in Example 27.

[0042] 24. The antibody according to any one of items 19-23, wherein it shows reduced binding to more than one shuffled construct compared to binding to human TF, such as reduced binding to the following shuffled constructs:

[0043] A shuffled construct 42-84 mm as defined in item 19 and a shuffled construct 85-122 mm as defined in item 20, or

[0044] The shuffled construct as defined in item 21 is 123-137 mm, and the shuffled construct as defined in item 22 is 185-225 mm, and the shuffled construct as defined in item 23 is 226-250 mm.

[0045] 25. The antibody of any one of the preceding items, wherein the antibody

[0046] - competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65, and

[0047] - does not compete with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93 for tissue factor binding.

[0048] 26. The antibody of item 25, wherein the antibody comprises a VH CDR3 region having

[0049] a) The sequence shown below

[0050] -SEQ ID No:12,

[0051] -SEQ ID No:16,

[0052] -SEQ ID No:20,

[0053] -SEQ ID No:24,

[0054] -SEQ ID No:28,

[0055] or

[0056] b) variants of any of the sequences described, for example variants having up to 1, 2, 3, 4, or 5 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0057] 27. The antibody of item 26, wherein the antibody comprises a VHCDR3 region having a sequence as shown in SEQ ID NO: 12 or a variant thereof, wherein the variant comprises a modification at one or more of positions 2, 3, 6, 9 and 11, preferably, wherein the modification is a substitution, more preferably, wherein the substitution is selected from the group consisting of:

[0058] a) R at position 2 is replaced by K,

[0059] b) S at position 3 is replaced by A or T,

[0060] c) G at position 6 is replaced by T,

[0061] d) L at position 9 is replaced by F, and

[0062] e) S at position 11 is replaced by Y.

[0063] 28. The antibody of item 1 or 26, wherein the antibody comprises:

[0064] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 10, 11 and 12, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 66, 67 and 68,

[0065] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 14, 15 and 16, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 70, 71 and 72,

[0066] c) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 18, 19 and 20, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 74, 75 and 76,

[0067] d) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 22, 23 and 24, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 78, 79 and 80,

[0068] e) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 26, 27 and 28, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 82, 83 and 84, or

[0069] f) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications in the sequence, more preferably amino acid substitutions, such as conservative amino acid substitutions.

[0070] 29. The antibody of item 26, comprising such a VH, which

[0071] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 9, 13, 17, 21 and 25, or

[0072] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 9, 13, 17, 21, 21 and 25.

[0073] 30. The antibody of item 26, comprising a VL,

[0074] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from SEQ ID NO: 65, 69, 73, 77 and 81, or

[0075] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 65, 69, 73, 77 and 81.

[0076] 31. The antibody of item 26, comprising:

[0077] a) a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65,

[0078] b) a VH region comprising the sequence of SEQ ID NO: 13 and a VL region comprising the sequence of SEQ ID NO: 69,

[0079] c) a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 73,

[0080] d) a VH region comprising the sequence of SEQ ID NO: 21 and a VL region comprising the sequence of SEQ ID NO: 77,

[0081] e) a VH region comprising the sequence of SEQ ID NO: 25 and a VL region comprising the sequence of SEQ ID NO: 81, or

[0082] f) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0083] 32. The antibody of any one of items 1-24, wherein the antibody

[0084] - competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65, and

[0085] - competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.

[0086] 33. The antibody of item 32, wherein the antibody comprises a VH CDR3 region having

[0087] a) The sequence shown below

[0088] -SEQ ID No:8,

[0089] -SEQ ID No:52,

[0090] or

[0091] b) variants of any of the sequences described, for example variants having up to 1, 2 or 3 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0092] 34. The antibody of claim 1 or 32, wherein the antibody comprises:

[0093] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 6, 7 and 8, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 62, 63 and 64,

[0094] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 50, 51 and 52, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 106, 107 and 108, or

[0095] c) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0096] 35. The antibody of item 32, comprising such a VH, which

[0097] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 5 and 49, or

[0098] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 5 and 49.

[0099] 36. The antibody of item 32, comprising a VL, wherein the VL

[0100] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from SEQ ID NOs: 61 and 105, or

[0101] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 61 and 105.

[0102] 37. The antibody of item 32, comprising:

[0103] a) a VH region comprising the sequence of SEQ ID NO: 5 and a VL region comprising the sequence of SEQ ID NO: 61,

[0104] b) a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 105, or

[0105] c) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0106] 38. The antibody of any one of items 1-24, wherein the antibody

[0107] - does not compete with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65 for tissue factor binding, and

[0108] - competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.

[0109] 39. The antibody of claim 38, wherein the antibody comprises a VH CDR3 region having

[0110] a) The sequence shown below

[0111] -SEQ ID No:32,

[0112] -SEQ ID No:36,

[0113] -SEQ ID No:40,

[0114] -SEQ ID No:56,

[0115] or

[0116] b) variants of any of the sequences described, for example variants having up to 1, 2 or 3 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0117] 40. The antibody of claim 1 or 38, wherein the antibody comprises:

[0118] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 30, 31 and 32, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 86, 87 and 88,

[0119] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 34, 35 and 36, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 90, 91 and 92,

[0120] c) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 38, 39 and 40, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 94, 95 and 96,

[0121] d) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 54, 55 and 56, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 110, 11 and 112, or

[0122] e) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0123] 41. The antibody of item 38, comprising a VH, wherein the VH

[0124] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 29, 33, 37 and 53, or

[0125] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 29, 33, 37 and 53.

[0126] 42. The antibody of item 38, comprising a VL, wherein the VL

[0127] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 85, 89, 93 and 109, or

[0128] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 85, 89, 93 and 109.

[0129] 43. The antibody of item 38, comprising:

[0130] a) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO: 85,

[0131] b) a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 89,

[0132] c) a VH region comprising the sequence of SEQ ID NO: 37 and a VL region comprising the sequence of SEQ ID NO: 93,

[0133] d) a VH region comprising the sequence of SEQ ID NO: 53 and a VL region comprising the sequence of SEQ ID NO: 109, or

[0134] e) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0135] 44. The antibody of any one of items 1 to 24 above, wherein the antibody competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 41 and a VL region comprising the sequence of SEQ ID NO: 97.

[0136] 45. The antibody of item 44, wherein the antibody comprises a VH CDR3 region having

[0137] a) The sequence shown below

[0138] -SEQ ID No:4,

[0139] -SEQ ID No:44,

[0140] -SEQ ID No:48,

[0141] or

[0142] b) variants of any of the sequences described, for example variants having up to 1, 2 or 3 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0143] 46. ​​The antibody of claim 1 or 44, wherein the antibody comprises:

[0144] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 2, 3 and 4, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 58, 59 and 60,

[0145] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 42, 43 and 44, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 98, 99 and 100,

[0146] c) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 46, 47 and 48, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 102, 103 and 104, or

[0147] d) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0148] 47. The antibody of item 44, comprising a VH, wherein the VH

[0149] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 1, 41 and 45, or

[0150] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 1, 41 and 45.

[0151] 48. The antibody of item 44, comprising a VL, wherein the VL

[0152] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from SEQ ID NO: 57, 97 and 101, or

[0153] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 57, 97 and 101.

[0154] 49. The antibody of item 44, comprising:

[0155] a) a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 57,

[0156] b) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97,

[0157] c) a VH region comprising the sequence of SEQ ID NO: 45 and a VL region comprising the sequence of SEQ ID NO: 101, or

[0158] d) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0159] 50. The antibody of any one of items 1-5, wherein the affinity of the antibody for tissue factor is less than 5 nM, such as less than 3.5 nM, such as less than 2 nM, when measured using the method described in Example 22 herein.

[0160] 51. The antibody of any one of items 1-5, wherein the kd of the antibody is greater than 10 when measured using the method described in Example 22 herein. -3 sec -1 , and / or when measured by the method described in Example 22 herein, the ka of the antibody is greater than 5x10 4 mol -1 sec -1 .

[0161] 52. The antibody of any one of items 1-5, wherein the kd of the antibody is greater than 10 when measured using the affinity method described in Example 22 herein. -3 sec -1 , and when measured using the affinity method described in Example 22 herein, the antibody has an affinity of less than 5 nM, such as less than 1 nM, such as less than 0.2 nM.

[0162] 53. An antibody according to any one of items 1-5 or 51 or 52, wherein the antibody does not show binding to healthy tissues, in particular does not show binding to human glomeruli, for example as measured by the assay method described in Example 23; but shows binding to pancreatic tumors, for example as measured by the assay method described in Example 23 of the present invention.

[0163] 54. The antibody of any one of items 1-5 or 51-53, wherein the antibody is effective in inhibiting the growth of established BX-PC3 tumors when measured using the method described in Example 26 herein.

[0164] 55. The antibody of any of the preceding items, wherein the antibody further has one or more of the following properties: inhibiting proliferation, inhibiting tumor angiogenesis, inducing tumor cell apoptosis, binding to alternatively spliced ​​tissue factor.

[0165] 56. The antibody of any one of items 1-5, which competes for tissue factor binding with an antibody comprising:

[0166] a) a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65,

[0167] b) a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 57,

[0168] c) a VH region comprising the sequence of SEQ ID NO: 5 and a VL region comprising the sequence of SEQ ID NO: 61,

[0169] d) a VH region comprising the sequence of SEQ ID NO: 13 and a VL region comprising the sequence of SEQ ID NO: 69,

[0170] e) a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 73,

[0171] f) a VH region comprising the sequence of SEQ ID NO: 21 and a VL region comprising the sequence of SEQ ID NO: 77,

[0172] g) a VH region comprising the sequence of SEQ ID NO: 25 and a VL region comprising the sequence of SEQ ID NO: 81,

[0173] h) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO: 85,

[0174] i) a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 89,

[0175] j) a VH region comprising the sequence of SEQ ID NO: 37 and a VL region comprising the sequence of SEQ ID NO: 93,

[0176] k) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97,

[0177] 1) a VH region comprising the sequence of SEQ ID NO: 45 and a VL region comprising the sequence of SEQ ID NO: 101,

[0178] m) a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 105, or

[0179] n) a VH region comprising the sequence of SEQ ID NO:53 and a VL region comprising the sequence of SEQ ID NO:109.

[0180] 57. The antibody of any one of items 1-5, which binds to the same epitope on tissue factor as an antibody having:

[0181] a) a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65,

[0182] b) a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 57,

[0183] c) a VH region comprising the sequence of SEQ ID NO: 5 and a VL region comprising the sequence of SEQ ID NO: 61,

[0184] d) a VH region comprising the sequence of SEQ ID NO: 13 and a VL region comprising the sequence of SEQ ID NO: 69,

[0185] e) a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 73,

[0186] f) a VH region comprising the sequence of SEQ ID NO: 21 and a VL region comprising the sequence of SEQ ID NO: 77,

[0187] g) a VH region comprising the sequence of SEQ ID NO: 25 and a VL region comprising the sequence of SEQ ID NO: 81,

[0188] h) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO: 85,

[0189] i) a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 89,

[0190] j) a VH region comprising the sequence of SEQ ID NO: 37 and a VL region comprising the sequence of SEQ ID NO: 93,

[0191] k) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97,

[0192] 1) a VH region comprising the sequence of SEQ ID NO: 45 and a VL region comprising the sequence of SEQ ID NO: 101,

[0193] m) a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 105, or

[0194] n) a VH region comprising the sequence of SEQ ID NO:53 and a VL region comprising the sequence of SEQ ID NO:109.

[0195] 58. The antibody of any of the preceding items, wherein the antibody comprises:

[0196] - derived from a human germline selected from the group consisting of V HThe heavy chain variable region of the sequences: IGHV1-18*01, IGHV3-23*01, IGHV3-30*01, IGHV3-33*01, IGHV3-33*03, IGHV1-69*02, IGHV1-69*04 and IGHV5-51*01, and / or

[0197] - derived from a human germline selected from the group consisting of V κ The sequences of light chain variable regions are: IGKV3-20*01, IGKV1-13*02, IGKV3-11*01 and IGKV1D-16*01.

[0198] 59. The antibody of any of the preceding items, wherein the antibody is a full-length antibody, preferably an IgG1 antibody, in particular an IgG1,κ antibody.

[0199] 60. The antibody of any of the preceding items, wherein the antibody is conjugated to another moiety, such as a cytotoxic moiety, a radioisotope or a drug.

[0200] 61. The antibody of any one of items 1-3 or 15-49, wherein the antibody is an effector function-deficient antibody.

[0201] 62. The antibody of claim 61, wherein the effector function-deficient anti-tissue factor antibody is a stabilized human IgG4 antibody, for example, an antibody in which the arginine at position 409 of the human IgG4 heavy chain constant region is replaced with lysine, threonine, methionine or leucine, preferably lysine, and / or in which the hinge region comprises a Cys-Pro-Pro-Cys sequence.

[0202] 63. The antibody of any one of items 1-3 or 15-49, wherein the antibody is a monovalent antibody.

[0203] 64. The antibody of claim 63, wherein the monovalent antibody is constructed by the following method, comprising:

[0204] i) providing a nucleic acid construct encoding the monovalent antibody light chain, the construct comprising a nucleotide sequence encoding the VL region of a selected antigen-specific antibody and a nucleotide sequence encoding the constant CL region of Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of Ig are operably linked together, and wherein, in the case of IgG1 subtype, the nucleotide sequence encoding the CL region is modified so that the CL region does not contain any amino acid as described below: in the presence of polyclonal human IgG or when administered to animals or humans, the amino acid is capable of forming a disulfide bond or a covalent bond with other peptides comprising the same amino acid sequence as the CL region;

[0205] ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VH region of a selected antigen-specific antibody and a nucleotide sequence encoding the constant CH region of human Ig, wherein the nucleotide sequence encoding the CH region is modified so that the region corresponding to the hinge region, and other regions of the CH region as required by the Ig subtype, such as the CH3 region, do not contain any amino acid residues as follows: in the presence of polyclonal human IgG or when administered to animals or humans, the amino acid residues participate in the formation of disulfide bonds or covalent or stable non-covalent heavy chain bonds with other peptides comprising the same amino acid sequence as the CH region of human Ig, wherein the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked together;

[0206] iii) providing a cell expression system for producing the monovalent antibody;

[0207] iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).

[0208] 65. The antibody of item 63, wherein the monovalent antibody comprises

[0209] (i) the variable region of an antibody according to items 1 to 26, or an antigen-binding portion of said region, and

[0210] (ii) Immunoglobulin C H Area or including C H 2 and C H 3 region fragment, where the C H The region or fragment thereof has been modified so that the region corresponding to the hinge region and, if the immunoglobulin is not of the IgG4 subtype, the C H Other areas of the zone, such as C H Region 3 does not contain any H disulfide bond or with the same C H The amino acid residues in the region form additional covalent or stable non-covalent inter-heavy chain bonds.

[0211] 66. The antibody of any one of items 63-65, wherein the heavy chain has been modified such that the entire hinge has been deleted.

[0212] 67. A bispecific molecule comprising the antibody of any one of items 1-62 and a second binding specificity, such as a binding specificity for human effector cells, human Fc receptors or T cell receptors.

[0213] 68. An expression vector comprising a nucleotide sequence encoding one or more amino acid sequences selected from SEQ ID NO: 1 to SEQ ID NO: 112.

[0214] 69. The expression vector according to item 68 further comprises a nucleotide sequence encoding the light chain constant region, the heavy chain constant region, or the constant regions of both the light chain and the heavy chain of a human antibody.

[0215] 70. A recombinant eukaryotic or prokaryotic host cell which produces an antibody as defined in any one of items 1-59 or 61-66.

[0216] 71. A pharmaceutical composition comprising an antibody as defined in any one of items 1 to 54, or a bispecific molecule as defined in item 67, and a pharmaceutically acceptable carrier.

[0217] 72. An antibody as defined in any one of items 1 to 66 or a bispecific molecule as defined in item 67 for use as a medicament.

[0218] 73. The antibody or bispecific molecule of claim 72, wherein the use is for treating cancer.

[0219] 74. The antibody or bispecific molecule of item 73, wherein the cancer is selected from the following group: central nervous system tumors, head and neck cancer, lung cancer, breast cancer, esophageal cancer, gastric cancer, liver and gallbladder cancer, pancreatic cancer, colorectal cancer, bladder cancer, kidney cancer, prostate cancer, endometrial cancer, ovarian cancer, malignant melanoma, sarcoma, tumor of unknown origin, bone marrow cancer, acute lymphocytic leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, skin cancer, glioma, brain cancer, uterine cancer and rectal cancer.

[0220] 75. The antibody or bispecific molecule of claim 72, wherein the use is for the treatment of pancreatic cancer.

[0221] 76. The antibody or bispecific molecule of claim 72, wherein the use is for the treatment of colorectal cancer.

[0222] 77. The antibody or bispecific molecule of claim 72, wherein the use is for the treatment of ovarian cancer.

[0223] 78. The antibody or bispecific molecule of item 72, wherein the use is for the treatment of breast cancer.

[0224] 79. The antibody or bispecific molecule of item 72, wherein the use is for the treatment of prostate cancer.

[0225] 80. The antibody or bispecific molecule of item 72, wherein the use is for treating bladder cancer.

[0226] 81. The antibody or bispecific molecule of claim 72, wherein the drug is used to treat cancer in combination with one or more other therapeutic agents such as chemotherapeutic agents.

[0227] 82. Use of the antibody of any one of items 1 to 66 or the bispecific molecule of item 697 in the manufacture of a medicament for treating cancer.

[0228] 83. The use of item 82, comprising the additional features of any one of items 74-81.

[0229] 84. A method for inhibiting the growth and / or proliferation of tumor cells expressing tissue factor, comprising administering to an individual in need thereof the antibody of any one of items 1 to 66 or the bispecific molecule as defined in item 67.

[0230] 85. A method for producing the antibody of any one of items 1-59 or 61-66, the method comprising the steps of:

[0231] a) culturing the host cell of item 70, and

[0232] b) Purification of antibodies from the culture medium.

[0233] 86. A diagnostic composition comprising an antibody as defined in any one of items 1-67.

[0234] 87. A method for detecting the presence of tissue factor in a sample, comprising:

[0235] - contacting the sample with the antibody of any one of items 1 to 66 or the bispecific molecule of item 67 under conditions that allow formation of a complex between said antibody or bispecific molecule and tissue factor; and

[0236] - Analyze whether a complex is formed.

[0237] 88. A kit for detecting the presence of tissue factor in a sample, comprising

[0238] - the antibody of any one of items 1 to 66 or the bispecific molecule of item 67; and

[0239] -Instructions for using the kit. Sequence Listing <110> Jianma Bao <120> Human antibodies against tissue factor <130> P57 <160> 112 <170> PatentIn version 3.2 <210> 1 <211> 126 <212> PRT <213> Person <400> 1 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Tyr 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg Ile His Arg Gly Ala Gly Tyr Ser Ser Ser Trp Pro Gly Ala 100 105 110 Phe Asp Ile Trp Gly Gln Gly Thr Met Val Thr Val Ser Ser 115 120 125 <210> 2 <211> 8 <212> PRT <213> Person <400> 2 Gly Tyr Ser Phe Thr Ser Tyr Trp 1 5 <210> 3 <211> 8 <212> PRT <213> person <400> 3 Ile Tyr Pro Gly Asp Ser Asp Thr 1 5 <210> 4 <211> 19 <212> PRT <213> person <400> 4 Ala Arg Ile His Arg Gly Ala Gly Tyr Ser Ser Ser Trp Pro Gly Ala 1 5 10 15 Phe Asp Ile <210> 5 <211> 119 <212> PRT <213> person <400> 5 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Val Ala Ser Gly Phe Thr Val Ser Asn Asp 20 25 30 Gly Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Leu Ile Trp Tyr Asp Gly Val Asn Lys Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Lys Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Arg Pro Gly Thr Phe Tyr Gly Leu Asp Val Trp Gly Gln Gly 100 105 110 Thr Thr Val Thr Val Ser Ser 115 <210> 6 <211> 8 <212> PRT <213> Human <400> 6 Gly Phe Thr Val Ser Asn Asp Gly 1 5 <210> 7 <211> 8 <212> PRT <213> Human <400> 7 Ile Trp Tyr Asp Gly Val Asn Lys 1 5 <210> 8 <211> 12 <212> PRT <213> Human <400> 8 Ala Arg Arg Pro Gly Thr Phe Tyr Gly Leu Asp Val 1 5 10 <210> 9 <211> 118 <212> PRT <213> Human <400> 9 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 <213> HumanSer Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Asp Tyr Thr Tyr Tyr Thr Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Ser Pro Trp Gly Tyr Tyr Leu Asp Ser Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 10 <211> 8 <212> PRT <213> Human <400> 10 Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 11 <211> 8 <212> PRT <213> Human <400> 11 Ile Ser Gly Ser Gly Asp Tyr Thr 1 5 <210> 12 <211> 11 <212> PRT <213> Person <400> 12 Ala Arg Ser Pro Trp Gly Tyr Tyr Leu Asp Ser 1 5 10 <210> 13 <211> 120 <212> PRT <213> Person <400> 13 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ala Ile Ser Gly Ser Gly Asp Ser Thr Asn Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Asp Gly Tyr Phe Leu Leu Trp Tyr Phe Asp Leu Trp Gly Arg 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 14 <211> 8 <212> PRT <213> Person <400> 14 Gly Phe Thr Phe Ser Ser Tyr Ala 1 5 <210> 15 <211> 8 <212> PRT <213> Person <400> 15 Ile Ser Gly Ser Gly Asp Ser Thr 1 5 <210> 16 <211> 13 <212> PRT <213> Person <400> 16 Ala Lys Asp Gly Tyr Phe Leu Leu Trp Tyr Phe Asp Leu 1 5 10 <210> 17 <211> 118 <212> PRT <213> Person <400> 17 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Ser Tyr 20 25 30 Gly Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Val Ile Ser Gly Ser Gly Gly Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Ala Pro Trp Thr Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 18 <211> 8 <212> PRT <213> Human <400> 18 Gly Phe Thr Phe Ser Ser Tyr Gly 1 5 <210> 19 <211> 8 <212> PRT <213> Human <400> 19 Ile Ser Gly Ser Gly Gly Thr Thr 1 5 <210> 20 <211> 11 <212> PRT <213> Human <400> 20 Ala Lys Ala Pro Trp Thr Tyr Tyr Phe Asp Tyr 1 5 10 <210> 21 <211> 118 <212> PRT <213> Human <400> 21 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asn Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ser Ser Ile Ser Gly Ser Gly Gly Arg Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Phe 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 22 <211> 8 <212> PRT <213> Human <400> 22 Gly Phe Thr Phe Asn Asn Tyr Ala 1 5 <210> 23 <211> 8 <212> PRT <213> Human <400> 23 Ile Ser Gly Ser Gly Gly Arg Thr 1 5 <210> 24 <211> 11 <212> PRT <213> Human <400> 24 Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr 1 5 10 <210> 25 <211> 118 <212> PRT <213> Human <400> 25 Glu Val Gln Leu Leu Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Gly 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asn Tyr 20 25 30 Ala Met Ser Trp Val Arg Gln Ala Pro Ala Lys Gly Leu Asp Trp Val 35 40 45 Ser Gly Ile Ser Gly Ser Gly Val Thr Thr Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Phe Cys 85 90 95 Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr Trp Gly Gln Gly Ile 100 105 110 Leu Val Ala Val Ser Ser 115 <210> 26 <211> 8 <212> PRT <213> Human <400> 26 Gly Phe Thr Phe Ser Asn Tyr Ala 1 5 <210> 27 <211> 8 <212> PRT <213> Human <400> 27 Ile Ser Gly Ser Gly Val Thr Thr 1 5 <210> 28 <211> 11 <212> PRT <213> Human <400> 28 Ala Lys Thr Pro Trp Gly Tyr Tyr Phe Asp Tyr 1 5 10 <210> 29 <211> 118 <212> PRT <213> Human <400> 29 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Pro Arg Gly Thr Phe Ser Tyr Tyr 20 25 30 Thr Ile Ser Trp Val Arg Gln Ala Pro Gly Gln Gly Leu Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Val Ala Asn Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Ser Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Gly Asp Arg Arg Tyr Phe Asp Tyr Trp Gly Gln Gly Thr 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 30 <211> 8 <212> PRT <213> Human <400> 30 Arg Gly Thr Phe Ser Tyr Tyr Thr 1 5 <210> 31 <211> 8 <212> PRT <213> Human <400> 31 Ile Ile Pro Ile Leu Gly Val Ala 1 5 <210> 32 <211> 11 <212> PRT <213> Human <400> 32 Ala Arg Glu Gly Asp Arg Arg Tyr Phe Asp Tyr 1 5 10 <210> 33 <211> 120 <212> PRT <213> Human <400> 33 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Arg Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Ser Asn Asp Gly Tyr Asn Asp Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Val Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Gln Leu Gly Arg Gly Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 34 <211> 8 <212> PRT <213> Human <400> 34 Gly Phe Thr Phe Ser Arg Tyr Ala 1 5 <210> 35 <211> 8 <212> PRT <213> Human <400> 35 Ile Ser Asn Asp Gly Tyr Asn Asp 1 5 <210> 36 <211> 13 <212> PRT <213> Human <400> 36 Ala Arg Asp Gly Gln Leu Gly Arg Gly Tyr Phe Asp Tyr 1 5 10 <210> 37 <211> 120 <212> PRT <213> Human <400> 37 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Pro Ala Ser Gly Phe Thr Phe Ser Ile Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Val Ser Asn Asp Gly Tyr Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asp Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp Gly Gln Leu Gly Arg Gly Tyr Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 38 <211> 8 <212> PRT <213> Human <400> 38 Gly Phe Thr Phe Ser Ile Tyr Ala 1 5 <210> 39 <211> 8 <212> PRT <213> Human <400> 39 Val Ser Asn Asp Gly Tyr Asn Lys 1 5 <210> 40 <211> 13 <212> PRT <213> Human <400> 40 Ala Arg Asp Gly Gln Leu Gly Arg Gly Tyr Phe Asp Tyr 1 5 10 <210> 41 <211> 118 <212> PRT <213> Human <400> 41 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Ser Asp Tyr 20 25 30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val 35 40 45 Ala Val Ile Pro Tyr Asp Gly Asp Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Glu Asp Trp Gly Leu Glu Val Asp Tyr Trp Gly Gln Gly Ala 100 105 110 Leu Val Thr Val Ser Ser 115 <210> 42 <211> 8 <212> PRT <213> Human <400> 42 Gly Phe Thr Phe Ser Asp Tyr Ala 1 5 <210> 43 <211> 8 <212> PRT <213> Human <400> 43 Ile Pro Tyr Asp Gly Asp Asn Lys 1 5 <210> 44 <211> 11 <212> PRT <213> Human <400> 44 Ala Arg Glu Asp Trp Gly Leu Glu Val Asp Tyr 1 5 10 <210> 45 <211> 121 <212> PRT <213> Human <400> 45 Glu Val Gln Leu Val Gln Ser Gly Ala Glu Val Lys Lys Pro Gly Glu 1 5 10 15 Ser Leu Lys Ile Ser Cys Lys Gly Ser Gly Tyr Ser Phe Thr Ser Cys 20 25 30 Trp Ile Gly Trp Val Arg Gln Met Pro Gly Lys Gly Leu Glu Trp Met 35 40 45 Gly Ile Ile Tyr Pro Gly Asp Ser Asp Thr Arg Tyr Ser Pro Ser Phe 50 55 60 Gln Gly Gln Val Thr Ile Ser Ala Asp Lys Ser Ile Ser Thr Ala Tyr 65 70 75 80 Leu Gln Trp Ser Ser Leu Lys Ala Ser Asp Thr Ala Met Tyr Tyr Cys 85 90 95 Ala Arg His Lys Leu Gly Met Asp His Asp Ala Phe Asp Ile Trp Gly 100 105 110 Gln Gly Thr Met Val Thr Val Ser Ser 115 120 <210> 46 <211> 8 <212> PRT <213> Human <400> 46 Gly Tyr Ser Phe Thr Ser Cys Trp 1 5 <210> 47 <211> 8 <212> PRT <213> Human <400> 47 Ile Tyr Pro Gly Asp Ser Asp Thr 1 5 <210> 48 <211> 14 <212> PRT <213> Human <400> 48 Ala Arg His Lys Leu Gly Met Asp His Asp Ala Phe Asp Ile 1 5 10 <210> 49 <211> 118 <212> PRT <213> Human <400> 49 Gln Val Gln Leu Val Gln Ser Gly Ala Glu Val Arg Lys Pro Gly Ser 1 5 10 15 Ser Val Lys Val Ser Cys Lys Ala Ser Gly Gly Ser Phe Asn Asn Tyr 20 25 30 Pro Ile Phe Trp Val Arg Gln Ala Pro Gly Gln Gly Phe Glu Trp Met 35 40 45 Gly Arg Ile Ile Pro Ile Leu Gly Ile Thr Ala Tyr Ala Gln Lys Phe 50 55 60 Gln Gly Arg Val Thr Ile Thr Ala Asp Lys Ser Thr Ser Thr Ala Tyr 65 70 75 80 Met Glu Leu Asn Ser Leu Arg Ser Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Gly Gly Asp Asp Leu Asp Ala Phe Asp Ile Trp Gly Gln Gly Thr 100 105 110 Met Val Ser Val Ser Ser 115 <210> 50 <211> 8 <212> PRT <213> Human <400> 50 Gly Gly Ser Phe Asn Asn Tyr Pro 1 5 <210> 51 <211> 8 <212> PRT <213> Human <400> 51 Ile Ile Pro Ile Leu Gly Ile Thr 1 5 <210> 52 <211> 11 <212> PRT <213> Human <400> 52 Ala Gly Gly Asp Asp Leu Asp Ala Phe Asp Ile 1 5 10 <210> 53 <211> 120 <212> PRT <213> Human <400> 53 Gln Val Gln Leu Val Glu Ser Gly Gly Gly Val Val Gln Pro Gly Arg 1 5 10 15 Ser Leu Arg Leu Ser Cys Ala Gly Ser Gly Phe Thr Phe Asn Arg Tyr 20 25 30 Ala Met Tyr Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Asp Trp Val 35 40 45 Ala Val Ile Ser Asn Asp Gly Ile Asn Lys Tyr Tyr Ala Asp Ser Val 50 55 60 Lys Gly Arg Phe Thr Ile Ser Arg Asp Asn Ser Lys Asn Thr Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Arg Asp His Thr Met Val Arg Gly Ala Phe Asp Tyr Trp Gly Gln 100 105 110 Gly Thr Leu Val Thr Val Ser Ser 115 120 <210> 54 <211> 8 <212> PRT <213> Human <400> 54 Gly Phe Thr Phe Asn Arg Tyr Ala 1 5 <210> 55 <211> 8 <212> PRT <213> Human <400> 55 Ile Ser Asn Asp Gly Ile Asn Lys 1 5 <210> 56 <211> 13 <212> PRT <213> Human <400> 56 Ala Arg Asp His Thr Met Val Arg Gly Ala Phe Asp Tyr 1 5 10 <210> 57 <211> 107 <212> PRT <213> Human <400> 57 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Arg Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asp Ser Asp Pro Ile 85 90 95 Thr Phe Gly Gln Gly Thr Arg Leu Glu Ile Lys 100 105 <210> 58 <211> 6 <212> PRT <213> Human <400> 58 Gln Gly Ile Ser Arg Trp 1 5 <210> 59 <211> 3 <212> PRT <213> Human <400> 59 Ala Ala Ser 1 <210> 60 <211> 9 <212> PRT <213> Human <400> 60 Gln Gln Tyr Asp Ser Asp Pro Ile Thr 1 5 <210> 61 <211> 107 <212> PRT <213> Human <400> 61 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 62 <211> 7 <212> PRT <213> Human <400> 62 Gln Ser Val Ser Ser Ser Tyr 1 5 <210> 63 <211> 3 <212> PRT <213> Human <400> 63 Gly Ala Ser 1 <210> 64 <211> 8 <212> PRT <213> Human <400> 64 Gln Gln Tyr Gly Ser Ser Leu Thr 1 5 <210> 65 <211> 107 <212> PRT <213> person <400> 65 Asp Ile Gln Met Thr Gln Ser Pro Pro Ser Leu Ser Ala Ser Ala Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 66 <211> 6 <212> PRT <213> person <400> 66 Gln Gly Ile Ser Ser Arg 1 5 <210> 67 <211> 3 <212> PRT <213> person <400> 67 Ala Ala Ser 1 <210> 68 <211> 9 <212> PRT <213> person <400> 68 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 69 <211> 108 <212> PRT <213> person <400> 69 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Arg Ser Ser 20 25 30 Tyr Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 70 <211> 7 <212> PRT <213> person <400> 70 Gln Ser Val Arg Ser Ser Tyr 1 5 <210> 71 <211> 3 <212> PRT <213> person <400> 71 Gly Ala Ser 1 <210> 72 <211> 9 <212> PRT <213> person <400> 72 Gln Gln Tyr Gly Ser Ser Pro Arg Thr 1 5 <210> 73 <211> 108 <212> PRT <213> person <400> 73 Glu Ile Val Leu Thr Gln Ser Pro Gly Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Gly Ser Ser 20 25 30 Ser Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu 35 40 45 Ile Tyr Gly Ala Ser Ser Arg Ala Thr Gly Ile Pro Asp Arg Phe Ser 50 55 60 Gly Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Arg Leu Glu 65 70 75 80 Pro Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Tyr Gly Ser Ser Pro 85 90 95 Arg Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys 100 105 <210> 74 <211> 7 <212> PRT <213> Human <400> 74 Gln Ser Val Gly Ser Ser Ser 1 5 <210> 75 <211> 3 <212> PRT <213> Human <400> 75 Gly Ala Ser 1 <210> 76 <211> 9 <212> PRT <213> Human <400> 76 Gln Gln Tyr Gly Ser Ser Pro Arg Thr 1 5 <210> 77 <211> 107 <212> PRT <213> Human <400> 77 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Arg 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 78 <211> 6 <212> PRT <213> Human <400> 78 Gln Gly Ile Ser Ser Arg 1 5 <210> 79 <211> 3 <212> PRT <213> Human <400> 79 Ala Ala Ser 1 <210> 80 <211> 9 <212> PRT <213> Human <400> 80 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 81 <211> 108 <212> PRT <213> Person <400> 81 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Leu 85 90 95 Tyr Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 82 <211> 6 <212> PRT <213> Person <400> 82 Gln Gly Ile Ser Ser Trp 1 5 <210> 83 <211> 3 <212> PRT <213> Human <400> 83 Ala Ala Ser 1 <210> 84 <211> 10 <212> PRT <213> Human <400> 84 Gln Gln Tyr Asn Ser Tyr Pro Leu Tyr Thr 1 5 10 <210> 85 <211> 107 <212> PRT <213> Human <400> 85 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Asp Ile Ser Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ile Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 86 <211> 6 <212> PRT <213> Human <400> 86 Gln Asp Ile Ser Ser Ala 1 5 <210> 87 <211> 3 <212> PRT <213> Human <400> 87 Asp Ala Ser 1 <210> 88 <211> 9 <212> PRT <213> Human <400> 88 Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5 <210> 89 <211> 107 <212> PRT <213> Human <400> 89 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 90 <211> 6 <212> PRT <213> Human <400> 90 Gln Ser Val Ser Ser Tyr 1 5 <210> 91 <211> 3 <212> PRT <213> Human <400> 91 Asp Ala Ser 1 <210> 92 <211> 9 <212> PRT <213> Human <400> 92 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 93 <211> 107 <212> PRT <213> Human <400> 93 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Ile Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 94 <211> 6 <212> PRT <213> Human <400> 94 Gln Ser Val Ser Ser Tyr 1 5 <210> 95 <211> 3 <212> PRT <213> Human <400> 95 Asp Ala Ser 1 <210> 96 <211> 9 <212> PRT <213> Human <400> 96 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 <210> 97 <211> 107 <212> PRT <213> Human <400> 97 Ala Ile Gln Leu Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Asn Ser Ala 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Ser Leu Glu Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Phe Asn Ser Tyr Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 98 <211> 6 <212> PRT <213> Human <400> 98 Gln Gly Ile Asn Ser Ala 1 5 <210> 99 <211> 3 <212> PRT <213> Human <400> 99 Asp Ala Ser 1 <210> 100 <211> 9 <212> PRT <213> Human <400> 100 Gln Gln Phe Asn Ser Tyr Pro Leu Thr 1 5 <210> 101 <211> 107 <212> PRT <213> Human <400> 101 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Pro 85 90 95 Thr Phe Gly Gln Gly Thr Thr Val Glu Val Lys 100 105 <210> 102 <211> 6 <212> PRT <213> Human <400> 102 Gln Gly Ile Ser Ser Trp 1 5 <210> 103 <211> 3 <212> PRT <213> Human <400> 103 Ala Ala Ser 1 <210> 104 <211> 9 <212> PRT <213> Human <400> 104 Gln Gln Tyr Asn Ser Tyr Pro Pro Thr 1 5 <210> 105 <211> 107 <212> PRT <213> Human <400> 105 Asp Ile Gln Met Thr Gln Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr Ile Thr Cys Arg Ala Ser Gln Gly Ile Ser Ser Trp 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Glu Lys Ala Pro Lys Ser Leu Ile 35 40 45 Tyr Ala Ala Ser Ser Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln Pro 65 70 75 80 Glu Asp Phe Ala Thr Tyr Tyr Cys Gln Gln Tyr Asn Ser Tyr Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Leu Glu Ile Lys 100 105 <210> 106 <211> 6 <212> PRT <213> Human <400> 106 Gln Gly Ile Ser Ser Trp 1 5 <210> 107 <211> 3 <212> PRT <213> Human <400> 107 Ala Ala Ser 1 <210> 108 <211> 9 <212> PRT <213> Human <400> 108 Gln Gln Tyr Asn Ser Tyr Pro Tyr Thr 1 5 <210> 109 <211> 107 <212> PRT <213> Person <400> 109 Glu Ile Val Leu Thr Gln Ser Pro Ala Thr Leu Ser Leu Ser Pro Gly 1 5 10 15 Glu Arg Ala Thr Leu Ser Cys Arg Ala Ser Gln Ser Val Ser Ser Tyr 20 25 30 Leu Ala Trp Tyr Gln Gln Lys Pro Gly Gln Ala Pro Arg Leu Leu Ile 35 40 45 Tyr Asp Ala Ser Asn Arg Ala Thr Gly Ile Pro Ala Arg Phe Ser Gly 50 55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Glu Pro 65 70 75 80 Glu Asp Phe Ala Val Tyr Tyr Cys Gln Gln Arg Ser Asn Trp Pro Leu 85 90 95 Thr Phe Gly Gly Gly Thr Lys Val Glu Ile Lys 100 105 <210> 110 <211> 6 <212> PRT <213> Person <400> 110 Gln Ser Val Ser Ser Tyr 1 5 <210> 111 <211> 3 <212> PRT <213> Person <400> 111 Asp Ala Ser 1 <210> 112 <211> 9 <212> PRT <213> people <400> 112 Gln Gln Arg Ser Asn Trp Pro Leu Thr 1 5 BRIEF DESCRIPTION OF THE DRAWINGS

[0241] Figure 1 : Sequence alignment of the antibodies of the present invention. The SEQ ID NO is given in brackets to the right of the sequence.

[0242] CDR1, CDR2 and CDR3 according to IMGT are highlighted: italicized sequences represent the CDR1 region, underlined sequences represent the CDR2 region, and bold sequences represent the CDR3 region.

[0243] Figure 2 :IgG4 sequence (SEQ ID NO:113-114)

[0244] SEQ ID NO: 113: Amino acid sequence of wild-type human IgG4 CH region. The italicized sequence represents the CH1 region, the highlighted sequence represents the hinge region, the regular sequence represents the CH2 region, and the underlined sequence represents the CH3 region.

[0245] SEQ ID NO: 114: Amino acid sequence of the hingeless CH region of human IgG4.

[0246] Figure 3 : Binding of anti-TF human monoclonal antibodies to the extracellular domain of TF The binding of anti-TF human monoclonal antibodies to the extracellular domain of TF in ELISA binding was determined by ELISA.

[0247] Figure 4 : Binding of anti-TF human mAbs to membrane-bound TF. Binding of anti-TF human mAbs to membrane-bound TF on MDA-MD-231. Binding was determined by FACS analysis.

[0248] Figure 5 : Inhibition of FVIIa binding to TF. Inhibition of FVIIa binding to TF. FVIIa binding and inhibition of this binding by TF-specific human monoclonal antibodies were measured by ELISA.

[0249] Figure 6 : Inhibition of FVIIa-induced ERK phosphorylation. Inhibition of FVIIIa-induced ERK phosphorylation was analyzed by Western blot.

[0250] Figure 7 : Inhibition of FVIIa-induced IL-8 release. Inhibition of FVIIa-induced IL-8 release. MDA-MB-231 were cultured in serum-free medium, supplemented with TF-specific antibodies and FVIIa. FVIIa-induced IL-8 was measured by ELISA.

[0251] Figure 8 : Inhibition of FXa production. Inhibition of FXa generation. The ability of TF-specific human mAbs to inhibit FXa generation was tested in an assay system using a colorimetric FXa-specific substrate to measure the conversion of FX to FXa under the action of TF / FVIIa.

[0252] Fig. 9 : Inhibition of blood coagulation. Coagulation inhibition. The inhibition of coagulation by TF-human mAb was measured using an assay system for measuring TF-induced coagulation time.

[0253] Fig.10 : TF-human monoclonal antibody induces Bx-PC3 cell lysis via ADCC. TF-human monoclonal antibody induces Bx-PC3 cell lysis via ADCC.

[0254] Fig.11 : Complement components C3c and C4c are deposited on target cells.

[0255] Fig.12 :Immunohistochemical analysis of TF-human monoclonal antibody binding to glomeruli. Immunohistochemical analysis of TF-human monoclonal antibody binding to normal human kidney.

[0256] Fig.13 :Immunohistochemical analysis of TF-human monoclonal antibody binding to pancreatic tumors. Immunohistochemical analysis of TF-human monoclonal antibody binding to pancreatic tumors.

[0257] Fig.14 : In vivo efficacy of TF-human mAb in established MDA-MB-231 tumor xenografts. In vivo efficacy of TF-human mAb in established MDA-MB-231 tumor xenografts in the mammary fat pad of SCID mice.

[0258] Fig.15 : Bleeding time determined after intravenous injection of TF-specific human monoclonal antibody 011 in cynomolgus monkeys. Antibodies were administered on days 1 (0 mg / kg), 8 (1 mg / kg), 15 (10 mg / kg) and 22 (100 mg / kg). Bleeding time (min), functional bleeding time and blood loss were measured 1, 24 and 120 hours after dosing.

[0259] Fig.16: In vivo efficacy of TF-human monoclonal antibody in preventive established BX-PC3 tumor xenografts. Treatment with 400 μg / mouse on day 0, two-way reconstituted measures ANOVA followed by Bonferroni post hoc test: 111 vs. KLH: from day 28 onwards: P < 0.05, from day 32 onwards: P < 0.01 (upper figure). Treatment with 300 μg / mouse on day 8, then 150 μg / mouse per week. Two-way reconstituted measures ANOVA followed by Bonferroni post hoc test: 111 vs. KLH: from day 35 onwards: P < 0.05 (lower figure).

[0260] Fig.17 : Shuffled constructs and TF domains. TFmm shuffled constructs, containing TFmm domain (upper panel). TFmm shuffled constructs, containing TFhs domain (lower panel).

[0261] Fig.18 : Binding of anti-TF antibodies to TF shuffled constructs. Binding of anti-TF human mAbs to TF shuffled constructs expressed on HEK293F cells. Shown is the binding of anti-TF human mAbs to different TF shuffled constructs expressed on HEK293F cells, as determined by FACS. Each panel shows data from one primary clone. The x-axis shows the different constructs, Mock, TFhs, TFmm, TFhsmm, TFmmhs, TFhs1-41mm, TFhs42-84mm, TFhs85-122mm, TFhs123-137mm, TFhs138-159mm, TFhs160-184mm, TFhs185-225mm, TFhs226-250mm, TFmm1-47hs, TFmm48-90hs, TFmm91-122hs, TFmm123-137hs, TFmm138-159hs, TFmm160-184hs, TFmm185-225hs, TFmm226-250hs.

[0262] Fig.19 : Binding of human monoclonal antibody-TF Fab fragment to the extracellular domain of TF, ELISA. The binding of human monoclonal antibody-TF Fab fragment to the extracellular domain of TF was determined by ELISA.

[0263] Fig. 20 : Binding of human monoclonal antibody-TF Fab fragment to TF extracellular domain, FACS. Binding of human monoclonal antibody-TF Fab fragment to cellular TF was determined by FACS on BxPC3 cells.

[0264] Fig.21: Binding profile of anti-TF human Mabs depending on the number of TF molecules expressed. Binding curves of anti-TF human Mabs depending on the number of TF molecules expressed. Binding of anti-TF human Mabs to cell lines expressing different levels of TF was determined by FACS.

[0265] DETAILED DESCRIPTION OF THE INVENTION

[0266] definition

[0267] The terms "tissue factor," "TF," "CD142," "tissue factor antigen," "TF antigen," and "CD142 antigen" are used interchangeably herein and, unless otherwise indicated, include any variants, isoforms, and species homologs of human tissue factor that are naturally expressed by cells or expressed on cells transfected with the tissue factor gene.

[0268] The term "immunoglobulin" refers to a class of structurally related glycoproteins composed of two pairs of polypeptide chains, a pair of light (L) low molecular weight chains and a pair of heavy (H) chains, all four of which can be interconnected by disulfide bonds. The structure of immunoglobulins has been well characterized. See, for example, Fundamental Immunology, Chapter 7 (Paul, W., ed., 2nd ed. Raven Press, NY (1989)). In short, each heavy chain typically consists of a heavy chain variable region (abbreviated herein as V H The heavy chain constant region is typically composed of three domains: C H 1,C H 2 and C H 3. Each light chain is typically composed of a light chain variable region (abbreviated herein as V L The light chain constant region is typically composed of one domain: C L . V H and V L The V domains can be further subdivided into regions of hypervariability (or hypervariable regions, which can be extremely variable in sequence and / or form of the structurally defined loops), also called complement determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). H and V L Typically, it is composed of three CDRs and four FRs, arranged from the amino terminus to the carboxyl terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4 (see also Chothia and Lesk J. Mol. Biol. 196, 901-917 (1987)). Typically, the numbering of amino acid residues in this region is according to IMGT., Sequences of Proteins of Immunological Interest, Fifth Edition, Public Health Service, National Institutes of Health, Bethesda, MD. (1991) (Phrases such as "variable region residues numbered the same as or according to Kabat" are used herein with reference to this numbering system for the heavy chain variable region or the light chain variable region). Using this numbering system, the actual linear amino acid sequence of a peptide may contain fewer or more amino acids, corresponding to a shortening or insertion of a variable region FR or CDR. For example, the heavy chain variable region may contain 1 in V H CDR2 contains a single amino acid insertion after residue 52 (residue 52a according to Kabat), and contains multiple insertion residues after heavy chain FR residue 82 (e.g., residues 82a, 82b and 82c according to Kabat, etc.). The Kabat numbering of residues in a given antibody can be determined by aligning homologous regions of the antibody sequence with a "standard" Kabat numbering sequence.

[0269] In the context of the present invention, the term "antibody" (Ab) refers to an immunoglobulin molecule, a fragment of an immunoglobulin molecule, or a derivative thereof, which has the ability to specifically bind to an antigen under typical physiological conditions, and has a half-life of a considerable time, such as at least about 30 minutes, at least about 45 minutes, at least about 1 hour, at least about 2 hours, at least about 4 hours, at least about 8 hours, at least about 12 hours, about 24 hours or longer, about 48 hours or longer, about 3, 4, 5, 6, 7 or more days, etc., or any other relevant functionally defined period (e.g., a time sufficient to induce, promote, enhance and / or regulate a physiological response associated with the binding of the antibody to the antigen, and / or a time sufficient for the antibody to recruit effector activity). The variable regions of the heavy and light chains of the immunoglobulin molecule contain a binding domain that interacts with the antigen. The constant region of the antibody (Abs) can mediate the binding of the immunoglobulin to host tissues or factors, including various cells of the immune system (e.g., effector cells) and components of the complement system, such as C1q, the first component in the classical pathway of complement activation. The anti-TF antibody may also be a bispecific antibody, a diabody or similar molecule (for a description of diabodies, see, for example, PNAS USA 90(14), 6444-8 (1993)). Indeed, the bispecific antibodies, diabodies, etc. provided by the present invention may bind to any suitable target in addition to binding to a portion of tissue factor or tissue factor FVIIa complex. As previously mentioned, unless otherwise indicated or clearly contradictory in context, the term "antibody" herein includes fragments of an antibody that retain the ability to specifically bind to an antigen. It has been shown that the antigen binding function of an antibody can be performed by fragments of a full-length antibody. Examples of binding fragments encompassed by the term "antibody" include (i) Fab' or Fab fragments, a V L ,V H ,C L and C H 1 domain, or a monovalent antibody as described in WO2007059782 (Genmab); (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments connected by a disulfide bridge in the hinge region; (iii) a fragment consisting essentially of V H and C H 1 domain; (iv) an Fd fragment consisting essentially of V L and V H (v) dAb fragments (Ward et al., Nature 341 , 544-546 (1989)), which is basically composed of V H Domain structure, also known as domain antibody (Holt et al; Trends Biotechnol. 2003 Nov; 21 (11): 484-90); (vi) camelid antibodies or nanobodies (Revets et al; Expert Opin Biol Ther. 2005 Jan; 5 (1): 111-24), and (vii) separate complement determining regions (CDRs). Moreover, although the two domains of the Fv fragment, V L and V H , are encoded by different genes, but they can be recombinantly linked by a synthetic linker that enables them to be produced as a single protein chain, with V L and V H The domains are paired to form monovalent molecules (called single-chain antibodies or single-chain Fv (scFv), see, e.g., Bird et al., Science 242 ,423-426(1988) and Huston et al.,PNAS USA 85, 5879-5883 (1988)). Unless otherwise indicated or the context clearly indicates, these single-chain antibodies are included in the scope of the term "antibody". Although these fragments are generally included in the meaning of antibodies, they constitute unique features of the present invention as a whole and independently, showing different biological properties and utilities. In the context of the present invention, these and other useful antibody fragments are further discussed. It should also be understood that, unless otherwise indicated, the term "antibody" also includes polyclonal antibodies, monoclonal antibodies (mAbs), antibody-like polypeptides, such as chimeric antibodies and humanized antibodies, and fragments of antibodies that retain the ability to specifically bind to antigens (antigen-binding fragments) using any known technology, such as enzymatic cleavage, peptide synthesis and recombinant technology. The antibodies produced can have any isotype.

[0270] An "anti-TF antibody" is an antibody as described above, which specifically binds to the antigen tissue factor.

[0271] As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions from human germline immunoglobulin sequences. Human antibodies of the present invention may include amino acid residues that are not encoded by human germline immunoglobulin sequences (e.g., by random or site-directed mutagenesis in vitro, or by mutations introduced during gene shuffling or by somatic mutations in vivo). However, as used herein, the term "human antibody" is not intended to include antibodies in which CDR sequences from another mammalian species (e.g., mouse) germline are grafted onto human framework sequences.

[0272] In a preferred embodiment, antibody of the present invention is separated.As used herein, "separated antibody" is intended to indicate the following antibody, which is substantially free of other antibodies with different antigenic specificities (for example, the separated antibody of specific binding tissue factor is substantially free of the antibody of specific binding antigens except tissue factor). However, the separated antibody of the epitope, isotype or variant of specific binding human tissue factor can have cross-reactivity with other related antigens, for example, from antigens of other species (for example tissue factor interspecies homologues). And the separated antibody can be substantially free of other cell materials and / or chemicals.In one embodiment of the invention, two or more "separated" monoclonal antibodies with different antigen binding specificities are combined in a well-defined composition.

[0273] When two or more antibodies are used in the context of this document, the term "competes with" or "cross-competes with" means that the two or more antibodies compete for binding to TF, for example, in the assay described herein in Example 6. For some antibody pairs, competition in the assay method of Example 6 can only be observed when one antibody is coated on the plate and the other is used for competition, but not vice versa. When used herein, the term "competes with" is also intended to cover these combination antibodies.

[0274] As used herein, the term "monoclonal antibody" or "monoclonal antibody composition" refers to a preparation of antibody molecules having a single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope. Thus, the term "human monoclonal antibody" refers to an antibody displaying a single binding specificity having variable and constant regions from human germline immunoglobulin sequences. Human monoclonal antibodies can be produced by hybridomas comprising B cells obtained from transgenic or transchromosomal non-human animals (e.g., transgenic mice) fused to immortalized cells having a genome comprising a human heavy chain transgene and a light chain transgene.

[0275] As used herein, in the context of the binding of an antibody to a predetermined antigen, the term "binding" typically refers to its binding in an amount equivalent to K D Approximately 10 -7 M or smaller, for example, about 10 -8 M or smaller, for example, about 10 -9 M or smaller, about 10 -10 M or smaller, or about 10 -11 The binding affinity of the antibody to the predetermined antigen is 240M or less (e.g., when measured by surface plasmon resonance (SPR) technology using an antigen as a ligand and an antibody as an analyte in a BIAcore 3000 instrument), and the binding affinity to the predetermined antigen is compared to the binding affinity to a nonspecific antigen (e.g., BSA or casein) other than the predetermined antibody or a closely related antigen, and the K of the predetermined antigen is 240M or less. D K D The amount by which the affinity is reduced depends on the K of the antibody. D , so when the antibody's K D When very low (ie, the antibody is highly specific), the amount by which affinity for the antigen is lower than affinity for a nonspecific antigen may be at least 10,000-fold.

[0276] As used herein, the term “k d ”(sec -1) refers to the dissociation rate constant for a specific antibody-antigen interaction. This value is also called k off value.

[0277] As used herein, the term “k a ”(M -1 x sec -1 ) refers to the association rate constant for a specific antibody-antigen interaction.

[0278] As used herein, the term “K D ”(M) refers to the dissociation equilibrium constant for a specific antibody-antigen interaction.

[0279] As used herein, the term “K A ”(M -1 ) refers to the binding equilibrium constant for a specific antibody-antigen interaction and is expressed by k a Divide by k d get.

[0280] The present invention also provides V comprising the antibody of the embodiment L Area V H V used in the context of anti-TF antibodies L 、V H Functional variants of the CDRs or CDRs still allow the antibody to retain at least a substantial portion (at least about 50%, 60%, 70%, 80%, 90%, 95% or more) of the affinity / avidity and / or specificity / selectivity of the antibody of the invention, and in some cases, such anti-TF antibodies may have greater affinity, selectivity and / or specificity than the parent antibody.

[0281] These functional variants typically retain significant sequence identity with the parent antibody. The percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % homology=number of identical positions / total number of positions x100), while taking into account the number of gaps and the length of each gap, the introduction of gaps is for the needs of optimal comparison between the two sequences. The sequence alignment between the two sequences and the determination of the percent identity can be realized with a mathematical algorithm, as described in the following non-limiting examples.

[0282] The percent identity between two nucleotide sequences can be determined using the GAP program in the GCG software package (available at http: / / www.gcg.com) using the NWSgapdna.CMP matrix, a gap weight of 40, 50, 60, 70 or 80, and a length weight of 1, 2, 3, 4, 5 or 6. The percent identity between two nucleotide or amino acid sequences can also be determined using the method of E. Meyers and W. Miller, Comput. Appl. Biosci4 , 11-17 (1988)), which has been incorporated into the ALIGN program (version 2.0), using the PAM120 weighted residue table, a gap length penalty of 12, and a gap penalty of 4. In addition, the percent identity between two amino acid sequences can be determined using Needleman and Wunsch, J. Mol. Biol. 48 , 444-453 (1970)) algorithm, which was introduced into the GAP program of the GCG software package (available from http: / / www.gcg.com), using the Blossum 62 matrix or the PAM250 matrix, gap weights of 16, 14, 12, 10, 8, 6, or 4, and length weights of 1, 2, 3, 4, 5, or 6.

[0283] The sequence of a CDR variant may differ from that of the parent antibody sequence by predominantly conservative substitutions, e.g., at least about 35%, about 50% or more, about 60% or more, about 70% or more, about 75% or more, about 80% or more, about 85% or more, about 90% or more, about 95% or more (e.g., about 65-99%, such as about 96%, 97% or 98%) of the substitutions in the variant are conservative substitutions of amino acid residues.

[0284] The sequence of a CDR variant may differ from that of the parent antibody sequence by predominantly conservative substitutions, e.g., at least 10 substitutions in the variant, e.g., at least 9, 8, 7, 6, 5, 4, 3, 2 or 1 substitution are conservative amino acid residue substitutions.

[0285] In the context of the present invention, conservative substitutions may be defined by substitutions within the amino acid classes reflected in one or more of the following three tables.

[0286] Classification of amino acid residues for conservative substitutions

[0287]

[0288]

[0289] Other conservative amino acid residue substitution classifications

[0290] 1 A S T 2 D E 3 N Q 4 R K 5 I L M 6 F Y W

[0291] Other physical and functional classifications of amino acid residues

[0292] Alcohol residue S and T Aliphatic residues I, L, V, and M Cyclic olefin related residues F, H, W, and Y Hydrophobic residues A, C, F, G, H, I, L, M, R, T, V, W, and Y Negatively charged residues D and E Polar residues C, D, E, H, K, N, Q, R, S, and T Positively charged residues H, K, and R Small residues A, C, D, G, N, P, S, T, and V Minimal residue A, G, and S Residues involved in turn formation A, C, D, E, G, H, K, N, Q, R, S, P, and T Flexible residues Q, T, K, S, G, P, D, E, and R

[0293] More conservative substitution groupings include: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and aspartate-glutamate.

[0294] Other amino acid groupings may also be established using the principles described in, for example, Creighton (1984) Proteins: Structure and Molecular Properties (2d Ed. 1993), WH Freeman and Company.

[0295] In one embodiment of the invention, the conservation of hydropathic / hydrophilic properties and residue weight / size is also substantially maintained in the variant CDRs compared to the CDRs of the antibodies of the embodiments (e.g., the weight class, hydropathic score, or both of the sequence are retained by at least about 50%, at least about 60%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or more (e.g., about 65-99%)). For example, conservative residue substitutions may also be, or alternatively may be, substitutions based on strong or weak or weight based conservation groups, as is known in the art.

[0296] Residues that retain similarity can also or alternatively be measured by a similarity score determined using the BLAST program (e.g., BLAST 2.2.8 accessible through NCBI, using standard settings BLOSUM62, open gap = 11 and extended gap = 1). Suitable variants exhibit at least about 45%, such as at least about 55%, at least about 65%, at least about 75%, at least about 85%, at least about 90%, at least about 95%, or more (e.g., about 70-99%) similarity compared to the parent peptide.

[0297] As used herein, "isotype" refers to the immunoglobulin family (eg, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM) encoded by the heavy chain constant region genes.

[0298] The term "epitope" refers to a protein determinant that can specifically bind to an antibody. An epitope is usually composed of surface groups of a molecule, such as amino acids or sugar side chains, and usually has specific three-dimensional structural characteristics, as well as specific charged characteristics. The difference between a conformational epitope and a non-conformational epitope is that, in the presence of a denaturant, the binding to the former disappears, while the binding to the latter does not disappear. An epitope may include amino acid residues that are directly involved in binding (also referred to as the immunodominant component of an epitope), and other amino acid residues that are not directly involved in binding, such as amino acid residues that can be effectively blocked by a specific antigen binding peptide (in other words, these amino acid residues are located within the footprint of the specific antigen binding peptide).

[0299] As used herein, if the antibody is obtained from a system using human immunoglobulin sequences (e.g., by immunizing a transgenic mouse carrying a human immunoglobulin gene or by screening a human immunoglobulin gene library), and wherein the selected human antibody V domain sequence has at least 90%, such as at least 95%, such as at least 96%, such as at least 97%, such as at least 98%, or such as at least 99% similarity in amino acid V domain sequence to the amino acid sequence encoded by the germline immunoglobulin gene, then the human antibody is said to be "derived from" a specific germline sequence. Typically, except for the heavy chain CDR3, human antibodies from specific human germline sequences show no more than 20 amino acid differences, such as no more than 10 amino acid differences, such as no more than 9, 8, 7, 6 or 5, such as no more than 4, 3, 2 or 1 amino acid differences compared to the amino acid sequence encoded by the germline immunoglobulin gene.

[0300] As used herein, the term "inhibit growth" (e.g., of cells, such as tumor cells) is intended to include any measurable decrease in cell growth when contacted with an anti-TF antibody, compared to the growth of the same cells not contacted with the anti-TF antibody, for example, inhibition of cell culture growth by at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 99%, or 100%. This decrease in cell growth can occur by a variety of mechanisms, such as effector cell phagocytosis, ADCC, CDC, and / or apoptosis.

[0301] The term "bispecific molecule" is intended to include any agent, such as a protein, peptide, or protein or peptide complex, that has two different binding specificities. For example, a molecule can bind to or interact with (a) a cell surface antigen and (b) an Fc receptor on the surface of an effector. The term "bispecific antibody" is intended to include any anti-TF antibody that is a bispecific molecule. The term "bispecific antibody" also includes diabodies. Diabodies are bivalent, bispecific antibodies in which VH and V L The domains are expressed on a single polypeptide chain, but are connected by a linker that is too short to allow pairing of the two domains on the same chain, thereby forcing the domains to pair with the complementary domains on another chain and create two antigen-binding sites (see, e.g., Holliger, P. et al., PNAS USA 90 ,6444-6448(1993),Poljak,RJetal.,Structure 2 ,1121-1123(1994)).

[0302] An "antibody with defective effector function" or "effector function defective antibody" refers to an antibody that has a significantly reduced or absent ability to activate one or more effector mechanisms (e.g., complement activation or Fc receptor binding). Thus, the ability of the effector function defective antibody to mediate antibody-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) is significantly reduced or absent. An example of such an antibody is IgG4.

[0303] The term "monovalent antibody" in the context of the present invention means that the antibody molecule is able to bind to a single antigen molecule and thus has no antigen cross-linking capability.

[0304] The term "stabilized IgG4 antibody" refers to an IgG4 antibody that has been modified to reduce half-molecule exchange (see van der Neut Kolfschoten M et al. (2007) Science 14; 317(5844) and references therein, see also Labrijn et al. (2009) Nature Biotechnology, 27, 767-771).

[0305] As used herein, the term "effector cell" refers to an immune cell that participates in the effector phase of an immune response (as opposed to the recognition phase and activation phase of an immune response). Examples of immune cells include cells of bone marrow or lymphoid origin, such as lymphocytes (e.g., B cells and T cells, including cytolytic T cells). Effector cells include, for example, cytotoxic T cells (CTLs), killer cells, natural killer cells, macrophages, monocytes, eosinophils, polymorphonuclear cells, such as neutrophils, granulocytes, mast cells, and basophils. Some effector cells express specific Fc receptors and perform specific immune functions. In some embodiments, effector cells are capable of inducing antibody-dependent cellular cytotoxicity (ADCC), such as natural killer cells, which are capable of inducing ADCC. For example, monocytes and macrophages expressing FcRs participate in the specific killing of target cells and present antigens to other components of the immune system, or bind to cells presenting antigens. In one embodiment, effector cells can phagocytose target antigens or target cells. The expression of specific FcRs on effector cells can be regulated by humoral factors, such as cytokines. For example, it has been found that the expression of FcγRI is upregulated by interferon γ (IFN-γ) and / or G-CSF. This increased expression can increase the cytotoxicity of cells carrying FcγRI to the target. Effector cells are capable of phagocytosis or lysis of target antigens or target cells.

[0306] As used herein, the term "vector" is intended to indicate a nucleic acid molecule capable of transporting another nucleic acid connected thereto. One type of vector is a "plasmid", which refers to a circular double-stranded DNA loop, in which additional DNA segments can be connected. Another type of vector is a viral vector, in which additional DNA segments can be connected to the viral genome. Certain vectors are capable of autonomous replication in the host cell into which they are introduced (e.g., bacterial vectors and episomal mammalian vectors with bacterial replication origins). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of the host cell after being introduced into the host cell, thereby replicating with the host genome. Moreover, certain vectors can guide the expression of genes operably connected thereto. These vectors are referred to herein as "recombinant expression vectors" (or simply "expression vectors"). Generally, useful expression vectors in recombinant DNA technology are often in the form of plasmids. In this specification, "plasmid" and "vector" can be used interchangeably because plasmids are the most frequently used vector forms. However, the present invention is intended to include these other forms of expression vectors, such as viral vectors (replication-defective retroviruses, adenoviruses, adeno-associated viruses), which perform equivalent functions.

[0307] As used herein, the term "recombinant host cell" (or simply "host cell") is intended to indicate a cell into which an expression vector has been introduced. It should be understood that these terms are intended to indicate not only the specific subject cell, but also the progeny of these cells. Because certain modifications may occur in subsequent generations due to mutations or environmental influences, these progeny may not actually be identical to the parent cell, but are still included in the scope of the term "host cell" used herein. Recombinant host cells include, for example, transfectomas, such as CHO cells, HEK293 cells, NS / 0 cells, and lymphocytes.

[0308] As used herein, the term "transfectoma" includes recombinant eukaryotic host cells, such as CHO cells, NS / 0 cells, HEK293 cells, plant cells or fungi, including yeast cells, that express the antibody.

[0309] The term "transgenic non-human animal" refers to a non-human animal whose genome includes one or more human heavy and / or light chain transgenes or transchromosomes (integrated or not integrated into the natural genomic DNA of the animal) and is capable of expressing fully human antibodies. For example, a transgenic mouse may have a human light chain transgene, as well as a human heavy chain transgene or a human heavy chain transchromosome, so that when immunized with TF antigen and / or cells expressing TF, the mouse can produce human anti-TF antibodies. The human heavy chain transgene can be integrated into the chromosomal DNA of the mouse, as in transgenic mice (such as human monoclonal antibody mice such as HCo7 or HCo12 mice), or the human heavy chain transgene can be maintained outside the chromosome, as in the transchromosomal KM mice described in WO02 / 43478. These transgenic and transchromosomal mice (collectively referred to herein as "transgenic mice") are capable of producing multiple isotypes (e.g., IgG, IgA, IgM, IgD and / or IgE) of human monoclonal antibodies against a given antigen through VDJ recombination and isotype switching. Transgenic non-human animals can also be used to produce antibodies against specific antigens by introducing genes encoding such specific antibodies, for example by operatively linking the gene to a gene that is expressed in the milk of the animal.

[0310] "Treatment" refers to the administration of an effective amount of a therapeutically active compound of the invention to alleviate, relieve, arrest or eradicate (cure) a disorder or disease state.

[0311] An "effective amount" refers to an amount effective, at dosages and for periods of time necessary, to achieve the desired therapeutic result. A therapeutically effective amount of an anti-TF antibody may vary depending on factors such as the disease state, age, sex, and weight of the individual, and the ability of the anti-TF antibody to elicit a desired response in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutic benefits thereof.

[0312]

[00136] "Anti-idiotypic" (Id) antibodies are antibodies that recognize unique determinants typically associated with the antigen binding site of an antibody.

[0313] Further aspects and embodiments of the invention

[0314] As mentioned above, in a first aspect, the present invention relates to a human antibody that binds to human tissue factor.

[0315] In one embodiment, the apparent affinity (EC) of an antibody binding to the extracellular domain of tissue factor when measured using the assay described in Example 13 is 50 ) is 3 nM or lower, such as 0.50 nM or lower, such as 0.35 nM or lower, such as 0.20 nM or lower, such as 0.1 nM or lower.

[0316] In another embodiment, the antibody binds to mammalian cells expressing tissue factor (e.g., A431 cells transfected with a construct encoding tissue factor), preferably with an apparent affinity (EC) of 0.001 when measured using the assay described in Example 14. 50 ) is preferably 10 nM or lower, such as 8 nM or lower, such as 5 nM or lower, such as 2 nM or lower, such as 1 nM or lower, such as 0.5 nM or lower, such as 0.3 nM or lower.

[0317] In another embodiment, the antibody is capable of inducing antibody-dependent cellular cytotoxicity in A431 cells, preferably, when measured using the assay described in Example 20, EC 50 The value is 2 nM or less, such as 1 nM or less, such as 0.7 nM or less, or 0.3 nM or less, such as 0.20 nM or less, or 0.1 nM or less, or 0.05 nM or less.

[0318] In another embodiment, the antibody is effective in inhibiting the growth of established MDA-MB-231 tumors when measured by the method described in Example 24 and / or inhibiting the growth of established BxPC3 tumors when measured by the method described in Example 26.

[0319] In another embodiment, the antibody can inhibit tissue factor-induced coagulation, preferably, when measured using the assay method described in Example 19, the median inhibitory concentration is less than 10nM, such as less than 5nM, such as less than 2nM, such as less than 1nM.

[0320] In another embodiment, the antibody does not inhibit coagulation.In one embodiment, coagulation is inhibited by up to 30%, such as 25%, such as 20%, such as 15%, such as 10% or such as 5%, compared to native levels.

[0321] In a further embodiment, the antibody can inhibit the binding of FVIIa to tissue factor, preferably with a maximum inhibition value of greater than 80%, such as greater than 90% inhibition, when measured using the assay described in Example 15.

[0322] In a further embodiment, the antibody can inhibit FVIIa-induced IL-8 release from MDA-MB-231 cells, preferably, when measured using the assay method described in Example 17, the maximum inhibition value is greater than 40%, such as greater than 50%, such as greater than 60% inhibition.

[0323] In a further embodiment, the antibody may inhibit the conversion of FX to Fxa by the TF / FVIIa complex, preferably by less than 50%, such as less than 40%, such as in the range of 1-30%, when measured using the assay described in Example 18.

[0324] In a further embodiment, the antibody competes for binding to tissue factor with an antibody comprising a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:65.

[0325] In a further embodiment, the binding of an antibody of the invention to tissue factor does not involve all of the following three residues: W at position 45, K at position 46, or Y at position 94 of tissue factor. In an even further embodiment, the binding does not involve any of the following residues: W at position 45, K at position 46, or Y at position 94 (these codes refer to mature TF, the equivalent positions in Genbank entry NP_001984 are 77, 78, and 126).

[0326] In another embodiment of the antibody of the invention, the antibody competes for binding to tissue factor with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.

[0327] In a further embodiment, the antibody may inhibit FVIIa-induced ERK phosphorylation, preferably with a median inhibitory concentration of less than 10 nM, such as less than 5 nM, such as less than 2 nM, when assayed using the assay described in Example 16.

[0328] In a further embodiment, the antibody can inhibit ERK phosphorylation, preferably, when measured using the assay method described in Example 16, its median inhibitory concentration is less than 10nM, such as less than 5nM, such as less than 2nM, and when measured using the assay method described in Example 17, the maximum inhibition of FVII-induced IL-8 release does not exceed 10%.

[0329] In a further embodiment, the antibody is capable of inducing C3c and C4c deposition, preferably, wherein the antibody is capable of inducing C3c and C4c deposition as determined as described in Example 21.

[0330] In a further embodiment, the antibody Fab fragment binds to the extracellular domain of tissue factor as described in Example 28 with an EC50 value of less than 0.1 μg / mL, such as less than 0.05 μg / mL, such as less than 0.04 μg / mL when measured by ELISA.

[0331] In a further embodiment, the antibody Fab fragment binds to the extracellular domain of tissue factor as described in Example 28 with an EC50 value higher than 1.0 μg / mL when measured by ELISA.

[0332] In a further embodiment, the antibody Fab fragment binds to the extracellular domain of tissue factor as described in Example 28 with an EC50 value of less than 10 μg / mL, such as less than 1 μg / mL, such as less than 0.5 μg / mL, or less than 0.2 μg / mL.

[0333] In a further embodiment, the antibody binds to human tissue factor but not to murine tissue factor, and has reduced binding to a shuffled construct 42-84mm comprising the human TF sequence except for amino acids 42-84, which are replaced by the mouse sequence, as described in Example 27, compared to binding to human TF.

[0334] In a further embodiment, the antibody binds to human tissue factor but not to murine tissue factor, and has reduced binding to a shuffled construct 85-122mm comprising the human TF sequence except for amino acids 85-122, which are replaced by the mouse sequence, as described in Example 27, compared to binding to human TF.

[0335] In a further embodiment, the antibody binds to human tissue factor but not to murine tissue factor, and has reduced binding to a shuffled construct 123-137mm comprising the human TF sequence except for amino acids 123-137, which are replaced by the mouse sequence, as described in Example 27, compared to binding to human TF.

[0336] In a further embodiment, the antibody binds to human tissue factor but not to murine tissue factor, and binding to a shuffled construct 185-225mm comprising the human TF sequence except for amino acids 185-225, which are replaced by the mouse sequence, as described in Example 27, is reduced compared to binding to human TF.

[0337] In a further embodiment, the antibody binds to human tissue factor but not to murine tissue factor, and has reduced binding to a shuffled construct 226-250mm comprising the human TF sequence except for amino acids 226-250, which are replaced by the mouse sequence, as described in Example 27, compared to binding to human TF.

[0338] In a further embodiment, the antibody exhibits reduced binding to more than one of the shuffled constructs compared to binding to human TF. In one embodiment, the antibody exhibits reduced binding to constructs 42-84mm and 85-122mm. In one embodiment, the antibody exhibits reduced binding to constructs 123-137mm and 185-225mm. In one embodiment, the antibody exhibits reduced binding to constructs 123-137mm and 185-225mm and construct 226-250mm.

[0339] In a further embodiment, the antibody is capable of inducing C3c and C4c deposition, preferably, the antibody is capable of inducing C3c and C4c deposition when assayed as described in Example 21.

[0340] In one embodiment of the antibody of the present invention, the antibody

[0341] - competes for binding to tissue factor with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65, and

[0342] - does not compete with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93 for binding to tissue factor.

[0343] In a further embodiment, the antibody comprises a VH CDR3 region having

[0344] a) The sequence shown below

[0345] -SEQ ID No:12,

[0346] -SEQ ID No:16,

[0347] -SEQ ID No:20,

[0348] -SEQ ID No:24,

[0349] -SEQ ID No:28,

[0350] or

[0351] b) variants of any of the sequences described, for example variants having up to 1, 2, 3, 4, or 5 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0352] In a further embodiment, the antibody comprises a VHCDR3 region having a sequence as shown in SEQ ID NO: 12 or a variant thereof, wherein the variant comprises a modification at one or more of positions 2, 3, 6, 9 and 11, preferably, wherein the modification is a substitution, more preferably, wherein the substitution is selected from the following group:

[0353] a) R at position 2 is replaced by K,

[0354] b) S at position 3 is replaced by A or T,

[0355] c) G at position 6 is replaced by T,

[0356] d) L at position 9 is replaced by F, and

[0357] e) S at position 11 is replaced by Y.

[0358] In another embodiment, the antibody comprises:

[0359] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 10, 11 and 12, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 66, 67 and 68,

[0360] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 14, 15 and 16, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 70, 71 and 72,

[0361] c) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 18, 19 and 20, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 74, 75 and 76,

[0362] d) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 22, 23 and 24, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 78, 79 and 80,

[0363] e) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 26, 27 and 28, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 82, 83 and 84, or

[0364] f) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0365] In a further embodiment, the antibody comprises a VH, which

[0366] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 9, 13, 17, 21 and 25, or

[0367] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 9, 13, 17, 21, 21 and 25.

[0368] In a further embodiment, the antibody comprises a VL, which

[0369] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from SEQ ID NO: 65, 69, 73, 77 and 81, or

[0370] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VL region sequence selected from SEQ ID NO: 65, 69, 73, 77 and 81.

[0371] In a further embodiment, the antibody comprises:

[0372] a) a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65,

[0373] b) a VH region comprising the sequence of SEQ ID NO: 13 and a VL region comprising the sequence of SEQ ID NO: 69,

[0374] c) a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 73,

[0375] d) a VH region comprising the sequence of SEQ ID NO: 21 and a VL region comprising the sequence of SEQ ID NO: 77,

[0376] e) a VH region comprising the sequence of SEQ ID NO: 25 and a VL region comprising the sequence of SEQ ID NO: 81, or

[0377] f) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0378] In a further embodiment, the antibody

[0379] - competes for binding to tissue factor with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65, and

[0380] - competes for binding to tissue factor with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.

[0381] In a further embodiment, the antibody comprises a VH CDR3 region having

[0382] a) The sequence shown below

[0383] -SEQ ID No:8,

[0384] -SEQ ID No:52,

[0385] or

[0386] b) variants of any of the sequences described, for example variants having at most 1, 2 or 3 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0387] In a further embodiment, the antibody comprises:

[0388] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 6, 7 and 8, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 62, 63 and 64,

[0389] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 50, 51 and 52, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 106, 107 and 108, or

[0390] c) a variant of any of the antibodies described above, wherein the variant preferably has 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0391] In a further embodiment, the antibody comprises a VH, which

[0392] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NOs: 5 and 49, or

[0393] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 5 and 49.

[0394] In a further embodiment, the antibody comprises a VL, which

[0395] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from SEQ ID NOs: 61 and 105, or

[0396] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VL region sequence selected from SEQ ID NO: 61 and 105.

[0397] In a further embodiment, the antibody comprises:

[0398] a) a VH region comprising the sequence of SEQ ID NO: 5 and a VL region comprising the sequence of SEQ ID NO: 61,

[0399] b) a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 105, or

[0400] c) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0401] In a further embodiment, the antibody

[0402] - does not compete with an antibody comprising a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65 for binding to tissue factor, and

[0403] - competes for binding to tissue factor with an antibody comprising a VH region comprising the sequence of SEQ ID NO:37 and a VL region comprising the sequence of SEQ ID NO:93.

[0404] In a further embodiment, the antibody comprises a VH CDR3 region having

[0405] a) The sequence shown below

[0406] -SEQ ID No:32,

[0407] -SEQ ID No:36,

[0408] -SEQ ID No:40,

[0409] -SEQ ID No:56,

[0410] or

[0411] b) variants of any of the sequences described, for example variants having at most 1, 2 or 3 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0412] In a further embodiment, the antibody comprises:

[0413] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 30, 31 and 32, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 86, 87 and 88,

[0414] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 34, 35 and 36, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 90, 91 and 92,

[0415] c) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 38, 39 and 40, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 94, 95 and 96,

[0416] d) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 54, 55 and 56, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 110, 111 and 112, or

[0417] e) A variant of any of the antibodies described, wherein the variant preferably has 1, 2 or 3 amino acid modifications in the sequence, more preferably, amino acid substitutions, such as conservative amino acid substitutions.

[0418] In a further embodiment, the antibody comprises a VH, which

[0419] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 29, 33, 37 and 53, or

[0420] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 29, 33, 37 and 53.

[0421] In a further embodiment, the antibody comprises a VL, which

[0422] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from SEQ ID NO: 85, 89, 93 and 109, or

[0423] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VL region sequence selected from SEQ ID NO: 85, 89, 93 and 109.

[0424] In a further embodiment, the antibody comprises:

[0425] a) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO: 85,

[0426] b) a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 89,

[0427] c) a VH region comprising the sequence of SEQ ID NO: 37 and a VL region comprising the sequence of SEQ ID NO: 93,

[0428] d) a VH region comprising the sequence of SEQ ID NO: 53 and a VL region comprising the sequence of SEQ ID NO: 109, or

[0429] e) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0430] In a further embodiment, the antibody competes for binding to tissue factor with an antibody comprising a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97.

[0431] In a further embodiment, the antibody comprises a VH CDR3 region having

[0432] a) The sequence shown below

[0433] -SEQ ID No:4,

[0434] -SEQ ID No:44,

[0435] -SEQ ID No:48,

[0436] or

[0437] b) variants of any of the sequences described, for example variants having at most 1, 2 or 3 amino acid modifications, preferably substitutions, such as conservative substitutions.

[0438] In a further embodiment, the antibody comprises:

[0439] a) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 2, 3 and 4, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 58, 59 and 60,

[0440] b) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 42, 43 and 44, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 98, 99 and 100,

[0441] c) a VH region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 46, 47 and 48, and a VL region comprising CDR1, 2 and 3 sequences of SEQ ID NOs: 102, 103 and 104, or

[0442] d) a variant of any of the antibodies described above, wherein the variant preferably has 1, 2 or 3 amino acid modifications in the sequence, more preferably, amino acid substitutions, such as conservative amino acid substitutions.

[0443] In a further embodiment, the antibody comprises a VH, which

[0444] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VH region sequence selected from SEQ ID NO: 1, 41 and 45, or

[0445] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VH region sequence selected from SEQ ID NO: 1, 41 and 45.

[0446] In a further embodiment, the antibody comprises a VL, which

[0447] a) has at least 80% identity, such as at least 90%, at least 95%, or at least 98% or 100% identity to a VL region sequence selected from SEQ ID NO: 57, 97 and 101, or

[0448] b) has up to 20, such as 15, or 10, or 5, 4, 3, 2 or 1 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, compared to the VL region sequence selected from SEQ ID NO: 57, 97 and 101.

[0449] In a further embodiment, the antibody comprises:

[0450] a) a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 57,

[0451] b) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97,

[0452] c) a VH region comprising the sequence of SEQ ID NO: 45 and a VL region comprising the sequence of SEQ ID NO: 101, or

[0453] d) a variant of any of the antibodies described, wherein the variant preferably has at most 1, 2 or 3 amino acid modifications, more preferably amino acid substitutions, such as conservative amino acid substitutions, in the sequence.

[0454] In a further embodiment, the affinity of the antibody of the invention for tissue factor is less than 5 nM, such as less than 3.5 nM, such as less than 2 nM, when measured using the method described in Example 22 herein.

[0455] One particularly interesting group of antibodies of the invention are characterized by normal or high affinity and high off-rate (kd) for binding to tissue factor. As demonstrated herein, such antibodies can show tumor-specific binding in that they bind to cancerous tissues but not or less to healthy tissues. Without being bound to any particular theory, we hypothesize that this group of antibodies is only able to bind well to cells that express high levels of TF because binding is only efficient when it is bivalent. Examples of these antibodies include antibodies 044, 098, and 111 as described herein.

[0456] Thus, in one embodiment, the kd of an antibody of the invention is greater than 10 when measured using the affinity method described in Example 22 herein. -3 sec -1, and the affinity is less than 5 nM, such as less than 1 nM, such as less than 0.2 nM when measured using the affinity method described in Example 22 herein.

[0457] In another embodiment, the kd of an antibody of the invention is greater than 10 when measured using the affinity method described in Example 22 herein. -3 sec -1 , and / or when measured using the affinity method described in Example 22 herein, ka is greater than 5x10 4 mol -1 sec -1 .

[0458] In a further embodiment, the antibody shows no binding to healthy tissue, particularly no binding to human glomeruli, when assayed using the assay described in Example 23, but shows binding to pancreatic tumors when assayed using the assay described in Example 23 herein.

[0459] In a further embodiment, the antibody is effective in inhibiting the growth of established BX-PC3 tumors when assayed by the method described in Example 26 herein.

[0460] In another embodiment, the antibody of the invention has one or more of the following properties: inhibiting proliferation, inhibiting tumor angiogenesis, inducing tumor cell apoptosis, and binding to alternatively spliced ​​tissue factor.

[0461] In a further embodiment, an antibody of the invention competes for binding to tissue factor with an antibody comprising:

[0462] a) a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65,

[0463] b) a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 57,

[0464] c) a VH region comprising the sequence of SEQ ID NO: 5 and a VL region comprising the sequence of SEQ ID NO: 61,

[0465] d) a VH region comprising the sequence of SEQ ID NO: 13 and a VL region comprising the sequence of SEQ ID NO: 69,

[0466] e) a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 73,

[0467] f) a VH region comprising the sequence of SEQ ID NO: 21 and a VL region comprising the sequence of SEQ ID NO: 77,

[0468] g) a VH region comprising the sequence of SEQ ID NO: 25 and a VL region comprising the sequence of SEQ ID NO: 81,

[0469] h) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO: 85,

[0470] i) a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 89,

[0471] j) a VH region comprising the sequence of SEQ ID NO: 37 and a VL region comprising the sequence of SEQ ID NO: 93,

[0472] k) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97,

[0473] 1) a VH region comprising the sequence of SEQ ID NO: 45 and a VL region comprising the sequence of SEQ ID NO: 101,

[0474] m) a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 105, or

[0475] n) a VH region comprising the sequence of SEQ ID NO:53 and a VL region comprising the sequence of SEQ ID NO:109.

[0476] In a further embodiment, an antibody of the invention binds to the same epitope on tissue factor as an antibody having:

[0477] a) a VH region comprising the sequence of SEQ ID NO: 9 and a VL region comprising the sequence of SEQ ID NO: 65,

[0478] b) a VH region comprising the sequence of SEQ ID NO: 1 and a VL region comprising the sequence of SEQ ID NO: 57,

[0479] c) a VH region comprising the sequence of SEQ ID NO: 5 and a VL region comprising the sequence of SEQ ID NO: 61,

[0480] d) a VH region comprising the sequence of SEQ ID NO: 13 and a VL region comprising the sequence of SEQ ID NO: 69,

[0481] e) a VH region comprising the sequence of SEQ ID NO: 17 and a VL region comprising the sequence of SEQ ID NO: 73,

[0482] f) a VH region comprising the sequence of SEQ ID NO: 21 and a VL region comprising the sequence of SEQ ID NO: 77,

[0483] g) a VH region comprising the sequence of SEQ ID NO: 25 and a VL region comprising the sequence of SEQ ID NO: 81,

[0484] h) a VH region comprising the sequence of SEQ ID NO: 29 and a VL region comprising the sequence of SEQ ID NO: 85,

[0485] i) a VH region comprising the sequence of SEQ ID NO: 33 and a VL region comprising the sequence of SEQ ID NO: 89,

[0486] j) a VH region comprising the sequence of SEQ ID NO: 37 and a VL region comprising the sequence of SEQ ID NO: 93,

[0487] k) a VH region comprising the sequence of SEQ ID NO:41 and a VL region comprising the sequence of SEQ ID NO:97,

[0488] 1) a VH region comprising the sequence of SEQ ID NO: 45 and a VL region comprising the sequence of SEQ ID NO: 101,

[0489] m) a VH region comprising the sequence of SEQ ID NO: 49 and a VL region comprising the sequence of SEQ ID NO: 105, or

[0490] n) a VH region comprising the sequence of SEQ ID NO:53 and a VL region comprising the sequence of SEQ ID NO:109.

[0491] In a further embodiment, the antibodies of the invention comprise:

[0492] - derived from a human germline selected from the group consisting of V H The heavy chain variable region of the sequences: IGHV1-18*01, IGHV3-23*01, IGHV3-30*01, IGHV3-33*01, IGHV3-33*03, IGHV1-69*02, IGHV1-69*04 and IGHV5-51*01, and / or

[0493] - derived from a human germline selected from the group consisting of V κ The sequences of the light chain variable regions are: IGKV3-20*01, IGKV1-13*02, IGKV3-11*01 and IGKV1D-16*01.

[0494] In a further aspect, the invention relates to a monoclonal anti-TF antibody comprising a VH region having a sequence as shown in SEQ ID NO: 9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49 or 53, or a variant of any of said sequences, said variant having up to 25 amino acid modifications, such as 20, such as up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions.

[0495] A variant of a sequence as shown in SEQ ID NO: 9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49 or 53 may have at least 80% identity, such as at least 85% identity, or 90% identity, or 95% identity, such as 96% identity, or 97% identity, or 98% identity, or 99% identity to any of said sequences.

[0496] In one aspect of the invention, an isolated monoclonal anti-TF antibody comprises a VL sequence having a sequence as shown in SEQ ID NO: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101 or 105, or a variant of any of said sequences, said variant having, for example, up to 25 amino acid modifications, such as 20, such as up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions.

[0497] A variant of a sequence as shown in SEQ ID NO: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101 or 105 may have at least 80% identity to any of the sequences, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity.

[0498] In another embodiment, the antibody comprises:

[0499] a), a VL region having a sequence selected from the group consisting of SEQ ID No: 65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101 or 105, and a VH region having a sequence selected from the group consisting of SEQ ID No: 9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49 or 53,

[0500] b) A variant of any of the above, wherein said variant preferably only has conservative substitutions in said sequence.

[0501] In a preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 65 and a VH region having a sequence as shown in SEQ ID No: 9, or a variant of either of these two sequences, wherein the variant

[0502] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0503] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO: 9 or SEQ ID NO: 65, respectively.

[0504] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 57 and a VH region having a sequence as shown in SEQ ID No: 1, or a variant of either sequence, wherein the variant

[0505] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0506] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO: 1 or SEQ ID NO: 57, respectively.

[0507] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 61 and a VH region having a sequence as shown in SEQ ID No: 5, or a variant of either sequence, wherein the variant

[0508] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0509] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO: 5 or SEQ ID NO: 61, respectively.

[0510] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 69 and a VH region having a sequence as shown in SEQ ID No: 13, or a variant of either sequence, the variant:

[0511] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0512] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO: 13 or SEQ ID NO: 69, respectively.

[0513] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 73 and a VH region having a sequence as shown in SEQ ID No: 17, or a variant of either sequence, the variant:

[0514] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0515] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO: 17 or SEQ ID NO: 73, respectively.

[0516] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 77 and a VH region having a sequence as shown in SEQ ID No: 21, or a variant of either sequence, the variant:

[0517] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0518] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO:21 or SEQ ID NO:77, respectively.

[0519] In another preferred embodiment, the antibody comprises a VL region having the sequence shown in SEQ ID No: 81 and a VH region having the sequence shown in SEQ ID No: 25, or a variant of either sequence, the variant:

[0520] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0521] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO:25 or SEQ ID NO:81, respectively.

[0522] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 85 and a VH region having a sequence as shown in SEQ ID No: 29, or a variant of either sequence, the variant:

[0523] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0524] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO:29 or SEQ ID NO:85, respectively.

[0525] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 89 and a VH region having a sequence as shown in SEQ ID No: 33, or a variant of either sequence, the variant:

[0526] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0527] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO:33 or SEQ ID NO:89, respectively.

[0528] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 93 and a VH region having a sequence as shown in SEQ ID No: 37, or a variant of either sequence, the variant:

[0529] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0530] b) has at least 80% identity, such as at least 85% identity, or 90% identity, or 95% identity, such as 96% identity, or 97% identity, or 98% identity, or 99% identity to SEQ ID NO:37 or SEQ ID NO:93, respectively.

[0531] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 97 and a VH region having a sequence as shown in SEQ ID No: 41, or a variant of either sequence, the variant:

[0532] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0533] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO:41 or SEQ ID NO:97, respectively.

[0534] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 101 and a VH region having a sequence as shown in SEQ ID No: 45, or a variant of either sequence, the variant:

[0535] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0536] b) has at least 80% identity, such as at least 85% identity, or 90% identity, or 95% identity, such as 96% identity, or 97% identity, or 98% identity, or 99% identity to SEQ ID NO:45 or SEQ ID NO:101, respectively.

[0537] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 105 and a VH region having a sequence as shown in SEQ ID No: 49, or a variant of either sequence, the variant:

[0538] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0539] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO:49 or SEQ ID NO:105, respectively.

[0540] In another preferred embodiment, the antibody comprises a VL region having a sequence as shown in SEQ ID No: 109 and a VH region having a sequence as shown in SEQ ID No: 53, or a variant of either sequence, the variant:

[0541] a) has up to 25 amino acid modifications, such as 20, for example up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or

[0542] b) has at least 80% identity, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity to SEQ ID NO:53 or SEQ ID NO:109, respectively.

[0543] The monoclonal antibodies of the invention can be produced, for example, by the hybridoma method, which was first described in Kohler et al., Nature 256 , 495 (1975); or they may be produced by recombinant DNA methods. Monoclonal antibodies may also be isolated from phage antibody libraries using techniques such as those described in the following literature: Clackson et al., Nature 352 , 624-628 (1991) and Marks et al., J. Mol. Biol. 222 , 581-597 (1991). Monoclonal antibodies can be obtained from any suitable source. Thus, for example, monoclonal antibodies can be obtained from hybridomas, wherein the hybridomas can be prepared from murine splenic B cells obtained from mice immunized with the target antigen. The target antigen can be in the form of, for example, cells expressing the antigen on the surface, or nucleic acid encoding the target antigen. Monoclonal antibodies can also be obtained from hybridomas derived from antibody-expressing cells of immunized humans or non-human mammals (e.g., rats, rabbits, dogs, primates, etc.).

[0544] In one embodiment, the antibodies of the present invention are human antibodies. Human monoclonal antibodies to tissue factor can be produced using transgenic or transchromosomal mice carrying part of the human immune system rather than the mouse system. Such transgenic or transchromosomal mice include the following mice, which are respectively referred to as human monoclonal antibody mice (HuMAb) and KM mice, and are collectively referred to as "transgenic mice" herein.

[0545] The human mAb mouse contains human immunoglobulin gene miniloci encoding unrearranged human heavy chain variable and constant chain (μ and γ) and light chain variable and constant chain (κ) immunoglobulin sequences and also has targeted mutations that inactivate the endogenous μ and κ chain loci (Lonberg, N. et al., Nature 368, 856-859 (1994)). As a result, the mice show reduced mouse IgM or κ expression in response to immunization, and the introduced human heavy and light chain transgenes undergo class switching and somatic mutation to produce high-affinity human IgG, κ monoclonal antibodies (Lonberg, N. et al. (1994), supra; for review, see Lonberg, N. Handbook of Experimental Pharmacology 113 ,49-101(1994),Lonberg,N.and Huszar,D.,Intern.Rev.Immunol.Vol. 13 65-93 (1995) and Harding, F. and Lonberg, N. Ann. NYAcad.Sci 764 536-546 (1995)). The preparation of human monoclonal antibody mice is described in detail in the following literature: Taylor, L. et al., Nucleic Acids Research 20 ,6287-6295(1992),Chen,J.et al.,International Immunology 5 ,647-656(1993),Tuaillon et al.,J.Immunol. 152 ,2912-2920(1994),Taylor,L.et al.,International Immunology 6 ,579-591(1994),Fishwild,D.et al.,Nature Biotechnology 14 ,845-851(1996). See also US 5,545,806, US 5,569,825, US 5,625,126, US 5,633,425, US 5,789,650, US 5,877,397, US 5,661,016, US 5,814,318, US 5,874,299, US 5,770,429, US 5,545,807, WO 98 / 24884, WO 94 / 25585, WO 93 / 1227, WO 92 / 22645, WO 92 / 03918 and WO 01 / 09187.

[0546] HCo7 mice have a JKD disruption in their endogenous light chain (kappa) gene (e.g., Chen et al., EMBO J. 12 , 821-830 (1993)), has a CMD interruption in its endogenous heavy chain gene (as described in Example 1 of WO 01 / 14424), and has a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14 , 845-851 (1996)) and an HCo7 human heavy chain transgene (as described in US 5,770,429).

[0547] HCo12 mice have a JKD disruption in their endogenous light chain (kappa) gene (as described in Chen et al., EMBO J. 12 , 821-830 (1993)), has a CMD interruption in its endogenous heavy chain gene (as described in Example 1 of WO 01 / 14424), and has a KCo5 human kappa light chain transgene (as described in Fishwild et al., Nature Biotechnology 14 , 845-851 (1996)) and an HCo12 human heavy chain transgene (as described in Example 2 of WO 01 / 14424).

[0548] In the KM mouse strain, the endogenous mouse kappa light chain gene is expressed as described by Chen et al., EMBO J. 12 , 811-820 (1993), and the endogenous mouse heavy chain gene is homozygously interrupted as described in Example 1 of WO01 / 09187. This mouse strain carries a human kappa light chain transgene, KCo5, as described in Fishwild et al., Nature Biotechnology 14 , 845-851 (1996). This mouse strain also carries a human heavy chain transchromosome consisting of chromosome 14 segment hCF (SC20), as described in WO 02 / 43478.

[0549] The spleen cells of these transgenic mice can be used to produce hybridomas secreting human monoclonal antibodies according to well-known techniques. Human monoclonal or polyclonal antibodies of the present invention, or antibodies of the present invention derived from other species can also be produced by transgenic means, by producing another non-human animal or plant transgenic with target immunoglobulin heavy chain and light chain sequences, and producing antibodies in a recyclable form. Regarding transgenic production in mammals, antibodies can be produced in the milk of goats, cattle and other mammals and recovered therefrom. See, for example, US 5,827,690, US 5,756,687, US5,750,172 and US 5,741,957.

[0550] Furthermore, human antibodies of the present invention or antibodies of the present invention from other species can be produced by display technology (including but not limited to phage display, retroviral display, ribosome display and other technologies) using techniques well known in the art, and the resulting molecules can be further subjected to additional maturation processes, such as affinity maturation, which techniques are well known in the art (see, for example, Hoogenboom et al., J. Mol. Biol. 227 , 381(1991)(phage display), Vaughan et al., Nature Biotech 14 , 309(1996)(phage display), Hanes and Plucthau, PNAS USA 94 , 4937-4942(1997)(ribosome display),Parmley and Smith,Gene 73 ,305-318(1988)(phage display),Scott TIBS 17,241-245(1992),Cwirla et al.,PNAS USA 87 ,6378-6382(1990),Russel et al.,Nucl.Acids Research 21 ,1081-1085(1993),Hogenboom et al.,Immunol.Reviews 130 ,43-68(1992),Chiswell and McCaffertyTIBTECH 10 , 80-84 (1992), and US 5,733,743). If non-human antibodies are generated using display technology, these antibodies can be humanized.

[0551] The antibodies of the present invention may be of any isotype. The choice of isotype typically follows the desired effector function, such as ADCC induction. Examples of isotypes are IgG1, IgG2, IgG3, and IgG4. Any of the human light chain constant regions, κ or λ, may be used. If desired, the type of the anti-TF antibody of the present invention may be converted by known methods. For example, an antibody of the present invention that was originally IgM may be type-converted to an IgG antibody of the present invention. Further, type conversion technology may be used to convert one IgG subclass to another IgG subclass, such as from IgG1 to IgG2. Therefore, the effector function of the antibodies of the present invention may be changed to, for example, IgG1, IgG2, IgG3, IgG4, IgD, IgA, IgE, or IgM antibodies by isotype conversion for different therapeutic uses. In one embodiment, the antibody of the present invention is an IgG1 antibody, such as IgG1, κ.

[0552] In one embodiment, the antibody of the present invention is a full-length antibody, preferably an IgG1 antibody, in particular an IgG1, κ antibody. In another embodiment, the antibody of the present invention is an antibody fragment or a single-chain antibody.

[0553] Antibody fragments can be obtained, for example, by fragmentation using conventional techniques, and the resulting fragments can be screened based on use in the same manner as described herein for intact antibodies. For example, F(ab')2 fragments can be produced by treating antibodies with pepsin. The resulting F(ab')2 fragments can be treated to reduce disulfide bridges to produce Fab' fragments. Fab fragments can be obtained by treating IgG antibodies with papain; Fab' fragments can be obtained by digesting IgG antibodies with pepsin. F(ab') fragments can also be produced by combining the following Fab' with thioether bonds or disulfide bonds. Fab' fragments are antibody fragments obtained by cutting off a disulfide bond in the hinge region of F(ab')2. Fab' fragments can be obtained by treating F(ab')2 fragments with a reducing agent, such as dithiothreitol. Antibody fragments can also be produced by expressing nucleic acids encoding these fragments in recombinant cells (see, for example, Evans et al., J. Immunol. Meth. 184 , 123-38 (1995)). For example, a chimeric gene encoding a portion of a F(ab')2 fragment may include: a chimeric gene encoding a portion of a H chain C H 1 domain and hinge region, followed by a translation stop codon to produce this truncated antibody fragment molecule.

[0554] In one embodiment, the anti-TF antibody is a monovalent antibody, preferably a monovalent antibody as described in WO2007059782 (Genmab) (incorporated herein by reference), which has a deletion in the hinge region. Thus, in one embodiment, the antibody is a monovalent antibody, wherein such an anti-TF antibody is constructed by a method comprising:

[0555] i) providing a nucleic acid construct encoding the monovalent antibody light chain, the construct comprising a nucleotide sequence encoding the VL region of a selected antigen-specific anti-TF antibody and a nucleotide sequence encoding the constant CL region of Ig, wherein the nucleotide sequence encoding the VL region of the selected antigen-specific antibody and the nucleotide sequence encoding the CL region of Ig are operably linked together, and wherein, in the case of IgG1 subtype, the nucleotide sequence encoding the CL region is modified so that the CL region does not contain any amino acid that can form a disulfide bond or a covalent bond with other peptides comprising the same amino acid sequence as the CL region when administered to animals or humans;

[0556] ii) providing a nucleic acid construct encoding the heavy chain of the monovalent antibody, the construct comprising a nucleotide sequence encoding the VH region of a selected antigen-specific antibody and a nucleotide sequence encoding the constant CH region of human Ig, wherein the nucleotide sequence encoding the CH region is modified so that the region corresponding to the hinge region, and other regions of the CH region, such as the CH3 region, as required by the Ig subtype, do not contain any amino acid residues as follows: in the presence of polyclonal human IgG or when administered to animals or humans, the amino acid residues participate in the formation of disulfide bonds or covalent or stable non-covalent heavy chain bonds with other peptides comprising the same amino acid sequence as the human Ig CH region, wherein the nucleotide sequence encoding the VH region of the selected antigen-specific antibody and the nucleotide sequence encoding the CH region of the Ig are operably linked together;

[0557] iii) providing a cell expression system for producing the monovalent antibody;

[0558] iv) producing the monovalent antibody by co-expressing the nucleic acid constructs of (i) and (ii) in cells of the cell expression system of (iii).

[0559] Similarly, in one embodiment, the anti-TF antibody is a monovalent antibody comprising:

[0560] (i) a variable region of an antibody of the invention as described herein, or an antigen-binding portion of such a region, and

[0561] (ii) Immunoglobulin C H Area or including C H 2 and CH 3 region fragment, where the C H The region or fragment thereof is modified so that the region corresponding to the hinge region and, if the immunoglobulin is not of the IgG4 subtype, the C H Other areas of the zone, such as C H Region 3 does not contain any H The amino acids in the region form disulfide bonds or other covalent or stable non-covalent inter-heavy chain bonds.

[0562] In a further embodiment, the heavy chain of the monovalent anti-TF antibody is modified such that the entire hinge is deleted.

[0563] In a further embodiment, the antibody is of IgG4 subtype (see SEQ ID NO: 114, a hingeless variant of SEQ ID NO: 113), but C H Region 3 is modified to produce one or more of the following amino acid substitutions: Thr (T) at position 234 is replaced by Ala (A); Leu (L) at position 236 is replaced by Ala (A); Leu (L) at position 236 is replaced by Val (V); Phe (F) at position 273 is replaced by Ala (A); Phe (F) at position 273 is replaced by Leu (L); Tyr (Y) at position 275 is replaced by Ala (A).

[0564] In another further embodiment, the sequence of the monovalent antibody is modified such that it does not contain any acceptor sites for N-linked glycosylation.

[0565] The anti-TF antibodies of the present invention also include single-chain antibodies. Single-chain antibodies are peptides in which the heavy chain and light chain Fv are linked. In one embodiment, the present invention provides a single-chain Fv (scFv), in which the heavy chain and light chain in the anti-TF antibody Fv of the present invention are linked in a single peptide chain with a flexible peptide linker (typically about 10, 12, 15 or more amino acid residues). Methods for producing such antibodies are described, for example, in the following documents: US 4,946,778, Pluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, New York, pp. 269-315 (1994), Bird et al., Science 242 ,423-426(1988),Huston et al.,PNAS USA 85,5879-5883(1988)and McCafferty et al.,Nature 348 , 552-554 (1990). Single-chain antibodies can be monovalent if only a single V H and V L If two V H and V L If more than two V H and V L words.

[0566] In one embodiment, the anti-TF antibodies of the invention are effector function deficient antibodies. Such antibodies are particularly useful when the antibodies are used to stimulate the immune system by blocking the inhibitory effects of TF. For such applications, it may be advantageous for the antibodies to lack effector functions (e.g., ADCC) since such functions may lead to undesirable cytotoxicity.

[0567] In one embodiment, the effector function-deficient anti-TF antibody is a stabilized IgG4 antibody. Examples of suitable stabilized IgG4 antibodies are antibodies wherein the arginine at position 409 (indicated by the EU index, see Kabat et al) of the human IgG4 heavy chain constant region is replaced by lysine, threonine, methionine or leucine, preferably by lysine (as described in WO2006033386 (Kirin)) and / or wherein the hinge region comprises a Cys-Pro-Pro-Cys sequence.

[0568] In a further embodiment, the stabilized IgG4 anti-TF antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met and Leu at a position corresponding to 409, and / or having a residue selected from the group consisting of Ala, Val, Gly, Ile and Leu at a position corresponding to 405, and wherein the antibody optionally comprises one or more other substitutions, deletions and / or insertions, but does not comprise a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, the antibody comprises a Lys or Ala residue at a position corresponding to 409, or the CH3 region of the antibody is replaced by the CH3 region of human IgG1, human IgG2 or human IgG3.

[0569] In a further embodiment, the stabilized IgG4 anti-TF antibody is an IgG4 antibody comprising a heavy chain and a light chain, wherein the heavy chain comprises a human IgG4 constant region having a residue selected from the group consisting of Lys, Ala, Thr, Met and Leu at a position corresponding to 409, and / or having a residue selected from the group consisting of Ala, Val, Gly, Ile and Leu at a position corresponding to 405, and wherein the antibody optionally comprises one or more other substitutions, deletions and / or insertions, and wherein the antibody comprises a Cys-Pro-Pro-Cys sequence in the hinge region. Preferably, the antibody comprises a Lys or Ala residue at a position corresponding to 409, or the CH3 region of the antibody is replaced by the CH3 region of human IgG1, human IgG2 or human IgG3.

[0570] In a further embodiment, the effector function-deficient anti-TF antibody is an antibody of non-IgG4 type, such as IgG1, IgG2 or IgG3, which has been mutated so that the ability to mediate effector function (such as ADCC) is reduced or even abolished. These mutations have been described in, for example, Dall'Acqua WF et al., J Immunol. 177 (2):1129-1138(2006) and Hezareh M, J Virol.; 75 (24):12161-12168(2001).

[0571] In a further embodiment, the antibody of the invention is coupled to another moiety, such as a cytotoxic moiety, a radioisotope or a drug. Such an antibody can be produced by chemically coupling another moiety to the N-terminus or C-terminus of an anti-TF antibody or a fragment thereof (e.g., an anti-TF antibody H chain, L chain or an anti-TF specific / selective fragment thereof) (see, e.g., Antibody Engineering Handbook, edited by Osamu Kanemitsu, published by Chijin Shokan (1994)). Such coupled antibody derivatives can also be produced by coupling at a suitable internal residue or sugar group).

[0572] In general, the anti-TF antibodies described herein may be modified by inclusion of any suitable number of such modified amino acids and / or in association with such conjugated substituents. Suitability in this context generally depends on the ability to retain at least substantially the relevant TF selectivity and / or specificity of the underivatized parent anti-TF antibody. For example, inclusion of one or more modified amino acids may be advantageous in terms of increasing the serum half-life of the polypeptide, reducing the antigenicity of the polypeptide, or increasing the storage stability of the polypeptide. For example, the amino acids are co-translationally modified or post-translationally modified during recombinant production (e.g., N-linked glycosylation at an NXS / T motif during expression in mammalian cells), or modified by synthetic methods. Non-limiting examples of modified amino acids include glycosylated amino acids, sulfated amino acids, prenylated (e.g., farnesylated, geranylgeranylated) amino acids, acetylated amino acids, acylated amino acids, PEGylated amino acids, biotinylated amino acids, carboxylated amino acids, phosphorylated amino acids, and the like. References sufficient to guide one skilled in the art of amino acid modification abound throughout the literature. Examples of protocols can be found in Walker (1998) Protein Protocols On Cd-Rom, Humana Press, Towata, NJ. The modified amino acid can be selected, for example, from the following amino acids: glycosylated amino acids, PEGylated amino acids, farnesylated amino acids, acetylated amino acids, biotinylated amino acids, amino acids coupled to lipid groups or amino acids coupled to organic derivatizing agents.

[0573] The anti-TF antibody can also be chemically modified by covalent coupling to a polymer, for example, to increase its circulating half-life. Examples of polymers and methods of attaching them to peptides are exemplified in, for example, US 4,766,106, US 4,179,337, US 4,495,285 and US 4,609,546. Other examples of polymers include polyoxyethylated polyols and polyethylene glycol (PEG) (e.g., PEG having a molecular weight of about 1,000 to about 40,000, such as about 2,000 to about 20,000, such as about 3,000-12,000 g / mol).

[0574] In one embodiment, the invention provides an anti-TF antibody conjugated to a second molecule selected from the group consisting of a radionuclide, an enzyme, an enzyme substrate, a cofactor, a fluorescent marker, a chemiluminescent marker, a peptide tag, or a magnetic particle. In one embodiment, the anti-TF antibody may be conjugated to one or more antibody fragments, nucleic acids (oligonucleotides), nucleases, hormones, immunomodulators, chelators, boron compounds, photoactive agents, dyes, and the like. These and other agents may be conjugated directly or indirectly to the anti-TF antibodies of the invention. An example of direct conjugation of a second agent is through a spacer moiety. These spacers may be insoluble or soluble (see, e.g., Diener et al., Science 231 , 148 (1986)), and the spacer can be selected to allow the drug to be released from the anti-TF antibody at the target site and / or under specific conditions. Other examples of agents that can be coupled to the anti-TF antibody include lectins and fluorescent peptides.

[0575] In one embodiment, an anti-TF antibody comprising one or more radiolabeled amino acids is provided. Radiolabeled anti-TF antibodies can be used for diagnostic and therapeutic purposes (coupling to a radiolabeled molecule is another possible feature). Non-limiting examples of labels for polypeptides include, but are not limited to, 3H, 14C, 15N, 35S, 90Y, 99Tc, and 125I, 131I, and 186Re. Methods for preparing radiolabeled amino acids and related peptide derivatives are known in the art (see, for example, Junghans et al., in Cancer Chemotherapy and Biotherapy 655-686 (2d edition, Chafner and Longo, eds., Lippincott Raven (1996)) and US 4,681,581, US 4,735,210, US 5,101,827, US 5,102,990 (US RE35,500), US 5,648,471 and US 5,697,902. For example, radioactive isotopes can be coupled by the chloramine T method.

[0576] In one embodiment, the anti-TF antibody of the invention comprises a conjugated nucleic acid or nucleic acid-related molecule. In this aspect of the invention, the conjugated nucleic acid is a cytotoxic ribonuclease. In one embodiment, the conjugated nucleic acid is an antisense nucleic acid (e.g., an S100A10 targeted antisense molecule, which can also be an independent component of the combination composition or combination method of the invention, see, e.g., Zhang et al., J Biol Chem. 279(3), 2053-62 (2004). In one embodiment, the coupled nucleic acid is an inhibitory RNA molecule (e.g., an siRNA molecule). In one embodiment, the coupled nucleic acid is an immunostimulatory nucleic acid (e.g., a DNA molecule containing an immunostimulatory CpG motif). In one embodiment, the coupled nucleic acid is an expression cassette encoding a tumor suppressor gene, an anticancer vaccine, an anticancer cell factor, or an apoptotic agent. These derivatives may also include coupled nucleic acids encoding the expression of one or more cytotoxic proteins (e.g., plant or bacterial toxins).

[0577] In one embodiment, the anti-TF antibody is coupled to a functional nucleic acid molecule. Functional nucleic acid molecules include antisense molecules, interfering nucleic acid molecules (e.g., siRNA molecules), aptamers, ribozymes, triplex forming molecules, and external guide sequences. Functional nucleic acid molecules can function as effectors, inhibitors, regulators, and stimulators with specific activities of target molecules, or functional nucleic acid molecules can have new activities independent of any other molecules.

[0578] In another embodiment, the anti-TF antibody of the invention is conjugated to an aptamer.

[0579] In another embodiment, the present invention provides an anti-TF antibody conjugated to a ribozyme.

[0580] Any method known in the art for coupling an anti-TF antibody to a coupling molecule, such as described above, may be used, including the methods described in Hunter et al., Nature 144 ,945(1962),David et al.,Biochemistry 13 ,1014(1974),Pain et al.,J.Immunol.Meth. 40 , 219 (1981) and Nygren, J. Histochem.and Cytochem. 30, 407 (1982). Many types of cytotoxic compounds can be linked to proteins by using reactive groups on the cytotoxic compound or by using cross-linking agents. A common reactive group that can form stable covalent bonds with amino groups in vivo is isothiocyanate (Means et al., Chemical modifications of proteins (Holden-Day, San Francisco 1971) pp.105-110). This group preferentially reacts with the ε-amino group of lysine. Maleimide is a common reactive group used to form stable in vivo covalent bonds with the sulfhydryl group on cysteine ​​(Ji., Methods Enzymol 91 , 580-609 (1983)). Monoclonal antibodies are usually unable to form covalent bonds with radioactive metal ions, but they can be indirectly attached to the antibody by using chelating agents covalently linked to the antibody. Chelating agents can be attached to the antibody through the amine groups of amino acid residues (Meares et al., Anal. Biochem. 142 , 68-78 (1984)) and thiol (Koyama, Chem. Abstr. 120 , 217262t (1994)) and can also be linked via sugar groups (Rodwell et al., PNAS USA 83 ,2632-2636(1986),Quadri et al.,Nucl.Med.Biol. 20 , 559-570 (1993)). Since these chelators contain two types of functional groups, one that binds the metal ion and the other that connects the chelator to the antibody, they are often referred to as bifunctional chelators (Sundberg et al., Nature 250 ,587-588(1974)).

[0581] In one embodiment, the invention provides an anti-TF antibody, such as a human anti-TF antibody, conjugated to a therapeutic moiety, such as a cytotoxin, a chemotherapeutic drug, an immunosuppressant, or a radioisotope. Such conjugates are referred to herein as "immunoconjugates." Immunoconjugates that include one or more cytotoxins are referred to as "immunotoxins."

[0582] Cytotoxins or cytotoxic agents include any agent that is detrimental to cells (e.g., kills cells). These types of drugs and their mechanisms of action are well known in the art and are described in Goodman et al., Goodman and Gilman's The Pharmacological Basis Of Therapeutics, 8th ed., Macmillan Publishing Co., 1990. In, for example, Vitetta, Immunol. Today 14 Additional techniques related to the preparation of antibody immunotoxins are provided in US Pat. No. 5,194,594.

[0583] Suitable therapeutic agents for use in forming immunoconjugates of the invention include taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicine, doxorubicin, daunorubicin, dihydroxy anthracindione, mitoxantrone, mithramycin, actinomycin D, D), 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine), alkylating agents (e.g., nitrogen mustard hydrochloride, thioepa, chlorambucil, melphalan, carbamazepine), The invention also includes but is not limited to nitrogen mustard (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives, such as carboplatin), antibiotics (such as actinomycin D (formerly known as actinomycin), bleomycin, daunorubicin (formerly known as daunomycin), doxorubicin, idarubicin, miramycin, mitomycin, mitoxantrone, plicamycin, anthramycin (AMC)), diphtheria toxin and related molecules (such as diphtheria A chain and its active fragments and hybrid molecules), ricin toxins (such as ricin toxin A or deglycosylated ricin A chain toxin), cholera toxin, Shiga-like toxins (SLT-I, SLT-II,The conjugated molecules include serotonin, ... See, e.g., Pastan et al., Cell, 47 ,641(1986) and Goldenberg,Calif.ACancer Journal for Clinicians 44 , 43 (1994). Therapeutic agents, which can be administered in combination with the anti-TF antibodies of the invention as described elsewhere herein, can also be candidates for therapeutic moieties that can be conjugated to the anti-TF antibodies of the invention.

[0584] In one embodiment, an anti-TF antibody of the invention is conjugated to a chelator linker (eg, tiuxetan) that allows coupling of the antibody to a radioisotope.

[0585] In a further aspect, the invention relates to a bispecific molecule comprising an anti-TF antibody of the invention as described herein above and a second binding specificity, e.g. a binding specificity for human effector cells, a human Fc receptor or a T-cell receptor, or a binding specificity for another epitope of TF.

[0586] In addition to the anti-TF binding specificity and the binding specificity for human effector cells, human Fc receptor or T cell receptor, the bispecific molecules of the invention may further comprise a third binding specificity.

[0587] Exemplary bispecific antibody molecules of the invention include (i) two antibodies, one with specificity for TF and the other with specificity for a second target, both coupled together, (ii) a single antibody having one chain specific for TF and a second chain specific for a second molecule, and (iii) a single chain antibody with specificity for both TF and a second molecule. Typically, the second target / second molecule is a molecule other than TF. In one embodiment, the second molecule is a cancer antigen / tumor-associated antigen, such as carcinoembryonic antigen (CEA), prostate-specific antigen (PSA), RAGE (renal antigen), alpha-fetoprotein, CAMEL (CTL-recognition antigen on melanoma), CT antigens (such as MAGE-B5, -B6, -C2, -C3, and D; Mage-12; CT10; NY-ESO-1, SSX-2, GAGE, BAGE, MAGE, and SAGE), mucin antigens (such as MUC1, mucin-CA125, etc.), ganglioside antigens, tyrosinase, gp75, C-myc, Mart1, MelanA, MUM-1, MUM-2, MUM-3, HLA-B7, and Ep-CAM. In one embodiment, the second molecule is a cancer-associated integrin, such as α5β3 integrin. In one embodiment, the second molecule is an angiogenic factor or other cancer-related growth factor, such as vascular endothelial growth factor (VEGF), fibroblast growth factor (FGF), epidermal growth factor (EGF), epidermal growth factor receptor (EGFR), angiogenin and its receptor, in particular, a receptor associated with cancer progression (e.g., one of the HER1-HER4 receptors, c-met or RON). Other cancer progression-related proteins discussed herein may also be suitable second molecules.

[0588] In one embodiment, the bispecific antibodies of the invention are diabodies. Bispecific antibodies also include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be coupled to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (see, e.g., US 4,676,980). Heteroconjugate antibodies can be prepared by any convenient cross-linking method.

[0589] In a further aspect, the invention relates to an expression vector encoding an antibody of the invention.

[0590] In one embodiment, the expression vector of the present invention comprises a nucleotide sequence encoding one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 1-112.

[0591] In another specific embodiment, the expression vector of the present invention comprises a nucleotide sequence encoding one or more VH amino acid sequences selected from the group consisting of SEQ ID NO:9, 1, 5, 13, 17, 21, 25, 29, 33, 37, 41, 45, 49 and 53.

[0592] In a specific embodiment, the expression vector of the present invention comprises a nucleotide sequence encoding one or more VH CDR3 amino acid sequences selected from the group consisting of SEQ ID NOs 4, 8, 12, 16, 20, 24, 28, 32, 36, 40, 44, 48, 52 and 56.

[0593] In another specific embodiment, the expression vector of the present invention comprises a nucleotide sequence encoding one or more VL amino acid sequences selected from the group consisting of SEQ ID NO:65, 57, 61, 69, 73, 77, 81, 85, 89, 93, 97, 101 and 105.

[0594] In another specific embodiment, the expression vector of the present invention comprises a nucleotide sequence encoding one or more VL CDR3 amino acid sequences selected from the group consisting of SEQ ID NO: 60, 64, 68, 72, 76, 80, 84, 88, 92, 96, 100, 104 and 108.

[0595] In a specific embodiment, the expression vector of the invention comprises a nucleotide molecule encoding a variant of one or more of the above amino acid sequences, wherein the variant has up to 25 amino acid modifications, such as 20, such as up to 15, 14, 13, 12 or 11 amino acid modifications, such as 10, 9, 8, 7, 6, 5, 4, 3, 2 or 1 amino acid modifications, such as deletions or insertions, preferably substitutions, such as conservative substitutions, or has at least 80% identity with any of the sequences, such as at least 85% identity or 90% identity or 95% identity, such as 96% identity or 97% identity or 98% identity or 99% identity with any of the aforementioned amino acid sequences.

[0596] In a further embodiment, the expression vector further comprises a nucleotide sequence encoding a light chain constant region, a heavy chain constant region, or both light and heavy chain constant regions of an antibody, such as a human antibody.

[0597] Such expression vectors can be used to recombinantly produce the antibodies of the invention.

[0598] The expression vector in the context of the present invention may be any suitable vector, including chromosomal, non-chromosomal, synthetic nucleic acid vectors (containing a nucleic acid sequence of a suitable set of expression control elements). Examples of such vectors include derivatives of SV40, bacterial plasmids, phage DNA, baculovirus, yeast plasmids, vectors derived from a combination of plasmids and phage DNA, and viral nucleic acid (RNA or DNA) vectors. In one embodiment, the nucleic acid encoding the anti-TF antibody is contained in a naked DNA or RNA vector, including, for example, a linear expression element (e.g., in Sykes and Johnston, Nat Biotech 17 , 355-59 (1997)), compact nucleic acid vectors (e.g., described in US 6,077,835 and / or WO 00 / 70087), plasmid vectors such as pBR322, pUC 19 / 18 or pUC 118 / 119, "midge" size-minimized nucleic acid vectors (e.g., described in Schakowski et al., Mol Ther 3 , 793-800 (2001)), or as a precipitated nucleic acid vector construct, such as a CaP04-precipitated construct (e.g., described in WO 00 / 46147, Benvenisty and Reshef, PNAS USA 83 ,9551-55(1986),Wigler et al.,Cell 14 , 725 (1978), and Coraro and Pearson, Somatic Cell Genetics 7 , 603 (1981)). Such nucleic acid vectors and their use are well known in the art (see, for example, US 5,589,466 and US 5,973,972).

[0599] In one embodiment, the vector is suitable for expressing the anti-TF antibody in bacterial cells. Examples of such vectors include expression vectors such as BlueScript (Stratagene), pIN vectors (Van Heeke & Schuster, J Biol Chem 264 , 5503-5509 (1989), pET vector (Novagen, Madison WI) etc.).

[0600] The expression vector may also be, or alternatively is, a vector suitable for expression in a yeast system. Any vector suitable for expression in a yeast system may be used. Suitable vectors include, for example, vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH (reviewed in: F. Ausubel et al., ed. Current Protocols in Molecular Biology, Greene Publishing and Wiley InterScience New York (1987), and Grant et al., Methods in Enzymol 153 ,516-544(1987)).

[0601] Nucleic acid and / or vector can also comprise the nucleic acid sequence of coding secretion / localization sequence, and secretion / localization sequence can target polypeptide, for example immature polypeptide chain, to periplasmic space or cell culture medium.These sequences are known in the art, and comprise secretion leader sequence or signal peptide, organelle targeting sequence (for example nuclear localization sequence, ER retention signal, mitochondrial transport sequence (transit sequence), chloroplast transport sequence), membrane localization / anchor sequence (for example stop transfer sequence, GPI anchor sequence) etc.

[0602] In the expression vector of the present invention, the nucleic acid encoding the anti-TF antibody may include or be associated with a suitable promoter, enhancer and other elements that facilitate expression. Examples of these elements include strong expression promoters (e.g., human CMV IE promoter / enhancer and RSV, SV40, SL3-3, MMTV, and HIV LTR promoters), effective poly(A) termination sequences, replication origins for E. coli plasmid products, antibiotic resistance genes as selection tags, and / or convenient cloning sites (e.g., polylinkers). The nucleic acid may also include an inducible promoter (as opposed to a constitutive promoter), such as CMV IE (the skilled artisan will recognize that these terms actually describe the expression level of a gene under certain conditions).

[0603] In one embodiment, an expression vector encoding an anti-TF antibody can be placed in or delivered into a host cell or host animal via a viral vector.

[0604] In a further aspect, the invention relates to a recombinant eukaryotic or prokaryotic host cell, such as a transfectoma, which produces an antibody of the invention as defined herein or a bispecific molecule of the invention as defined herein. Examples of host cells include yeast, bacteria and mammalian cells, such as CHO or HEK cells. For example, in one embodiment, the invention provides a cell comprising a nucleic acid stably integrated into the genome of the cell and comprising a coding sequence for expressing an anti-TF antibody of the invention. In another embodiment, the invention provides a cell comprising a non-integrated nucleic acid, such as a plasmid, cosmid, phagemid or linear expression element, comprising a coding sequence for expressing an anti-TF antibody of the invention.

[0605] In a further aspect, the invention relates to a hybridoma that produces an antibody of the invention as defined herein. In a further aspect, the invention relates to a transgenic non-human animal comprising a nucleic acid encoding a human heavy chain and a human light chain, wherein the animal or plant produces an antibody of the invention. The production of such hybridomas and transgenic animals has been described above.

[0606] In a further aspect, the present invention relates to a method for producing an anti-TF antibody of the present invention, the method comprising the steps of:

[0607] a) culturing a hybridoma or host cell of the invention as described herein above, and

[0608] b) purifying the antibody of the present invention from the culture medium.

[0609] In a further main aspect, the invention relates to an anti-TF antibody as defined herein or a bispecific molecule as defined herein for use as a medicament.

[0610] In a further aspect, the present invention relates to a pharmaceutical composition comprising:

[0611] - an anti-TF antibody as defined herein or a bispecific molecule as defined herein, and

[0612] - A pharmaceutically acceptable carrier.

[0613] The pharmaceutical composition can be formulated with a pharmaceutically acceptable carrier or diluent and any other known adjuvants and excipients according to conventional techniques, such as those disclosed in Remington: The Science and Practice of Pharmacy, 19th Edition, Gennaro, Ed., Mack Publishing Co., Easton, PA, 1995.

[0614] The pharmaceutically acceptable carrier or diluent and any other known adjuvant and excipient should be suitable for the selected compound of the present invention and the selected mode of administration. The suitability of the carrier and other components used in the pharmaceutical composition is determined based on the following factors, i.e., there is no significant adverse effect on the desired biological properties of the selected compound of the present invention or pharmaceutical composition (e.g., no substantial effect on antigen binding (e.g., 10% or less relative inhibition, 5% or less relative inhibition, etc.)).

[0615] The pharmaceutical compositions of the present invention may also include diluents, fillers, salts, buffers, detergents (e.g., non-ionic detergents such as Tween 20 or Tween 80), stabilizers (e.g., sugars or protein-free amino acids), preservatives, tissue fixatives, solubilizers, and / or other materials suitable for inclusion in a pharmaceutical composition.

[0616] It has been reported that in cancer cells, such as human colorectal cancer cells, TF expression is controlled by two major transformation events that drive disease progression (K-ras oncogene activation and p53 tumor suppressor inactivation) in a manner that is dependent on MEK / mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3'-kinase (PI3K) (Yu et al. (2005) Blood 105:1734).

[0617] Cancer cells that overexpress TF may be particularly good targets for the anti-TF antibodies of the invention, since more antibodies can be bound per cell. Thus, in one embodiment, a cancer patient to be treated with an anti-TF antibody of the invention is a patient, such as a pancreatic, lung or colorectal cancer patient, who has been diagnosed with one or more mutations in K-Ras and / or one or more mutations in p53 in their tumor cells.

[0618] In an alternative embodiment, the cancer patient to be treated with the anti-TF antibody of the invention is a patient, such as a pancreatic cancer, lung cancer or colorectal cancer patient, who does not have a mutation in K-Ras. Without being bound by any particular theory, some tumor cells with K-Ras activation may be less susceptible to anti-TF antibody treatment because the effectiveness of anti-TF antibodies in affecting intracellular signaling mechanisms may be lower in cells with activated K-Ras.

[0619] Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention may be varied to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition or mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors, including the activity of the particular composition of the present invention employed or its amide, the route of administration, the time of administration, the rate of excretion of the particular compound employed, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health and previous medication history of the patient being treated, and similar factors well known in the medical field.

[0620] The pharmaceutical composition can be administered by any suitable route and mode. Suitable routes for administering the compounds of the invention in vivo and in vitro are well known in the art and can be selected by those skilled in the art.

[0621] In one embodiment, the pharmaceutical compositions of the invention are administered parenterally.

[0622] As used herein, the terms "parenteral administration" and "administered parenterally" refer to modes of administration other than enteral and topical administration, usually by injection, and include intraepithelial, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, intratendinous, transtracheal, subcutaneous, subticular, intraarticular, subcapsular, subarachnoid, intraspinal, intracranial, intrathoracic, epidural, and intrasternal injection and infusion.

[0623] In one embodiment, the pharmaceutical composition is administered by intravenous or subcutaneous injection or infusion.

[0624] Pharmaceutically acceptable carriers include any and all suitable solvents, dispersion media, coatings, bactericidal and fungicidal agents, isotonic agents, antioxidants and absorption delaying agents, and the like that are physiologically compatible with the compounds of the invention.

[0625] Examples of suitable aqueous and non-aqueous carriers that can be used in the pharmaceutical composition of the present invention include water, saline, phosphate buffered saline, ethanol, dextrose, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, such as olive oil, corn oil, peanut oil, cottonseed oil, and sesame oil, carboxymethylcellulose colloid solution, tragacanth gum, and injectable organic esters, such as ethyl oleate, and / or various buffers. Other carriers are well known in the pharmaceutical field.

[0626] Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for immediate preparation of sterile injectable solutions or dispersions. The use of these media and agents on pharmaceutically active substances is known in the art. Unless any conventional media or agents are incompatible with the active compound, their use in the pharmaceutical compositions of the present invention is contemplated herein.

[0627] Proper fluidity can be maintained, for example, by the use of coating materials, such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants.

[0628] The pharmaceutical composition of the present invention may also include pharmaceutically acceptable antioxidants, such as (1) water-soluble antioxidants, such as ascorbic acid, cysteine ​​hydrochloride, sodium bisulfate, sodium metabisulfite, sodium sulfite and the like; (2) oil-soluble antioxidants, such as ascorbyl palmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), lecithin, propyl gallate, α-tocopherol, and the like; and (3) metal chelators, such as citric acid, ethylenediaminetetraacetic acid (EDTA), sorbitol, tartaric acid, phosphoric acid, and the like.

[0629] The pharmaceutical composition of the present invention may also include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, glycerol or sodium chloride in the composition.

[0630] The pharmaceutical composition of the present invention may also contain one or more adjuvants suitable for the selected route of administration, such as preservatives, wetting agents, emulsifiers, dispersants, preservatives or buffers, which can improve the shelf life or effectiveness of the pharmaceutical composition. The compound of the present invention can be prepared with a carrier that protects the compound from rapid release, such as a sustained-release formulation, including implants, transdermal patches and microencapsulated delivery systems. Such carriers may include gelatin, glyceryl monostearate, glyceryl distearate, biodegradable, biocompatible polymers, such as ethylene vinyl acetate copolymers, polyanhydrides, polyglycolic acid, collagen, polyorthoesters and polylactic acid, alone or with wax, or other materials well known in the art. Methods for preparing such preparations are generally known to those skilled in the art. See, for example, Sustained and Controlled Release Drug Delivery Systems, edited by JR Robinson, Marcel Dekker, Inc., New York, 1978.

[0631] In one embodiment, the compounds of the present invention can be formulated to ensure proper distribution in vivo. Pharmaceutically acceptable carriers for parenteral administration include sterile aqueous solutions or dispersions for the immediate preparation of sterile injectable solutions or dispersions of sterile powders. The use of these media and agents for pharmaceutically active substances is known in the art. Any conventional media or agents, as long as they are not incompatible with the active compound, are contemplated for use in the pharmaceutical compositions of the present invention. Supplementary active compounds may also be incorporated into the composition.

[0632] Pharmaceutical compositions for injection typically must be sterile and stable under the conditions of manufacture and storage. The composition can be formulated into a solution, microemulsion, liposome or other ordered structure suitable for high drug concentration. The carrier can be an aqueous or non-aqueous solvent or dispersion medium, containing, for example, water, ethanol, polyols (e.g., glycerol, propylene glycol, polyethylene glycol, etc.), and suitable mixtures thereof, vegetable oils, such as olive oil, and injectable organic esters, such as ethyl oleate. Suitable fluidity can be maintained, for example, by the use of a coating, such as lecithin; by maintaining the desired particle size in the case of a dispersion; and by the use of a surfactant. In many cases, it is preferred to contain an isotonic agent, such as a sugar, a polyol such as glycerol, mannitol, sorbitol, or sodium chloride in the composition. Prolonged absorption of the injectable composition can be achieved by including an agent that can delay absorption, such as monostearate and gelatin in the composition. Sterile injectable solutions can be prepared in this way: the required amount of the active compound is incorporated into a suitable solvent containing one or a combination of multiple ingredients (e.g., as listed above) as required, followed by aseptic microfiltration. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle containing a basic dispersion medium and the desired other ingredients, such as those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which processes produce the active ingredient plus any additional powder from a previously sterile-filtered solution.

[0633] The preparation of sterile injectable solutions can be by adding the required amount of active compound to a suitable solvent with one or more of the combinations of the above-listed ingredients, followed by sterile microfiltration, as needed. Generally, dispersions are prepared by adding the active compound to a sterile medium containing a basic dispersion medium and other necessary components from the above-listed ingredients. In the case of sterile powders for the preparation of sterile injectable solutions, examples of preparation methods are vacuum drying and freeze drying (lyophilization), which produce a powder of the active ingredient plus any additional desired ingredients from a previously sterile filtered solution.

[0634] The pharmaceutical composition of the present invention may contain one compound of the present invention or a combination of multiple compounds of the present invention.

[0635] As mentioned above, in another aspect the invention relates to an antibody of the invention as defined herein or a bispecific molecule of the invention as described herein for use as a medicament.

[0636] The anti-TF antibodies of the invention can be used for a variety of purposes. In particular, the antibodies of the invention can be used to treat various forms of cancer. In one aspect, the anti-TF monoclonal antibodies of the invention are used to treat various solid cancer types, such as tumors of the central nervous system, head and neck cancer, lung cancer (e.g., non-small cell lung cancer), breast cancer, esophageal cancer, gastric cancer, liver and biliary cancer, pancreatic cancer, colorectal cancer, bladder cancer, kidney cancer, prostate cancer, endometrial cancer, ovarian cancer, malignant melanoma, sarcoma (soft tissue, such as bone and muscle), tumors of unknown primary origin (i.e., of unknown origin), leukemias, bone marrow cancers (e.g., multiple myeloma), acute lymphocytic leukemia, chronic lymphocytic leukemia, and non-Hodgkin's lymphoma, skin cancer, glioma, cancers of the brain, uterus, and rectum.

[0637] Furthermore, autoimmune inflammatory diseases, such as myopathy or multiple sclerosis, can serve as targets for the anti-TF monoclonal antibodies of the present invention.

[0638] The anti-TF monoclonal antibodies of the present invention can also be used for the treatment of hemostasis.

[0639] Cancer-associated hemostatic disorders may also be targets for intervention according to the present invention.

[0640] Further, diseases with inflammation, such as myopathy, rheumatoid arthritis, osteoarthritis, ankylosing spondylitis, gout, spondylarthropathris, ankylosing spondylitis, Reiter's syndrome, psoriatic arthropathy, enteropathic spondylitis, juvenile arthropathy, reactive arthropathy, infectious or post-infectious arthritis, tuberculous arthritis, viral arthritis, fungal arthritis, syphilitic arthritis, glomerulonephritis, end-stage renal disease, systemic lupus erythematosus, mb. Crohn's, ulcerative colitis, inflammatory bowel disease, cystic fibrosis, chronic obstructive pulmonary disease (COPD), asthma, allergic asthma, bronchitis, acute bronchitis, chronic bronchitis, idiopathic pulmonary fibrosis, or multiple sclerosis, can be targets of the anti-TF monoclonal antibodies of the present invention.

[0641] The anti-TF monoclonal antibody of the present invention can also be used for the treatment of hemostasis.

[0642] Cancer-associated hemostatic disorders may also serve as targets for intervention in accordance with the present invention.

[0643] Additionally, vascular diseases, such as vascular restenosis, myocardial vascular disease, cerebrovascular disease, retinopathia, and macular degeneration, including but not limited to wet AMD, can be treated with anti-TF monoclonal antibodies.

[0644] The anti-TF monoclonal antibodies of the present invention can also be used to treat patients with cardiovascular risk factors, such as atherosclerosis, hypertension, diabetes, dyslipidemia, and acute coronary syndromes, including but not limited to, acute myocardial infarction and stroke.

[0645] The anti-TF monoclonal antibodies of the present invention can also be used to inhibit thrombosis, such as DVT, renal embolism, pulmonary embolism, arterial thrombosis, or to treat thrombosis after arterial surgery, peripheral vascular bypass graft or coronary artery bypass graft, arteriovenous shunt, removal of devices such as stents or catheters.

[0646] The anti-TF monoclonal antibody of the present invention can also be used to inhibit renal ischemia-reperfusion injury.

[0647] The anti-TF monoclonal antibody of the present invention can also be used to treat hyperlipoproteinemia and hyperparathyroidism.

[0648] The anti-TF monoclonal antibody of the present invention can also be used to treat vasculitis, ANCA-positive vasculitis, and Behcet's disease.

[0649] The anti-TF monoclonal antibodies of the present invention can also be used to block trauma-induced respiratory failure, such as acute respiratory distress syndrome and acute lung injury.

[0650] The anti-TF monoclonal antibodies of the present invention can also be used to block infection-induced organ dysfunction, such as renal failure, acute respiratory distress syndrome, and acute lung injury.

[0651] The anti-TF monoclonal antibodies of the present invention can also be used to treat various thromboembolic diseases, such as thromboembolic diseases caused by angioplasty, myocardial infarction, unstable angina and coronary artery stenosis.

[0652] The anti-TF monoclonal antibodies of the invention may also be used in a prophylactic setting, for the treatment of TF-mediated complications of systemic infection, such as sepsis or pneumonia.

[0653] The anti-TF monoclonal antibodies of the present invention may also be used for the prophylactic treatment of patients with atherosclerotic blood vessels who are at risk of thrombosis.

[0654] The anti-TF monoclonal antibodies of the present invention can also be used to treat graft-versus-host disease.

[0655] The anti-TF monoclonal antibodies of the present invention can also be used to increase beta cell engraftment in pancreatic islet transplantation, prevent cardiac allograft vasculopathy (CAV), and prevent acute graft rejection.

[0656] The anti-TF monoclonal antibodies of the present invention can also be used to treat diseases involving circulating tissue-factor exposing microparticles, such as but not limited to vascular thrombosis, type II diabetes, AMI, and pulmonary hypertension.

[0657] Similarly, the invention relates to a method for inhibiting the growth and / or proliferation of tumor cells expressing TF, comprising administering to an individual in need thereof an antibody or bispecific molecule of the invention. In one embodiment, the tumor cells are involved in cancer, such as prostate cancer, lung cancer (e.g., non-small cell lung cancer), breast cancer, colorectal cancer (e.g., metastatic colorectal cancer), pancreatic cancer, endometrial cancer, ovarian cancer, cutaneous melanoma, leukemic myeloid cancer (e.g., multiple myeloma), acute lymphocytic leukemia, chronic lymphocytic leukemia and non-Hodgkin's lymphoma, skin cancer, prostate cancer, glioma, cancer of the brain, kidney, uterus, bladder and rectum.

[0658] In addition, the present invention relates to the use of a monoclonal antibody that binds to human TF in the preparation of a medicament for the treatment of cancer, such as one of the specific cancer indications mentioned above.

[0659] In one embodiment, the selection of patients to be treated with anti-TF antibodies is based on the level of tissue factor (TF) in their urine and / or blood. In a specific embodiment, the patient to be treated has relatively high levels of TF in urine and / or blood. For example, the TF level in the urine of the patient to be treated may be greater than 20 ng / ml, such as greater than 40 ng / ml, such as greater than 100 ng / ml, such as greater than 200 ng / ml. Alternatively, or in addition, the TF level in the patient's serum may be greater than 100 pg / ml, such as greater than 200 pg / ml. This can be determined, for example, using ELISA.

[0660] In a further embodiment of the treatment methods of the invention, the efficacy of the treatment is monitored during the treatment, e.g., at predetermined time points. In one embodiment, efficacy can be monitored by measuring TF levels in urine or blood, e.g., by ELISA. In another embodiment, efficacy can be determined by visualizing the disease area, e.g., by performing one or more PET-CT scans, e.g., using a labeled anti-TF antibody, such as a labeled anti-TF antibody of the invention. Furthermore, a labeled anti-TF antibody, such as a labeled anti-TF antibody of the invention, can be used to detect TF-producing tumors, e.g., using a PET-CT scan.

[0661] The dosage regimen in the above-described methods of treatment and uses is adjusted to provide the optimal desired response (e.g., a therapeutic response). For example, a single bolus may be given, several divided doses may be given over a period of time, or the dose may be proportionally reduced or increased as the exigencies of the therapeutic situation dictate. Parenteral compositions may be formulated in unit dosage form to facilitate administration and maintain uniformity of dosage. As used herein, unit dosage form refers to physically discrete units suitable as unitary dosages for subjects to be treated; each unit contains a predetermined amount of active compound and a necessary pharmaceutical carrier, the predetermined amount of the active compound being calculated to produce the desired therapeutic effect. The specifications of the unit dosage forms of the present invention are governed and directly determined by the following factors: (a) the unique characteristics of the active compound and the specific therapeutic effect to be achieved, and (b) the limitations inherent in the prior art to the compatibility of such active compounds for treating individual sensitivities.

[0662] The effective dose and dose range of the anti-TF antibody depends on the disease or condition to be treated and can be determined by one skilled in the art. An exemplary non-limiting range of a therapeutically effective amount of a compound of the invention is about 0.1-100 mg / kg, such as about 0.1-50 mg / kg, such as about 0.1-20 mg / kg, such as 0.1-10 mg / kg, such as about 0.5, such as about 0.3, such as about 1, or about 3 mg / kg.

[0663] A physician or veterinarian with ordinary skills in the art can easily determine and prescribe the effective amount of the pharmaceutical composition required. For example, for the anti-TF antibody contained in the pharmaceutical composition, the physician or veterinarian can start with a dosage level lower than the amount required to achieve the desired therapeutic effect, and gradually increase the dosage until the desired effect is achieved. In general, a suitable daily dose of the composition of the present invention is such an amount of compound that it is the lowest dose that can effectively produce a therapeutic effect. Such an effective dose generally depends on the factors mentioned above. Administration can be, for example, intravenous, intramuscular, intraperitoneal or subcutaneous, for example, applied near the target site. If desired, the effective daily dose of the pharmaceutical composition can be administered as 2, 3, 4, 5, 6 or more subdoses divided at appropriate intervals throughout the day, optionally in unit dosage form. Although it is possible for the compound of the present invention to be administered alone, it is preferably administered in the form of a pharmaceutical composition as described above.

[0664] In one embodiment, the anti-TF antibody may be administered at 10-500 mg / m 2 , for example 200-400 mg / m 2 The dosage can be infused and administered. This administration can be repeated, for example 1-8 times, for example 3-5 times. Administration can be performed by continuous infusion over 2-24 hours, for example 2-12 hours.

[0665] In one embodiment, the anti-TF antibody can be administered by slow continuous infusion over a longer period of time, such as more than 24 hours, to reduce toxic side effects.

[0666] In one embodiment, the anti-TF antibody may be administered at a dose of 250 mg-2000 mg, e.g., 300 mg, 500 mg, 700 mg, 1000 mg, 1500 mg or 2000 mg per week, up to 8 times, e.g., 4-6 times. Administration may be performed by continuous infusion over a period of 2-24 hours, e.g., 2-12 hours. This dosing regimen may be repeated one or more times as needed, e.g., one or more times after 6 or 12 months. The dose may be determined or adjusted by measuring the amount of the compound of the invention in the blood after administration, e.g., by removing a biological sample and administering an anti-idiotypic antibody that targets the antigen binding region of the anti-TF antibody of the invention.

[0667] In one embodiment, the anti-TF antibody may be administered via maintenance therapy, such as once per week for a period of 6 months or longer.

[0668] In one embodiment, the anti-TF antibody can be administered by a regimen comprising a single infusion of an anti-TF antibody of the invention followed by an infusion of an anti-TF antibody of the invention conjugated to a radioisotope. This regimen can be repeated, for example, after 7-9 days.

[0669] As a non-limiting example, treatment according to the present invention may be provided as a daily dose of a compound of the invention in an amount of about 0.1-100 mg / kg per day, such as 0.5, 0.9, 1.0, 1.1, 1.5, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 45, 50, 60, 70, 80, 90 or 100 mg / kg per day, starting 1, 2, 3, 4, 5, 6, 7, 8, 90, 100 mg / kg per day. , 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 days, or at least one of weeks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20, or any combination thereof, using a single dose or divided doses every 24, 12, 8, 6, 4, or 2 hours, or any combination thereof.

[0670] An "effective amount" for tumor treatment can also be measured by its ability to stabilize the course of the disease. The ability of a compound to inhibit cancer can be evaluated in an animal model system that predicts its effectiveness in human tumors. Alternatively, this property of the composition can be evaluated by examining the ability of the compound to inhibit cell growth or induce apoptosis by in vitro assays known to technicians. A therapeutically effective amount of a therapeutic compound can reduce tumor size or otherwise alleviate the subject's symptoms. One of ordinary skill in the art can determine these amounts based on factors such as the subject's physique, the severity of the subject's symptoms, and the specific composition or route of administration selected.

[0671] Anti-TF antibodies can also be administered prophylactically to reduce the risk of developing cancer, delay the onset of events in cancer progression, and / or reduce the risk of recurrence during cancer remission. This is particularly useful for patients whose tumors are known to exist due to other biological factors but are difficult to locate.

[0672] Anti-TF antibodies may also be administered in combination therapy, i.e., in combination with other therapeutic agents relevant to the disease or condition being treated. Thus, in one embodiment, a medicament containing the antibody is used in combination with one or more other therapeutic agents, such as a cytotoxic agent, a chemotherapeutic agent, or an anti-angiogenic agent.

[0673] Such combined administration may be simultaneous, separate or sequential. For simultaneous administration, the agents may suitably be administered as one composition or as separate compositions. The present invention therefore also provides a method for treating a disease involving TF-expressing cells as described above, the method comprising administering an anti-TF antibody of the present invention in combination with one or more other therapeutic agents as described below.

[0674] In one embodiment, the invention provides a method for treating a disease involving cells expressing TF in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody of the invention and at least one chemotherapeutic agent.

[0675] In one embodiment, the present invention provides a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody of the present invention and at least one chemotherapeutic agent.

[0676] In one embodiment, the invention provides use of an anti-TF antibody of the invention for the preparation of a pharmaceutical composition for administration with at least one chemotherapeutic agent for the treatment of cancer.

[0677] In one embodiment, such chemotherapeutic agents can be selected from antimetabolites such as methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, fludarabine, 5-fluorouracil, decarbazine, hydroxyurea, asparaginase, gemcitabine, cladribine, and similar agents.

[0678] In one embodiment, such chemotherapeutic agents can be selected from alkylating agents such as mechlorethamine, thioepa, chlorambucil, melphalan, carbendazim (BSNU), lomustine (CCNU), cyclophosphamide, busulfan, dibromomannitol, streptozotocin, dacarbazine (DTIC), procarbazine, mitomycin C, cisplatin and other platinum derivatives such as carboplatin, and similar drugs.

[0679] In one embodiment, such chemotherapeutic agents may be selected from anti-mitotic agents, such as taxanes, such as docetaxel and paclitaxel, and vinca alkaloids, such as vindesine, vincristine, vinblastine, and vinorelbine.

[0680] In one embodiment, the chemotherapeutic agent may be selected from a topoisomerase inhibitor, such as topotecan or irinotecan.

[0681] In one embodiment, the chemotherapeutic agent may be selected from cytostatic drugs such as etoposide and teniposide.

[0682] In one embodiment, such chemotherapeutic agents may be selected from growth factor inhibitors such as ErbBl (EGFR) inhibitors (eg, Iressa, Erbitux (cetuximab), Tarceva and similar drugs), ErbB2 (Her2 / neu) inhibitors (eg, Herceptin and similar drugs), and the like.

[0683] In one embodiment, such chemotherapeutic agents may be selected from tyrosine kinase inhibitors such as imatinib (Glivec, Gleevec STI571), lapatinib, PTK787 / ZK222584, and similar drugs.

[0684] In one embodiment, the invention provides a method for treating a disease involving cells expressing TF in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody of the invention and at least one angiogenesis, neovascularization and / or other vascularization inhibitor.

[0685] Examples of such angiogenesis inhibitors are urokinase inhibitors, matrix metalloproteinase inhibitors (e.g., marimastat, novamastat, BAY 12-9566, AG 3340, BMS-275291 and similar drugs), endothelial cell migration and proliferation inhibitors (e.g., TNP-470, squalamine, 2-methoxyestradiol, combretastatins, endostatin, angiostatin, penicillamine, SCH66336 (Schering-Plough Corp, Madison, NJ), R115777 (Janssen Pharmaceutica, Inc, Titusville, NJ) and similar drugs), angiogenic growth factor antagonists (e.g., ZD6474, SU6668, antibodies to angiogenic agents and / or their receptors (e.g., VEGF, bFGF, and angiopoietin-1), thalidomide, thalidomide analogs (e.g., CC-5013), Sugen 5416, SU5402, anti-angiogenic ribozymes (e.g., angiozyme), interferon α (e.g., interferon α2a), suramin and similar drugs), VEGF-R kinase inhibitors and other anti-angiogenic tyrosine kinase inhibitors (e.g., SU011248), endothelial-specific integrin / survival signaling inhibitors (e.g., vitaxin and similar drugs), copper antagonists / chelators (e.g., tetrathiomolybdate, captopril and similar drugs), carboxyamido-triazole (CAI), ABT-627, CM101, interleukin-12 (IL-12), IM862, PNU145156E, and nucleic acid molecules that inhibit angiogenesis (e.g., antisense-VEGF-cDNA, cDNA encoding angiostatin, cDNA encoding p53, and cDNA encoding defective VEGF receptor-2) and similar drugs.

[0686] Other examples of these angiogenesis, neovascularization and / or other vascularization inhibitors are anti-angiogenic heparin derivatives and related molecules (e.g., heperinase III), temozolomide, NK4, macrophage migration inhibitory factor (MIF), cyclooxygenase-2 inhibitors, hypoxia-inducible factor 1, anti-angiogenic soy isoflavones, oltipraz, fumagillin and its analogs, somatostatin analogs, pentosan polysulfate, teicogalan sodium, dalteparin, tumstatin, thrombospondin, NM-3, combrestatin, canstatin, avastatin, antibodies against other related targets (e.g., anti-α-v / beta-3 integrin and anti-kininostatin monoclonal antibodies) and similar drugs.

[0687] In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody for treating a disease as described above can be an anti-cancer immunogen, such as a cancer antigen / tumor-associated antigen (e.g., epithelial cell adhesion molecule (EpCAM / TACSTD1), mucin 1 (MUC1), carcinoembryonic antigen (CEA), tumor-associated glycoprotein 72 (TAG-72), gp100, Melan-A, MART-1, KDR, RCAS1, MDA7, cancer-associated virus vaccines (e.g., human papillomavirus vaccines), tumor-derived heat shock proteins, and the like. Many other suitable cancer antigens / tumor-associated antigens described elsewhere herein and similar molecules known in the art can also be further or alternatively used in such an embodiment. Anti-cancer immunogenic peptides also include anti-idiotypic "vaccines", such as BEC2 anti-idiotypic antibodies, mitumomab, CeaVac and related anti-idiotypic antibodies, anti-idiotypic antibodies to MG7 antibodies, and other anti-cancer anti-idiotypic antibodies (see, e.g., Birebent et al., Vaccine. 21 (15),1601-12(2003),Li et al.,Chin Med J(Engl). 114 (9),962-6(2001),Schmitt et al.,Hybridoma. 13 (5),389-96(1994),Maloney et al.,Hybridoma. 4 (3),191-209(1985),Raychardhuri et al.,J Immunol. 137 (5), 1743-9(1986), Pohl et al., Int J Cancer. 50 (6), 958-67(1992), Bohlen et al., Cytokines Mol Ther. 2 (4), 231-8 (1996) and Maruyama, J Immunol Methods. 264 (1-2), 121-33 (2002)). The anti-idiotypic antibody may also be optionally coupled to a carrier, which may be a synthetic (usually inert) molecular carrier, a protein (e.g., keyhole limpet hemocyanin (KLH) (see, e.g., Ochi et al., Eur J Immunol. 17 (11), 1645-8 (1987)) or cells (e.g., erythrocytes - see, e.g., Wi et al., J Immunol Methods. 122(2), 227-34(1989)).

[0688] In one embodiment, the therapeutic agent for use in combination with an anti-TF antibody for treating a condition as described above may be an anticancer cytokine, a chemokine, or a combination thereof. Examples of suitable cytokines and growth factors include IFNγ, IL-2, IL-4, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, IL-18, IL-23, IL-24, IL-27, IL-28a, IL-28b, IL-29, KGF, IFNα (e.g., INFα2b), IFNβ, GM-CSF, CD40L, Flt3 ligand, stem cell factor, anciplostatin, and TNFα. Suitable chemokines include Glu-Leu-Arg (ELR)-negative chemokines, such as IP-10, MCP-3, MIG, and SDF-1α from the human CXC and CC chemokine families. Suitable cytokines include cytokine derivatives, cytokine variants, cytokine fragments, and cytokine fusion proteins. Additionally or alternatively, these and other methods or uses herein involving naturally occurring nucleic acids encoding peptides may be performed by "gene activation" and homologous recombination gene upregulation techniques as described in US 5,968,502, US 6,063,630 and US 6,187,305 and EP 0505500.

[0689] In one embodiment, the therapeutic agent used in combination with an anti-TF antibody for treating a condition as described above may be a cell cycle control / apoptosis regulator (or "regulator"). Cell cycle control / apoptosis regulators may include molecules that target and regulate cell cycle control / apoptosis regulators, such as (i) cdc-25 (e.g., NSC 663284), (ii) cyclin-dependent kinases that overstimulate the cell cycle (e.g., flavopiridol (L868275, HMR1275), 7-hydroxy-staurosporine (UCN-01, KW-2401), and roscovitine (R-roscovitine, CYC202)), and (iii) telomerase regulators (e.g., BIBR1532, SOT-095, GRN163 and compositions described in, e.g., US 6,440,735 and US 6,713,055). Non-limiting examples of molecules that can interfere with the apoptotic pathway include TNF-related apoptosis-inducing ligand (TRAIL) / apoptosis-2 ligand (Apo-2L), antibodies that activate the TRAIL receptor, IFN, and antisense Bcl-2.

[0690] In one embodiment, the therapeutic agent used in combination with the anti-TF antibody for the treatment of the above diseases can be a hormone modulator, such as drugs used for anti-androgen and anti-estrogen therapy. Examples of these hormone modulators are tamoxifen, iodoxifene, fulvestrant, droloxifene, toremifene, raloxifene, diethylstilbestrol, ethinyl estradiol / ethinyl estradiol, anti-androgens (e.g., flutaminde / eulexin), progesterone (e.g., hydroxyprogesterone caproate, medroxyprogesterone / Provera, megestrol acetate / Megs), adrenocortical steroids (e.g., hydrocortisone, prednisone), luteinizing hormone-releasing hormone (and its analogs or other LHRH agonists, such as buserelin and goserelin), aromatase inhibitors (e.g., anastrozole / Arimidex, aminoglutethimide / cytraden, exemestane), hormone inhibitors (e.g., octreotide / sandodecanedine), and the like.

[0691] In one embodiment, the therapeutic agent used in combination with the anti-TF antibody for treating the above diseases can be an anti-anergic drug (e.g., a small molecule compound, a protein, a glycoprotein, or an antibody that blocks tolerance to tumor and cancer antigens). Examples of these compounds are molecules that block CTLA-4 activity, such as MDX-010 (ipilimumab) (Phan et al., PNAS USA 100 ,8372(2003)).

[0692] In one embodiment, the therapeutic agent used in combination with the anti-TF antibody for treating the above diseases can be a nucleic acid or vector containing a tumor suppressor gene, such as a replication-deficient adenovirus encoding human recombinant wild-type p53 / SCH58500, etc.; an antisense nucleic acid targeting an oncogene, a mutated or unregulated gene; or an siRNA targeting a mutated or unregulated gene. Examples of tumor suppressor targets include, for example, BRCA1, RB1, BRCA2, DPC4 (Smad4), MSH2, MLH1, and DCC.

[0693] In one embodiment, the therapeutic agent used in combination with the anti-TF antibody for treating the above diseases can be an anti-cancer nucleic acid, such as genasense (augmerosen / G3139), LY900003 (ISIS 3521), ISIS 2503, OGX-011 (ISIS 112989), LE-AON / LEraf-AON (liposome-encapsulated c-raf antisense oligonucleotide / ISIS-5132), MG98, and other antisense nucleic acids targeting PKCα, clusterin, IGFBP, protein kinase A, cyclin D1, or Bcl-2h.

[0694] In one embodiment, the therapeutic agent used in combination with the anti-TF antibody for treating the above diseases can be an anti-cancer inhibitory RNA molecule (see, e.g., Lin et al., Curr Cancer Drug Targets. 1 (3),241-7(2001),Erratum in:Curr Cancer Drug Targets. 3 (3), 237(2003), Lima et al., Cancer GeneTher. 11 (5),309-16(2004),Grzmil et al.,Int J Oncol. 4 (1), 97-105(2004), Collis et al., Int J Radiat Oncol Biol Phys. 57 (2Suppl),S144(2003),Yang et al.,Oncogene. 22 (36), 5694-701 (2003) and Zhang et al., Biochem Biophys Res Commun. 303 (4),1169-78(2003)).

[0695] The compositions and combined administration methods of the present invention also include the administration of nucleic acid vaccines, such as naked DNA vaccines encoding these cancer antigens / tumor-associated antigens (see, for example, US 5,589,466, US 5,593,972, US 5,703,057, US 5,879,687, US 6,235,523, and US 6,387,888). In one embodiment, the combined administration method and / or combined composition comprises an autologous vaccine composition. In one embodiment, the combined composition and / or combined administration method comprises a whole cell vaccine or a cytokine expressing cell (e.g., a fibroblast expressing recombinant IL-2, a dendritic cell expressing a recombinant cytokine, etc.) (see, for example, Kowalczyk et al., Acta Biochim Pol. 50 (3), 613-24(2003), Reilly et al., Methods Mol Med. 69 , 233-57(2002) and Tirapu et al., Curr Gene Ther. 2 (1), 79-89 (2002)). Another example of such an autologous cell method that can be used in the combined methods of the present invention is Personalized immunotherapy approach (formerly known as GTOP-99) (Genitope, Inc. - Redwood City, CA, USA).

[0696] In one embodiment, the present invention provides a combined composition and a combined administration method, wherein an anti-TF antibody is combined with a virus, a viral protein, or the like or co-administered. For example, a replication-deficient virus, which is generally capable of one or a few rounds of replication in vivo and targets tumor cells, can be used as a component of such a composition and method. Such viral agents may contain nucleic acids encoding immunostimulants (e.g., GM-CSF and / or IL-2), or be accompanied therewith. Natural oncolytic viruses and these recombinant oncolytic viruses (e.g., HSV-1 virus, reovirus, replication-deficient and replication-sensitive adenoviruses, etc.) can be used as components of these methods and compositions. Therefore, in one embodiment, the present invention provides a combined composition and a combined administration method, wherein an anti-TF antibody is combined with an oncolytic virus or co-administered. Examples of these viruses include oncolytic adenoviruses and herpes viruses, which may or may not be modified viruses (see, e.g., Shah et al., J Neurooncol. 65 (3),203-26(2003),Stiles et al.,Surgery. 134 (2), 357-64(2003), Sunarmura et al., Pancreas. 28(3),326-9(2004),Teshigahara et al.,J Surg Oncol. 85 (1), 42-7(2004), Varghese et al., Cancer Gene Ther. 9 (12),967-78(2002),Wildner et al.,Cancer Res. 59 (2),410-3(1999),Yamanaka,Int J Oncol. 24 (4), 919-23 (2004) and Zwiebel et al., Semin Oncol. 28 (4), 336-43(2001)).

[0697] The combined compositions and combined administration methods of the present invention may also involve "whole cell" and "adoptive" immunotherapy methods. For example, these methods may include infusing or re-infusing immune system cells (e.g., tumor infiltrating lymphocytes (TILs), such as CD4 + and / or CD8 + T cells (e.g., T cells amplified with tumor-specific antigens and / or genetic enhancers), B cells expressing antibodies or other antibody producing / presenting cells, dendritic cells (e.g., recombinant dendritic cells expressing anti-cytokines, dendritic cells cultured with DC expanders such as GM-CSF and / or Flt3-L, and / or dendritic cells loaded with tumor-associated antigens), anti-tumor NK cells, so-called hybrid cells, or combinations thereof. Cell lysates can also be used in these methods and compositions. Cellular "vaccines" that are in clinical trials for these aspects include Canvaxin TM ,APC-8015(Dendreon),HSPPC-96(Antigenics), and Cell lysates. Antigens shed from cancer cells and mixtures thereof (see, e.g., Bystryn et al., Clinical Cancer Research Vol. 7, 1882-1887, July 2001), optionally mixed with an adjuvant such as alum, may also be used as components of these methods and combination compositions.

[0698] In one embodiment, the anti-TF antibody can be delivered to the patient in combination with an internal vaccination method. In vivo vaccination refers to the induced tumor or cancer cell death in the patient, such as drug-induced or radiation-induced cell death of tumor cells, which typically results in the initiation of an immune response against (i) the whole tumor cell or (ii) a part of the tumor cell, including (a) secreted proteins, glycoproteins or other products, (b) membrane-associated proteins or glycoproteins or other components associated with or inserted into the membrane, and / or (c) intracellular proteins or other intracellular components. The immune response induced by in vivo vaccination can be humoral (i.e., antibody-complement mediated) or cell-mediated (e.g., the occurrence and / or increase of endogenous cytotoxic T lymphocytes that can recognize killed tumor cells or parts thereof in vivo). In addition to radiotherapy, non-limiting examples of drugs and agents that can be used to induce tumor cell death and in vivo vaccination are conventional chemotherapeutic agents, cell cycle inhibitors, anti-angiogenic drugs, monoclonal antibodies, apoptosis inducers, and signal transduction inhibitors.

[0699] Other examples of anticancer agents that may be used as therapeutic agents in combination with anti-TF antibodies to treat the above diseases are differentiation inducing agents, retinoic acid analogs (e.g., all-trans retinoic acid, 13-cis retinoic acid, and similar drugs), vitamin D analogs (e.g., seocalcitol and similar drugs), inhibitors of ErbB3, ErbB4, IGF-IR, insulin receptor, PDGFRa, PDGFRbeta, Flk2, Flt4, FGFR1, FGFR2, FGFR3, FGFR4, TRKA, TRKC, c-met, Ron, Sea, Tie, Tie2, Eph, Ret, Ros, Alk, LTK, PTK7, and the like.

[0700] Other examples of anticancer agents that may be used as therapeutic agents in combination with anti-TF antibodies to treat the above diseases are modulators of cathepsin B, cathepsin D dehydrogenase activity, glutathione-S-transferases (e.g., glutacylcysteine ​​synthetase and lactate dehydrogenase), and similar drugs.

[0701] Other examples of anticancer agents that may be used as therapeutic agents in combination with anti-TF antibodies to treat the above diseases are estramustine and epirubicin.

[0702] Other examples of anticancer agents that may be used as therapeutic agents in combination with anti-TF antibodies to treat the above diseases are HSP90 inhibitors, such as 17-allylaminogeldanamycin, antibodies against tumor antigens such as PSA, CA125, KSA, etc., integrins such as integrin β1, VCAM inhibitors and similar drugs.

[0703] Other examples of anticancer agents that may be used as therapeutic agents in combination with anti-TF antibodies to treat the above diseases are calcineurin inhibitors (e.g., valspodar, PSC 833 and other MDR-1 or p-glycoprotein inhibitors), TOR inhibitors (e.g., sirolimus, everolimus and rapamycin), and inhibitors of the "lymphocyte homing" mechanism (e.g., FTY720), and agents that have an effect on cell signaling, such as adhesion molecule inhibitors (e.g., anti-LFA, etc.).

[0704] In one embodiment, the anti-TF antibodies of the invention are used in combination with one or more other therapeutic antibodies, such as bevacizumab (Acrivastin ), zalutumumab, cetuximab Vectibix TM ), ofatumumab, zanolimumab, daratumumab, ranibizumab Zenapax, Simulect, Remicade, Humira, Tysabri, Xolair, raptiva, nimotuzumab, rituximab, and / or trastuzumab (Herceptin Other therapeutic antibodies that can be used in combination with the antibodies of the present invention are disclosed in the following documents: WO98 / 40408 (antibodies capable of binding to native human TF), WO04 / 094475 (antibodies capable of binding to human tissue factor that do not inhibit factor-mediated coagulation compared to normal plasma controls), WO03 / 093422 (binding affinity to the TF:VIIa complex is greater than affinity to TF alone), or WO03 / 037361 (TF agonists or antagonists for apoptosis-related treatments).

[0705] In another embodiment, two or more different antibodies of the present invention as described herein are used in combination to treat disease. Particularly interesting combinations include two or more non-competitive antibodies. Therefore, in one embodiment, the patient is treated with an antibody combination of an antibody in a cross-block group I as defined herein and an antibody combination in a group II or III as defined herein. In another embodiment, the patient is treated with an antibody combination of an antibody in a group II as defined below and an antibody combination in a group III. These combination therapies can result in each cell combining more number of antibody molecules, thereby providing higher efficacy, for example, by activating complement-mediated dissolution.

[0706] In one embodiment, administration of an anti-TF antibody can be combined with the delivery of one or more agents as described below that can facilitate the delivery of the anti-TF antibody or combination composition to the interior of the tumor. This approach can be combined, for example, with the delivery of relaxin, which can relax tumors (see, e.g., US 6,719,977). In one embodiment, an anti-TF antibody of the invention can be conjugated to a cell penetrating peptide (CPP). Cell penetrating peptides and related peptides (e.g., engineered cell penetrating antibodies) are described, for example, in Zhao et al., J Immunol Methods. 254 (1-2),137-45(2001),Hong et al.,Cancer Res. 60 (23),6551-6(2000).Lindgren et al.,Biochem J. 377 (Pt 1),69-76(2004),Buerger et al.,J Cancer Res Clin Oncol. 129 (12), 669-75(2003), Pooga et al., FASEB J. 12 (1), 67-77 (1998) and Tseng et al., Mol Pharmacol. 62 (4),864-72(2002).

[0707] In one embodiment, the invention relates to a method for treating a disease involving cells expressing TF in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody and at least one anti-inflammatory agent.

[0708] In one embodiment, such anti-inflammatory agents may be selected from the group consisting of aspirin and other salicylates, Cox-2 inhibitors (e.g., rofecoxib and celecoxib), NSAIDs (e.g., ibuprofen, fenoprofen, naproxen, sulindac, diclofenac, piroxicam, ketoprofen, diflunisal, nabumetone, etodolac, oxaprozin and indomethacin), anti-IL6R antibodies, anti-IL8 antibodies (e.g., antibodies described in WO2004058797, e.g., 10F8), anti-IL15 antibodies (e.g., antibodies described in WO03017935 and WO2004076620), anti-IL15 R antibodies, anti-CD4 antibodies (e.g., zanolimumab), anti-CD11a antibodies (e.g., efalizumab), anti-α-4 / β-1 integrin (VLA4) antibodies (e.g., natalizumab), CTLA4-Ig for the treatment of inflammatory diseases, prednisolone, prednisone, disease-modifying antirheumatic drugs (DMARDs) such as methotrexate, hydroxychloroquine, sulfasalazine, pyrimidine synthesis inhibitors (e.g., leflunomide), IL-1 receptor blockers (e.g., anakinra), TNF-α blockers (e.g., etanercept, infliximab, and adalimumab), and similar drugs.

[0709] In one embodiment, such immunosuppressive and / or immunomodulatory agents may be selected from the group consisting of cyclosporine, azathioprine, mycophenolic acid, mycophenolate mofetil, corticosteroids such as prednisone, methotrexate, gold salts, sulfasalazine, antimalarials, brequinar, leflunomide, mizoribine, 15-deoxyspergualine, 6-mercaptopurine, cyclophosphamide, rapamycin, tacrolimus (FK-506), OKT3, anti-thymocyte globulin, thymopentin, thymosin-α and similar drugs.

[0710] In one embodiment, such immunosuppressive and / or immunomodulatory agents can be selected from immunosuppressive antibodies, such as antibodies that bind to p75 of the IL-2 receptor, antibodies against CD25 (e.g., as described in WO2004045512, such as AB1, AB7, AB11 and AB12), or antibodies that bind to, for example, MHC, CD2, CD3, CD4, CD7, CD28, B7, CD40, CD45, IFNγ, TNF-α, IL-4, IL-5, IL-6R, IL-7, IL-8, IL-10, CD11a or CD58, or antibodies that bind to their ligands.

[0711] In one embodiment, such immunosuppressive and / or immunomodulatory agents may be selected from: soluble IL-15R, IL-10, B7 molecules (B7-1, B7-2, variants thereof and fragments thereof), ICOS and OX40, negative T cell regulator inhibitors (e.g. antibodies against CTLA4) and similar drugs.

[0712] In one embodiment, the invention provides a method for treating a disease involving cells expressing TF in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody and an anti-C3b(i) antibody.

[0713] In one embodiment, the therapeutic agent used in combination with the anti-TF antibody for treating the above diseases can be selected from the group consisting of histone deacetylase inhibitors (e.g., phenylbutyrate) and / or DNA repair agents (e.g., DNA repair enzymes and related compositions, such as dimericine.

[0714] The method of the present invention comprising administering a therapeutically effective amount of an anti-TF antibody for treating the above-mentioned diseases may also include anti-cancer directed photodynamic therapy (e.g., anti-cancer laser therapy—which may optionally be implemented using a photosensitizer, see, e.g., Zhang et al., J Control Release. 93 (2), 141-50 (2003)), anticancer sound and vibration wave therapy (see, for example, Kambe et al., Hum Cell. 10 (1), 87-94 (1997)), and / or anticancer nutritional therapy (see, for example, Roudebush et al., Vet Clin North Am Small Anim Pract. 34 (1), 249-69, viii (2004) and Rafi, Nutrition. 20 (1), 78-82 (2004)). Similarly, anti-TF antibodies can be used to prepare pharmaceutical compositions for administration together with anti-cancer directed photodynamic therapy (e.g., anti-cancer laser therapy - which optionally can use a photosensitizer), anti-cancer sound and shock wave therapy, and / or anti-cancer nutritional therapy to treat the above diseases.

[0715] In one embodiment, the invention provides a method for treating a disease involving TF-expressing cells in a subject, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody, such as an anti-TF antibody of the invention, and radiation therapy.

[0716] In one embodiment, the invention provides a method for treating or preventing cancer, the method comprising administering to a subject in need thereof a therapeutically effective amount of an anti-TF antibody, such as an anti-TF antibody of the invention, and radiation therapy.

[0717] In one embodiment, the invention provides an anti-TF antibody, such as an anti-TF antibody of the invention, for use in the manufacture of a pharmaceutical composition for treating cancer for administration in combination with radiation therapy.

[0718] Radiotherapy can include radiation or the associated administration of a radiotherapeutic drug to a patient. The radiation source can be external to or internal to the patient to be treated (radiation therapy can be, for example, in the form of external beam radiation therapy (EBRT) or brachytherapy (BT)). Radioactive elements that can be used to implement these methods include, for example, radium, cesium-137, iridium-192, americium-241, gold-198, cobalt-57, copper-67, technetium-99, iodine-123, iodine-131, and indium-111.

[0719] In a further embodiment, the invention provides a method for treating or preventing cancer, the method comprising administering a therapeutically effective amount of an anti-TF antibody, such as an anti-TF antibody of the invention, to a subject in need thereof in combination with surgery.

[0720] As described above, the pharmaceutical compositions of the present invention can be used in combination therapy, i.e., in combination with one or more medicaments associated with the condition to be treated, or as separate pharmaceutical compositions, or the compounds of the present invention are co-formulated with one or more additional therapeutic agents as described above. The dosage of the compounds of the present invention and / or co-administered medicaments required for these combination therapies can be lower, thereby avoiding possible toxicity or complications associated with various monotherapies.

[0721] In addition to the above, other combination therapies of interest include the following:

[0722] For pancreatic cancer treatment, anti-TF antibody in combination with antimetabolites (e.g., 5-fluorouracil and / or gemcitabine), possibly in combination with one or more compounds selected from the group consisting of 90Y-hPAM4, ARC-100, ARQ-197, AZD-6244, bardoxolone methyl, cixutumumab, (IMC-A12), folitixorin calcium, GVAX, ipilimumab, KRX-0601, mebarone, MGCD-0103, MORAb-009, PX-12, Rh-Apo2L, TLN-4601, trabedersen, volociximab (M200), WX-671, pemetrexed, rubitecan, ixabepilone, OCX-0191Vion, 216586-46-8, lapatinib, matuzumab, imatinib, sorafinib, trastuzumab, exabepilone, erlotinib, acrivastin and cetuximab.

[0723] For colorectal cancer treatment, an anti-TF antibody is combined with one or more compounds selected from gemcitabine, bevacizumab, FOLFOX, FOLFIRI, XELOX, IFL, oxaliplatin, irinotecan, 5-FU / LV, capecitabine, UFT, EGFR targeting agents such as cetuximab, panitumumab, zalutumumab, nimotuzumab; VEGF inhibitors, or tyrosine kinase inhibitors such as sunitinib.

[0724] For breast cancer treatment, anti-TF antibody is combined with one or more compounds selected from the group consisting of antimetabolites, anthracyclines, taxanes, alkylating agents, epothilone antihormones (letrozole (femar), tamoxifen, etc.), ErbB2 (Her2 / neu) inhibitors (e.g. Herceptin and similar drugs), CAF / FAC (cyclofosfamide, doxorubicin, 5FU) AC (cyclo, doxo), CMF (cyclo, methotrexate, 5FU), docetaxel + capecitabine, GT (paclitaxel, gemcitabine) FEC (cyclo, epi, 5FU) in combination with Herceptin, paclitaxel + / - carboplatin, vinorelbine, docetaxel, CT in combination with lapatinib; capecitabine.

[0725] For bladder cancer treatment, an anti-TF antibody is combined with one or more compounds selected from the group consisting of antimetabolites (gemcitabine, alimta, methotrexate), platinum analogs (cisplatin, carboplatin), EGFr inhibitors (e.g., cetuximab or zalutumumab), VEGF inhibitors (e.g., acrivastine), doxorubicin, tyrosine kinase inhibitors such as gefitinib, trastuzumab, antimitotic agents such as taxanes, e.g., paclitaxel, and vinca alkaloids such as vinblastine.

[0726] For prostate cancer treatment, an anti-TF antibody is combined with one or more compounds selected from: hormone / anti-hormone therapy; e.g., anti-androgens, luteinizing hormone releasing hormone (LHRH) agonists, and chemotherapeutic agents such as taxanes, mitoxantrone, estramustine, 5FU, vinblastine, ixabepilone.

[0727] • For ovarian cancer treatment, anti-TF antibody is combined with one or more compounds selected from: anti-mitotic agents, such as taxanes, and vinca alkaloids, caelyx, topotecan.

[0728] Diagnostic Uses

[0729] The anti-TF antibodies of the invention may also be used for diagnostic purposes. Thus, in a further aspect, the invention relates to a diagnostic composition comprising an anti-TF antibody as defined herein.

[0730] In one embodiment, the anti-TF antibodies of the invention can be used to diagnose diseases in vivo or in vitro in which activated TF-expressing cells play an active role in their pathogenesis by detecting the level of TF or the level of cells containing TF on their membrane surface. This can be achieved, for example, by contacting the test sample, optionally together with a control sample, with the anti-TF antibody under conditions that allow the formation of a complex between the antibody and TF. The formation of the complex is then detected (for example using ELISA). When a control sample is used together with a test sample, the complex is detected for both samples, and any statistically significant difference in complex formation between the samples indicates the presence of TF in the test sample.

[0731] Therefore, in a further aspect, the present invention relates to a method for detecting the presence of TF antigen or cells expressing TF in a sample, comprising:

[0732] - contacting the sample with an anti-TF antibody of the invention or a bispecific molecule of the invention under conditions that allow formation of a complex between the antibody and TF; and

[0733] - Analyze whether a complex is formed.

[0734] In one embodiment, the method is performed in vitro.

[0735] More specifically, the invention provides methods for identifying and diagnosing invasive cells and tissues, as well as other cells targeted by the anti-TF antibodies of the invention, and for monitoring the progress of therapeutic treatment, post-treatment status, risk of developing cancer, cancer progression, etc.

[0736] In one example of such a diagnostic assay, the invention provides a method for diagnosing the level of invasive cells in a tissue, comprising forming an immune complex between an anti-TF antibody and a potential TF-containing tissue, and detecting the formation of the immune complex, wherein the formation of the immune complex is associated with the presence of invasive cells in the tissue. The contacting can be performed in vivo using a labeled isolated antibody and standard imaging techniques, or can be performed in vitro on a tissue sample.

[0737] Anti-TF antibodies can be used to detect peptides and peptide fragments containing TF in any suitable biological sample by any suitable technique. Examples of conventional immunoassays provided herein include, but are not limited to, ELISA, RIA, FACS assays, plasma resonance assays, chromatographic assays, tissue immunohistochemistry, western blotting, and / or immunoprecipitation using anti-TF antibodies. The anti-TF antibodies of the present invention can be used to detect TF and TF fragments from humans. Suitable labels for anti-TF antibodies and / or secondary antibodies used in these techniques include, but are not limited to, various enzymes, prosthetic groups, fluorescent substances, luminescent substances, and radioactive substances. Examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, β-galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent substances include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylaminefluorescein / dansyl chloride or phycoerythrin; examples of luminescent substances include luminol; and examples of suitable radioactive substances include 125 I, 131 I, 35 S, and 3 H.

[0738] Anti-TF antibodies can also be measured in biological samples by competitive immunoassay using TF peptide standards labeled with a detectable substrate and unlabeled anti-TF antibodies. In this assay, the biological sample, labeled TF peptide standards, and anti-TF antibodies are mixed, and the amount of labeled TF standard peptide bound to the unlabeled anti-TF antibody is detected. The amount of TF antibodies in the biological sample is inversely proportional to the amount of labeled TF standard bound to the anti-TF antibody.

[0739] Anti-TF antibodies are particularly useful in in vivo imaging of tumors. In vivo imaging of TF-associated tumors can be performed by any suitable technique. For example, 99 Anti-TF antibodies or a second labeled (e.g., FITC-labeled) anti-TF antibody:TF complex in the tumor are labeled with Tc-labeling or labeling with another gamma-emitting isotope and imaged with a gamma scintillation camera (e.g., the Elscint Apex 409 ECT device), typically using a low energy, high resolution collimator or a low energy all-around collimator. The stained tissue can then be assessed for radioactivity counts as an indication of the amount of TF-related peptides within the tumor. Images obtained using these techniques can be used to assess the biodistribution of TF within a patient, mammal, or tissue (e.g., when using TF or TF fragments as a biomarker for the presence of invasive cancer cells). Variations on this technique include the use of magnetic resonance imaging (MRI) to provide imaging superior to gamma camera techniques. Similar immunoscintigraphy methods and principles are described, for example, in Srivastava (ed.), Radiolabeled Monoclonal Antibodies For Imaging And Therapy (Plenum Press 1988), Chase, "Medical Applications of Radioisotopes," in Remington's Pharmaceutical Sciences, 18th ed., Gennaro et al., (ed.), pp. 624-652 (Mack Publishing Co., 1990), and Brown, "Clinical Use of Monoclonal Antibodies," in Biotechnology And Pharmacy 227-49, Pezzuto et al., (ed.) (Chapman & Hall 1993). These images can also be used for targeted delivery of other anticancer agents, examples of which are described herein (e.g., apoptotic agents, toxins, or CHOP chemotherapy compositions). And / or these images can also serve as the basis for surgical techniques to remove tumors. Furthermore, such in vivo imaging techniques may allow identification and localization of tumors in cases where a patient is identified as having a tumor (due to the presence of other biomarkers, metastases, etc.), but the tumor cannot be identified using conventional analytical techniques. All of these methods are features of the present invention.

[0740] The in vivo imaging and other diagnostic methods provided by the present invention are particularly useful for detecting micrometastases in human patients (e.g., patients who have not been previously diagnosed with cancer or patients who are in recovery / remission from cancer). For example, carcinoma cancer cells, which account for up to 90% of all cancer cells, have been shown to be well stained with anti-TF antibody conjugate compositions. Detection with the monoclonal anti-TF antibodies described herein can indicate the presence of aggressive / invasive epithelial tumors, or / and provide an indication of the feasibility of using the relevant monoclonal anti-TF antibodies against these micrometastases.

[0741] In one embodiment, the invention provides an in vivo imaging method in which an anti-TF antibody of the invention is conjugated to a detection-promoting radio-opaque agent, the conjugated antibody is administered to a host (e.g., by injection into the bloodstream), and the presence and localization of the labeled antibody in the host is determined. By this technique and any other diagnostic method provided herein, the invention provides a method for screening a biological sample obtained from a human patient or in a human patient for the presence of disease-associated cells.

[0742] For diagnostic imaging, the radioisotope can be linked to the anti-TF antibody either directly or indirectly through the use of an intermediate functional group. Useful intermediate functional groups include chelators such as ethylenediaminetetraacetic acid and diethylenetriaminepentaacetic acid (see, for example, US 5,057,313).

[0743] In addition to radioisotopes and radiopaque agents, diagnostic methods can be performed with anti-TF antibodies coupled to dyes (e.g., coupled to biotin-streptavidin complexes), contrast agents, fluorescent compounds or molecules, and enhancers for magnetic resonance imaging (MRI), such as paramagnetic ions (see, e.g., U.S. Pat. No. 6,331,175, which describes MRI techniques and the preparation of antibodies coupled to MRI enhancers). Such diagnostic / detection agents can be selected from agents for magnetic resonance imaging and fluorescent compounds. In order to load the anti-TF antibody with radioactive metals or paramagnetic ions, it may be necessary to react it with an agent having a long tail to which are attached a plurality of chelating groups for binding to the ions. Such a tail may be a polymer, such as polylysine, a polysaccharide, or other derivatizable or derivatizable chain having pendant groups to which chelating groups can be attached, such as porphyrins, polyamines, crown ethers, bithiosemicarbazones, polyoximes, and similar groups known to be useful for this purpose. Chelating agents can be coupled to anti-TF antibodies using standard chemistry.

[0744] Thus, the present invention provides diagnostic anti-TF antibody conjugates, wherein the anti-TF antibody is conjugated to a contrast agent (e.g., for magnetic resonance imaging, computed tomography or ultrasound contrast enhancement agents) or a radionuclide, which may be, for example, an isotope that emits gamma, beta, alpha, Auger electrons, or positrons.

[0745] In a further aspect, the present invention relates to a kit for detecting the presence of TF antigen or cells expressing TF in a sample, comprising

[0746] - an anti-TF antibody of the invention or a bispecific molecule of the invention; and

[0747] -Instructions for use of the kit.

[0748] In one embodiment, the present invention provides a kit for diagnosing cancer, comprising a container containing an anti-TF antibody, and one or more reagents for detecting the binding of the anti-TF antibody to the TF peptide. The reagents may include, for example, fluorescent labels, enzyme labels, or other detectable labels. The reagents may also include a second or third antibody or reagent for an enzyme reaction, wherein the enzyme reaction produces a product that can be visualized. In one embodiment, the present invention provides a diagnostic kit, which includes one or more anti-TF antibodies of the present invention in a labeled or unlabeled form, a reagent for incubation in an indirect assay, and a substrate or derivatization reagent for detection in such an assay, depending on the nature of the label. Control reagents and instructions for use may also be included.

[0749] Diagnostic kits can also be provided for using anti-TF antibodies, such as conjugated / labeled anti-TF antibodies, to detect cell activity in a tissue sample or host or to detect the presence of TF peptides. In such diagnostic kits, as well as kits for therapeutic applications described elsewhere herein, the anti-TF antibody is typically placed in a container in lyophilized form, provided alone or with additional antibodies specific for the target cell or peptide. Typically, a pharmaceutically acceptable carrier (e.g., an inert diluent) and / or its components such as Tris, phosphate or carbonate buffers, stabilizers, preservatives, biocides, biocides, inert proteins such as serum albumin, etc. are also included (typically placed in a separate container for mixing), and additional reagents (typically also placed in a separate container). In some kits, a second antibody capable of binding to the anti-TF antibody is also included, which is usually placed in a separate container. The second antibody is usually conjugated to a label and formulated in a manner similar to the anti-TF antibodies of the present invention. Using the methods described above and elsewhere herein, anti-TF antibodies can be used to define subsets of cancer / tumor cells, and to characterize these cells and associated tissues / growths.

[0750] In situ detection can be achieved by removing a tissue sample from a patient and providing such sample with a combination of the labeled anti-TF antibodies of the present invention. The provision of the anti-TF antibodies of the present invention can be achieved by applying or overlaying the labeled anti-TF antibodies of the present invention to the biological sample. By using this procedure, it is possible to determine not only the presence of TF or TF fragments, but also the distribution of these peptides in the tissue being tested (e.g., when assessing the spread of cancer cells). Using the present invention, one of ordinary skill will readily appreciate that any of a number of histological methods (e.g., staining procedures) can be modified to achieve such in situ detection.

[0751] In a further aspect, the invention relates to an anti-idiotypic antibody which binds to an anti-TF antibody of the invention as described herein.

[0752] Anti-idiotypic (Id) antibodies are antibodies that recognize unique determinants generally associated with the antigen binding site of an antibody. Id antibodies can be prepared by immunizing an animal with an anti-TF monoclonal antibody to be prepared for Id, wherein the species and genetic type of the animal are the same as the species and genetic type from which the anti-TF monoclonal antibody is derived. The immunized animal is generally able to recognize the idiotypic determinants of the antibody used for immunization and respond to them by producing antibodies to these idiotypic determinants (anti-Id antibodies). Such antibodies are described, for example, in US 4,699,880. Such antibodies are a further feature of the present invention.

[0753] Anti-Id antibodies can also be used as an "immunogen" to induce an immune response in another animal, producing so-called anti-anti-Id antibodies. Anti-anti-Id antibodies may be epitopically identical to the original mAb that induced the anti-Id antibodies. Thus, by using antibodies directed against the idiotypic determinants of a certain mAb, it is possible to identify other clones expressing antibodies with the same specificity. Anti-Id antibodies can be altered (thereby producing anti-Id antibody variants) and / or derivatized by any suitable technique, such as those described elsewhere herein with respect to the anti-TF antibodies of the invention. For example, an anti-Id mAb can be coupled to a carrier such as keyhole limpet hemocyanin (KLH) and used to immunize BALB / c mice. Sera from these mice typically contain anti-anti-Id antibodies that have similar (even if not identical) binding properties to the original / parent TF antibody.

[0754] The present invention is further illustrated by the following examples, which should not be construed as further limiting. Example

[0755] Example 1

[0756] Tissue Factor (TF) expression construct

[0757] Complete codon optimized constructs for expressing TF or its extracellular domain in HEK, NS0 or CHO cells were generated. The protein encoded by these constructs is identical to the Genbank accession number NP_001984 of TF. The construct contains suitable restriction sites and optimal Kozak sequences (Kozak, 1987) for cloning. The construct was cloned in the mammalian expression vector pEE13.4 (Lonza Biologics) (Bebbington, Renner et al. 1992) to obtain pEE13.4TF. PCR was used to amplify the portion encoding the TF extracellular domain (ECD) (amino acids 1-251) from the synthetic construct, while adding a C-terminal His tag (TFECDHis) containing 6 histidine residues. The construct was cloned into pEE13.4 and fully sequenced to confirm the correctness of the construct.

[0758] Example 2

[0759] Transient expression in HEK-293F cells

[0760] Freestyle TM 293-F (a HEK-293 subclone adapted to suspension growth and chemically defined Freestyle medium, (HEK-293F)) cells were obtained from Invitrogen and transfected with appropriate plasmid DNA using 293fectin (Invitrogen) according to the manufacturer's instructions. In the case of antibody expression, appropriate heavy and light chain vectors as described in Example 10 were co-expressed.

[0761] Example 3

[0762] Semi-stably expressed in NS0 cells

[0763] pEE13.4TF was stably transfected into NS0 cells and stable clones were selected by growth in the absence of glutamine and in the presence of 7.5 μM methylsulfoximine (MSX). Pools of clones were grown in suspension medium while maintaining selection pressure. TF expression in the pools was verified by FACS analysis and stored for future use.

[0764] Example 4

[0765] Stable expression in CHO cells

[0766] pEE13.4TF was stably transfected into CHO-K1SV (Lonza Biologics) cells and stable clones were selected by growth in the absence of glutamine and in the presence of 50 μM MSX. Single clones were picked, expanded, and tested for TF expression by FACS analysis as described below. Highly expressing clones were selected and preserved for future use.

[0767] Example 5

[0768] Purification of His-tagged TF

[0769] TFECDhis was expressed in HEK-293F cells. The his tag in TFECDhis enables purification using immobilized metal affinity chromatography. In this process, the chelating agent immobilized on the chromatography resin is charged with Co 2+ The supernatant containing TFECDHis is incubated with the resin in batch mode (i.e., solution). The His-tagged proteins bind strongly to the resin beads, while other proteins present in the culture supernatant do not bind strongly. After incubation, the beads are recovered from the supernatant and filled into the column. The column is washed to remove weakly bound proteins. The strongly bound TFECDHis protein is then eluted with a buffer containing imidazole, which competes with His for binding to Co 2+ The eluate is removed from the protein by buffer exchange on a desalting column.

[0770] Example 6

[0771] Transgenic mouse immunization program

[0772] Human monoclonal antibody mice were alternately treated with 5x10 6 Semi-stable transfected NS0-TF cells or immunized with 20 μg TFECDHis protein. A total of 8 immunizations were implemented, 4 intraperitoneal (IP) immunizations and 4 tail root subcutaneous (SC) immunizations. The first immunization using cells was completed in complete Freund's adjuvant (CFA; Difco Laboratories, Detroit, MI, USA). For all other immunizations, cells were injected IP in PBS, and TFECDHis was injected SC with incomplete Freund's adjuvant (IFA; Difco Laboratories, Detroit, MI, USA). When the serum titer is sufficient (serum with a dilution of 1 / 50 or less is found to be positive in at least 2 consecutive biweekly screening events in the antigen-specific screening assay method as described in Example 7), two additional intravenous (IV) boosted immunizations were performed 4 and 3 days before fusion with 10 μg TFECDHis protein dissolved in 100 μl PBS.

[0773] The first immunization with cells was performed in CFA, for all other immunizations (7), cells were injected IP in PBS. When serum titers were found to be adequate, mice were treated with 1x10 40 μL of 1 ... 6 Transiently and semi-stably transfected NS0-TF cells were immunized with IV for two additional times.

[0774] Example 7

[0775] Cognate antigen-specific screening assay

[0776] The presence of anti-TF antibodies in sera of immunized mice or in culture supernatants of human MAb (human monoclonal antibody) hybridomas or transfectomas was determined by cognate antigen-specific screening assay (quadrant) using Fluorometric Micro volume Assay Technology (FMAT; Applied Biosystems, Foster City, CA, USA).

[0777] To this end, 3 cell-based assay systems and 1 bead-based assay system were used in combination. In the cell-based assay, binding to TH1015-TF (HEK-293F cells transiently expressing TF; generated as described above) and A431 (which express TF on the cell surface) as well as HEK293 wild-type cells (not expressing TF, negative control) was determined. In the bead-based assay, binding to biotinylated TF (SB1015-TF) coupled to streptavidin beads was determined.

[0778] Samples were added to cells / beads for TF binding. Subsequently, binding to human mAbs was detected with a fluorescent conjugate (goat anti-human IgG-Cy5; Jackson ImmunoResearch). Mouse anti-human TF antibody (ERL; conjugated to Alexa-647 at Genmab) was used as a positive control, and human mAb-mouse pooled serum and mouse-chrompure-Alexa647 antibody were used as negative controls. Samples were scanned with an Applied Biosystems 8200 Cell Detection System (8200CDS) and "Count x Fluorescence" was used as a readout.

[0779] Example 8

[0780] Human mAb hybridoma generation

[0781] The human monoclonal antibody mice with sufficient antigen-specific titer (as defined above) were euthanized, and the spleen and lymph nodes on both sides of the abdominal aorta and vena cava were collected. The fusion of splenocytes and lymph node cells with mouse myeloma cell lines was performed by electrofusion using CEEF 50 electrofusion system (Cyto Pulse Sciences, Glen Burnie, MD, USA) in accordance with the manufacturer's instructions. The selection and cultivation of the obtained human monoclonal antibody hybridomas were carried out according to standard methods (for example, as described in the following documents: Coligan JE, Bierer, BE, Margulies, DH, Shevach, EM and Strober, W., eds. Current Protocols in Immunology, John Wiley & Sons, Inc., 2006).

[0782] Example 9

[0783] Mass spectrometry of purified antibodies

[0784] Small portions of antibody-containing supernatants (0.8 ml each) from the 6-well or Hyperflask platforms were purified using PhyTip columns (PhyNexus Inc., San Jose, USA) containing protein G resin on a Sciclone ALH 3000 workstation (Caliper Lifesciences, Hopkinton, USA). The PhyTtip columns were used according to the manufacturer's instructions, but the buffers were PBS (B. Braun, Medical BV, Oss, Netherlands) for binding buffer and 0.1 M glycine-HCl pH 2.7 (Fluka Riedel-de Buchs, Germany). After purification, the samples were neutralized with 2M Tris-HCl pH 9.0 (Sigma-Aldrich, Zwijndrecht, Netherlands). Alternatively, in some cases, larger volumes of culture supernatant were purified using protein A affinity column chromatography.

[0785] After purification, the samples were placed in a 384-well plate (Waters, 100ul square well plate, part number 186002631). The samples were deglycosylated with N-glycosylase F (Roche lot number 11365177001) at 37°C overnight. DTT (15mg / ml) was added (1μl / well) and incubated at 37°C for 1h. The samples (5 or 6ul) were chromatographed on an Acquity UPLC TMDesalting was performed on a BEH300 C18, 1.7 μm, 2.1 x 50 mm column (Waters, Milford, USA) at 60° C. MQ water and LC-MS grade acetonitrile (Biosolve, lot 01204101, Valkenswaard, The Netherlands) containing 0.1% formic acid (Fluka, lot 56302, Buchs, Germany) were used as eluents A and B, respectively. TM Time-of-flight electrospray ionization mass spectra were recorded online on a mass spectrometer (Bruker, Bremen, Germany). Prior to analysis, the 900-3000 m / z scale was calibrated using ES tuning mix (Agilent Technologies, Santa Clara, USA). TM Deconvolution was performed using the software v.3.4 (Bruker) using the Maximum Entropy algorithm to find molecular weights between 5 and 80 kDa.

[0786] After deconvolution, the resulting heavy and light chain masses of all samples were compared to find duplicate antibodies. In the comparison of the heavy chains, the possible presence of C-terminal lysine variants was taken into account. The result was a series of unique antibodies, where "unique" was defined as a unique combination of heavy and light chains. In the case of duplicate antibodies found, the results from other tests were used to decide which was the best material to proceed with the experiment.

[0787] MS analysis of the molecular weight of the heavy and light chains of 118 TF-specific hybridomas yielded 70 unique antibodies (unique heavy / light chain combinations). These were characterized in several functional tests, identifying 14 lead candidates, TF-specific antibodies.

[0788] Example 10

[0789] Sequence analysis of the variable domain of anti-TF human monoclonal antibody and cloning into expression vector

[0790] From 5x10 6Total RNA of anti-TF human mAbs was prepared from 100 ng of total RNA, and 5'-RACE-complementary DNA (cDNA) was prepared from 100 ng of total RNA using the SMARTRACE cDNA Amplification Kit (Clontech) according to the manufacturer's instructions. VH (heavy chain variable region) and VL (light chain variable region) coding regions were amplified by PCR and cloned into the pCR-Blunt II-TOPO vector (Invitrogen) using the Zero Blunt PCR Cloning Kit (Invitrogen). For each human mAb, 16 VL clones and 8 VH clones were sequenced. The sequences are listed in the sequence listing and Figure 1 Given in.

[0791] Table 1A and Table 1B (below) give an overview of antibody sequence information and the most homologous germline sequences.

[0792] Table 1A Heavy chain homologs

[0793]

[0794] Table 1B Light chain homologs

[0795]

[0796] Sequence Listing Reference:

[0797]

[0798]

[0799]

[0800]

[0801]

[0802]

[0803]

[0804] Embodiment 11

[0805] Antibody purification

[0806] The culture supernatant was filtered through a 0.2 μm dead-end filter and loaded onto a 5 ml protein A column (rProtein A FF, Amersham Bioscience) and eluted with 0.1 M citric acid-NaOH, pH 3. The eluate was immediately neutralized with 2 M Tris-HCl, pH 9 and dialyzed overnight into 12.6 mM NaH2PO4, 140 mM NaCl, pH 7.4 (B. Braun). After dialysis, the sample was sterile filtered through a 0.2 μm dead-end filter. Purity was determined by SDS-PAGE, and concentration was measured by turbidimetry and 280 nm absorbance. The purified antibody was divided into equal parts and stored at -80 ° C. Once melted, the purified antibody was stored in equal parts at 4 ° C. Mass spectrometry was performed to identify the molecular weight of the heavy and light chains of the antibodies expressed by the hybridoma as described in Example 9.

[0807] Example 12

[0808] Antibody cross-competition studies using sandwich-ELISA

[0809] ELISA plate wells were coated with each anti-TF human monoclonal antibody diluted in PBS (0.5 or 2 μg / ml 100 μL / well) overnight at +4°C. The ELISA wells were washed with PBS, blocked with 2% (v / v) chicken serum (Gibco, Paisley, Scotland) dissolved in PBS for 1 hour at room temperature, and washed again with PBS. Subsequently, 50 μL of anti-TF human monoclonal antibody (10 μg / mL) was added, followed by 50 μL of TFECDHis (0.5 or 1 μg / ml) (generated at Genmab; Example 5) and incubated at RT for 1 hour (while shaking). The plate was washed 3 times with PBST (PBS + 0.05% Tween) and incubated with anti-his biotin BAM050 diluted 1:2000 at RT for 1 hour (while shaking). The plate was washed and incubated with streptavidin-poly HRP (Sanquin, Amsterdam, The Netherlands) at RT for 20 minutes and washed again. The reaction was further developed with ABTS (Roche Diagnostics) at RT in the dark and stopped after 15 min by adding 2% (w / v) oxalic acid and measuring the absorbance at 405 nm.

[0810] Table 2 shows that 3 cross-block groups (antibody groups that compete with each other for binding to TFECDHis) can be identified, where antibodies 013, 044 and 087-Lg63 belong to one cross-block group (group I), antibodies 011, 017-D12, 42, 092-A09 and 101 belong to another cross-block group (group II), and antibodies 003, 025, 109 and 111 belong to the third cross-block group (group III). Antibody 114 was found to compete with antibodies from cross-blocking groups II and III for binding to TFECDHis. The binding of antibody 098 to TFECDHis can be competed with antibodies from cross-blocking groups II and III.

[0811]

[0812] Table 2 – Competition of anti-TF antibodies for binding to TFECDHis.

[0813] White boxes indicate no competition for the binding of TFECDHis, light grey boxes indicate partial competition for the binding of TFECDHis, and dark grey boxes indicate competition for the binding of TFECDHis.

[0814] Embodiment 13

[0815] Binding of anti-TF human monoclonal antibody to TF extracellular domain in ELISA

[0816] The specificity of the anti-TF human mAbs obtained was evaluated by ELISA. ELISA plates (Microlon; Greiner Bio-One) were coated with 0.5 μg / mL TFECDHis in PBS, pH 7. at +4°C overnight. The coated ELISA plates were emptied and blocked with 2% (v / v) chicken serum (Gibco, Paisley, Scotland) in PBS for 1 hour at room temperature and washed with PBS containing 0.05% Tween 20 (PBST). Subsequently, serially diluted human mAbs in PBSTC (PBS supplemented with 2% (v / v) chicken serum and 0.05% (v / v) Tween-20) were incubated at RT for 1 hour under shaking (300 rpm). Bound human mAbs were detected by incubation with HRP-conjugated goat-anti-human IgG antibody (Jackson ImmunoResearch) diluted 1:5,000 in PBSTC for 1 hour under shaking (300 rpm). The reaction was further developed with ABTS (Roche Diagnostics) at RT in the dark and the reaction was terminated after 15-30 minutes by adding 2% (w / v) oxalic acid and then measuring the absorbance at 405 nm. Human monoclonal antibody-KLH (human monoclonal antibody against KLH (keyhole limpet hemocyanin)) was used as a negative control. Mouse anti-human TF (ERL) was used as a positive control (HRP-labeled anti-mouse IgG as a conjugate). Binding curves were analyzed using GraphPad Prism V4.03 software with nonlinear regression (variable slope sigmoidal dose-response).

[0817] like Figure 3 As can be seen, all anti-TFs bound to TFECDHis. 50 Values ​​are the average of 3 experiments and range from 0.09-0.46 nM (Table 3, see below).

[0818] Table 3:

[0819]

[0820] Embodiment 14

[0821] Binding of anti-TF human mAb to membrane-bound TF

[0822] Binding of anti-TF human mAbs to membrane-bound TF was determined by FACS analysis using TF-transfected CHO cells or TF-expressing tumor cell lines MDA-MB-231, (fluorescein-transfected) A431 and Bx-PC3.

[0823] The cells were suspended in PBS (2x10 6Cells / ml) were placed in a 96-well V-bottom plate (50 μl / well). 50 μl of human monoclonal antibodies serially diluted in FACS buffer (PBS with 0.1% BSA and 0.02% sodium azide) were added to the cells and incubated on ice for 30 minutes. After washing 3 times with FACS buffer, 50 μl of phycoerythrin (PE)-coupled goat-anti-human IgGFc (Jackson ImmunoResearch) (diluted 1:100 in FACS buffer) was added. After 30 minutes on ice (in the dark), the cells were washed 3 times and the specific binding of human monoclonal antibodies was detected by flow cytometry on a FACSCalibur (BD Biosciences). Human monoclonal antibody-KLH was used as a negative control. Mouse anti-TF and subsequent PE-coupled anti-mouse IgGFc were used as positive controls. Binding curves were analyzed with nonlinear regression (sigmoidal dose-response with variable slope) using GraphPad Prism V4.03 software (GraphPad Software, San Diego, CA, USA).

[0824] Figure 4 An example of a binding curve of a TF-specific human mAb to MDA-MB-231 cells is shown. Table 4 gives an overview of the EC50 values ​​for binding of TF-specific human mAbs to TF-transfected CHO cells (S1015-TF), MDA-MB-231, A431 and Bx-PC3 cells.

[0825]

[0826] Table 4 - Overview of EC50 and maximum mean fluorescence index (max MFI) values ​​for binding of TF-specific human mAbs to different cell types determined by FACS analysis.

[0827] EC50 values ​​are in nM. The maximum MFI was 30 μg / mL antibody for MDA-MB-231, BxPC3 and A431 cells and 7.5 μg / mL antibody for S1015-TF.

[0828] Embodiment 15

[0829] Inhibits the binding of FVIIa to TF

[0830] The inhibition of TF-human monoclonal antibody on the binding of FVIIa to TFECDHis was measured by ELISA. ELISA plates were coated with TFECDHis (0.5 μg / mL, 100 μl / well) overnight. The plates were emptied, blocked with PBS containing 2% (v / v) chicken serum (1 hour, RT), and emptied again. 4-fold serial dilutions of TF-human monoclonal antibody or human monoclonal antibody-KLH (negative control) were added to the wells, followed by the addition of FVIIa at an EC50 concentration (100 nM), and the plates were incubated at RT for 1 hour (while shaking, 300 rpm). The plates were washed and incubated with rabbit-anti-FVIIa (2.5 μg / mL; Abcam) as above. The plates were washed and incubated with pig-anti-rabbit IgG-HRP antibody (1:2,500; DAKO). After washing, the immune complexes were visualized using ABTS as a substrate. The reaction was terminated by adding 2% v / v oxalic acid, and the optical density at 405 nm was then measured with an ELISA reader. The antibody concentration required to obtain 50% inhibition (IC50) was calculated using GraphPad prism (non-linear regression analysis).

[0831] Figure 5 It was shown that antibodies from cross-blocking groups II and III efficiently inhibited the binding of FVIIa to TF, whereas antibodies from cross-blocking group I were unable to inhibit FVIIa binding (or inhibited to a much lower extent).

[0832] Table 5 shows the IC50 and maximum inhibition value (percentage) of the inhibition of FT-specific human monoclonal antibodies on the binding of FVIIa to TF.

[0833]

[0834] Table 5 - IC50 values ​​and maximum inhibition values ​​(percentage) of TF-specific human monoclonal antibodies against the binding of FVIIa to TF

[0835] Example 16

[0836] Inhibition of FVIIa-induced ERK phosphorylation

[0837] When coagulation factor VIIa (FVIIa) binds to TF, phosphorylation of mitogen-activated kinases (p42 and p44 MAPK or ERK1 and ERK2) is triggered. The squamous cell carcinoma cell line A431 expresses high levels of TF and induces optimal (3-5-fold) ERK phosphorylation (ERK-P) within 10 minutes after stimulation with FVIIa, as measured by the AlphaScreenSurefire ERK assay system (Perkin Elmer).

[0838] A431 cells (30,000 cells / well) were seeded in 96-well TC plates and cultured overnight (37°C, 5% CO2, 85% humidity) in serum-free medium (RPMI containing 20% ​​HSA and penicillin / streptomycin). The medium was then replaced with DMEM (without additives) and the cells were incubated for 1.5 hours. TF-human monoclonal antibody or human monoclonal antibody-KLH were added in a 3-fold serial dilution and the cells were incubated for 0.5 hours. Then, the cells were stimulated with FVIIa at an EC80 concentration (50nM; 10 minutes; 37°C, 5% CO2, 85% humidity). The cells were washed once with PBS and lysed with 25 μL lysis buffer (Perkin Elmer, Surefire kit). The lysate was centrifuged (3 minutes, 330xg, RT). 4 μL of supernatant was transferred to a 384-well Proxiplate (PerkinElmer). 7 μL of reaction buffer / activation buffer premix containing AlphaScreen beads (Perkin Elmer, Surefire kit) was added and the plate was incubated for 2 hours at RT in the dark. The plate was read using the "SurefirePlus" protocol of EnVision technology.

[0839] Figure 6 It was shown that antibody 013 did not inhibit FVIIa-induced ERK phosphorylation, 044 and 111 moderately inhibited ERK phosphorylation, and all other antibodies efficiently blocked ERK phosphorylation as measured by the AlphaScreen Surefire ERK Assay System.

[0840] Table 6 shows the IC50 values ​​and maximum inhibition values ​​(percentage) of TF-specific human mAbs on FVIIa-induced ERK phosphorylation, measured using the AlphaScreen Surefire ERK Assay System.

[0841]

[0842] Table 6 - IC50 values ​​and maximum inhibition values ​​(percentage) of TF-specific human monoclonal antibodies on FVIIa-induced ERK phosphorylation (measured using the AlphaScreen Surefire ERK assay system)

[0843] Results obtained in the AlphaScreen Surefire ERK assay were confirmed by Western blot analysis using HaCaT and BxPC3 cell lines. 30,000 cells / well were seeded in DMEM with minimal serum (starvation medium) and cultured overnight. Cells were further cultured in serum-free DMEM for 2 hours, and anti-TF antibody was added during the last 30 minutes of culture. Cells were stimulated with 0, 10 or 50 nM FVIIa for 10 minutes (37°C) and subsequently lysed in cell lysis buffer (50 μL lysis buffer per well, lysis for 30-60 minutes under shaking conditions, RT). Sample buffer containing 25 μL SDS was added to each sample. Samples were loaded onto SDS-PAGE gels, run, and blotted using standard western blot procedures. Blots were blocked with TBST 1x containing 5% irrelevant protein (ELK) for 1 hour at RT. Blots were incubated with rabbit anti-ERK-P antibody (O / N, 4°C). Blots were washed with TBST 1x and incubated with anti-rabbit IgG HRP (1 hr, RT), washed, developed with HRP substrate, and imaged using the Optigo Ultima imaging system (Isogen Life Sciences).

[0844] Figure 6 Results for a sub-panel of antibodies in BxPC3 cells are shown. ERK phosphorylation induced by 10 nM FVIIa was not inhibited by antibody 013, but was effectively inhibited by antibodies 111, 044 and 025 (the latter being an example of all other TF-specific human mAbs described here). More strongly induced ERK phosphorylation (50 nM FVIIa) was not inhibited by antibodies 013, 111 and 044, but was inhibited by antibody 025.

[0845] Embodiment 17

[0846] Inhibition of FVIIa-induced IL-8 release

[0847] The ability of TF-specific human monoclonal antibodies to inhibit FVIIa-induced IL-8 release was tested using MDA-MB-231 cells. Cells were seeded in 96-well plates (60,000 cells / well) and cultured in DMEM containing CS, sodium pyruvate, l-glutamine, MEM NEAA and penicillin / streptomycin (O / N, 37°C, 5% CO2). The tissue culture medium was removed, washed twice in serum-free, high calcium medium (DMEM containing penicillin / streptomycin), and cultured in this medium for another 105 minutes. Serial dilutions of antibodies were added and the cells were cultured for 15 minutes. FVIIa (Novo Nordisk; final concentration 10nM) was added and the cells were cultured for 5 hours. The supernatant was removed and centrifuged (300 xg, RT). The concentration of IL-8 in the supernatant was measured using an IL-8 ELISA kit according to the manufacturer's protocol (Sanquin).

[0848] Figure 7 It was shown that antibodies from cross-blocking groups II and III efficiently inhibited FVIIa-induced IL-8 release from MDA-MB-231 cells, except for antibody 111 from interaction group III. Antibodies from cross-blocking group I (013, 044 and 87-Lg6) were all unable to inhibit FVIIa-induced IL-8 release.

[0849] Table 7 shows the IC50 values ​​and maximum inhibition values ​​(percentage) of TF-specific human mAbs for inhibiting FVIIa-induced IL-8 release.

[0850]

[0851] Table 7 - IC50 values ​​and maximum inhibition values ​​(percentage) of TF-specific human mAbs for inhibiting FVIIa-induced IL-8 release.

[0852] Embodiment 18

[0853] Inhibits FXa production

[0854] The ability of TF-specific human mAbs to inhibit FXa generation was tested in the following assay system, in which the conversion of FX to FXa under the action of TF / FVIIa complex was measured with a colorimetric FXa-specific substrate. TF (Innovin) and serial dilutions of TF-specific human mAbs, positive controls (mouse anti-TF) and negative controls (human mAbs-KLH) were added to a flat-bottom 96-well plate (all diluted in Hepes buffer containing 3 mM CaCl2). The plate was incubated for 30 minutes at RT, and FVIIa (final concentration 1 nM) and FX (ERL; final concentration 200 nM) were added. The plate was incubated at 37°C for 30 minutes. 50 μl was transferred from each well to a 96-well plate containing stop buffer (5 mM EDTA dissolved in 100 ml Hepes buffer) (preheated, 37°C). FXa-specific substrate Chromogenix-2765 (Instrumation Laboratory Company) was added, the plate was incubated at 37°C for 60 minutes, and OD405 nm at 37°C was measured.

[0855] Figure 8 It was shown that antibody 017-D12 strongly inhibited FXa production, 013 showed moderate inhibition, and the other antibodies showed lower inhibition or no inhibition on FXa production.

[0856] Table 8 shows the IC50 values ​​and maximum inhibition values ​​(percentage) of TF-specific human mAbs for inhibiting FXa generation.

[0857] Grouping Human monoclonal antibody TF IC50 nM %Maximum Inhibition I 13 0.05 31 I 44 NA 3 I 87-Lg6 nt nt II 11 0.05 26 II 017-D12 0.28 84 II 42 nt nt II 092-A09 0.30 21 II 101 nt nt II / III 98 0.43 14 II / III 114 0.24 21 III 3 0.07 21 III 25 0.30 19 III 109 0.09 18 III 111 0.07 7

[0858] Table 8 - IC50 values ​​and maximum inhibition values ​​(percentage) of TF-specific human mAbs for inhibition of FXa production.

[0859] Embodiment 19

[0860] Inhibits blood coagulation

[0861] The inhibition of coagulation by TF-human monoclonal antibodies was measured using an assay system for determining TF-induced coagulation time. The following mixture was prepared in a 96-well plate: 17 μl 100 mM CaCl2 (final concentration 17 mM), 10 μl 1:100 innovin (final concentration 1:1000), 23 μl 1x HEPES buffer and 50 μl serially diluted antibodies. 50 μl of pooled human plasma was added to the wells of an Immulon 2B plate (ThermoElectron). 50 μl of the prepared antibody mixture was added to the Immulon 2b plate, and coagulation development was measured at 405 nm every 15 seconds for 25 minutes using a kinetic plate reader. The increase in optical density was plotted against time, and the coagulation time (t1 / 2) was calculated. The coagulation time was plotted against the antibody concentration. The IC50 for antibody-induced inhibition of coagulation was calculated from the results by nonlinear regression analysis using GraphPad Prism.

[0862] Fig. 9 It was shown that antibodies 044, 087 and 111 did not inhibit TF-induced coagulation, whereas all other antibodies did.

[0863] Table 9 shows the IC50 values ​​of TF-specific human mAbs for inhibiting coagulation.

[0864]

[0865] Table 9 - IC50 values ​​of TF-specific human monoclonal antibodies for inhibiting coagulation.

[0866] Embodiment 20

[0867] Antibody-dependent cell-mediated cytotoxicity

[0868] Target cell preparation

[0869] Collect target cells expressing TF (5x10 6 Bx-PC3 cells, MDA-MB-231 cells or A431 cells) were washed (2 times in PBS, 1500 rpm, 5 min), and collected in 1 ml of RPMI 1640 medium supplemented with cosmic calf serum, sodium pyruvate, L-glutamic acid, MEM NEAA and penicillin / streptomycin, and 100 μCi 51Cr (chromium-51; Amersham Biosciences Europe GmbH, Roosendaal, The Netherlands). The mixture was incubated in a shaking water bath at 37°C for 1 hour. After washing the cells (2 times in PBS, 1500 rpm, 5 min), the cells were resuspended in culture medium and viable cells were counted by trypan blue exclusion. The viable cell concentration was adjusted to 1×10 5 cells / ml.

[0870] Preparation of effector cells:

[0871] Peripheral blood mononuclear cells (PBMCs) were isolated from fresh buffy coats (Sanquin, Amsterdam, The Netherlands) by standard Ficoll density centrifugation according to the manufacturer's instructions (lymphocyte separation medium; Lonza, Verviers, France). After resuspending the cells in culture medium, the cells were counted by trypan blue exclusion and the concentration was adjusted to 1×10 7 cells / ml.

[0872] ADCC settings:

[0873] 50 μl 51 Cr-labeled target cells were transferred to microtiter wells and 50 μl of serially diluted antibodies (diluted in culture medium) were added. The cells were incubated (RT, 15 min) and 50 μl of effector cells were added to obtain an effector to target ratio of 100:1. To determine the maximum lysis level, 100 μl of 5% Triton-X100 was added instead of the effector cells; to determine the spontaneous lysis level, 100 μl of culture medium was added; to determine the antibody-independent lysis level, 50 μl of effector cells and 50 μl of culture medium were added. Subsequently, the cells were incubated overnight at 37°C, 5% CO2. After the cells were centrifuged (1200 rpm, 3 min), 75 μl of the supernatant was transferred to a MICRONIC tube. The released 51 Cr, such as the percentage of antibody-mediated lysis calculated according to the following formula:

[0874] ((cpm sample – cpm antibody-independent lysis) / (cpm maximum lysis – cpm spontaneous lysis)) x 100%

[0875] Where cpm is counts per minute.

[0876] Fig.10It was shown that all tested TF-human mAbs induced ADCC against lysis of Bx-PC3 cells, albeit with different potencies (EC50).

[0877] Table 10 shows the EC50 values ​​(nM) of ADCC of TF-specific human mAbs against different cell lines.

[0878]

[0879]

[0880] Table 10 - EC50 values ​​(nM) of ADCC of TF-specific human mAbs on different cell lines.

[0881] Embodiment 21

[0882] Complement deposition

[0883] The deposition of complement fragments C3c and C4c on target cells incubated with TF-human monoclonal antibodies was measured by FACS analysis. Target cells expressing TF (Bx-PC3 or MDA-MB-231 cells) were seeded in 96-well round-bottom plates (1x10e5 cells / well) in RPMI containing 1% BSA. Antibodies (30 μg / mL) were added and the cells were incubated at RT for 15 minutes. 25 μL of combined human serum was added as a complement source, and spontaneous complement binding was determined using heat-inactivated human serum. The cells were incubated at 37°C for 45 minutes. The cells were washed once and incubated with anti-human C3c FITC or anti-human C4c FITC (DAKO) in FACS buffer and incubated on ice for 30 minutes. The samples were analyzed with FACS Canto.

[0884] Fig.11 It was shown that antibodies from cross-block group I were unable to induce deposition of C3c or C4c on BxPC3 or MDA-MB-231 cells. All antibodies from cross-block group II tested induced deposition of C3c and C4c, as did antibodies from cross-block group III, with the exception of antibody 003.

[0885] Embodiment 22:

[0886] Affinity / affinity studies

[0887] Affinity determination

[0888] The binding of antibodies to TF was analyzed by surface plasmon resonance in BIAcore 3000 (GE Healthcare). TFECDHis was used for analysis. Human monoclonal antibodies (500 resonance units) were fixed on CM-5 sensor chips according to the experimental protocol recommended by the manufacturer. In brief, after surface activation with EDC and NHS, human monoclonal antibodies were injected onto the activated CM-5 surface, where the antibodies were placed in 10mM sodium acetate pH 4.0-5.5 at a speed of 5μl / min, followed by deactivation with 1M ethanolamine. TFECDHis of a series of concentrations dissolved in HBS-EP buffer was injected onto the fixed antibody at a flow rate of 30μl / min for 180 seconds. Human monoclonal antibody surface regeneration was performed by injection of 10mM glycine-HCl pH 2.0 or 10mM sodium acetate pH 3.0. Kinetic analysis was performed using double reference subtraction and model 1:1 (Langemuir) binding analysis.

[0889] Table 11 shows that for most human mAbs, the measured affinities are in the (sub) nanomolar range. However, kinetic parameters cannot be determined from all antibodies. 044 does have a high variation in the dissociation rate (kd) and has a high residual, which means that the curve fit is not good. The dissociation rates of 098, 111 and 087-Lg6 are too high to be measured by Biacore 3000.

[0890]

[0891] na cannot be evaluated => 10 -3 sec -1

[0892] Table 11. Kinetic constants for the reactivity of TF-human mAbs to TFECDHis - affinity measurements

[0893] Affinity determination

[0894] The determination of TF (TFECDHis) binding to TF-specific human monoclonal antibodies was essentially as described above, wherein TFECDHis was immobilized on a CM-5 sensor chip (300 resonance units) and a series of concentrations of human monoclonal antibodies were used for kinetic analysis. The kinetic analysis was performed using double reference subtraction and model 1:1 (Langmuir) binding analysis.

[0895] Table 12 shows the affinity measurements of antibodies 11, 98, 109 and 111. Although the affinity measurements of 98 and 111 showed high off rates (exceeding the assay limit of Biacore (ie >10 -3 )), and affinity measurements revealed interactions in the nanomolar range.

[0896] Grouping Human monoclonal antibody TF Affinity nM II 11 0.47 II / III 98 4.85 III 109 0.01 III 111 0.11

[0897] Table 12. Kinetic constants of TFECDHIS to TF-human mAb-affinity measurement reaction

[0898] Embodiment 23:

[0899] Immunohistochemical analysis of binding to normal human tissue and pancreatic tumors

[0900] The binding of TF-human mAb to various human tissues known to express TF (colon, heart, kidney, skin, lung and brain) was determined by immunohistochemistry (IHC).

[0901] IHC on frozen tissue

[0902] Frozen tissue sections (4-6 μm thick) were cut and fixed in acetone. Endogenous tissue peroxidase (PO) was blocked and tissue sections were pre-incubated with normal human serum to remove non-specific binding of the antibodies applied later to endogenous Fc receptors. Mouse antibodies against human TF (and negative control mouse antibodies) were applied to the tissue at optimal dilutions and subsequently detected with Powervision-PO (goat anti-mouse / -rabbit IgG)-PO. Human monoclonal antibodies specific for TF were coupled to Fab' goat anti-human IgG (Fc)-FITC and then applied to frozen tissue sections at 3 dilutions, including a predetermined optimal dilution. Subsequently, human monoclonal antibody-Fab-FITC complexes were detected by rabbit anti-FITC and Powervision-PO. PO activity was visualized using AEC as a substrate and cell nuclei were visualized with hematoxylin. Staining was analyzed by bright field microscopy.

[0903] IHC using mouse antibodies on formalin-fixed and paraffin-embedded (FFPE) tissues

[0904] FFPE tissue biopsies were sliced ​​at 4 μm, dewaxed, blocked for endogenous tissue peroxidase, and subjected to antigen retrieval (pH 6, citrate buffer). Tissue sections were pre-incubated in normal human serum before incubation with mouse antibodies to prevent nonspecific binding to endogenous Fc receptors. Mouse antibodies against human TF (and negative control mouse antibodies) were applied to tissue sections at optimal dilutions and subsequently detected with Powervision-PO (goat anti-mouse / -rabbit IgG)-PO. PO activity was visualized using AEC as a substrate and nuclei were visualized using hematoxylin. Staining was analyzed by bright field microscopy.

[0905] Fig.12 Examples of binding of antibodies 013 (positive staining), 011 (positive staining), 114 (positive staining) and 111 (intermediate staining) to glomeruli are shown. Antibodies 098 and 044 did not bind to glomeruli.

[0906] Table 13 gives an overview of the staining results of all TF-human mAbs in all tested tissues in human kidney.

[0907]

[0908] Table 13. IHC staining of human glomeruli

[0909] Table 14 shows the staining results of selected TF-specific human mAbs in human kidney, colon, heart, brain and skin, and human pancreatic tumors

[0910] Ab Human kidney Human colon Human Heart Human brain Human skin Pancreatic tumors 13 Nephron+ Basement membrane++ - + Epidermis+ +++ 114 Nephron++ Basement membrane++ - ++ Epidermis++ ++++ 11 Nephron+ Basement membrane++ - ++ Na(+) +++ 44 - Basement membrane+ - + / - na ++ 98 - Basement membrane+ - + / - Na(+) +++ 111 Nephron + / - Basement membrane+ - + na +++

[0911] Table 14. IHC staining of normal human tissues and pancreatic tumors

[0912] IHC analysis of TF-human monoclonal antibodies binding to human pancreatic tumors showed positive staining for all TF-human monoclonal antibodies ( Fig.13 ).

[0913] Embodiment 24:

[0914] Treatment of established MDA-MB-231 tumor xenografts in the mammary fat pad of SCID mice

[0915] The in vivo efficacy of TF-human mAb was determined in established orthotopic MDA-MB-231 xenograft tumors in SCID mice. 6Tumor cells in PBS were injected subcutaneously into the second mammary fat pad of female SCID mice, and then treatment with TF-human mAb or control mAb (human mAb-KLH) was started when the tumor size became measurable. Antibodies were injected on days 21 (260 μg / mouse), 28 (130 μg / mouse), and 42 (130 μg / mouse). Tumor volume was measured at least twice a week. Volume (mm) was calculated from caliper (PLEXX) measurements. 3 ): 0.52x(length)x(width) 2 .

[0916] Fig.14 It was shown that antibodies 114, 111, 013, 098, 011 and 044 were all able to effectively inhibit the growth of established orthotopic MDA-MB-231 tumors.

[0917] Embodiment 25:

[0918] Pilot repeat dosing of TF-specific human monoclonal antibodies in cynomolgus monkeys

[0919] To obtain preliminary information on the toxicology of the TF-specific human mAb, including an assessment of the ability of the antibody to interfere with the coagulation cascade and thus potentially increase the risk of bleeding in exposed animals, an experimental repeated-dose study was performed in cynomolgus monkeys.

[0920] Two male and two female cynomolgus monkeys (Macaca fascicularis), approximately 2 years old, received intravenous injections of antibody 011:

[0921] -Study Day 1: 0 mg / kg (vehicle only)

[0922] - Day 8: 1 mg / kg; 1 mL / minute

[0923] -Day 15: 10 mg / kg; 1 mL / minute

[0924] - Day 22: 100 mg / kg; 1 mL / minute

[0925] Animals were followed for 27 days, at which time they were euthanized for necropsy and organ histological evaluation.

[0926] The primary endpoints of the study were:

[0927] - Clinical observation: Determine the signs of bleeding from gums and eyes every day.

[0928] - Functional bleeding time and blood loss: measured on days 1, 8, 15 and 22 (1, 24 and 120 h after dosing) and at 2 pre-study time points.

[0929] - Blood / blood traces / blood clots: HE staining of all tissues (determined on tissues obtained at the final sacrifice)

[0930] - Blood in urine, feces, vomitus: Determine daily / weekly.

[0931] No apparent toxicity was observed with repeated or increased doses of antibody 011. Animals showed no clinical signs, no signs of cytokine release. In addition, there were no apparent clinical signs indicating impaired coagulation system or systemic bleeding. At the time point of 1 hour after administration, the average bleeding time on the 22nd day was significantly longer than that observed on the 1st day (p=0.012). There were no other statistically significant differences on the 8th, 15th and 22nd days compared to the 1st day. Moreover, it was found that there was no apparent toxicity to major organs and no adverse hematological effects. The preliminary conclusion of the histological evaluation of the tissue obtained from this study was that there were no histological findings attributable to the test substance treatment in the 4 treated animals.

[0932] Fig.15 Individual data points (duplicate samples) for each animal are shown as a function of time. Bleeding times were determined for 4 animals on days 1, 8, 15 and 22 (1, 24 and 120 h) and at two pre-trial time points.

[0933] Embodiment 26

[0934] Prophylactic and therapeutic treatment of BxPC3 tumor xenografts in SCID mice

[0935] The in vivo efficacy of TF-human mAb in the prophylactic and therapeutic treatment of BxPC3 cell xenografts in SCID mice was determined. 6 BxPC3 tumor cells placed in PBS were injected subcutaneously in female SCID mice and then treated with TF-human mAb or control mAb (human mAb-KLH). For preventive treatment, the antibody (400 μg / mouse) was injected intraperitoneally 1 hour after tumor induction. For therapeutic treatment, antibody injections (300 μg / mouse) were started 8 days after tumor induction, and then antibody injections (150 μg / mouse) were performed weekly. Tumor volume was measured at least twice a week. Volume (mm 3 ) Calculated from caliper (PLEXX) measurement: 0.52x(length)x(width) 2 .

[0936] Fig.16 The results showed that TF-specific human mAbs can preventively and therapeutically treat BxPC3 xenograft tumors.

[0937] Embodiment 27

[0938] DNA shuffling between mouse and human TF to identify domains important for binding of anti-TF human mAbs

[0939] To determine the domains important for binding of anti-TF human mAbs to human TF, DNA shuffling was performed between human and mouse TF. Shuffled constructs were prepared from DNA encoding human TF by replacing human domains with mouse domains, and shuffled constructs were prepared from DNA encoding mouse TF by replacing mouse domains with human domains. If a domain in human TF is important for binding of anti-TF human mAbs, binding will be lost when replaced with mouse domains. Human and mouse TF have 57% homology at the protein level. Fig.17 Human TF constructs containing murine TF domains (TFhs, containing TFmm domains), and murine TF constructs containing human TF domains are shown. HEK293F cells were transiently transfected with the constructs or vector alone (pcDNA3.3SP; mock). FACS analysis was performed essentially as described above, ...

Claims

1. A human antibody that binds to human tissue factor.

2. The antibody of claim 1, wherein the antibody has an apparent affinity (EC) of 3 nM or less, such as 0.50 nM or less, such as 0.35 nM or less, such as 0.20 nM or less, such as 0.1 nM or less, when measured using the assay method described in Example 13. 50 ) binds to the extracellular domain of tissue factor.

3. An antibody according to any one of the preceding claims, wherein the antibody binds to mammalian cells expressing tissue factor, such as A431 cells transfected with a construct encoding tissue factor, preferably with an apparent affinity (EC) of 0.1% when measured using the assay described in Example 14. 50 ) is 10 nM or lower, such as 8 nM or lower, such as 5 nM or lower, such as 2 nM or lower, such as 1 nM or lower, such as 0.5 nM or lower, such as 0.3 nM or lower.

4. The antibody of any one of the preceding claims, wherein the antibody is capable of inducing antibody-dependent cellular cytotoxicity in A431 cells, preferably, when measured using the assay method described in Example 20, its EC 50 The value is 2 nM or less, such as 1 nM or less, such as 0.7 nM or less, or 0.3 nM or less, such as 0.20 nM or less, or 0.1 nM or less, or 0.05 nM or less.

5. The antibody of any of the preceding claims, wherein the antibody is effective in inhibiting the growth of established MDA-MB-231 tumors when measured by the method described in Example 24 and / or is effective in inhibiting the growth of established BxPC3 tumors when measured by the method described in Example 26.

6. The antibody of any of the preceding claims, wherein the antibody inhibits tissue factor-induced coagulation, preferably with a median inhibitory concentration of less than 10 nM, such as less than 5 nM, such as less than 2 nM, such as less than 1 nM, when determined using the assay method described in Example 19.

7. The antibody of any of the preceding claims, wherein the antibody inhibits the binding of FVIIa to tissue factor, preferably with a maximum inhibition value of greater than 80%, such as greater than 90%, when measured using the assay described in Example 15.

8. An antibody according to any one of the preceding claims, wherein the antibody inhibits FVIIa-induced IL-8 release from MDA-MB-231 cells, preferably to a maximum inhibition value of greater than 40%, such as greater than 50%, such as greater than 60%, when measured using the assay method described in Example 17.

9. The antibody of any of the preceding claims, wherein the antibody inhibits the conversion of FX to FXa by the TF / FVIIa complex, preferably by less than 50%, such as less than 40%, such as in the range of 1-30%, when measured using the assay described in Example 18.

10. The antibody of any one of the preceding claims, wherein the antibody competes for tissue factor binding with an antibody comprising a VH region comprising the sequence of SEQ ID NO:9 and a VL region comprising the sequence of SEQ ID NO:65.

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