Antibody for factor xa inhibitor and method for using the same

Abstract

To provide an antibody for factor xa inhibitor and method for using the same.SOLUTION: One of the major limitations of anticoagulant therapy is the limitation of the ability to rapidly reverse anticoagulant activity in the case of treatment-related bleeding risk and in cases of overdosage or required emergency surgical intervention. Therefore, a specific effective antidote for all forms of anticoagulant therapy is highly desirable. The present invention relates to an antidote to an anticoagulant that targets factor Xa. The antidotes are factor Xa protein derivatives that bind to the factor Xa inhibitors thereby substantially neutralizing them but do not assemble into the prothrombinase complex. The derivatives described herein lack or have reduced intrinsic coagulant activity. Disclosed herein are methods of stopping or preventing bleeding in a patient that is currently undergoing anticoagulant therapy with a factor Xa inhibitor.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under U.S.C. § 119(e) of U.S. Provisional Patent Application No. 60 / 976,343, filed September 28, 2007, and U.S. Provisional Patent Application No. 61 / 090,574, filed August 20, 2008. Both U.S. Provisional Patent Application Nos. 60 / 976,343 and 61 / 090,574 are incorporated by reference in their entireties.

[0002] The present invention relates to the use of factor Xa (fXa) derivatives that have reduced or lacking intrinsic procoagulant activity, but are also capable of binding and / or neutralizing fXa inhibitors, thereby acting as antidotes to anticoagulants that target fXa. [Background technology]

[0003] Anticoagulants meet a market demand for treating or preventing unwanted thrombosis in patients prone to forming blood clots, such as those suffering from coagulation disorders, those under a period of immobilization or those undergoing medical surgery. However, one of the major limitations of anticoagulant therapy is the risk of bleeding associated with the treatment and the limited ability to rapidly reverse anticoagulant activity in cases of overdosage or when emergency surgical procedures are required. Therefore, specific and effective antidotes for all forms of anticoagulant therapy are highly desirable. For safety considerations, it is also advantageous to have anticoagulant-antidote pairs when developing new anticoagulants.

[0004] The currently available anticoagulant-antidote pairs for over-anticoagulation are heparin-protamine and warfarin-vitamin K. Fresh frozen plasma and recombinant factor VIIa (rfVIIa) have also been used as non-specific antidotes in patients under low molecular weight heparin therapy, in patients suffering from major trauma or severe bleeding (Non-Patent Document 1). Also, protamine fragments (U.S. Pat. No. 6,624,141) and small synthetic peptides (U.S. Pat. No. 6,200,955) have been reported as antidotes for heparin or low molecular weight heparin; and thrombin muteins (U.S. Pat. No. 6,060,300) have been reported as antidotes for thrombin inhibitors. Intermediates and derivatives of prothrombin have been reported as antidotes for hirudin and synthetic thrombin inhibitors (U.S. Pat. Nos. 5,817,309 and 6,086,871).

[0005] One promising form of anticoagulant therapy targets factor Xa (fXa), and indeed, several direct fXa inhibitors are currently in various stages of clinical development for use in anticoagulant therapy. Many of these are small molecules. Although these new fXa inhibitors show therapeutic promise, there remains a need for specific and effective antidotes. When surgery is required in patients who are over-anticoagulated or treated with these fXa inhibitors, agents may be required to substantially neutralize the administered fXa inhibitor or inhibitors and restore normal hemostasis.

[0006] Currently available agents such as recombinant factor VIIa (rfVIIa) are mechanistically limited and not specific for reversing fXa inhibitors, and thus improved options for clinicians are highly desirable. In human studies, rfVIIa has been used to reverse the effects of indirect antithrombin III-dependent fXa inhibitors such as fondaparinux and idraparinux (Non-Patent Document 2; Non-Patent Document 3). The mechanism of action of factor VIIa (fVIIa) is to interact with tissue factor to convert factor X (fX) present in the blood circulation to fXa in order to restore normal hemostasis in patients. This mode of action necessarily dictates that the highest potential concentration of fXa that can be achieved to neutralize active site-specific fXa inhibitors is limited by the circulating plasma concentration of fX. Thus, the potential of using rfVIIa to reverse the effects of direct fXa inhibitors is mechanistically limited. Since the circulating plasma concentration of fX is 150 nanomolar ("nM"), the maximum amount of fXa produced by this mode would be 150 nM. Thus, the potential of using rfVIIa to reverse the effects of direct fXa inhibitors is mechanistically limited. The reported therapeutic concentrations of small molecule fXa inhibitors such as rivaroxaban are higher than the potential amount of fXa produced by rfVIIa (approximately 600 nM, Non-Patent Document 4). Thus, the use of rfVIIa to reverse therapeutic or supratherapeutic levels of anticoagulation by fXa inhibitors results in an insufficient level of efficacy. As shown in Figure 4, the use of rfVIIa had only limited effect in neutralizing the anticoagulant activity of the factor Xa inhibitor betrixaban (described below). Recombinant fVIIa showed dose-responsive detoxification activity at 50-100 nM, but the effect plateaued at 100-200 nM, indicating that its detoxification effect is limited by factors other than its concentration. At all rfVIIa concentrations tested, betrixaban still showed dose-responsive inhibition of fXa, with inhibition of up to 75% at a concentration of 250 nM. This observation is consistent with the proposed mechanism of action of fVIIa.This is also supported by studies showing that rfVIIa did not completely reverse the inhibitory effects of fondaparinux on parameters of thrombin generation and prothrombin activation (Non-Patent Document 5).

[0007] Exogenous active fXa cannot be administered directly to a subject in the same manner as rfVIIa. Unlike rfVIIa, which has very low procoagulant activity in the absence of its cofactor tissue factor, native fXa is a potent enzyme and carries the potential risk of causing thrombosis. Thus, there are disadvantages to using either rfVIIa or active fXa as an antidote to fXa anticoagulant therapy.

[0008] Thus, there is a need for improved antidotes that do not cause undesirable thrombosis and that are effective to substantially neutralize the anticoagulant activity of fXa inhibitors in cases of fXa inhibitor overdose or when normal hemostasis needs to be restored to prevent or stop bleeding.

[0009] Any and all publications, patents, and patent applications mentioned herein are hereby incorporated by reference in their entirety. [Prior art documents] [Non-patent literature]

[0010] [Non-Patent Document 1] Lauritzen, B. et al., Blood, 2005, pp. 607A-608A. [Non-Patent Document 2] Bijsterveld, NR et al., Circulation, 2002, 106:2550-2554 [Non-Patent Document 3] Bijsterveld, NR et al., British Journal of Haematology, 2004(124):653-658. [Non-Patent Document 4] Kubitza D et al., Eur. J. Clin. Pharmacol. 2005, 61:873-880 [Non-Patent Document 5] Gerotiafas, GT et al., Thrombosis & Haemostasis, 2204(91):531-537 Summary of the Invention [Problem to be solved by the invention]

[0011] It has now been discovered that administering modified derivatives of fXa protein is useful as an antidote to anticoagulants that target fXa. The modified derivatives of fXa protein do not compete with fXa to assemble and form the prothrombinase complex, but instead bind and / or substantially neutralize anticoagulants such as fXa inhibitors. Derivatives useful as antidotes are modified to retain the ability to bind inhibitors while reducing or eliminating intrinsic procoagulant and anticoagulant activity. It is contemplated that the derivatives of the present invention include active site modifications, or altering or removing the entire Gla domain from fXa, or various combinations thereof. Furthermore, since active site modified full-length fXa is known to be an anticoagulant, it is contemplated that modification of the Gla domain reduces or eliminates the anticoagulant effect of fXa derivatives on normal hemostasis.

[0012] It is further contemplated that modification of the EGF domain of fXa (either by deletion or substitution of the EGF1, EGF2, or both EGF1 and EGF2 domains) will result in derivatives useful in the methods of the invention. Modification of the EGF domain may occur alone or in addition to modification of the Gla domain.

[0013] In one embodiment, the derivative maintains the necessary structural features to bind to an anticoagulant that targets fXa (or a fXa inhibitor), such that the derivative binds, directly or indirectly, to the inhibitor, thereby substantially neutralizing the inhibitor. [Means for solving the problem]

[0014] In one aspect, the present invention provides a method for preventing or reducing bleeding in a subject undergoing anticoagulant therapy with a factor Xa inhibitor, comprising administering to the subject an effective amount of a factor Xa protein derivative that binds to the factor Xa inhibitor but does not assemble to form a prothrombinase complex. In one embodiment, the derivative has one or more of the following properties: reduced or no procoagulant activity; a modified or removed Gla domain; and a modified active site. The derivative of the present invention selectively binds to and inhibits exogenously administered factor Xa inhibitors, thereby substantially neutralizing the anticoagulant activity of the fXa inhibitor. Both in vitro and in vivo methods are contemplated in this method. Various additional modifications to the factor Xa protein contemplated by the present invention can be found throughout the detailed description.

[0015] It is to be understood that the derivatives contemplated by the present invention are not plasma-derived Factor VIIa, recombinant Factor VIIa, fresh frozen plasma, prothrombin complex concentrate, or whole blood.

[0016] One aspect of the invention is the use of factor Xa derivatives and compositions containing same to treat patients who have undergone or are undergoing over-anticoagulation therapy with factor Xa inhibitors, or who have previously been administered a factor Xa inhibitor and subsequently require hemostasis, such as may be required by elective or emergency surgery. In one aspect, the modified fXa protein is distinguished from naturally occurring fXa in that it has reduced or eliminated intrinsic procoagulant activity and does not interfere with the function of physiological fXa in hemostasis, while still being able to bind and substantially neutralize fXa inhibitors.

[0017] In another embodiment, the modified factor Xa protein is co-administered with an agent capable of extending the plasma half-life (or circulating half-life) of the factor Xa derivative. In yet another embodiment, the antidote is conjugated to a moiety to extend its plasma half-life.

[0018] Also provided is a pharmaceutical composition containing a factor Xa derivative that binds to (and / or substantially neutralizes) a factor Xa inhibitor but does not assemble to form a prothrombinase complex. The pharmaceutical composition optionally includes a pharma- ceutical acceptable carrier.

[0019] In another aspect, the invention provides a kit comprising a fXa inhibitor for use in anticoagulation and a fXa inhibitor antidote (or factor Xa derivative) for use when substantial neutralization of the anticoagulant activity of the fXa inhibitor is required.

[0020] One embodiment of the present invention is directed to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 12, or a polypeptide having at least 80% homology to SEQ ID NO: 12. Another embodiment is directed to an isolated double-chain polypeptide comprising the amino acid sequence of SEQ ID NO: 13, or a polypeptide having at least 80% homology to SEQ ID NO: 13. Yet another embodiment is directed to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 15, or a polypeptide having at least 80% homology to SEQ ID NO: 15.

[0021] Also provided is a pharmaceutical composition comprising a carrier and a polypeptide as described immediately above.

[0022] Also provided are polynucleotides encoding the immediately above described polypeptides.

[0023] Further provided herein is a peptide conjugate comprising a carrier covalently or non-covalently bound to the immediately preceding polypeptide, The carrier may be a liposome, a micelle, a pharma- ceutically acceptable polymer, or a pharma-ceutically acceptable carrier.

[0024] The present invention is also directed to an antibody that binds to the immediately preceding polypeptide. The antibody may be a polyclonal, monoclonal, chimeric or humanized antibody. Biologically active fragments of the antibody are also provided. The antibody may be detectably labeled. The antibody (or antibody fragment) is also provided as part of a composition that further comprises a carrier.

[0025] In one embodiment, an antibody-peptide complex is provided that comprises an antibody of the invention and a polypeptide that specifically binds to the antibody. The polypeptide is the polypeptide against which the antibody was raised. The antibody can be a polyclonal antibody, a monoclonal antibody, a chimeric antibody, or a humanized antibody.

[0026] In another embodiment, a method for preparing the polypeptide of the present invention is provided, comprising expressing a polynucleotide encoding the polypeptide in a prokaryotic or eukaryotic host cell. In one embodiment, the host cell is a eukaryotic cell, in particular a Chinese Hamster Ovary cell. In one embodiment, the heavy chain (SEQ ID NO: 15) is expressed in a prokaryotic cell, such as E. coli. In another embodiment, the polypeptide is isolated.

[0027] In yet another embodiment, an isolated prokaryotic or eukaryotic host cell is provided comprising a polynucleotide encoding a polypeptide of the invention. In one embodiment, the host cell is in a composition further comprising a carrier.

[0028] In yet another embodiment, there is provided a method for preventing or reducing bleeding in a subject undergoing anticoagulant therapy with a factor Xa inhibitor, comprising administering to the subject an effective amount of a composition comprising a polypeptide of the invention and a carrier. Also provided is a method for selectively binding to and inhibiting an exogenously administered factor Xa inhibitor in a subject undergoing anticoagulant therapy, comprising administering to the subject an effective amount of a composition as described immediately above. In a preferred embodiment of the invention, for example, the following is provided: (Item 1) A method for preventing or reducing bleeding in a subject undergoing anticoagulant therapy with a factor Xa inhibitor, comprising administering to the subject an effective amount of a factor Xa protein derivative that binds to the factor Xa inhibitor and does not assemble to form a prothrombinase complex. (Item 2) A method for selectively binding to and inhibiting an exogenously administered factor Xa inhibitor in a subject, comprising administering to the subject an effective amount of an Xa protein derivative that binds to the factor Xa inhibitor and does not assemble to form a prothrombinase complex. (Item 3) 3. The method according to claim 1 or 2, wherein the factor Xa protein derivative is directly or indirectly bound to the factor Xa inhibitor. (Item 4) 4. The method of claim 3, wherein the derivative has reduced or no procoagulant activity. (Item 5) 5. The method according to any of items 1 to 4, wherein the derivative is a Gla-deficient factor Xa protein derivative or a des-Gla factor Xa protein. (Item 6) 6. The method according to any of items 1 to 5, wherein the derivative has a modified active site. (Item 7) 7. The method according to any of items 1 to 6, wherein the derivative comprises at least amino acid residues 46 to 448 of SEQ ID NO: 3 or an equivalent thereof. (Item 8) 8. The method according to any of items 1 to 7, wherein the derivative comprises at least amino acid residues 46 to 139 and 195 to 448 of SEQ ID NO: 3 or an equivalent thereof. (Item 9) 9. The method according to any of items 1 to 8, wherein the derivative lacks the light chain of fXa and contains the serine protease catalytic domain present in the heavy chain. (Item 10) 10. The method according to any of items 1 to 9, wherein the derivative is a polypeptide comprising the amino acid sequence of SEQ ID NO:5. (Item 11) The derivative comprises a catalytic domain that lacks proteolytic activity but is optionally chemically or recombinantly modified to maintain structural features required for binding, the domain comprising: a. a mammalian protease selected from the group consisting of plasma kallikrein, thrombin, and trypsin; or B bacterial protease subtilisin 11. The method according to any of items 1 to 10, (Item 12) 12. The method according to any of items 1 to 11, wherein the derivative comprises the amino acid sequence of SEQ ID NO: 7 or an equivalent thereof having a modified light chain. (Item 13) 13. The method according to any of items 1 to 12, wherein the derivative comprises a light chain modified to reduce phospholipid membrane binding. (Item 14) 14. The method according to any of items 1 to 13, wherein the modified light chain comprises at least one amino acid substitution, addition or deletion in the Gla-domain compared to the Gla-domain of a wild-type human factor Xa protein. (Item 15) 15. The method according to any of items 1 to 14, wherein the derivative is selected from the group consisting of uncarboxylated, undercarboxylated, and decarboxylated factor Xa proteins. (Item 16) 16. The method according to any of items 1 to 15, wherein the derivative comprises the heavy chain of SEQ ID NO: 7 or an equivalent thereof which has been modified. (Item 17) 17. The method according to any of items 1 to 16, wherein the derivative is modified by at least one amino acid substitution of an amino acid selected from the group consisting of Glu216, Glu218, Arg332, Arg347, Lys351, and Ser379. (Item 18) 18. The method of any of items 1 to 17, wherein Ser379 is modified to dehydro-alanine or alanine. (Item 19) 19. The method according to any of items 1 to 18, wherein the derivative is des-Gla anhydro-fXa having the amino acid sequence of SEQ ID NO: 10 or an equivalent thereof. (Item 20) 20. The method according to any of items 1 to 19, wherein the derivative is des-Gla fXa-S379A having the amino acid sequence of SEQ ID NO: 11 or an equivalent thereof. (Item 21) 21. The method according to any of items 1 to 20, wherein the derivative has a reduced interaction with ATIII, cofactors fV / fVa and / or fVIII / fVIIIa and comprises the amino acid sequence of SEQ ID NO: 7 or an equivalent thereof. (Item 22) 22. The method according to any of items 1 to 21, wherein the derivative comprises the amino acid sequence of SEQ ID NO: 12 or 13 or 15 or a polypeptide having at least 80% homology to SEQ ID NO: 12 or 13 or 15. (Item 23) 23. The method according to any of items 1 to 22, wherein the derivative has at least one amino acid substitution at amino acid position Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424. (Item 24) 24. The method according to any of items 1 to 23, wherein the derivative has a modification in the EGF domain, the modification being selected from the group consisting of a deletion of the EGF1 domain, a deletion of the EGF2 domain, and a deletion of both the EGF1 and EGF2 domains. (Item 25) 25. The method of any of items 1 to 24, wherein the derivative is conjugated to a moiety capable of increasing the circulatory half-life of the derivative. (Item 26) 26. The method of claim 25, wherein the moiety is selected from the group consisting of polyethylene glycol, acyl groups, liposomes, encapsulating agents, carrier proteins, artificial phospholipid membranes, nanoparticles, Fc peptide fragments, Fc carrier domains, and chimeric proteins comprising fXa derivatives, and combinations thereof. (Item 27) 27. The method of any of items 1 to 26, further comprising administering an effective amount of an agent that increases the circulating half-life of the derivative. (Item 28) 28. The method of claim 27, wherein the agent is an anti-fXa antibody that specifically recognizes and binds to the non-active site of fXa or a fXa derivative bound to alpha-2-macroglobulin. (Item 29) 29. The method of any of items 1 to 28, wherein the Factor Xa inhibitor is selected from the group consisting of fondaparinux, idraparinux, biotinylated idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor, DX-9065a, YM-60828, YM-150, apixaban, rivaroxaban, PD-348292, otamixaban, DU-176b, LY517717, GSK913893, razaxaban, low molecular weight heparin, betrixaban or a pharma- ceutically acceptable salt thereof, and combinations thereof. (Item 30) 30. The method of claim 29, wherein the factor Xa inhibitor is selected from betrixaban, ribalaxaban, apixaban, low molecular weight heparin, and combinations thereof. (Item 31) 31. The method according to any of items 1 to 30, wherein the subject is experiencing a clinical major bleeding event selected from the group consisting of hemorrhage, hemorrhage into a vital organ, hemorrhage requiring reoperation or a new therapeutic procedure, and a bleeding index of 2.0 or greater with associated overt hemorrhage. (Item 32) 32. The method of any of items 1 to 31, wherein the subject is experiencing a bleeding event selected from the group consisting of persistent or recurrent nosebleeds, rectal or urinary tract bleeding not requiring a therapeutic procedure, substantial injection site hematoma, spontaneous non-injection site hematoma, hematoma occurring following minor trauma, substantial blood loss, and bleeding requiring unscheduled blood transfusion. (Item 33) 33. The method according to any of items 1 to 32, wherein the derivative is administered preoperatively. (Item 34) A pharmaceutical composition comprising a factor Xa derivative protein which binds to a factor Xa inhibitor and does not assemble to form a prothrombinase complex, and a pharma- ceutically acceptable carrier. (Item 35) An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:12, or a polypeptide having at least 80% homology to SEQ ID NO:12. (Item 36) An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:12. (Item 37) An isolated double-chain polypeptide comprising the amino acid sequence of SEQ ID NO:13 or a polypeptide having at least 80% homology to SEQ ID NO:13. (Item 38) An isolated polypeptide comprising the amino acid sequence of SEQ ID NO:15 or a polypeptide having at least 80% homology to SEQ ID NO:15. (Item 39) 39. A peptide conjugate comprising a carrier covalently or non-covalently bound to the polypeptide according to any one of items 35 to 38. (Item 40) 40. The peptide conjugate according to item 39, wherein the carrier is a liposome, a micelle, a pharma- ceutically acceptable polymer, or a pharma- ceutically acceptable carrier. (Item 41) A pharmaceutical composition comprising a carrier and a polypeptide according to any one of items 35 to 38. (Item 42) 39. A polynucleotide encoding the polypeptide of any one of items 35 to 38. (Item 43) 39. An antibody that binds to the polypeptide of any one of items 35 to 38. (Item 44) 44. The antibody of item 43, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanized antibody. (Item 45) A biologically active fragment of the antibody according to item 43. (Item 46) 44. The antibody of item 43, which is detectably labeled. (Item 47) 44. A composition comprising the antibody of item 43 and a carrier. (Item 48) A composition comprising the antibody fragment of item 45. (Item 49) 47. An antibody-peptide complex comprising the antibody of item 46 and a polypeptide that specifically binds to the antibody. (Item 50) 50. The complex of item 49, wherein the polypeptide is a polypeptide against which an antibody has been raised. (Item 51) 50. The conjugate of item 49, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a chimeric antibody or a humanized antibody. (Item 52) A method for preparing a polypeptide according to any one of items 35 to 38, comprising expressing a polynucleotide encoding the polypeptide according to any one of items 35 to 38 in a prokaryotic or eukaryotic host cell. (Item 53) 53. The method of claim 52, wherein the polypeptide comprises the amino acid sequence of SEQ ID NO: 12 and the polynucleotide comprises the nucleotide sequence of SEQ ID NO: 16. (Item 54) 53. The method of claim 52, wherein the host cell is a Chinese hamster ovary cell. (Item 55) 55. The method of any one of items 52 to 54, further comprising isolating the polypeptide. (Item 56) 39. An isolated prokaryotic or eukaryotic host cell comprising a polynucleotide encoding a polypeptide according to any one of items 35 to 38. (Item 57) A composition comprising a carrier and a prokaryotic or eukaryotic host cell according to item 56. (Item 58) 42. A method for preventing or reducing bleeding in a subject undergoing anticoagulant therapy with a factor Xa inhibitor, comprising administering to the subject an effective amount of the composition according to item 41. (Item 59) 42. A method for selectively binding to and inhibiting an exogenously administered factor Xa inhibitor in a subject undergoing anticoagulant therapy with a factor Xa inhibitor, comprising administering to the subject an effective amount of the composition according to item 41. (Item 60) 60. The method of item 58 or 59, wherein the Factor Xa inhibitor is selected from the group consisting of fondaparinux, idraparinux, biotinylated idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor, DX-9065a, YM-60828, YM-150, apixaban, rivaroxaban, PD-348292, otamixaban, DU-176b, LY517717, GSK913893, razaxaban, low molecular weight heparin, betrixaban or a pharma- ceutically acceptable salt thereof, and combinations thereof. (Item 61) 61. The method of claim 60, wherein the factor Xa inhibitor is selected from betrixaban, ribaraxaban, apixaban, low molecular weight heparin, and combinations thereof. (Item 62) 61. The method according to item 60, wherein the derivative is administered preoperatively. [Brief description of the drawings]

[0029] [Figure 1]FIG. 1 is a schematic diagram showing the domain structure of human factor X (SEQ ID NO: 1) shown in Table 1, as reported in Leytus et al., Biochem., 1986, vol. 25, pp. 5098-5102. SEQ ID NO: 1 is the amino acid sequence of human fX encoded by the nucleotide sequence of human fX shown in Table 2 (SEQ ID NO: 2), which is known in the prior art. For example, the translated amino acid sequence is reported in Leytus et al., Biochem., 1986, vol. 25, pp. 5098-5102,<http: / / www.ncbi.nlm.nih.gov / entrez / viewer.fcgi?db=nuccore&id=89142731> and can be found in GenBank under "NM_000504." The numbering of the amino acids in this sequence is based on the fX sequence. The human fX precursor (SEQ ID NO:1) contains a prepro leader sequence (amino acids 1-40 of SEQ ID NO:1), followed by a sequence corresponding to the fX light chain (LC) (amino acids 41-179 of SEQ ID NO:1), the RKR triplet (amino acids 180-182 of SEQ ID NO:1) that is removed during secretion of fX, and the fX heavy chain (amino acids 183-488 of SEQ ID NO:1) that contains the activation peptide (AP) (amino acids 183-234 of SEQ ID NO:1), as well as the catalytic domain (amino acids 235-488 of SEQ ID NO:1). [Diagram 2]FIG. 3 shows the amino acid sequence of mature human factor X (SEQ ID NO: 3). The numbering of the amino acids in this figure is based on the mature fX sequence starting from the N-terminus of the fX light chain. Factor X circulates in plasma as a two-chain molecule linked by disulfide bonds. The light chain (LC) has 139 amino acid (amino acids 41-179 of SEQ ID NO: 1) residues and contains a gamma-carboxyglutamic acid (Gla)-rich domain (amino acids 1-45 of SEQ ID NO: 3) with a short aromatic stack (AS) (amino acids 40-45 of SEQ ID NO: 3), followed by two epidermal growth factor (EGF)-like domains (EGF1: amino acids 46-84 of SEQ ID NO: 3, EGF2: amino acids 85-128 of SEQ ID NO: 3). The heavy chain (HC) has 306 amino acids and contains a 52 amino acid activation peptide (AP: amino acids 143-194 of SEQ ID NO: 3), followed by a catalytic domain (amino acids 195-448 of SEQ ID NO: 3). The catalytic triad corresponds to H57-D102-S195 in the chymotrypsin numbering and is located at His236, Asp282, and Ser379 in the fX sequence and is underlined (amino acids 236, 282, and 379 of SEQ ID NO:3). [Diagram 3] FIG. 3 is a schematic diagram showing the domain structure of mature human factor X shown in FIG. The amino acid numbering in this diagram is based on the mature fX sequence. The cleavage sites for chymotrypsin digestion to remove the Gla-domain containing fragment (amino acids 1-44 of SEQ ID NO:3) and fX activation to remove the activation peptide are highlighted. Chymotrypsin digestion of fXa results in Gla-domain-free fXa, which lacks amino acid residues 1-44 (SEQ ID NO:4). [Figure 4] FIG. 1 shows the effect of various concentrations of rfVIIa in the presence of tissue factor on the anticoagulant activity of the fXa inhibitor betrixaban on thrombin generation (described below), expressed as a relative fluorescence unit (RFU) assay (described in Example 2). The data show that the combination of rfVIIa and tissue factor was not able to completely neutralize the anticoagulant activity of the fXa inhibitor betrixaban at concentrations up to 200 nM. [Diagram 5]FIG. 1 shows that, compared to active fXa, anhydro-fXa with its Gla-domain intact reverses fXa inhibition by betrixaban in a purified system containing active fXa and betrixaban (open circles), while anhydro-fXa alone has negligible procoagulant activity (open triangles). FXa chromogenic activity was normalized to active fXa in the absence of any inhibitor (open squares). This is described in more detail in Example 2. The data show that anhydro-fXa is inactive towards fXa substrates, but retains the ability to bind fXa inhibitors. [Figure 6] 5 shows that anhydro-fXa with an intact Gla domain is a potent inhibitor in the plasma thrombin generation (expressed as relative fluorescence units (RFU)) assay (described in Example 2). It almost completely inhibited thrombin generation at about 115 nM. The data indicate that anhydro-fXa with an unmodified Gla-domain is not suitable for use as a fXa inhibitor antidote. [Figure 7] Figure 1 shows a comparison of the clotting activity of active fXa in a 96-well plate format before chymotrypsin digestion and after 15 and 30 minutes of chymotrypsin digestion. As shown in this figure, the clotting time (change in OD405) was significantly delayed after 15 minutes of digestion of fXa with chymotrypsin, and no clotting was observed by 20 minutes when fXa was digested for 30 minutes. This result was also used to establish conditions for chymotrypsin digestion of anhydro-fXa, since it has no activity that can be monitored during digestion. This is described more fully in Example 3. [Figure 8] FIG. 1 shows the binding affinity of des-Gla anhydro-fXa with the factor Xa inhibitor betrixaban, as described in Example 4. The data show that des-Gla anhydro-fXa, prepared by chymotrypsin digestion of anhydro-fXa to remove the Gla-domain-containing fragment (residues 1-44), can bind betrixaban with an affinity similar to that of native fXa (fXa: Ki=0.12 nM, des-Gla anhydro-fXa: Kd=0.32 nM). [Figure 9] FIG. 1 shows the reversal of anticoagulant activity of various concentrations of betrixaban by addition of a 680 nM concentration of antidote (des-Gla anhydro-fXa) in the thrombin generation assay of Example 2. At a concentration of 680 nM, des-Gla anhydro-fXa was able to produce a virtually complete restoration of fXa activity. [Figure 10] FIG. 1 shows the reversal of anticoagulant activity of 250 nM betrixaban by various concentrations of antidote (des-Gla anhydro-fXa) in a clot prolongation assay using aPTT reagent in a 96-well plate format (described in Example 3). The data show that when approximately 608 nM of antidote was used to neutralize 250 nM of the fXa inhibitor betrixaban, clotting times were comparable to that of control platelet poor plasma. [Figure 11] FIG. 1 shows the effect of 563 nM of antidote (des-Gla anhydro-fXa) on the anticoagulant activity of enoxaparin (0.3125-1.25 U / mL) in a clot prolongation assay using aPTT reagent in a 96-well plate format, expressed as fold change after normalization. The assay protocol is described in Example 3. The data show that the addition of 563 nM of antidote significantly neutralized the activity of enoxaparin, a low molecular weight heparin. [Figure 12] FIG. 1 shows the effect of the antidote des-Gla anhydro-fXa on the activity of thrombin (5 nM) and its inhibition by the specific thrombin inhibitor argatroban at 50 nM in a chromogenic assay. As expected, the fXa inhibitor antidote did not detectably affect either the activity of thrombin or its inhibition by the specific inhibitor argatroban at concentrations up to 538 nM. This is described more fully in Example 14. [Figure 13]Figure 1 shows the effect of various concentrations of antidote des-Gla anhydro-fXa on the anticoagulant activity of 400nM betrixaban in an aPTT assay using a standard clotting timer. The assay protocol is described in Example 3. The data show that the antidote of the fXa inhibitor substantially reverses the inhibition of fXa by 400nM betrixaban. The EC50 of the antidote was estimated to be about 656nM at 400nM betrixaban. [Figure 14] Figure 1 shows a map of the DNA construct for expressing the fXa triple mutant (SEQ ID NO: 12) in CHO cells. Plasmid DNA was linearized and transfected into CHO dhfr(-) cells. Cells were selected using tetrahydrofolate (HT)-deficient medium and methotrexate (MTX). Stable clones were screened for high protein expression by ELISA. The fXa triple mutant was produced in serum-free medium and purified by a combination of ion exchange and affinity columns. The numbering in the map is based on the polynucleotide sequence encoding human fX, SEQ ID NO: 1. For example, the alanine mutation at active site S419 (SEQ ID NO: 1) corresponds to the mutation at S379 (SEQ ID NO: 3) of mature human fX, described throughout this application and more specifically in Example 7. [Figure 15] Western blot of purified r-antidote with monoclonal antibodies that recognize the heavy and heavy chains of fX, respectively. Upon reduction of the disulfide bond linking the light and heavy chains, the heavy chain of r-antidote migrates at the predicted molecular weight similar to plasma-derived fXa. Deletion of amino acids 6-39 in the Gla-domain of the fXa mutant results in a lower molecular weight band for the light chain of r-antidote compared to normal FXa. [Figure 16]Figure 1 shows plasma levels of betrixaban in mice (n=7-10 / group) after oral administration of betrixaban alone (15 mg / kg) or after oral administration of betrixaban (15 mg / kg) followed by intravenous injection of plasma-derived antidote (pd-antidote) prepared according to Example 1 (300 μg, IV). pd-antidote was administered 5 min prior to the 1.5 hour time point, and mouse blood samples (0.5 mL) were collected 1.5, 2.0, and 4.0 hours after oral administration of betrixaban. Whole blood INR, betrixaban and antidote plasma levels were analyzed. Betrixaban levels (mean ± SEM) in mouse plasma were plotted as a function of time for mice at 15 mg / kg (open squares) and 15 mg / kg followed by antidote injection (open circles). The PK-PD correlations for the antidote treatment groups at 1.5 hours (5 minutes after antidote injection) are summarized in Table 13. A single injection of antidote resulted in a >50% reduction in functional betrixaban based on INR measurements, as described more fully in Example 8. [Figure 17] Figure 17 shows the results of mouse experiments with purified r-antidote (n=4-10 mice / group). Betrixaban levels in mouse plasma (Figure 17A) and whole blood INR (Figure 17B) were compared after oral administration of betrixaban alone (15 mg / kg) or oral administration of betrixaban (15 mg / kg) followed by intravenous injection of r-antidote (300 μg). Mean values ​​for each treatment group are shown. As summarized in Table 14, a single IV injection of r-antidote resulted in >50% correction of the ex vivo whole blood INR, justifying effective neutralization of fXa inhibitors by antidote, by multiple injections or other regimes. These results demonstrate that the fXa mutants of the present invention have the potential to act as universal antidotes to reverse the anticoagulant effects of fXa inhibitors in patients undergoing bleeding or other medical emergencies. This is more fully described in Example 8. [Figure 18]Figure 13 shows the reversal of the inhibitory effect of enoxaparin by r-Antidote in a 96-well turbidity change clotting assay. The results are essentially similar to pd-Antidote (Figure 11), indicating that both fXa derivatives have comparable functional antidote activity. 50 nM of r-Antidote substantially corrected (>75%) the inhibitory effect of 1.25 U / mL of enoxaparin. The assay protocol is shown in Example 11. [Figure 19] 18 and 19 are both described in Example 11, showing the reversal of the inhibitory effect of low molecular weight heparins (LMWHs) by r-antidote as tested in a human plasma clotting assay. [Figure 20] 1 shows the reversal of the anticoagulant effect of rivaroxaban by r-antidote, which is more fully described in Example 12. [Figure 21] FIG. 1 shows an alignment of the r-antidote polynucleotide and translated polypeptide sequences. [Figure 22] Figure 22 shows the results of mouse experiments using a single IV injection (1 injection) or two injections (2 injections) of r-Antidote (n=5 mice / group, 312ug / 200ul of r-Antidote). Plasma betrixaban levels (Figure 22A) were compared after oral administration of betrixaban (15mg / kg) followed by intravenous injection of vehicle or r-Antidote (see Example 8 for details). As shown in Figure 22A, a single IV injection of r-Antidote increased plasma betrixaban levels by more than 8-fold compared to the vehicle control (Control_1), indicating the ability of the antidote to effectively capture betrixaban in vivo. A second injection of the antidote further increased betrixaban levels by less than 2-fold compared to the single injection, indicating the limited amount of betrixaban in mouse blood and possibly the reversal of its anticoagulant effect by the antidote. FIG. 22B demonstrates that the measured INR decreases as the ratio of antidote / betrixaban increases in mouse plasma after a single and two injections of antidote. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0030] I. Definition The practice of the present invention will employ, unless otherwise indicated, conventional techniques of tissue culture, immunology, molecular biology, microbiology, cell biology, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Russell (eds.) (2001) Molecular Cloning: A Laboratory Manual, 3rd ed.; Ausubel et al. (eds.) (2007) Current Protocols in Molecular Biology; Methods in Enzymology (Academic Press, Inc., NY); MacPherson et al. (1991) PCR 1: A Practical Approach (IRL Press at Oxford University Press); MacPherson et al. (1995) PCR 2: A Practical Approach; Harlow and Lane (eds.) (1999) Antibodies, A Laboratory Manual; Freshney (2005) Culture of Animal Cells: A Manual of Basic Technique, 5th ed.; Gait ed. (1984) Oligonucleotide Synthesis; U.S. Patent No. 4,683,195; Hames and Higgins (eds.) (1984) Nucleic Acid Hybridization; Anderson (1999) Nucleic Acid Hybridization; edited by Hames and Higgins (1984) Transcription and Translation; Immobilized Cells and Enzymes (IRL Press (1986)); Perbal (1984) A Practical Guide to Molecular Cloning; edited by Miller and Calos (1987) Gene Transfer Vectors for See Mammalian Cells (Cold Spring Harbor Laboratory); Gene Transfer and Expression in Mammalian Cells, ed. Makrides (2003); Immunochemical Methods in Cell and Molecular Biology, eds. Mayer and Walker (1987) (Academic Press, London); Weir's Handbook of Experimental Immunology, eds. Herzenberg et al. (1996); Manipulating the Mouse Embryo: A Laboratory Manual, 3rd Edition (Cold Spring Harbor Laboratory Press (2002)).

[0031] All numerical designations, including ranges, such as pH, temperature, time, concentration, and molecular weight, are approximations that vary by (+) or (-) in increments of 0.1. It is to be understood, although not always explicitly stated, that all numerical designations are preceded by the term "about." It is also to be understood, although not always explicitly stated, that the reagents described herein are merely illustrative, and that equivalents of such are known in the art.

[0032] As used in the specification and claims, the singular forms "a," "an," and "the" include plural references unless the content clearly dictates otherwise. For example, the term "a pharma- ceutically acceptable carrier" includes a plurality of pharma- ceutically acceptable carriers, including mixtures thereof.

[0033] The term "comprising" as used herein is intended to mean that the compositions and methods include the recited elements, but do not exclude others. "Consisting essentially of" as used to define compositions and methods means excluding all other elements that are of essential significance to the combination of intended use. Thus, a composition consisting essentially of the elements defined herein does not exclude trace contaminants from the isolation and purification methods, as well as pharma- ceutically acceptable carriers such as phosphate buffered saline, preservatives, etc. "Consisting of" means excluding other than trace elements of other components and substantial method steps for administering the compositions of the invention. The embodiments defined by each of these variable terms are within the scope of the invention.

[0034] A "subject" of diagnosis or therapy is a cell or a mammal, including a human. Non-human animals of diagnostic or therapeutic interest include, for example, murines, e.g., rats, mice, canines, e.g., dogs, lagomorphs, e.g., rabbits, farm animals, sport animals, and pets.

[0035] The terms "protein" and "polypeptide" are used interchangeably in their broadest sense and refer to a compound of two or more subunits of amino acids, amino acid analogs, or peptidomimetics. The subunits may be linked by peptide bonds. In another embodiment, the subunits may be linked by other bonds, such as esters, ethers, etc. A protein or peptide must contain at least two amino acids, and there is no limit to the maximum number of amino acids that may make up a protein or peptide sequence. As used herein, the term "amino acid" refers to either natural and / or unnatural or synthetic amino acids, including glycine and both the D and L optical isomers, amino acid analogs, and peptidomimetics. The one-letter and three-letter abbreviations of naturally occurring amino acids are listed below. Peptides of three or more amino acids are commonly called oligopeptides if the peptide chain is short. If the peptide chain is long, the peptide is commonly called a polypeptide or protein.

[0036] [ka] "Factor Xa" or "fXa" or "fXa protein" refers to a serine protease in the blood coagulation pathway that arises from inactive factor X (fX). Factor Xa is activated by factor IXa with its cofactor factor VIIIa in a complex known as intrinsic Xase, or by factor VIIa with its cofactor tissue factor in a complex known as extrinsic Xase. fXa forms a membrane-bound prothrombinase complex with factor Va and is the active component in the prothrombinase complex that catalyzes the conversion of prothrombin to thrombin. Thrombin is the enzyme that catalyzes the conversion of fibrinogen to fibrin, which ultimately leads to the formation of a blood clot. Thus, the biological activity of fXa is sometimes referred to herein as "procoagulant activity."

[0037] The nucleotide sequence encoding human factor X ("fX") is<http: / / www.ncbi.nlm.nih.gov / entrez / viewer.fcgi?db=nuccore&id=89142731> fX can be found in GenBank under "NM_000504" and is set forth in FIG. 1b and SEQ ID NO:2. The corresponding amino acid sequence and domain structure of fX are described in Leytus et al., Biochemistry, 1986, 25:5098-5102. The domain structure of mature fX is also described in Venkateswarlu, D. et al., Biophysical Journal, 2002, 82:1190-1206. After catalytic cleavage of the first 52 residues of the heavy chain (amino acids 143-194 of SEQ ID NO:3), fX is activated to fXa (SEQ ID NO:6). FXa contains a light chain (SEQ ID NO:8) and a heavy chain (SEQ ID NO:9). The first 45 amino acid residues of the light chain (residues 1-45 of SEQ ID NO:6) are referred to as the Gla domain because they contain 11 post-translationally modified γ-carboxyglutamic acid residues (Gla). It also contains a short (6 amino acid residues) aromatic stack sequence (residues 40-45 of SEQ ID NO:6). Chymotrypsin digestion selectively removes residues 1-44, resulting in Gla-domain-less fXa (SEQ ID NO:4). The serine protease catalytic domain of fXa is located in the C-terminal heavy chain. The heavy chain of fXa is highly homologous to other serine proteases such as thrombin, trypsin, and activated protein C.

[0038] The domain structure of mature factor X can be found in Venkateswarlu D. et al., Biophysical J., 2002, vol. 82, pp. 1190-1206, which is incorporated herein by reference in its entirety. The numbering of the amino acids in this figure is the same as in FIG. 3. The tripeptide Arg140-Lys141-Arg142 (RKR triplet shown in FIG. 1), which connects the light chain and the activation peptide, is not shown because a form lacking the tripeptide predominates in circulating plasma. The individual domains are shown in boxes, including amino acids 1-45 in FIG. 2 (SEQ ID NO: 3). Functionally important catalytic residues are circled, with "γ" representing a Gla (γ-carboxyglutamic acid) residue.

[0039] "Native fXa" or "wild-type fXa" refers to fXa naturally occurring in plasma or isolated in its original, unmodified form that retains the biological activity of activating prothrombin, thus promoting the formation of blood clots. The term includes naturally occurring polypeptides isolated from tissue samples and recombinantly produced fXa. "Active fXa" refers to fXa that has the biological activity of activating prothrombin. "Active fXa" can be native fXa or modified fXa that retains procoagulant activity.

[0040] "fXa derivative" or "modified fXa" or "derivative of factor Xa protein" refers to a fXa protein that has been modified so that it does not directly or indirectly bind to and assemble with a factor Xa inhibitor to form a prothrombinase complex. Structurally, the derivative is modified to provide no or reduced procoagulant activity. "Procoagulant activity" refers herein to the ability of an agent to cause blood clotting or clot formation. Reduced procoagulant activity means that the procoagulant activity is reduced by at least about 50%, or more than about 90%, or more than about 95% compared to wild-type fXa. For example, recombinant fX-S395A has essentially no procoagulant activity as measured by an in vitro assay, such as a fXa activity assay.

[0041] The derivatives have either a modified active site or a modified Gla domain, or both. Further modifications are also contemplated. It is contemplated that such modifications may be made in one or more of the following ways: deleting one or more of the amino acids in the sequence, substituting one or more amino acid residues with one or more different amino acid residues, and / or manipulating one or more amino acid side chains or the "C" or "N" termini thereof.

[0042] The term "active site" refers to the portion of an enzyme or antibody where a chemical reaction occurs. A "modified active site" refers to an active site that has been structurally modified to result in an active site with increased or decreased chemical reactivity or specificity. Examples of active sites include, but are not limited to, the catalytic domain of human factor X comprising amino acid residues 235-488 (FIG. 1), and the catalytic domain of human factor Xa comprising amino acid residues 195-448 (FIGS. 2 and 3). Examples of modified active sites include, but are not limited to, the catalytic domain of human factor Xa comprising amino acid residues 195-448 in SEQ ID NOs: 10, 11, 12, 13, or 15, with at least one amino acid substitution at positions Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424.

[0043] As mentioned above, the derivatives of the invention may have modified Gla domains or the Gla domain may be removed entirely. Examples of fXa derivatives suitable as antidotes in the methods of the invention are Gla-domain-free fXa (SEQ ID NO: 4 or 5), Gla-deficient fXa (SEQ ID NO: 7 with modifications described herein), fXa with modifications in the catalytic site (SEQ ID NO: 10 or 11), and fXa with modifications in sites known to be important for fV / fVa or fVIII / fVIIIa interactions (SEQ ID NO: 4, 5, 7, 10, or 11 with at least one amino acid substitution at positions Arg306, Glu310, Arg347, Lys351, Lys414, or Arg424), as detailed herein. Further examples of fXa derivatives contemplated by the invention are provided below.

[0044] "Gla-domain-free fXa" or "des-Gla fXa" refers to fXa that does not have a Gla-domain and includes fXa derivatives that possess other modification(s) in addition to the removal of the Gla-domain. Examples of Gla-domain-free fXa in the present invention include, but are not limited to, a fXa derivative lacking amino acid residues 1-39 of SEQ ID NO:3; a fXa derivative lacking amino acid residues 6-39 of SEQ ID NO:3, corresponding to a fXa mutant expressed in CHO cells described in more detail below (SEQ ID NO:12, Table 12); a fXa derivative lacking amino acid residues 1-44 of SEQ ID NO:3, corresponding to des-Gla fXa after chymotrypsin digestion of human fXa (SEQ ID NO:4, FIG. 3); and a fXa derivative lacking the entire Gla-domain residues 1-45 of SEQ ID NO:3 (SEQ ID NO:5) as described in Padmanabhan et al., Journal Mol. Biol., 1993, 232:947-966. Other examples include des-Gla anhydro fXa (SEQ ID NO: 10, Table 10) and des-Gla fXa-S379A (SEQ ID NO: 11, Table 11).

[0045] In some embodiments, des-Gla fXa comprises at least amino acid residues 40-448 of SEQ ID NO: 3, or an equivalent thereof. In some embodiments, des-Gla fXa comprises at least amino acid residues 45-488 (SEQ ID NO: 4) or 46-488 (SEQ ID NO: 5) of SEQ ID NO: 3, or an equivalent thereof.

[0046] In some embodiments, des-Gla fXa comprises at least amino acid residues 40-139 and 195-448 of SEQ ID NO: 3, or an equivalent thereof. In some embodiments, des-Gla fXa comprises at least amino acid residues 45-139 and 195-448 of SEQ ID NO: 3, or an equivalent thereof. In other embodiments, des-Gla fXa comprises at least amino acid residues 46-139 and 195-448 of SEQ ID NO: 3, or an equivalent thereof.

[0047] "Gla-deficient fXa" refers to fXa with a reduced number of free side chain γ-carboxyl groups in its Gla-domain. Like Gla-domain-free fXa, Gla-deficient fXa can carry other modifications. Gla-deficient fXa includes uncarboxylated, undercarboxylated and decarboxylated fXa. "Uncarboxylated fXa" or "decarboxylated fXa" refers to fXa derivatives that do not have the γ-carboxy groups of the γ-carboxyglutamic acid residues of the Gla domain, e.g., fXa in which all the γ-carboxyglutamic acids of the Gla domain have been replaced by different amino acids, or fXa in which all the side chain γ-carboxyls have been removed or masked by amination, esterification or other means. In recombinantly expressed proteins, uncarboxylated fXa is sometimes also referred to as so-called non-carboxylated fXa. "Undercarboxylated fXa" refers to a fXa derivative that has a reduced number of γ-carboxy groups in the Gla domain compared to wild-type fXa, e.g., a fXa in which one or more, but not all, of the γ-carboxyglutamic acids in its Gla domain have been replaced with one or more different amino acids, or a fXa in which at least one, but not all, of its side chain γ-carboxyls have been removed or masked by means such as amination and esterification.

[0048] The domain structure of human Gla-domain-free factor Xa can be found in Padmanabhan et al., J. Mol. Biol., 1993, vol. 232, pp. 947-966, which is incorporated herein by reference in its entirety. Amino acid numbering is based on homology with chymotrypsin, e.g., Ser195 corresponds to Ser379 in FIG. 2 when the numbering of human mature fX is used. Insertions are indicated by letters and deletions by two consecutive numbers. 300 is added to the light chain numbers to distinguish them from the heavy chain numbers. β363 is β-hydroxyaspartic acid. The diagonal line indicates the proteolytic cleavage observed in the crystalline material. The sequence of Gla-domain-free fXa, lacking amino acid residues 1-45 based on mature fX (SEQ ID NO: 3), is set forth in SEQ ID NO: 5.

[0049] In one embodiment, a fXa derivative may lack the light chain of fXa but still contain the serine protease catalytic domain present in the heavy chain. Additionally, chimeras with other serine protease catalytic domains may be used to make substitutions in the heavy chain.

[0050] "pd-antidote" or "plasma-derived antidote" refers to the des-Gla anhydro fXa derivative, which has the amino acid residues set forth in SEQ ID NO:10.

[0051] "r-antidote" or "recombinant antidote" refers to a fXa derivative lacking amino acid residues 6-39 of SEQ ID NO:3 (SEQ ID NO:12, Table 12), which corresponds to the fXa mutant expressed in CHO cells described in more detail below.

[0052] An "anticoagulant agent" or "anticoagulant" is an agent that inhibits the formation of blood clots. Examples of anticoagulants include, but are not limited to, specific inhibitors of thrombin, factor IXa, factor Xa, factor XIa, factor XIIa, or factor VIIa, heparin and derivatives, vitamin K antagonists, and anti-tissue factor antibodies. Examples of specific inhibitors of thrombin include hirudin, bivalirudin (Angiomax®), argatroban, and lepirudin (Refludan®). Examples of heparin and derivatives include unfractionated heparin (UFH), low molecular weight heparins (LMWHs), such as enoxaparin (Lovenox®), dalteparin (Fragmin®), and danaparoid (Orgaran®); and synthetic pentasaccharides, such as fondaparinux (Arixtra®). Examples of vitamin K antagonists include warfarin (Coumadin®), phenocoumarol, acenocoumarol (Sintrom®), chlorindione, dicumarol, diphenadione, ethyl viscoum acetate, phenprocoumon, phenindione, and thiochromarol. In one embodiment, the anticoagulant is an inhibitor of factor Xa. In one embodiment, the anticoagulant is betrixaban.

[0053] "Anticoagulant therapy" refers to a therapeutic regime administered to a patient to prevent unwanted blood clots or thrombosis. Anticoagulant therapy includes the administration of one or a combination of two or more anticoagulants or other drugs at doses and schedules appropriate to treat or prevent unwanted blood clots or thrombosis in a patient.

[0054] The term "factor Xa inhibitor" or "factor Xa inhibitor" refers to a compound capable of directly or indirectly inhibiting the activity of coagulation factor Xa, which catalyzes the conversion of prothrombin to thrombin in vitro and / or in vivo. Examples of known fXa inhibitors include, but are not limited to, fondaparinux, idraparinux, biotin-labeled idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor, DX-9065a (e.g., as described in Herbert, JM et al., J Pharmacol Exp Ther., 1996, 276(3):1030-8), YM-60828 (e.g., as described in Taniuchi, Y. et al., Thromb Haemost., 1998, 79(3):543-8), YM-150 (e.g., Eriksson, BI et al., Blood, 2005;106(11):Abstract1865), apixaban, rivaroxaban, PD-348292 (e.g., Pipeline Insight: Antithrombotics - Reaching the Untreated Prophylaxis Market, 2007), otamixaban, razaxaban (DPC906), BAY59-7939 (e.g., Turpie, AG et al., J. Thromb. Haemost., 2005, 3(11):2479-86), DU-176b (e.g., Hylek EM, Curr Opin Invest Drugs, 2007, 8(9):778-783), LY517717 (e.g., Agnelli, G. et al., J. Thromb. Haemost., 2007, 5(4):746-53), GSK913893, betrixaban (described below) and its derivatives. Low molecular weight heparins ("LMWHs") are also considered to be factor Xa inhibitors.

[0055] In one embodiment, the factor Xa inhibitor is selected from betrixaban, rivaroxaban, LMWH, and combinations thereof.

[0056] The term "betrixaban" refers to the compound "[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide" or a pharma- ceutically acceptable salt thereof. "[2-({4-[(dimethylamino)iminomethyl]phenyl}carbonylamino)-5-methoxyphenyl]-N-(5-chloro(2-pyridyl))carboxamide" refers to the compound having the following structure or a tautomer or a pharma-ceutically acceptable salt thereof.

[0057] [ka] Betrixaban is described in U.S. Patent Nos. 6,376,515 and 6,835,739, and U.S. Patent Application Publication No. 2007 / 0112039, filed November 7, 2006, the contents of which are incorporated herein by reference. Betrixaban is known to be a specific inhibitor of factor Xa.

[0058] The term "antidote" or "antidote to factor Xa inhibitors" as used herein refers to a molecule, such as a derivative of fXa, that can substantially neutralize or reverse the coagulation inhibitory activity of a fXa inhibitor by competing with active fXa for binding to available fXa inhibitors. Examples of antidotes of the invention are fXa derivatives with reduced phospholipid membrane binding, such as des-Gla fXa or Gla-deficient fXa, and fXa derivatives with reduced catalytic activity, such as active site modified fXa derivatives, and derivatives with reduced interaction with fV / Va or fVIII / fVIIIa. Examples of antidotes of the invention with reduced membrane binding and reduced catalytic activity include, but are not limited to, des-Gla anhydro-fXa by chymotrypsin digestion of anhydro-fXa (described in Example 1); des-Gla fXa-S379A (S195A in chymotrypsin numbering) by mutagenesis (described in Example 6).

[0059] Other examples of antidotes of the present invention include proteins or polypeptides containing a serine protease catalytic domain that share sufficient structural similarity with the fXa catalytic domain and thus are capable of binding to small molecule fXa inhibitors. Examples include, but are not limited to, thrombin, which binds to the fXa inhibitor GSK913893 (Young R. et al., Bioorg. Med. Chem. Lett., 2007, vol. 17(10):2927-2930); plasma kallikrein, which binds to the fXa inhibitor apixaban (Luettgen J. et al., Blood, 2006, vol. 108(11) abstract 4130); and trypsin (or its bacterial homologue subtilisin), which binds to the fXa inhibitor C921-78 with subnanomolar affinity (Kd=500 pM) (Betz A et al., Biochem., 1999, vol. 38(44):14582-14591).

[0060] In one embodiment, the derivatives of the invention bind directly or indirectly to factor Xa inhibitors. The terms "binding", "binding", "recognition" or "recognizing" as used herein are meant to include interactions between molecules that can be detected, for example, using a hybridization assay. The terms are also intended to include "binding" interactions between molecules. The interactions can be, for example, protein-protein, protein-nucleic acid, protein-small molecule or small molecule-nucleic acid in nature. Binding can be "direct" or "indirect". "Direct" binding includes direct physical contact between molecules. "Indirect" binding between molecules includes molecules being in direct physical contact with one or more intermediate molecules at the same time. For example, it is contemplated that the derivatives of the invention indirectly bind and substantially neutralize low molecular weight heparin and other indirect inhibitors of factor Xa. This binding may result in the formation of a "complex" that includes the interacting molecules. A "complex" refers to the association of two or more molecules that are held together by covalent or non-covalent bonds, interactions or forces.

[0061] "Neutralizing," "reversing," or "counteracting" the activity of an inhibitor of fXa, or similar phrases refer to the inhibition or blocking of the factor Xa inhibitory or anticoagulant function of an Xa inhibitor. Such phrases refer to partial inhibition or blocking of function, as well as inhibition or blocking of most or all of the fXa inhibitory activity, in vitro and / or in vivo.

[0062] In certain embodiments, a factor Xa inhibitor is substantially neutralized, meaning that its ability to directly or indirectly inhibit factor Xa is reduced by at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0063] The term "phospholipid membrane bound" refers to the binding of active fXa to Ca 2+ Its ability to bind to negatively charged phospholipid membranes or other cell membranes such as platelets in the presence of ions is mediated by gamma-carboxyglutamic acid residues in the Gla domain of fXa.

[0064] The term "reduced interaction" refers to the ability of an attenuated fXa derivative to bind or complex with ions or other cofactors that normally bind or complex with wild-type fXa. Examples of such interactions include, but are not limited to, the interaction of fXa with Ca 2+ These include binding to ions and phospholipid membranes, interactions with fV / fVa, or fVIII / f / VIIIa, etc. Preferably, the interaction of the fXa derivative with ions or other cofactors is reduced to 50% of that of wild-type fXa. More preferably, the interaction is reduced to 10%, 1%, and 0.1% of that of wild-type fXa. This refers to the ability of the derivative to "assemble to form the prothrombinase complex."

[0065] "fXa inhibitor binding activity" refers to the ability of a molecule to bind to an inhibitor of fXa. The antidotes of the present invention possess fXa inhibitor binding activity, whether direct or indirect.

[0066] The term "circulating half-life" or "plasma half-life" refers to the time required for the plasma concentration of an antidote circulating in the plasma to decrease to half of its initial concentration after a single administration.

[0067] The term "conjugated moiety" refers to a moiety that can be added to a fXa derivative by forming a covalent bond with a residue of the fXa derivative. The moiety can be directly attached to the residue of the fXa derivative or it can form a covalent bond with a linker, which in turn forms a covalent bond with a residue of the fXa derivative.

[0068] As used herein, "antibody" includes whole antibodies and any antigen-binding fragments or single chains thereof.Thus, the term "antibody" includes any protein or peptide containing molecule that includes at least a portion of an immunoglobulin molecule.Examples of such include, but are not limited to, the complementarity determining region (CDR) of heavy or light chain or a ligand-binding portion thereof, the variable region of heavy or light chain, the constant region of heavy or light chain, the framework (FR) region or any portion thereof, or at least a portion of a binding protein.

[0069] Antibodies can be polyclonal or monoclonal and can be isolated from any suitable biological source, for example, murine, rat, ovine, and canine.

[0070] A "composition" is intended to mean a combination of an active agent with another compound or composition, inert (eg, a detectable agent or label) or active, such as an adjuvant.

[0071] A "pharmaceutical composition" is intended to include a combination of an active agent with a carrier, inert or active, that renders the composition suitable for diagnostic or therapeutic use in vitro, in vivo or ex vivo.

[0072] An "effective amount" refers to an amount of derivative sufficient to induce a desired biological and / or therapeutic result. The result may be the alleviation of signs, symptoms, or causes of a disease, or any other desired alteration of a biological system. In the present invention, the result typically includes one or more of the following: neutralization of the fXa inhibitor administered to the patient, reversal of the anticoagulant activity of the fXa inhibitor, removal of the fXa inhibitor from the plasma, restoration of hemostasis, and reduction or cessation of bleeding. The effective amount will vary depending on the particular antidote used, the particular fXa inhibitor administered to the subject, the dosing regimen of the fXa inhibitor, the timing of administration of the antidote, the subject and condition being treated, the weight and age of the subject, the severity of the condition, the mode of administration, etc., all of which can be readily determined by one of skill in the art.

[0073] As used herein, the terms "treating," "treatment," and the like are used herein to mean obtaining a desired pharmacological and / or physiological effect. The effect can be prophylactic, in terms of completely or partially preventing the disorder or its signs or symptoms, and / or therapeutic, in terms of partially or completely curing the disorder and / or adverse effects resulting from the disorder.

[0074] "Treating" also encompasses any treatment of a disorder in a mammal, including (a) preventing the occurrence of the disorder in a subject who may be susceptible to the disorder but not yet diagnosed as having it, e.g., preventing bleeding in a patient with an anticoagulant overdose; (b) inhibiting the disorder, i.e., arresting its development, e.g., inhibiting bleeding; or (c) alleviating or ameliorating the disorder, e.g., reducing bleeding.

[0075] As used herein, "treating" further includes systemic remission of symptoms associated with the pathology and / or delay in the onset of symptoms. Clinical and subclinical evidence of "treatment" varies with pathology, individual, and treatment.

[0076] "Administration" can be provided in one dose, continuously or intermittently throughout the entire treatment period. Methods for determining the most effective means and dosage of administration are known to those skilled in the art and vary with the composition used in the treatment, the purpose of the treatment, the target cells being treated, and the subject being treated. Single or multiple administrations can be performed with the dosage level and pattern selected by the treating physician. Appropriate dosage forms and methods for administering drugs are known in the art.

[0077] The agents and compositions of the present invention can be used in the manufacture of medicinal products and for the treatment of humans and other animals by administration in accordance with conventional procedures, such as the active ingredients in a pharmaceutical composition.

[0078] The agents of the present invention can be administered for treatment by any suitable route, in particular parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. It will also be appreciated that the preferred route will vary with the condition and age of the recipient, and the disease being treated.

[0079] A number of in vitro assays, such as thrombin generation assays, as well as clinical clotting assays such as aPTT, PT and ACT, can determine whether this method, i.e., inhibition or reversal of factor Xa inhibitors, has been achieved.

[0080] The term "isolated" as used herein with respect to nucleic acids, such as DNA or RNA, refers to a molecule that is separated from other DNA or RNA, respectively, present in the natural source of the macromolecule. The term "isolated nucleic acid" is meant to include nucleic acid fragments that are not naturally occurring as fragments and are not found in the natural state. The term "isolated" is also used herein to refer to polypeptides and proteins that are isolated from other cellular proteins, and is meant to encompass both purified and recombinant polypeptides. In other embodiments, the term "isolated" means that a cell, tissue, polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof, is separated from cellular or other components with which it is normally associated in nature. For example, an isolated cell is one that is separated from tissues or cells of a different phenotype or genotype. As will be apparent to one of skill in the art, a non-naturally occurring polynucleotide, peptide, polypeptide, protein, antibody, or fragment thereof does not require "isolation" to distinguish it from its naturally occurring counterpart.

[0081] The term "equivalent thereof" as used herein when referring to a reference protein, polypeptide or nucleic acid is intended to have minimal homology while still maintaining the desired functionality. Any modified protein referred to herein is also intended to include its equivalent. For example, homology may be at least 75% homology, or at least 80%, or at least 85%, or at least 90%, or at least 95%, or 98% % homology, and exhibits substantially equivalent biological activity to the reference polypeptide or protein. A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (e.g., 80%, 85%, 90%, or 95%) of "sequence identity" with another sequence means that the percentage of bases (or amino acids) are the same when the two sequences are compared when aligned. It is noted that when only the heavy chain of fXa (or related serine protease) is used, the overall homology may be less than 75%, such as 65% or 50%, while still retaining the desired functionality. The alignment and % homology or sequence identity can be determined using software programs known in the art, such as those described in CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (ed. FM Ausubel et al., 1987), Supplement 30, section 7.7.18, table 7.7.1. Preferably, default parameters are used for alignment. A preferred alignment program is BLAST with default parameters. In particular, preferred programs are BLASTN and BLASTP with the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; prediction=10; matrix=BLOSUM62; explanation=50 sequences; sort order=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR.Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / cgi-bin / BLAST.

[0082] The terms "polynucleotide" and "oligonucleotide" are used interchangeably and refer to a polymeric form of nucleotides of any length, either deoxyribonucleotides or ribonucleotides or analogs thereof. Polynucleotides can have any three-dimensional structure and can perform any function, known or unknown. Non-limiting examples of polynucleotides are genes or gene fragments (e.g., probes, primers, EST or SAGE tags), exons, introns, messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. Polynucleotides can contain modified nucleotides, such as methylated nucleotides and nucleotide analogs. Modifications to the nucleotide structure, if present, can be provided before or after assembly of the polynucleotide. The sequence of nucleotides can be interrupted by non-nucleotide components. Polynucleotides can be further modified after polymerization, such as by conjugation with a label component. The term also refers to both double-stranded and single-stranded molecules. Unless otherwise specified or required, any embodiment of the invention that is a polynucleotide encompasses both the double-stranded form and each of the two complementary single-stranded forms that are known or predicted to constitute the double-stranded form.

[0083] A polynucleotide consists of a specific sequence of the four nucleotide bases: adenine (A); cytosine (C); guanine (G); thymine (T); and, if the polynucleotide is RNA, uracil (U) in place of thymine. Thus, the term "polynucleotide sequence" is an alphabetical representation of a polynucleotide molecule. This alphabetical representation can be entered into a database in a computer with a central processing unit and used for bioinformatics applications such as functional genomics and homology searching.

[0084] "Homology" or "identity" or "similarity" refers to the degree of sequence similarity between two peptides or two nucleic acid molecules. Homology can be determined by comparing positions in each sequence, which can be aligned for purposes of comparison. If a position in the compared sequences is occupied by the same base or amino acid, the molecules are homologous at that position. The degree of homology between sequences is a function of the number of matching or homologous positions shared by the sequences. An "unrelated" or "non-homologous" sequence shares less than 40% identity, or less than 25% identity, with one of the sequences of the present invention.

[0085] A polynucleotide or polynucleotide region (or a polypeptide or polypeptide region) having a certain percentage (e.g., 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 98% or 99%) of "sequence identity" with another sequence means that, when aligned, the percentage of bases (or amino acids) are the same when comparing the two sequences. This alignment and percent homology or sequence identity can be determined using software programs known in the art, such as those described in Current Protocols in Molecular Biology, eds. Ausubel et al. (2007). Preferably, default parameters are used for the alignment. One alignment program is BLAST, using default parameters. In particular, the programs are BLASTN and BLASTP using the following default parameters: genetic code=standard; filter=none; strand=both; cutoff=60; prediction=10; matrix=BLOSUM62; explanation=50 sequences; sort order=high score; database=non-redundant, GenBank+EMBL+DDBJ+PDB+GenBank CDS translation+SwissProtein+SPupdate+PIR. Details of these programs can be found at the following internet address: http: / / www.ncbi.nlm.nih.gov / blast / Blast.cgi, last accessed on November 26, 2007. Biologically equivalent polynucleotides are those that have a particular % homology and encode polypeptides having the same or similar biological activity.

[0086] The term "homolog of a nucleic acid" refers to a nucleic acid having a nucleotide sequence that has a certain degree of homology with the nucleotide sequence of the nucleic acid or its complement. A homolog of a double-stranded nucleic acid is intended to include a nucleic acid having a nucleotide sequence that has a certain degree of homology with it or with its complement. In one aspect, a homolog of a nucleic acid can hybridize with a nucleic acid or its complement.

[0087] "Gene" refers to a polynucleotide that contains at least one open reading frame (ORF) that can encode a particular polypeptide or protein after being transcribed and translated. Any of the polynucleotide or polypeptide sequences described herein can be used to identify larger fragments or full-length coding sequences of the gene with which it is associated. Methods for isolating sequences of larger fragments are known to those skilled in the art.

[0088] The term "express" refers to the production of a gene product.

[0089] As used herein, "expression" refers to the process by which a polynucleotide is transcribed into mRNA and / or the transcribed mRNA is subsequently translated into a peptide, polypeptide, or protein. If the polynucleotide is derived from genomic DNA, expression may include splicing of the mRNA in eukaryotic cells.

[0090] The term "encodes" as applied to a polynucleotide is said to "encode" a polypeptide if, in its natural state or when manipulated by methods well known to those of skill in the art, it can be transcribed and / or translated to produce mRNA for the polypeptide and / or fragments thereof. The antisense strand is the complement of such a nucleic acid, and the coding sequence can be deduced therefrom.

[0091] "Peptide conjugate" refers to the association of one or more polypeptides with another chemical or biological compound by covalent or non-covalent bonding. In a non-limiting example, the "conjugation" of a polypeptide with a chemical leads to improved stability or efficacy of the polypeptide for its intended purpose. In one embodiment, the peptide is conjugated to a carrier, and the carrier is a liposome, a micelle, or a pharma- ceutically acceptable polymer.

[0092] "Liposomes" are microscopic vesicles consisting of concentric lipid bilayers. Structurally, liposomes range in size and shape from long tubes to spheres, with dimensions ranging from a few hundred angstroms to fractions of a millimeter. The vesicle-forming lipids are selected to achieve a defined degree of fluidity or rigidity of the final complex providing the lipid composition of the outer layer. They are neutral (cholesterol) or bipolar, with hydrocarbon chain lengths ranging from 14 to 22, saturated or with one or more double C=C bonds, and include phospholipids such as phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylinositol (PI), and sphingomyelin (SM), as well as other types of bipolar lipids, including but not limited to dioleoylphosphatidylethanolamine (DOPE). Examples of lipids which can be used alone or in combination with other lipid components to produce stable liposomes are phospholipids such as hydrogenated soy phosphatidylcholine (HSPC), lecithin, phosphatidylethanolamine, lysolecithin, lysophosphatidylethanol-amine, phosphatidylserine, phosphatidylinositol, sphingomyelin, cephalin, cardiolipin, phosphatidic acid, cerebrosides, distearoylphosphatidylethane-olamine (DSPE), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoylphosphatidylethanolamine (POPE) and dioleoylphosphatidylethanolamine 4-(N-maleimido-methyl)cyclohexane-1-carbo-oxylate (DOPE-mal).Additional phosphorus-free lipids that can be incorporated into liposomes include stearylamine, dodecylamine, hexadecylamine, isopropyl myristate, triethanolamine-lauryl sulfate, alkyl-aryl sulfates, acetyl palmitate, glycerol ricinoleate, hexadecyl stereate, amphoteric acrylic polymers, polyethyloxylated fatty acid amides, and the cationic lipids mentioned above (DDAB, DODAC, DMRIE, DMTAP, DOGS, DOTAP (DOTMA), DOSPA, DPTAP, DSTAP, DC-Chol). Negatively charged lipids include phosphatidic acid (PA), dipalmitoyl phosphatidylglycerol (DPPG), dioleoyl phosphatidylglycerol and (DOPG), dicetyl phosphate, which can form vesicles. Typically, liposomes can be divided into three categories based on the nature of their overall size and lamellar structure. The New York. Academy Sciences Meeting, “Liposomes and Their Use in Biology and Medicine, December 1977, are multilamellar vesicles (MLVs), small unilamellar vesicles (SUVs) and large unilamellar vesicles (LUVs).

[0093] A "micelle" is an aggregate of surface-active molecules dispersed in a liquid colloid. Micelles in a typical aqueous solution form aggregates with the hydrophilic "head" regions in contact with the surrounding solvent and the hydrophobic tail regions sequestered in the center of the micelle. This type of micelle is known as a normal phase micelle (oil-in-water micelle). Reverse micelles have the head groups in the center and the tails extended outward (water-in-oil micelle). Micelles can be used to bind polynucleotides, polypeptides, antibodies or compositions described herein to facilitate efficient delivery to target cells or tissues.

[0094] The phrase " pharmaceutically acceptable polymer " refers to a group of compounds that can be conjugated with one or more polypeptides described herein.It is contemplated that the conjugation of polymer with polypeptide can extend the half-life of polypeptide in vivo and in vitro.Non-limiting examples include polyethylene glycol, polyvinylpyrrolidone, polyvinyl alcohol, cellulose derivatives, polyacrylates, polymethacrylates, sugars, polyols and mixtures thereof.

[0095] "Gene delivery vehicle" is defined as any molecule that can carry inserted polynucleotide into host cell.Examples of gene delivery vehicle are liposome, micelle biocompatible polymers, including natural and synthetic polymers; lipoproteins; polypeptides; polysaccharides; lipopolysaccharides; artificial virus envelopes; metal particles; bacteria or viruses, such as baculovirus, adenovirus and retrovirus, bacteriophage, cosmids, plasmids, fungal vectors, and other recombinant vehicles typically used in the art, described for expression in various eukaryotic and prokaryotic hosts, and can be used for gene therapy and simple protein expression.

[0096] The polynucleotides of the present invention can be delivered to cells or tissues using gene delivery vehicles. As used herein, "gene delivery," "gene transfer," "transduction," and the like are terms that refer to the introduction of an exogenous polynucleotide (sometimes called a "transgene") into a host cell, regardless of the method used for the transfer. Such methods include a variety of well-known techniques, such as vector-mediated gene transfer (e.g., by viral infection / transfection, or various other protein- or lipid-based gene delivery complexes) as well as techniques that facilitate the delivery of "naked" polynucleotides, such as electroporation, "gene gun" delivery, and various other techniques used to transfer polynucleotides. The introduced polynucleotide can be stably or transiently maintained in the host cell. Stable maintenance typically requires that the introduced polynucleotide contains an origin of replication compatible with the host cell or is integrated into a replicon, such as an extrachromosomal replicon (e.g., a plasmid) or a nuclear or mitochondrial chromosome, of the host cell. As known in the art and described herein, several vectors are known to be capable of mediating the transfer of genes into mammalian cells.

[0097] A "viral vector" is defined as a recombinantly produced virus or virus particle that contains a polynucleotide for delivery into a host cell in vivo, ex vivo, or in vitro. Examples of viral vectors include retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphavirus vectors, and the like. Alphavirus vectors, such as Semliki Forest virus-based vectors and Sindbis virus-based vectors, have also been developed for use in gene therapy and immunotherapy. Schlesinger and Dubensky (1999) Curr. See Opin. Biotechnol. 5:434-439 and Ying et al. (1999) Nat. Med. 5(7):823-827. In embodiments where gene transfer is mediated by a retroviral vector, the vector construct refers to a polynucleotide comprising a retroviral genome or a portion thereof and a therapeutic gene. As used herein, "retroviral-mediated gene transfer" or "retroviral transduction" have the same meaning and refer to the process of stably transferring a gene or nucleic acid sequence into a host cell by a virus entering the cell and integrating its genome into the host cell genome. The virus can enter the host cell by its normal infection mechanism or can be modified to bind to a different host cell surface receptor or ligand and enter the cell. As used herein, retroviral vector refers to a viral particle that can introduce exogenous nucleic acid into a cell by a viral or viral-like entry mechanism.

[0098] Retroviruses carry their genetic information in the form of RNA, but after the virus infects a cell, the RNA is reverse transcribed into a DNA form that integrates into the genomic DNA of the infected cell. The integrated DNA form is called a provirus.

[0099] In the embodiment where gene transfer is mediated by a DNA virus vector such as adenovirus (Ad) or adeno-associated virus (AAV), vector construct refers to a polynucleotide that includes the viral genome or a part thereof and a transgene. Adenovirus (Ad) is a relatively well-characterized homogeneous group of viruses that includes more than 50 serotypes. See, for example, International PCT Application WO95 / 27071. Ad does not require integration into the host cell genome. Recombinant Ad-derived vectors have also been constructed, particularly those that reduce the potential for recombination to produce wild-type virus. See International PCT Application WO95 / 00655 and WO95 / 11984. Wild-type AAV has high infectivity and specificity to integrate into the genome of host cells. See Hermonat and Muzyczka (1984) Proc. Natl. Acad. Sci. USA 81:6466-6470 and Lebkowski et al. (1988) Mol. Cell. Biol. 8:3988-3996.

[0100] Vectors that contain both a promoter and a cloning site into which a polynucleotide can be operably linked are well known in the art. Such vectors can transcribe RNA in vitro or in vivo and are commercially available from sources such as Stratagene (La Jolla, Calif.) and Promega Biotech (Madison, Wis.). To optimize expression and / or in vitro transcription, it may be necessary to remove, add, or modify the 5' and / or 3' untranslated portions of the clone to eliminate extra, potentially inappropriate alternative translation initiation codons, or other sequences that may disrupt or reduce expression at either the transcription or translation level. Alternatively, a consensus ribosome binding site can be inserted immediately 5' to the initiation codon to enhance expression.

[0101] Gene delivery vehicles also include DNA / liposome complexes, micelles and targeting viral protein-DNA complexes. Liposomes that also contain targeting antibodies or fragments thereof can be used in the methods of the present invention. To enhance delivery to cells, the nucleic acids or proteins of the present invention can be conjugated to antibodies or binding fragments thereof that bind to cell surface antigens, such as cell surface markers found on stem cells or cardiomyocytes. In addition to delivering polynucleotides to cells or cell populations, direct introduction of the proteins described herein into cells or cell populations can be performed by non-limiting techniques of protein transfection, or culture conditions that can enhance expression and / or promote the activity of the proteins of the present invention are other non-limiting techniques.

[0102] The term "solid support" refers to a non-aqueous surface such as a "culture plate," "gene chip," or "microarray." Such gene chips or microarrays can be used by several techniques known to those skilled in the art for diagnostic and therapeutic purposes. In one technique, oligonucleotides are arrayed on the gene chip for DNA sequence determination by hybridization techniques such as those outlined in U.S. Pat. Nos. 6,025,136 and 6,018,041. The polynucleotides of the invention can be modified into probes, which in turn can be used to detect gene sequences. Such techniques are described, for example, in U.S. Pat. Nos. 5,968,740 and 5,858,659. Probes can also be immobilized on electrode surfaces for electrochemical detection of nucleic acid sequences such as those described in U.S. Pat. No. 5,952,172 to Kayem et al. and Kelley et al. (1999) Nucleic Acids Res., 27:4830-4837.

[0103] A variety of "gene chips" or "microarrays" and similar technologies are known in the art. Examples of such include, but are not limited to, LabCard (ACLARA Bio Sciences Inc.); GeneChip (Affymetric, Inc); LabChip (Caliper Technologies Corp); Low Density Arrays with Electrochemical Sensing (Clinical Micro Sensors); LabCD System (Gamera Bioscience Corp.); Omni Grid (Gene Machines;Q Array (Genetix Ltd.);High-throughput automated mass spectrometry system with liquid-phase expression technology (Gene Trace Systems, Inc.);Thermal jet spotting system (Hewlett Packard Company);Hyseq HyChip (Hyseq, Inc.);BeadArray (Illumina, Inc.);GEM (Incyte Microarray Systems);High-throughput microarray system capable of dispensing 12 to 64 spots onto multiple glass slides (Intelligent Bio-Instruments);Molecular biology workstation and NanoChip (Nanogen, Inc.);Microfluidic glass chips (Orchid biosciences, Inc.);Biochip array machine with four PiezoTip piezoelectric drop-on-demand chips (Packard Instruments, Inc.);FlexJet (Rosetta Inpharmatic, Inc.);MALDI-TOF mass spectrometer (Sequnome);ChipMaker2 and ChipMaker3 (TeleChem International, Inc.); and GenoSensor (Vysis, Inc.). Examples of "gene chips" or "microarrays" are also described in U.S. Patent Publication Nos. 2007-0111322, 2007-0099198, 2007-0084997, 2007-0059769 and 2007-0059765, and U.S. Patent Nos. 7,138,506, 7,070,740, and 6,989,267.

[0104] In one embodiment, a "gene chip" or "microarray" containing probes or primers homologous to the polynucleotides, polypeptides or antibodies described herein is prepared. A suitable sample is obtained from the patient, and extraction of genomic DNA, RNA, protein or any combination thereof is performed and, if necessary, amplified. The sample is contacted with a panel of gene chips or microarrays under conditions suitable for hybridization of the gene or gene product of interest with the probe or primer contained on the gene chip or microarray. The probe or primer may be detectably labeled, thereby identifying the gene of interest. Alternatively, a chemical or biological reaction may be used to identify the probe or primer that has hybridized with the DNA or RNA of the gene of interest. The genotype or phenotype of the patient is then determined with the aid of the aforementioned devices and methods.

[0105] Other non-limiting examples of solid supports include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, gabbro, and magnetite. The nature of the support can be soluble to some extent or insoluble. The support material can have virtually any possible structural configuration so long as the coupled molecule is capable of binding to a polynucleotide, polypeptide, or antibody. Thus, the configuration of the support can be spherical, such as a bead, or cylindrical, such as the inner surface of a test tube or the outer surface of a rod. Alternatively, the surface can be flat, such as a sheet, test strip, or polystyrene beads. Those of skill in the art will know many other suitable carriers for binding antibodies or antigens, or can ascertain such by use of routine experimentation.

[0106] "Eukaryotic cells" include all kingdoms of life except Monera. They can be easily identified by a membrane-bound nucleus. Animals, plants, fungi, and protists are eukaryotes or organisms whose cells are organized into complex structures by internal membranes and a cytoskeleton. The most characteristic membrane-bound structure is the nucleus. For example, from eukaryotic hosts including yeast, higher plants, insects, and mammalian cells, or from prokaryotic cells, as described above. Non-limiting examples include monkeys, cows, pigs, mice, rats, birds, reptiles, and humans.

[0107] "Prokaryotic cells", which usually lack a nucleus or all other membrane-bound organelles, are divided into two ranges: bacteria and archaea. Furthermore, instead of having chromosomal DNA, the genetic information of these cells is in circular loops called plasmids. Bacterial cells are very small, roughly the size of an animal mitochondrion (approximately 1-2 μM in diameter and 10 μM in length). Prokaryotic cells feature three main shapes: rod-shaped, spherical, and spiral. Instead of going through a complex replication process like eukaryotes, bacterial cells divide by binary fission. Examples include, but are not limited to, bacillus bacteria, E. coli bacteria, and Salmonella bacteria.

[0108] The term "human antibody" as used herein is intended to include antibodies having variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the invention may include amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo). However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, such as a mouse, have been grafted onto human framework sequences. Thus, as used herein, the term "human antibody" refers to an antibody in which substantially all portions of the protein (e.g., CDRs, framework, CDRs ... L , C H Domain (for example, C H1, C H2 , C H3 A human antibody refers to an antibody in which the base sequence, hinge, (VL, VH) are substantially non-immunogenic in humans and have only minor sequence changes or mutations. Similarly, antibodies designated as primates (monkeys, baboons, chimpanzees, etc.), rodents (mouse, rats, rabbits, guinea pigs, hamsters, etc.) and other mammals indicate antibodies specific to such species, subgenus, genus, subfamily, family. Furthermore, chimeric antibodies include any combination of the above. Such changes or mutations optionally and preferably retain or reduce immunogenicity in humans or other species compared to the unmodified antibody. Thus, a human antibody is distinct from a chimeric or humanized antibody. It is noted that a human antibody can be produced by non-human animals or prokaryotic or eukaryotic cells capable of expressing functionally rearranged human immunoglobulin (e.g., heavy and / or light chain) genes. Furthermore, when the human antibody is a single-chain antibody, it can include a linker peptide not found in a natural human antibody. For example, an Fv can include a linker peptide, such as two to about eight glycine or other amino acid residues, connecting the variable region of the heavy chain and the variable region of the light chain. Such linker peptides are considered to be of human origin.

[0109] As used herein, a human antibody is "derived from" a particular germline sequence when the antibody is obtained from a system with human immunoglobulin sequences, for example, by immunizing a transgenic mouse carrying human immunoglobulin genes or by screening a human immunoglobulin gene library. A human antibody "derived from" a human germline immunoglobulin sequence can be identified as such by comparing the amino acid sequence of the human antibody to the amino acid sequence of a human germline immunoglobulin. The selected human antibody typically contains amino acid residues whose amino acid sequence is at least 90% identical to the amino acid sequence encoded by a human germline immunoglobulin gene and which identify the human antibody as human when compared to the amino acid sequence of a germline immunoglobulin of another species (e.g., a murine germline sequence). In certain cases, a human antibody may be at least 95%, or even at least 96%, 97%, 98%, or 99% identical to the amino acid sequence encoded by a germline immunoglobulin gene. Typically, a human antibody derived from a particular human germline sequence displays no more than 10 amino acid differences from the amino acid sequence encoded by the human germline immunoglobulin gene. In certain cases, a human antibody may display no more than 5, or even no more than 4, 3, 2, or 1 amino acid difference from the amino acid sequence encoded by the germline immunoglobulin gene.

[0110] "Human monoclonal antibody" refers to antibodies displaying a single binding specificity which have variable and constant regions derived from human germline immunoglobulin sequences. The term also contemplates recombinant human antibodies. Methods for making these antibodies are described herein.

[0111] The term "recombinant human antibody" as used herein includes all human antibodies prepared, expressed, produced or isolated by recombinant means, e.g., antibodies isolated from animals (e.g., mice) transgenic or transchromosomal for human immunoglobulin genes or hybridomas prepared therefrom, e.g., antibodies isolated from host cells transformed to express antibodies from transfectomas, antibodies isolated from recombinant combinatorial human antibody libraries, and antibodies prepared, expressed, produced or isolated by any other means, including splicing human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies may be subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used) such that the amino acid sequences of the VH and VL regions of the recombinant antibodies, while derived from and related to human germline VH and VL sequences, are sequences that may not naturally occur in vivo in the human antibody germline repertoire. Methods for making these antibodies are described herein.

[0112] As used herein, "isotype" refers to the antibody class that is encoded by the heavy chain constant region genes (eg, IgM or IgG1).

[0113] As used herein, the term "polyclonal antibody" or "polyclonal antibody composition" refers to a preparation of antibodies derived from different B-cell lines. These are a mixture of immunoglobulin molecules secreted against a specific antigen, each recognizing a different epitope.

[0114] The terms "monoclonal antibody" or "monoclonal antibody composition" as used herein refer to a preparation of antibody molecules of single molecular composition. A monoclonal antibody composition displays a single binding specificity and affinity for a particular epitope.

[0115] The term "label" as used herein refers to a directly or indirectly detectable compound or composition that is conjugated directly or indirectly to a composition to be detected, e.g., a protein such as a polynucleotide or an antibody, to make a "labeled" composition. The term also includes sequences conjugated to a polynucleotide that provide a signal when the inserted sequence is expressed, such as green fluorescent protein (GFP). The label may be detectable itself (e.g., radioisotope label or fluorescent label) or, in the case of an enzymatic label, may catalyze a chemical change in a substrate compound or composition that is detectable. The label may be suitable for small-scale detection or may be more suitable for high-throughput screening. Suitable labels thus include, but are not limited to, radioisotopes, fluorescent dyes, chemiluminescent compounds, dyes, and proteins, including enzymes. The label may be simply detected or quantified. A simply detected response generally includes a response that simply confirms its presence, while a quantified response generally includes a response that has a quantifiable (e.g., numerically reportable) value, such as intensity, polarization, and / or other properties. In luminescent or fluorescent assays, the detectable response may be generated directly, using a luminophore or fluorophore associated with the assay component actually involved in binding, or indirectly, using a luminophore or fluorophore associated with another (e.g., reporter or indicator) component.

[0116] Examples of luminescent labels that produce a signal include, but are not limited to, bioluminescence and chemiluminescence. A detectable luminescent response generally includes a change in or the occurrence of a luminescent signal. Suitable methods and luminophores for luminescent labeling of assay components are known in the art and are described, for example, in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th Edition). Examples of luminescent probes include, but are not limited to, aequorin and luciferase.

[0117] Examples of suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Handbook of Fluorescent Probes and Research Chemicals (6th Edition).

[0118] In another embodiment, the fluorescent label is functionalized to facilitate covalent binding with cellular components present in or on the surface of cells or tissues, such as cell surface markers.Suitable functional groups include, but are not limited to, isothiocyanate groups, amino groups, haloacetyl groups, maleimides, succinimidyl esters, and sulfonyl halides, all of which can be used to bind the fluorescent label with a second molecule.The choice of functional group of the fluorescent label depends on the binding site with either linker, drug, marker, or second labeling agent. II. Methods of the Invention One aspect of the invention relates to a method of preventing or reducing bleeding in a subject undergoing anticoagulant therapy by administering to the subject an effective amount of a factor Xa protein derivative. In one embodiment, the derivative has a modified active site and / or a modified Gla-domain and thus has reduced or no procoagulant activity. The derivative acts as an antidote and substantially neutralizes the anticoagulant activity of the inhibitor. In one embodiment, the derivative is either Gla-deficient or Gla-domain-less. The subject may be a mammal, or more particularly a human.

[0119] In another embodiment, the present invention is directed to a method of selectively binding and inhibiting an exogenously administered factor Xa inhibitor in a subject, comprising administering to the patient an effective amount of a factor Xa derivative as described above. The subject may be a cell or a mammal, such as a human.

[0120] Patients suitable for this treatment have previously undergone anticoagulant therapy, for example, have been administered one or more of the following anticoagulants, such as inhibitors of factor Xa. Examples of anticoagulants that are factor Xa inhibitors include, but are not limited to, fondaparinux, idraparinux, biotin-labeled idraparinux, enoxaparin, fragmin, NAP-5, rNAPc2, tissue factor pathway inhibitor, DX-9065a, YM-60828, YM-150, apixaban, rivaroxaban, PD-348292, otamixaban, DU-176b, LY517717, GSK913893, low molecular weight heparin, and betrixaban, or any combination thereof. Sources of various anticoagulants can be found throughout this specification.

[0121] In one embodiment, the derivative has a modified active site and / or a modified or removed Gla domain. In one embodiment, the factor Xa derivative has or does not exhibit procoagulant activity. In this embodiment, the derivative comprises at least amino acid residues 40-448, 45-448, and 46-448 of SEQ ID NO:3, or equivalents. In another embodiment, the derivative comprises at least amino acid residues 45-139 and 195-448 or 46-139 and 195-448 of SEQ ID NO:3, or equivalents.

[0122] In another embodiment of the invention, the fXa derivative retains the three-dimensional structure of the active site of the fXa protein. Information regarding the three-dimensional structure of des-Gla fXa can be found in Brandstetter, H. et al., J. Bio. Chem., 1996, 271:29988-29992.

[0123] In another embodiment of the invention, the fXa derivative may lack the Gla domain and either one of the two EGF domains. In another embodiment of the invention, the fXa derivative completely lacks the light chain. Other modifications of the heavy chain may include the catalytic domain of a related serine protease that can bind an inhibitor. The related serine protease has a catalytic domain that bears sufficient structural similarity to the fXa catalytic domain and thus can bind a small molecule fXa inhibitor. Examples of related serine proteases include, but are not limited to, mammalian proteases such as plasma kallikrein, thrombin and trypsin or the bacterial protease subtilisin. These derivatives further include modifications at amino acid residues corresponding to the active site serine (SER379) or aspartic acid (ASP282) residues described herein.

[0124] In some embodiments, the factor Xa protein with reduced procoagulant activity comprises a modified light chain, the modification being a substitution, addition or deletion of the Gla-domain to reduce phospholipid membrane binding of fXa. In some embodiments, the primary amino acid sequence of fXa is unchanged, but the side chains of certain amino acids are altered. An example of a modified Gla-domain that reduces phospholipid membrane binding of fXa comprises a polypeptide or protein having a primary amino acid sequence of SEQ ID NO: 3 or an equivalent thereof, in which at least one amino acid is substituted, added or deleted compared to the Gla-domain of wild-type human factor Xa protein. In some embodiments, the at least one amino acid substituted or deleted is gamma-carboxyglutamic acid (Gla). Gla residues are shown in SEQ ID NO: 3 at amino acid positions 6, 7, 14, 16, 19, 20, 25, 26, 29, 32 and 39. In some embodiments, the primary amino acid sequence of the antidote is identical to SEQ ID NO: 3 or an equivalent thereof, but is an uncarboxylated, undercarboxylated or decarboxylated factor Xa protein. In some embodiments, the antidote is des-Gla anhydro-fXa or des-Gla fX-S379A. In some embodiments, the factor Xa protein with reduced phospholipid membrane binding further comprises a modification or deletion of EGF1 and / or EGF2 (shown in FIG. 3 as amino acids 46-84 and 85-128, respectively), or a portion, i.e., a fragment, of the EGF1 and / or EGF2 domain. In some embodiments, the entire light chain or substantially the entire light chain is modified or removed. For example, a modified fXa protein with reduced phospholipid membrane binding may contain only the heavy chain, or the modified fXa may contain a heavy chain and a fragment of the light chain containing Cys132, the amino acid residue that forms a single disulfide bond with Cys302 of the heavy chain in SEQ ID NO:3. In some embodiments, the derivative comprises the amino acid sequence of SEQ ID NO:12. In some embodiments, the derivative is a double-chain polypeptide comprising SEQ ID NO:13. In other embodiments, the derivative is a polypeptide of SEQ ID NO:15.

[0125] In some embodiments, the factor Xa protein derivative comprises a modified heavy chain containing the catalytic domain of said factor Xa protein. In some embodiments, at least one amino acid substitution is present at one or more amino acid positions of fXa selected from the group consisting of Glu216, Glu218, Arg332, Arg347, Lys351, and Ser379 (Glu37, Glu39, Arg150, Arg165, Lys169, and Ser195 in chymotrypsin numbering, respectively) in SEQ ID NOs: 3 and 7. In some embodiments, the antidote is a factor Xa protein in which the active site serine (Ser379 in SEQ ID NOs: 3 and 7, Ser195 in chymotrypsin numbering) residue has been modified to dehydro-alanine or alanine. Such modifications may be made to wild-type fXa protein or any of the modified fXa proteins or fragments described above. For example, des-Gla anhydro-fXa, described in Example 1, in which the active site serine residue is replaced by dehydro-alanine, exhibited detoxifying activity.

[0126] In other embodiments, the derivatives have reduced interaction with ATIII, cofactors fV / fVa and fVIII / fVIIIa compared to wild-type or naturally occurring factor Xa. In some embodiments, at least one amino acid substitution is present at amino acid position Arg306, Glu310, Arg347, Lys351, Lys414 or Arg424 (Arg125, Glu129, Arg165, Lys169, Lys230 or Arg240 in chymotrypsin numbering, respectively) in SEQ ID NOs: 3 and 7. Such modifications may be made to wild-type fXa protein or any of the modified fXa proteins or fragments described above.

[0127] In other embodiments, the antidote is a protein that contains the amino acid sequence of a serine protease catalytic domain capable of mimicking the inhibitor-binding ability of fXa heavy chain. Such proteins can include mammalian proteases such as plasma kallikrein, thrombin, trypsin (or its bacterial homolog subtilisin) that have been recombinantly modified to lack serine protease activity capable of cleaving protein substrates, yet retain structural features of the active site cleft.

[0128] The present invention also provides pharmaceutical compositions containing one or more of the modified factor Xa derivatives and a pharma- ceutically acceptable carrier. The compositions are administered to a subject in need thereof in an amount that provides the desired benefit, i.e., reduction or cessation of bleeding. The compositions can be co-administered with any suitable agent or treatment that complements or enhances the activity of the factor Xa derivative. An example of such is a second agent that can extend the plasma half-life of the antidote. Examples of suitable second agents include, but are not limited to, anti-fXa antibodies that recognize the non-active site of the fXa heavy chain or the fXa derivative bound to α-2-macroglobulin. The formation of a complex between the fXa derivative and the second agent (non-active site antibody or α-2-macroglobulin) blocks the macromolecular interaction, but retains the ability for active site-dependent inhibitor binding. Examples of anti-fXa antibodies suitable for co-administration include, but are not limited to, those described in Yang YH et al., J. Immunol., 2006, 1;177(11):8219-25; Wilkens, M and Krishnaswamy, S., J. Bio. Chem., 2002, 277(11), 9366-9374; and Church WR et al., Blood, 1988, 72(6), 1911-1921.

[0129] In some embodiments, the factor Xa protein is modified by chemical, enzymatic or recombinant means. For example, the active site Ser379 may be chemically modified to dehydroalanine and the Gla domain may be enzymatically removed by chymotrypsin digestion, as described in Example 1. The modified fXa described herein may also be produced by recombinant means by modifying the sequence of a cDNA encoding wild-type fX (SEQ ID NO:2) to directly express a recombinant antidote (r-antidote), as further detailed in Example 7, or alternatively, a fX protein with the desired modification may be produced by recombinant means and then activated to modified fXa by an activator, such as a snake venom, e.g., Russell's viper venom, and a complex of fVIIa / tissue factor or fIXa / fVIIIa.

[0130] Subjects who may benefit from the administration of the compositions and associated methods described herein include those who have or are susceptible to clinically significant or non-hemorrhagic events. Examples of clinically significant bleeding events are selected from the group consisting of hemorrhage, bleeding into vital organs, bleeding requiring reoperation or new treatment procedures, and bleeding index of ≧2.0 with obvious bleeding. (Turpie AGG et al., NEJM, 2001, vol. 344:619-625.) Additionally, subjects may have or be susceptible to non-hemorrhagic events selected from the group consisting of persistent or recurrent and substantial epistaxis or bleeding that does not stop without treatment, rectal or urinary tract bleeding that does not rise to a level that requires treatment procedures, substantial hematomas at injection sites or other locations that occur spontaneously or following minor trauma, substantial blood loss that is greater than that normally associated with surgical procedures that do not require drainage, and bleeding that requires unplanned blood transfusion.

[0131] In some embodiments, the antidote is administered after administration of an overdose of an fXa inhibitor or prior to surgery that may expose the subject to the risk of bleeding.

[0132] In any of the methods described herein, it is understood that an effective amount of the derivative is administered to the subject, even if not necessarily explicitly stated. This amount can be empirically determined by the treating physician and will vary with the age, sex, weight and health of the subject. Additional factors that the treating physician should consider include, but are not limited to, the identity and / or amount of factor Xa inhibitor that may be administered, the method or manner of administering the antidote to the subject, the formulation of the antidote, and the therapeutic end point of the patient. Taking these variables into account, the skilled artisan will administer a therapeutically effective amount to the subject being treated. It is contemplated that a therapeutically effective amount of the antidote described herein sufficient to counteract or substantially neutralize the anticoagulant in the subject may contain the antidote at about 0.01 milligram of antidote per kilogram of subject body weight to 1 gram of antidote per kilogram of subject body weight. It is further contemplated that the antidote may be provided to a subject at a concentration ranging from about 10 nanomolar to about 100 micromolar, or from about 10 nanomolar to about 5 micromolar, or from about 100 nanomolar to about 2.5 micromolar.

[0133] The compositions can be administered in an amount in which the antidotes are effective to selectively recognize and bind, directly or indirectly, to the factor Xa inhibitor in the subject, and they can also be administered in an amount that substantially inhibits or substantially neutralizes an exogenously administered factor Xa inhibitor in the subject.

[0134] In yet another aspect, the present invention relates to a pharmaceutical composition for reversing or neutralizing the anticoagulant activity of a factor Xa inhibitor administered to a subject, comprising administering an effective amount of an antidote to the factor Xa inhibitor and a pharma- ceutically acceptable carrier, with the proviso that the antidote is not plasma-derived factor VIIa, recombinant factor VIIa, fresh frozen plasma, prothrombin complex concentrate, or whole blood.

[0135] In some embodiments, the antidote is any one of the antidotes described above. In some embodiments, the antidote is conjugated to a moiety that can extend the circulating half-life of the antidote. In some embodiments, the moiety is selected from the group consisting of polyethylene glycol, acyl groups, liposomes, carrier proteins, artificial phospholipid membranes, and nanoparticles. For example, the inactive site lysine or cysteine ​​residues of the fXa derivatives described herein can be chemically modified to attach a polyethylene glycol molecule. Other methods provided in Werle, M. & Bernkop-Schnurch, A. Strategies to Improve Plasma Half Life Time of Peptide and Protein Drugs, Amino Acids, 2006, 30(4):351-367 can be used to extend the plasma half-life of the antidote of the present invention.

[0136] In another embodiment of the invention, the half-life of the fXa derivative is improved by coupling an antidote to the Fc carrier domain. In one embodiment, the antidote is coupled to an immunoglobulin peptide portion or an Fc fragment, such as an IgG1 fragment. In one embodiment, a chimeric protein comprising an fXa derivative and an immunoglobulin peptide portion is contemplated. In yet another embodiment, the fXa derivative and the immunoglobulin peptide are coupled by a chemical reaction, such as a disulfide bond, using the heavy and kappa light chain constant regions of human IgG.

[0137] In some embodiments, the pharmaceutical composition further comprises an agent capable of extending the plasma half-life of the antidote. In another aspect, the pharmaceutical composition is co-formulated with an agent capable of extending the plasma half-life of the antidote. In some embodiments, the co-administered or co-formulated agent is an anti-fXa antibody that recognizes the non-active site of fXa or a fXa derivative bound to alpha-2-macroglobulin. III. Antidotes Factor Xa derivative One aspect of the present invention is the use of fXa derivatives, such as Gla-domain deleted fXa or des-Gla fXa, as safe and effective antidotes to substantially neutralize the activity of inhibitors of coagulation fXa to prevent or stop bleeding. It is contemplated that the antidotes of the present invention are effective to reverse the anticoagulant effects of fXa inhibitors, particularly active site-directed small molecule inhibitors.

[0138] It is contemplated that an antidote to a fXa inhibitor has reduced or no procoagulant activity, but can bind to a fXa inhibitor. It is contemplated that such limited activity allows the antidote to be administered at a higher level than circulating wild-type fXa. Certain fXa derivatives, such as des-Gla fXa and Gla-deficient fXa, are suitable antidotes of the present invention. In addition to having reduced or attenuated procoagulant activity, the antidote of the present invention should also be substantially non-immunogenic to a subject. An antidote may contain a combination of two or more of the above mutations and / or modifications. Furthermore, any of the above fXa derivatives may be administered alone or in combination with each other.

[0139] Factor Xa is a serine protease in the blood coagulation pathway responsible for converting prothrombin to thrombin. It is generated from inactive factor X upon activation by either intrinsic Xase (complex formed by factor IXa and its cofactor factor VIIIa) or extrinsic Xase (complex formed by factor VIIa and its cofactor tissue factor). Activated fX (fXa) can further undergo autocatalytic cleavage at the C-terminus of its heavy chain, converting fXaα to the secondary form fXaβ (Jesty, J et al., J. Biol. Chem., 1975, 250(12):4497-4504). Both fXaα and fXaβ are suitable substances of the present invention. fXa itself converts prothrombin at a slow rate that is insufficient to support clotting. Cofactor Ca 2+Only when fXa forms a prothrombinase complex with phospholipids and factor Va can it activate prothrombin at a rate fast enough to support coagulation (Skogen, WF et al., J. Biol. Chem., 1984, 259(4):2306-10). The complex is formed by the Ca2+ complex in the Gla domain of fXa. 2+ It requires a link between a negatively charged phospholipid and a γ-carboxyglutamic acid residue via a bridge.

[0140] Thus, the Gla domain does not contain the active site of fXa, but allows fXa to form the prothrombinase complex through the γ-carboxyglutamic acid residue. This is demonstrated by the selective removal of the fXa Gla-domain by chymotrypsin digestion (see FIG. 7 and Example 1). Clotting assays were performed with fXa during a time course of Gla-domain cleavage by chymotrypsin digestion. It has been reported that a reconstituted prothrombinase complex containing no Gla-domain fXa, fVa, phospholipids and calcium ions generates thrombin at a significantly reduced rate (0.5% of the product was produced compared to the control complex containing native fXa) (Skogen et al. J. Biol. Chem., 1984, 259(4):2306-10). As shown in FIG. 7, the activity of fXa in the formation of blood clots was partially reduced after 15 min of digestion of fXa with chymotrypsin, and after 30 min of digestion, activity was completely lost. Thus, undercarboxylated or decarboxylated fXa, which lacks the appropriate γ-carboxyglutamic acid residues required for calcium ion-dependent membrane binding, was found to be incapable of assembly of membrane-dependent coagulation complexes and did not support blood clotting (Mann, KG et al., Blood, 1990, 76:1-16).

[0141] It has also been established that Gla-domain-deleted fXa can bind active site-specific inhibitors of fXa. (Brandstetter, H. et al., J. Bio. Chem., 1996, 271:29988-29992). Crystal structure analysis of a small molecule fXa inhibitor bound to des-Gla human fXa has been reported, providing a structural description of the active site cleft (Brandstetter, J. Bio. Chem., 1996, 271:29988-29992 and Roehrig, J. Med. Chem., 2005, 48(19):5900-8). Figure 8 shows that des-Gla anhydro-fXa exhibits a binding affinity of 0.319 nM with the fXa inhibitor betrixaban, which is comparable to that of native fXa.

[0142] It has now been discovered that des-Gla fXa, and other fXa derivatives with reduced procoagulant activity but capable of fXa inhibitor binding, can be used as antidotes to fXa inhibitors. As shown in Figure 9, des-Gla anhydro-fXa showed complete reversal of the anticoagulant activity of betrixaban at a concentration of 680 nM. As detailed in Example 2, thrombin generation was initiated by adding a TF-containing reagent (Innovin), and thus is an indicator of clotting factor function in the extrinsic coagulation pathway. It is also demonstrated in Examples 9-13 that the recombinant antidote is useful for reversing a variety of anticoagulants.

[0143] Clotting prolongation assays using activated partial thromboplastin time (aPTT) reagents (Actin FS), which determine the function of coagulation factors in the intrinsic coagulation pathway, also show that des-Gla anhydro-fXa possesses antidote activity. Figure 10 shows the dose-responsive antidote effect of des-Gla anhydro-fXa against betrixaban at 250 nM, which is fully reversed at 600 nM. Figure 11 shows that des-Gla anhydro-fXa can also reverse the anticoagulant activity of another fXa inhibitor, enoxaparin. Figure 12 shows that des-Gla anhydro-fXa does not exhibit significant antidote activity against the direct thrombin inhibitor argatroban. Thus, des-Gla anhydro-fXa is a selective antidote for fXa inhibitors and can restore fXa procoagulant activity initiated by either the extrinsic or intrinsic pathways.

[0144] Furthermore, the antidote activity of des-Gla anhydro-fXa was demonstrated by an aPTT prolongation assay measured with a conventional coagulation timer. As shown in FIG. 13, des-Gla anhydro-fXa by itself had no effect on the aPTT of control plasma at the highest concentration tested (2576 nM). Betrixaban at 400 nM prolonged the aPTT by more than two-fold. This anticoagulant effect of betrixaban was reversed in a dose-responsive manner by des-Gla anhydro-fXa, restoring the aPTT to near-normal levels in control plasma at antidote concentrations higher than 1610 nM.

[0145] It is contemplated that further truncations of the fXa light chain, for example further deletions of the EGF1 domain, the EGF1+EGF2 domains, or fragments thereof, and inactive fXa having only the heavy chain may be useful antidotes in the present invention.

[0146] Gla-domain-deficient fXa does not support normal coagulation under physiologically relevant concentrations. However, the protein has the ability to cleave many substrates and cause coagulation at higher concentrations. For example, Skogen et al. (Skogen, WF et al., J. Biol. Chem., 1984, vol. 259(4):2306-10) showed that bovine des-Gla fXa has about 0.5-1.0% of the prothrombinase complex activity compared to wild-type fXa. Thus, modifications that further reduce or completely eliminate the procoagulant activity of fXa derivatives are contemplated by the methods of the present invention. Such modifications can be, for example, within the catalytic domain of fXa.

[0147] Several methods are contemplated for modifying the catalytic domain in the fXa heavy chain to reduce its procoagulant activity. For example, the active site residue S379 of fXa (as shown in SEQ ID NO: 7) can be selectively replaced with dehydro-alanine (see Example 1) or alanine (see Example 6) to reduce or eliminate the procoagulant activity. It is also known that complex formation between fXa and a reagent that targets the non-active site of fXa can block the macromolecule binding capacity of fXa, thus reducing its procoagulant activity while retaining the small molecule binding capacity in the active site. The non-active site targeting reagent includes, but is not limited to, monoclonal antibodies that target a region removed from the active site (Wilkens, M and Krishnaswamy, S, J. Bio. Chem., 2002, vol. 277(11), pp. 9366-9374), or alpha-2-macroglobulin. It is known that α-2-macroglobulin-serine protease complexes with trypsin, thrombin, or fXa, etc., can bind small molecule substrates (Kurolwa, K. et al., Clin. Chem., 1989, 35(11), 2169-2172).

[0148] It is also known that inactive fXa, which has modifications only in its heavy chain while its light chain remains unchanged, acts as an inhibitor of prothrombinase, since it interferes with the procoagulant activity of normal fXa, as shown in FIG. 6 (Hollenbach, S. et al., Thromb. Haemost., 1994, 71(3), 357-62). Thus, in one embodiment, the fXa derivative has modifications in its light and heavy chains. These modifications have been found to reduce or eliminate both the procoagulant and anticoagulant activities, while preserving the inhibitor binding ability of the fXa derivative.

[0149] Several methods can be used to generate Gla-domain-deficient fXa derivatives or other fXa derivatives described herein. For example, the Gla-domain can be completely removed by chymotrypsin cleavage to generate Gla-domain-free fXa. Alternatively, Gla-domain-free fX can be generated by chymotrypsin cleavage of native fX. The Gla-domain-free fX can then be converted to Gla-domain-free fXa by a fX activator. fX can be isolated from the plasma of the same or different species as the subject to be treated. For example, bovine fX has been shown to be functional in human plasma assays. Examples of fX activators include, but are not limited to, snake venoms such as Russell's viper venom, and complexes of fVIIa / tissue factor or fIXa / fVIIIa. Such means are known to those skilled in the art. For example, Rudolph AE et al. report recombinant fXa produced from recombinant factor X (fX) with a single substitution of Arg347 by glutamine (fXR347N) (Biochem., 2000, vol. 39(11):2861-2867). In one embodiment, the fXa derivative produced from a non-human source is non-immunogenic or substantially non-immunogenic. Example 7 also provides a method for producing a recombinant antidote having the amino acid sequence of SEQ ID NO:12.

[0150] Alternatively, fXa derivatives may be purified from human plasma or produced by recombinant DNA methods, where the appropriate gene for the fXa derivative is expressed in a suitable host organism. Expression and purification of recombinant fXa has been reported by several groups, see, for example, Larson, PJ et al., Biochem., 1998, 37:5029-5038, and Camire, RM et al., Biochem., 2000, 39:14322-14329 for the production of recombinant fXa; Wolf, DL et al., J. Bio. Chem., 1991, 266(21):13726-13730 for the production of recombinant fXa. Modified fXa may be prepared according to these procedures using a generally modified cDNA having a nucleotide sequence encoding the desired fXa mutant. Example 6 provides further details of directly expressing a Gla-domainless fXa-S379 mutant that has functional activity as an antidote.

[0151] It is contemplated that active site mutated or modified fXa lacking the Gla-domain, such as undercarboxylated fXa, may also be useful as a fXa inhibitor antidote. Undercarboxylated fXa may be prepared by recombinant means, by withholding vitamin K derivatives during protein expression (which are necessary for post-translational modification to form Gla residues), or by adding vitamin K antagonists such as warfarin during tissue culture. Decarboxylated fXa may be prepared by heating (Bajaj P., J. Biol. Chem., 1982, 257(7):3726-3731) or proteolytic digestion with chymotrypsin (Morita T. et al., J. Biol. Chem., 1986, 261(9):4015-4023). Antidotes may also be made in prokaryotic systems followed by in vitro refolding or construction of the fXa inhibitor binding site.

[0152] Gla residues can also be chemically modified to remove the carboxyl group responsible for calcium ion-dependent membrane binding. For example, the carboxyl group on Gla residues can be selectively removed under decarboxylating conditions or capped, for example, by esterification or amination. It is desirable that such esterification or amination be resistant to hydrolysis in vivo so that the modified fXa is not readily converted to active fXa, which can cause thrombosis.

[0153] Other mutants or derivatives of fXa may also be useful antidotes of the present invention. In one embodiment, the present invention encompasses the use of the mutants described in Peter J. Larson et al., Biochem., 1998, 37:5029-5038, as fXa inhibitor antidotes.

[0154] In another embodiment, the invention encompasses the use of catalytically inactive fXa mutants to prepare fXa inhibitor antidotes. See, e.g., Sinha, U. et al., Protein Antidote. Expression and Purif., 1992, 3:518-524, and mutants with chemical modifications as described by rXai, Nogami et al., J. Biol. Chem., 1999, 274(43):31000-7, such as dehydro-alanine (anhydro fXa). FXa in which the active site serine (Ser379 in the fX numbering shown in SEQ ID NO:7, and Ser195 in the chymotrypsin numbering) is replaced with an alanine (fXa-S379A in the fX numbering, or fXa-S195A in the chymotrypsin numbering), which eliminates the procoagulant activity, may also be used as a fXa inhibitor antidote. The present invention also contemplates fXa derivatives in which the active site serine residue is irreversibly acylated, but which are still capable of binding small molecule inhibitors. FXa reversibly acylated at the active site serine has been reported by Wolf et al., Blood, 1995, 86(11):4153-7. However, such reversible acylation can result in a time-dependent production of active fXa, leading to excess active fXa over time. The deacylation rate can be reduced by a strategy similar to that described by Lin PH et al., Thrombosis Res., 1997, 88(4):365-372. For example, fXa molecules acylated at Ser379 (Ser195 in chymotrypsin numbering) with 4-methoxybenzyl and 3-bromo-4-methoxybenzyl groups regain less than 50% of their original activity when incubated at 37° C. for 4 hours in a buffer having a pH of 7.5.

[0155] One embodiment is directed to the use of fXa derivatives having mutations at fXa residues known to be important for the interaction of fXa with cofactors fV / fVa. Such residues include, but are not limited to, Arg306, Glu310, Arg347, Lys351, or Lys414 (SEQ ID NOs: 3 and 7, which correspond to Arg125, Glu129, Arg165, Lys169, Lys230 in the chymotrypsin numbering). Examples of such mutants are reported in Rudolph, AE et al., J. Bio. Chem., 2001, 276:5123-5128. Additionally, mutations at fXa residues known to be important for fVIII / fVIIIa interaction, such as Arg424 in SEQ ID NOs: 3 and 7 (Arg240 in the chymotrypsin numbering), may also be used as fXa inhibitor antidotes. Examples of such mutants are described in Nogami, K. et al., J. Biol. Chem., 2004, vol. 279(32):33104-33113.

[0156] Other modifications of fXa active site residues or residues known to be important for serine protease interactions may also result in useful antidotes of the invention, examples being the replacement of Glu216, Glu218, and Arg332 (Glu37, Glu39, and Arg150, respectively, in chymotrypsin numbering) in SEQ ID NOs: 3 and 7 with other amino acid residues.

[0157] In one embodiment, the residual procoagulant activity of the antidote, as assessed by amidolytic substrate cleavage assay, is <1%, preferably <0.1%, more preferably <0.05% of native fXa from human plasma, e.g., when active site Ser379 (S195 in chymotrypsin numbering) is replaced by an alanine residue, recombinant fXa-S379A has no measurable procoagulant activity as measured by clotting assays.

[0158] The present invention further relates to nucleic acid sequences, in particular DNA sequences, which code for the above-mentioned fXa derivatives. These can be easily determined by back-translating the polypeptide sequence into the corresponding DNA sequence according to the genetic code. The codons preferably used are those which provide good expression in the required host organism. The nucleic acid sequences can be prepared by site-directed mutagenesis starting from the natural fXa gene sequence or by complete DNA synthesis. Polypeptides of the Invention In certain aspects, the present invention relates to an isolated polypeptide comprising the amino acid sequence of SEQ ID NO: 12, 13 or 15. The present invention also includes polypeptides having at least 80% homology to SEQ ID NO: 12, 13 or 15.

[0159] Polypeptides comprising the amino acid sequence of the invention can be prepared by expressing a polynucleotide encoding the polypeptide sequence of the invention in a suitable host cell. This can be accomplished by recombinant DNA technology methods known to those skilled in the art. Thus, the invention also provides a method for recombinantly producing the polypeptide of the invention in a eukaryotic or prokaryotic host cell. The proteins and polypeptides of the invention can also be obtained by chemical synthesis using a commercially available automated peptide synthesizer, such as those manufactured by Perkin Elmer / Applied Biosystems, Inc., Model 430A or 431A, Foster City, California, USA. The synthesized protein or polypeptide can be precipitated and further purified, for example, by high performance liquid chromatography (HPLC). Thus, the invention also provides a method for chemically synthesizing the protein of the invention by providing a sequence of the protein and reagents, such as amino acids and enzymes, and linking the amino acids together in the proper orientation and linear sequence.

[0160] It is known to those skilled in the art that modifications can be made to any peptide to provide altered properties. The polypeptides of the present invention can be modified to include unnatural amino acids. Thus, the peptides can include D-amino acids, combinations of D- and L-amino acids, and various "designer" amino acids (e.g., β-methyl amino acids, C-α-methyl amino acids, and N-α-methyl amino acids, etc.) to impart special properties to the peptide. Furthermore, by assigning specific amino acids at specific coupling steps, peptides with α-helices, β-turns, β-sheets, α-turns, and cyclic peptides can be created. In general, α-helical or random secondary structures are considered to be preferred.

[0161] In further embodiments, subunits of the polypeptide are selected that confer useful chemical and structural properties. For example, peptides containing D-amino acids can be resistant in vivo to proteases specific for L-amino acids. Modified compounds with D-amino acids can be synthesized with amino acids aligned in reverse order to produce the peptides of the invention as retro-inverso peptides. Furthermore, the present invention contemplates the preparation of peptides with better defined structural properties, as well as the use of peptidomimetics and peptidomimetic bonds, such as ester bonds, to prepare peptides with novel properties. In another embodiment, peptides incorporating reduced peptide bonds can be produced, i.e., R1-CH2NH-R2, where R1 and R2 are amino acid residues or sequences. Reduced peptide bonds can be introduced as dipeptide subunits. Such molecules are resistant to peptide bond hydrolysis, e.g., protease activity. Such molecules provide ligands with unique functions and activities, such as extended in vivo half-life due to metabolic degradation or resistance to protease activity. Moreover, it is well known that peptides that are constrained in certain systems exhibit enhanced functional activity (Hruby (1982) Life Sciences 31:189-199 and Hruby et al. (1990) Biochem J. 268:249-262). The present invention provides methods for producing constrained peptides that incorporate random sequences at all other positions.

[0162] The following non-classical amino acids may be incorporated into the peptides of the invention to introduce specific conformational motifs: 1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Kazrnierski et al. (1991) J. Am. Chem. Soc. 113:2275-2283); (2S,3S)-methyl-phenylalanine, (2S,3R)-methyl-phenylalanine, (2R,3S)-methyl-phenylalanine and (2R,3R)-methyl-phenylalanine (Kazmierski and Hruby (1991) Tetrahedron Lett. 32(41):5769-5772); 2-aminotetrahydronaphthalene-2-carboxylic acid (Landis (1989) Ph.D. Thesis, University of Arizona); hydroxy-1,2,3,4-tetrahydroisoquinoline-3-carboxylate (Miyake et al. (1989) J. Takeda Res. Labs. 43:53-76) histidine isoquinoline carboxylic acid (Zechel et al. (1991) Int. J. Pep. Protein Res. 38(2):131-138); and HIC (histidine cyclic urea), (Dharanipragada et al. (1993) Int. J. Pep. Protein Res. 42(1):68-77) and (Dharanipragada et al. (1992) Acta. Crystallogr. C. 48:1239-1241).

[0163] The following amino acid analogs and peptidomimetics may be incorporated into peptides to induce or favor particular secondary structures: LL-Acp (LL-3-amino-2-propenidone-6-carboxylic acid), β-turn-inducing dipeptide analogs (Kemp et al. (1985) J. Org. Chem. 50:5834-5838); β-sheet-inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5081-5082); β-turn-inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:5057-5060); α-helix-inducing analogs (Kemp et al. (1988) Tetrahedron Lett. 29:4935-4938); α-turn-inducing analogs (Kemp et al. (1989) J. Org. Chem., 54:109-115); analogues provided by the following references: Nagai and Sato (1985) Tetrahedron Lett. 26:647-650; and DiMaio et al. (1989) J. Chem. Soc. Perkin Trans. p. 1687; Gly-Ala turn analogs (Kahn et al. (1989) Tetrahedron Lett. 30:2317); amide bond isosteres (Clones et al. (1988) Tetrahedron Lett. 29:3853-3856); tetrazoles (Zabrocki et al. (1988) J. Am. Chem. Soc. 110:5875-5880); DTC (Samanen et al. (1990) Int. J. Protein Pep. Res. 35:501-509); and analogs taught in Olson et al. (1990) J. Am. Chem. Sci. 112:323-333 and Garvey et al. (1990) J. Org. Chem. 56:436. Conformationally restricted mimetics of β-turns and β-bulges, and peptides containing them, are described in U.S. Patent No. 5,440,013, issued Aug. 8, 1995 to Kahn.

[0164] Those skilled in the art know that any peptide can be modified by replacing one or more amino acids with one or more functionally equivalent amino acids that do not change the biological function of the peptide. In one embodiment, an amino acid is replaced by an amino acid that possesses similar intrinsic properties, including but not limited to hydrophobicity, size, or charge. Methods to use to determine the appropriate amino acid to replace which amino acid with which are known to those skilled in the art. Non-limiting examples include those described in Dahoff et al. (1978), Atlas of of Protein Sequence and Structure, Vol. 5, Supplement 2 (ed. MO Dayhoff), pp. 345-352. empirical substitution models described by the National Biomedical Research Foundation, Washington DC; the Dahoff matrix (PAM matrix, as described in Dahoff et al. (1978), supra, or the JTT matrix, as described by Jones et al. (1992) Comput. Appl. Biosci. 8:275-282 and Gonnet et al. (1992) Science 256:1443-1145; empirical models described by Adach and Hasegawa (1996) J. Mol. Evol. 42:459-468; the block substitution matrix (BLOSUM), as described by Henikoff and Henikoff (1992) Proc. Natl. Acad. Sci. USA 89:10915-10919; Nei (1987) Molecular Evolutionary Genetics. Columbia University Press, New York, 1987. Examples of methods that can be used include the Poisson model described by York; and the maximum likelihood (ML) method described by Muller et al. (2002) Mol. Biol. Evol. 19:8-13. Polypeptide Conjugates The polypeptides and polypeptide complexes of the present invention can be used in various formulations that may vary depending on the intended use. For example, one or more can be covalently or non-covalently bound (complexed) to various other molecules whose properties may vary depending on the specific purpose. For example, the peptides of the present invention can be covalently or non-covalently complexed to macromolecular carriers, including but not limited to natural and synthetic polymers, proteins, polysaccharides, polypeptides (amino acids), polyvinyl alcohol, polyvinylpyrrolidone, and lipids. The peptides can be conjugated with fatty acids for incorporation into liposomes, see U.S. Pat. No. 5,837,249. The peptides of the present invention can be covalently or non-covalently complexed to solid carriers, a variety of which are known in the art and described herein. The antigenic peptide epitopes of the present invention can be associated with antigen-presenting matrices, such as MHC complexes, with or without costimulatory molecules.

[0165] Examples of protein carriers include, but are not limited to, superantigens, serum albumin, tetanus toxoid, ovalbumin, thyroglobulin, myoglobulin, and immunoglobulins.

[0166] The peptide-protein carrier polymer may be formed using conventional cross-linking agents such as carbodiimides. Examples of carbodiimides are 1-cyclohexyl-3-(2-morpholinyl-(4-ethyl)carbodiimide (CMC), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) and 1-ethyl-3-(4-azonia-4-dimethylpentyl)carbodiimide.

[0167] Examples of other suitable cross-linking agents are cyanogen bromide, glutaraldehyde and succinic anhydride.In general, any of several homobifunctional agents may be used, including homobifunctional aldehydes, homobifunctional epoxides, homobifunctional imide esters, homobifunctional N-hydroxysuccinimide esters, homobifunctional maleimides, homobifunctional alkyl halides, homobifunctional pyridyl disulfides, homobifunctional aryl halides, homobifunctional hydrazides, homobifunctional diazonium derivatives and homobifunctional photoreactive compounds.Also included are heterobifunctional compounds, such as compounds with amine reactive groups and sulfhydryl reactive groups, compounds with amine reactive groups and photoreactive groups, and compounds with carbonyl reactive groups and sulfhydryl reactive groups.

[0168] Specific examples of such homobifunctional crosslinkers include the bifunctional N-hydroxysuccinimide esters dithiobis(succinimidyl propionate), disuccinimidyl suberate, and disuccinimidyl tartrate; the bifunctional imidoesters dimethyl adipimidate, dimethyl pimelimidate, and dimethyl suberimidate; the bifunctional sulfhydryl-reactive crosslinkers 1,4-di-[3'-(2'-pyridyldithio)propionamido]butane, bismaleimidohexane, and bis-N-maleimido-1,8-octane; the bifunctional aryl halides 1,5-difluoro-2,4-dinitrobenzene and 4,4'-difluoro-3,3'-dinitrophenyl sulfone; and bifunctional photoreactive agents such as bis-[b-(4-azidosalicylamido)ethyl]disulfide. difunctional aldehydes such as formaldehyde, malondialdehyde, succinaldehyde, glutaraldehyde, and adipaldehyde; difunctional epoxides such as 1,4-butanodiol diglycidyl ether; difunctional hydrazides such as adipic dihydrazide, carbohydrazide, and succinic dihydrazide; difunctional diazoniums such as o-tolidine, diazotized and bis-diazotized benzidine; difunctional alkyl halides such as N1N'-ethylene-bis(iodoacetamide), N1N'-hexamethylene-bis(iodoacetamide), N1N'-undecamethylene-bis(iodoacetamide), as well as benzyl halides and halo mustards such as a1a'-diiodo-p-xylenesulfonic acid and tri(2-chloroethyl)amine, respectively.

[0169] Examples of common heterobifunctional cross-linking agents that can be used to effect protein and peptide conjugation include, but are not limited to, SMCC (succinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate), MBS (m-maleimidobenzoyl-N-hydroxysuccinimide ester), SIAB (N-succinimidyl(4-iodoactyl)aminobenzoate), SMPB (succinimidyl-4-(p-maleimidomethyl)cyclohexane-1-carboxylate), and SMCC (succinimidyl-4-(p-maleimidomethyl)cyclohexane-1-carboxylate). dophenyl)butyrate), GMBS (N-(γ-maleimidobutyryloxy)succinimide ester), MPBH (4-(4-N-maleimidophenyl)butyric acid hydrazide), M2C2H (4-(N-maleimidomethyl)cyclohexane-1-carboxyl-hydrazide), SMPT (succinimidyloxycarbonyl-α-methyl-α-(2-pyridyldithio)toluene), and SPDP (N-succinimidyl 3-(2-pyridyldithio)propionate).

[0170] Cross-linking can be achieved by coupling carbonyl groups to amine or hydrazide groups by reductive amination.

[0171] The peptides of the invention may also be formulated as non-covalent attachment of monomers through ionic, adsorptive, or biospecific interactions. Complexes of peptides with highly positively or negatively charged molecules may be made by salt bridge formation in a low ionic strength environment such as deionized water. Larger complexes can be made with charged polymers such as poly-(L-glutamic acid) or poly-(L-lysine), which contain multiple negative and positive charges, respectively. Adsorption of peptides may be made to surfaces such as particulate latex beads or other hydrophobic polymers, forming non-covalently associated peptide-superantigen complexes that effectively mimic cross-linked or chemically polymerized proteins. Finally, peptides may be non-covalently attached by using biospecific interactions between other molecules. For example, exploitation of the strong affinity of biotin for proteins such as avidin or streptavidin or their derivatives can be used to form peptide complexes. These biotin-binding proteins contain four binding sites that can interact with biotin in solution or can be covalently attached to another molecule. (See Wilchek (1988) Anal. Biochem. 171:1-32.) Peptides can be modified to carry a biotin group using common biotinylation reagents such as the N-hydroxysuccinimidyl ester of D-biotin (NHS-biotin), which reacts with available amine groups on proteins. Biotin-labeled peptides can then be incubated with avidin or streptavidin to form large complexes. The molecular mass of such polymers can be tuned by carefully controlling the molar ratio of biotin-labeled peptide to avidin or streptavidin.

[0172] The present application also provides peptides and polypeptides described herein conjugated with a label, e.g., a fluorescent or bioluminescent label, for use in diagnostic methods. For example, detectably labeled peptides and polypeptides can be bound to a column and used for antibody detection and purification. Suitable fluorescent labels include, but are not limited to, fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methylcoumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue™, and Texas Red. Other suitable optical dyes are described in Haugland, Richard P. (1996) Molecular Probes Handbook.

[0173] The polypeptide of the present invention can also be combined with various liquid phase carriers, such as sterile or aqueous solutions, pharma- ceutical acceptable carriers, suspensions and emulsions. Examples of non-aqueous solvents include propyl ethylene glycol, polyethylene glycol and vegetable oils. When used to prepare antibodies, carriers can also include adjuvants that are useful for non-specifically enhancing specific immune responses. Those skilled in the art can easily determine whether an adjuvant is necessary and select one. However, for illustrative purposes only, suitable adjuvants include, but are not limited to, complete Freund's adjuvant, incomplete Freund's adjuvant and mineral salts. host cell Also provided is a host cell comprising one or more of the polypeptides of the present invention. In one embodiment, the polypeptide is expressed and present on the cell surface (extracellular). Suitable cells containing the polypeptide of the present invention include prokaryotic and eukaryotic cells, including but not limited to bacterial cells, yeast cells, insect cells, animal cells, mammalian cells, murine cells, rat cells, sheep cells, monkey cells and human cells. Examples of bacterial cells include Escherichia coli, Salmonella enterica and Streptococcus gordonii. Cells can be purchased from commercial vendors such as the American Type Culture Collection (ATCC, Rockville, MD, USA) or cultured from isolates using methods known in the art. Examples of suitable eukaryotic cells include, but are not limited to, 293T HEK cells, and hamster cell lines CHO, BHK-21; NIH3T3, NS0, a murine cell line called C127, monkey cell lines COS, Vero; and human cell lines HeLa, PER.C6 (sold by Crucell) U-937 and Hep G2. Non-limiting examples of insect cells include Spodoptera frugiperda. Examples of yeasts useful for expression include, but are not limited to, Saccharomyces, Schizosaccharomyces, Hansenula, Candida, Torulopsis, Yarrowia, or Pichia. See, for example, U.S. Patent Nos. 4,812,405; 4,818,700; 4,929,555; 5,736,383; 5,955,349; 5,888,768 and 6,258,559.

[0174] In addition to species specificity, the cells may be of any particular tissue type, such as somatic or embryonic stem cells, such as neural cells or stem cells that may or may not differentiate into neural cells, e.g., embryonic stem cells, adipose stem cells, neural stem cells and hematopoietic stem cells. The stem cells may be of human or animal origin, such as mammalian origin. Isolated Polynucleotides and Compositions The present invention also provides complementary polynucleotides to the above-identified sequences or their complements. Complementarity can be determined by conventional hybridization under moderate or high stringency conditions. The term polynucleotide as used herein contemplates DNA and RNA as well as modified nucleotides. For example, the present invention also provides antisense polynucleotide strands, such as antisense RNA to these sequences or their complements.

[0175] Also provided are polynucleotides encoding polypeptides that are substantially homologous and biologically equivalent to the polypeptides and polypeptide complexes of the invention. Substantially homologous and biologically equivalent are intended to mean those with various degrees of homology, e.g., at least 65%, alternatively at least 70%, alternatively at least 75%, alternatively at least 80%, alternatively at least 85%, alternatively at least 90%, alternatively at least 95%, alternatively at least 97% homology as defined above, and that encode polypeptides that have biological activity binding to factor Xa inhibitors and do not assemble to form prothrombinase complexes, as described herein. Although not necessarily explicitly stated, it will be understood that the embodiments relating to substantially homologous polypeptides and polynucleotides are directed to each aspect of the invention, e.g., polypeptides, polynucleotides and antibodies.

[0176] The polynucleotides of the present invention can be replicated using conventional recombinant techniques. Alternatively, the polynucleotides can be replicated using PCR techniques. PCR is the subject of U.S. Patent Nos. 4,683,195; 4,800,159; 4,754,065; and 4,683,202, and is described in PCR: The Polymerase Chain Reaction (Mullis et al., eds., Birkhauser Press, Boston (1994)) and references cited therein. Furthermore, one skilled in the art can replicate DNA using the sequences provided herein and a commercially available DNA synthesizer. Thus, the present invention also provides a method of obtaining the polynucleotides of the present invention by providing the linear sequence of the polynucleotide, suitable primer molecules, compounds such as enzymes, and instructions for replicating it and chemically replicating or linking the nucleotides in the appropriate orientation to obtain the polynucleotide. In another embodiment, these polynucleotides are further isolated. Furthermore, one skilled in the art can operably link the polynucleotide with a regulatory sequence for its expression in a host cell. The polynucleotide and the regulatory sequence are inserted into a host cell (prokaryotic or eukaryotic) for replication and amplification. The DNA thus amplified can be isolated from the cell by methods well known to those skilled in the art. The method of obtaining the polynucleotide by this method and the polynucleotide thus obtained are further provided herein.

[0177] RNA can be obtained by first inserting a DNA polynucleotide into a suitable prokaryotic or eukaryotic host cell. The DNA can be inserted by any suitable method, for example, by using a suitable gene delivery vehicle (e.g., liposome, plasmid or vector) or by electroporation. When the cell replicates and the DNA is transcribed into RNA, the RNA can then be isolated using methods well known to those skilled in the art, for example, those described in Sambrook and Russell (2001) supra. For example, mRNA can be isolated by extraction with a nucleic acid binding resin following the instructions provided by the manufacturer, using various lytic enzymes or chemical solutions, following the procedures described in Sambrook and Russell (2001) supra.

[0178] In one embodiment, RNA is small interfering RNA, also known as siRNA.The method of preparing and screening interfering RNA and the method of selecting for the ability to block polynucleotide expression are known in the art, and non-limiting examples are given below.These interfering RNA are provided by the present invention.

[0179] The siRNA sequence can be designed by obtaining the target mRNA sequence and determining the appropriate siRNA complementary sequence. The siRNA of the present invention is designed to interact with the target sequence, which means that they are sufficiently complementary to hybridize with the target sequence. The siRNA can be 100% identical to the target sequence. However, the homology of the siRNA sequence to the target sequence can be less than 100%, as long as the siRNA can hybridize with the target sequence. Thus, for example, the siRNA molecule can be at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% identical to the target sequence or the complement of the target sequence. Thus, siRNA molecules with insertions, deletions or single point mutations compared to the target can also be used. It is recommended to make several different siRNA sequences for one target mRNA to allow screening of the optimal target sequence. A homology search, such as a BLAST search, should be performed to ensure that the siRNA sequence does not contain homology to any known mammalian genes.

[0180] In general, it is preferred that the target sequence be located at least 100-200 nucleotides from the AUG start codon and at least 50-100 nucleotides away from the stop codon of the target mRNA (Duxbury (2004) J. Surgical Res. 117:339-344).

[0181] Researchers have determined specific characteristics shared by siRNA molecules that effectively silence their target genes (Duxbury (2004) J. Surgical Res. 117:339-344; Ui-Tei et al. (2004) Nucl. Acids Res. 32:936-48). As a general guideline, siRNAs that include one or more of the following criteria are particularly useful for gene silencing in mammalian cells: 45-55% GC ratio, no run of more than 9 G / C residues, G / C at the 5' end of the sense strand; A / U at the 5' end of the antisense strand; and at least 5 A / U residues in the first 7 bases at the 5' end of the antisense strand.

[0182] siRNAs are generally about 10 to about 30 nucleotides in length. For example, siRNAs can be 10 to 30 nucleotides in length, 12 to 28 nucleotides in length, 15 to 25 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 23 nucleotides in length. When an siRNA contains two strands of different lengths, the longer of the strands indicates the length of the siRNA. In this situation, the unpaired nucleotides of the longer strand form an overhang.

[0183] The term siRNA includes short hairpin RNA (shRNA). shRNA comprises an RNA strand that forms a single stem-loop structure, where the stem is made up of complementary sense and antisense strands that comprise a double-stranded siRNA, and the loop is a linker of various sizes. The stem structure of shRNA is generally about 10 to about 30 nucleotides in length. For example, the stem can be 10 to 30 nucleotides in length, 12 to 28 nucleotides in length, 15 to 25 nucleotides in length, 19 to 23 nucleotides in length, or 21 to 23 nucleotides in length.

[0184] Tools to assist in siRNA design are readily publicly available. For example, a computer-based siRNA design tool is available on the Internet at www.dharmacon.com, accessed November 26, 2007. Synthesis of dsRNA and siRNA dsRNA and siRNA have been described in Micura (2002) Agnes Chem. They can be chemically or enzymatically synthesized in vitro, as described in Int. Ed. Emgl., 41:2265-2269; Betz (2003) Promega Notes, 85:15-18; and Paddison and Hannon (2002) Cancer Cell., 2:17-23. Chemical synthesis can be performed by manual or automated methods, both of which are well known in the art, as described in Micura (2002), supra. siRNAs can also be expressed endogenously inside cells in the form of shRNAs, as described in Yu et al. (2002) Proc. Natl. Acad. Sci. USA, 99:6047-6052; and McManus et al. (2002) RNA, 8:842-850. Endogenous expression has been achieved using plasmid-based expression systems using small nuclear RNA promoters, such as RNA polymerase III U6 or H1, or RNA polymerase II U1, as described in Brummelkamp et al. (2002) Science 296:550-553 (2002); and Novarino et al. (2004) J. Neurosci. 24:5322-5330.

[0185] In vitro enzymatic dsRNA and siRNA synthesis can be performed using an RNA polymerase-mediated process as described in Fire et al. (1998) Nature 391:806-811; Donze and Picard (2002) Nucl. Acids Res. 30(10):e46; Yu et al. (2002); and Shim et al. (2002) J. Biol. Chem. 277:30413-30416 to generate individual sense and antisense strands that are annealed in vitro before delivery into the cells of choice. Several manufacturers (Promega, Ambion, New England Biolabs, and Stragene) produce transcription kits useful for performing in vitro synthesis.

[0186] In vitro synthesis of siRNA can be accomplished, for example, by using as DNA templates a pair of short double-stranded oligonucleotides containing T7 RNA polymerase promoters upstream of the sense and antisense RNA sequences. Each oligonucleotide of the duplex is a separate template for the synthesis of one strand of the siRNA. Then, as described in Protocols and Applications, Chapter 2: RNA interference, Promega Corporation, (2005) as described, separate short RNA strands are synthesized and annealed to form siRNA.

[0187] In vitro synthesis of dsRNA can be achieved, for example, by using a T7 RNA polymerase promoter at the 5' end of either strand of the DNA target sequence. This is achieved using separate DNA templates, each containing the target sequence in a different orientation relative to the T7 promoter, that are transcribed in two separate reactions. The resulting transcripts are mixed and annealed after transcription. The DNA templates used in this reaction can be generated by PCR or by using two linearized plasmid templates, each containing a T7 polymerase promoter at a different end of the target sequence. Protocols and Applications, Chapter 2: RNA interference, Promega Corporation, (2005).

[0188] To express the proteins described herein, delivery of the nucleic acid sequence encoding the gene of interest can be delivered by several techniques. Examples include viral techniques (e.g., retroviral vectors, adenoviral vectors, adeno-associated viral vectors, alphaviral vectors, etc.) and non-viral techniques (e.g., DNA / liposome complexes, micelles, and targeted viral protein-DNA complexes) as described herein. After entering the cells of interest, expression of the transgene can be under the control of a ubiquitous promoter (e.g., EF-1α) or a tissue-specific promoter (e.g., calcium calmodulin kinase 2 (CaMKI) promoter, NSE promoter, and human Thy-1 promoter). Alternatively, expression levels can be controlled by using an inducible promoter system (e.g., Tet on / off promoter) as described in Wiznerowicz et al. (2005) Stem Cells, 77:8957-8961.

[0189] Non-limiting examples of promoters include, but are not limited to, the cytomegalovirus (CMV) promoter (Kaplitt et al. (1994) Nat. Genet. 8:148-154), the CMV / human β3-globin promoter (Mandel et al. (1998) J. Neurosci. 18:4271-4284), the NCX1 promoter, the αMHC promoter, the MLC2v promoter, the GFAP promoter (Xu et al. (2001) Gene Ther. 8:1323-1332), the 1.8 kb neuron-specific enolase (NSE) promoter (Klein et al. (1998) Exp. Neurol., 150:183-194), chicken beta actin (CBA) promoter (Miyazaki (1989) Gene, 79:269-277) and beta-glucuronidase (GUSB) promoter (Shipley et al. (1991) Genetics, 10:1009-1018), human serum albumin promoter, alpha-1-antitrypsin promoter. To improve expression, other regulatory elements may be further operably linked to the transgene, such as, for example, the woodchuck hepatitis virus postregulatory element (WPRE) (Donello et al. (1998) J. Virol., 72:5085-5092) or the bovine growth hormone (BGH) polyadenylation site.

[0190] Also provided herein is a polynucleotide probe or primer comprising at least 10, or at least 17, or at least 20, or at least 50, or at least 75, or at least 100 polynucleotides encoding SEQ ID NOs: 12-15, or their complements. Suitable probes and primers are as described above. It is known in the art that a "perfectly matched" probe is not necessary for specific hybridization. Minor changes in the probe sequence, achieved by substitution, deletion, or insertion of a small number of bases, do not affect the specificity of hybridization. Generally, up to 20% base pair mismatch (when optimally aligned) is tolerated. Probes useful for detecting the aforementioned mRNAs are at least about 80% identical to a homologous region of comparable size contained in the previously identified sequence (identified above) corresponding to the previously characterized polynucleotide of the present invention. Alternatively, the probe is 85% identical to the corresponding gene sequence after alignment of the homologous region, which further exhibits 90% identity, or even at least 95% identity.

[0191] These probes can be used in radioactive assays (e.g., Southern and Northern blot analyses) to detect or monitor expression of the polynucleotides or polypeptides of the invention. The probes can also be attached to solid supports, such as chips, or arrays for use in high throughput screening assays to detect expression of genes corresponding to one or more polynucleotides of the invention.

[0192] The polynucleotides and fragments of the polynucleotides of the present invention can also serve as primers for detecting genes or gene transcripts expressed in neural cells, for example, to confirm the transduction of the polynucleotide into the host cell. In this context, amplification means any method using a primer-dependent polymerase that can replicate the target sequence with reasonable fidelity. Amplification can be performed by natural or recombinant DNA polymerases such as T7 DNA polymerase, Klenow fragment of E.coli DNA polymerase, and reverse transcriptase. The length of the primers is the same as that identified for the probe above.

[0193] The present invention further provides an isolated polynucleotide of the present invention operably linked to an RNA transcription promoter and other regulatory sequences for DNA or RNA replication and / or transient or stable expression. As used herein, the term "operably linked" means that the promoter is located in such a manner that it directs the transcription of RNA from the DNA molecule. Examples of such promoters are SP6, T4 and T7. In certain embodiments, a cell-specific promoter is used for cell-specific expression of the inserted polynucleotide. Vectors containing a promoter or promoter / enhancer, a stop codon and a selection marker sequence, and a cloning site that allows an inserted piece of DNA to be operably linked to the promoter are well known in the art and are commercially available. For general methods and cloning strategies, see Gene Expression Technology (Goeddel, ed., Academic Press, Inc. (1991)) and references cited therein, as well as Vectors: Essential Data Series (Gacesa and Ramji, eds., John Wiley & Sons, NY (1994)), which contains references to maps, functional characteristics, commercial suppliers, and GenEMBL accession numbers of a variety of suitable vectors. Preferably, these vectors are capable of transcribing RNA in vitro or in vivo.

[0194] Expression vectors containing these nucleic acids are useful for obtaining host vector systems for producing proteins and polypeptides. It is implied that these expression vectors must be replicable in the host organism, either as episomes or as an integrated part of chromosomal DNA. Suitable expression vectors include plasmids and viral vectors, including adenoviruses, adeno-associated viruses, retroviruses, cosmids, and the like. Adenovirus vectors are particularly useful for introducing genes into tissues in vivo, due to their high expression levels and efficient cell transformation both in vitro and in vivo. When the nucleic acid is inserted into a suitable host cell, e.g., a prokaryotic or eukaryotic cell, and the host cell replicates, the protein can be produced recombinantly. Suitable host cells depend on the vector and may include mammalian cells, animal cells, human cells, monkey cells, insect cells, yeast cells, and bacterial cells, as described above, and are constructed using well-known methods. See Sambrook and Russell (2001), supra. In addition to using viral vectors to insert exogenous nucleic acid into cells, nucleic acid can be inserted into host cells by methods well known in the art, such as transformation for bacterial cells, transfection using calcium phosphate precipitation for mammalian cells, DEAE-dextran, electroporation, or microinjection, see Sambrook and Russell (2001), supra.

[0195] The present invention also provides a delivery vehicle suitable for delivering the polynucleotide of the present invention into cells (whether in vivo, ex vivo, or in vitro).The polynucleotide of the present invention can be contained in a gene delivery vehicle, a cloning vector, or an expression vector.These vectors (especially expression vectors) can then be manipulated to take any of several forms, which can, for example, facilitate delivery and / or entry into cells.

[0196] These isolated host cells containing the polynucleotides of the invention are useful for the recombinant replication of the polynucleotides and for the recombinant production and high throughput screening of peptides.

[0197] The polynucleotides of the present invention can be conjugated with a detectable label or combined with a carrier, such as a solid support or a pharma- ceutically acceptable carrier. Suitable solid supports are those described above and carrying a suitable label. Methods for attaching a label to a polynucleotide are known to those skilled in the art. See Sambrook and Russell (2001), supra. therapeutic antibody compositions The present invention also provides antibodies capable of specifically forming complexes with the proteins or polypeptides of the present invention, which are useful in the therapeutic methods of the present invention. The term "antibody" includes polyclonal and monoclonal antibodies, antibody fragments, and derivatives thereof (as described above). Antibodies include, but are not limited to, mouse, rat, and rabbit or human antibodies. Antibodies can be produced in cell culture, in phages, or in a variety of animals, including, but not limited to, cows, rabbits, goats, mice, rats, hamsters, guinea pigs, sheep, dogs, cats, monkeys, chimpanzees, apes, and the like. Antibodies are also useful for identifying and purifying therapeutic polypeptides.

[0198] The present invention also provides an antibody-peptide complex comprising the above-mentioned antibody and a polypeptide that specifically binds to the antibody. In one embodiment, the polypeptide is a polypeptide against which the antibody was raised. In one embodiment, the antibody-peptide complex is an isolated complex. In a further embodiment, the antibody of the complex is, but is not limited to, a polyclonal antibody, a monoclonal antibody, a humanized antibody, or an antibody derivative as described herein. Either or both of the antibody or peptide of the antibody-peptide complex can be detectably labeled. In one embodiment, the antibody-peptide complex of the present invention can be used as a control or reference sample in a diagnostic or screening assay.

[0199] The polyclonal antibodies of the present invention can be produced using conventional techniques known in the art and well documented in the literature. There are several methods of producing polyclonal antibodies. For example, polyclonal antibodies are typically produced by immunization of a suitable mammal, such as, but not limited to, chicken, goat, guinea pig, hamster, horse, mouse, rat, and rabbit. An antigen is injected into the mammal, which induces the production of IgG immunoglobulin specific to the antigen by B lymphocytes. The IgG is purified from the serum of the mammal. Variations of this method include alterations in adjuvants, route and site of administration, injection volume per site, and number of sites per animal for optimal production and humane treatment of the animal. For example, adjuvants are typically used to improve or enhance the immune response to an antigen. Most adjuvants provide an antigen depot at the injection site, which allows for the slow release of antigen into the draining lymph nodes. Other adjuvants include surfactants that facilitate the concentration of protein antigen molecules and immunostimulatory molecules over a large surface area. Non-limiting examples of adjuvants for producing polyclonal antibodies include Freund's adjuvant, Ribi adjuvant system, and Titermax.Polyclonal antibodies can be produced using the methods described in U.S. Patent Nos. 7,279,559; 7,119,179; 7,060,800; 6,709,659; 6,656,746; 6,322,788; 5,686,073; and 5,670,153.

[0200] The monoclonal antibodies of the invention can be produced using conventional hybridoma technology, which is known in the art and well documented in the literature. For example, hybridomas can be produced using suitable immortal cell lines (e.g., myeloma cell lines, including but not limited to, Sp2 / 0, Sp2 / 0-AG14, NSO, NS1, NS2, AE-1, L.5, >243, P3X63Ag8.653, Sp2SA3, Sp2MAI, Sp2SS1, Sp2SA5, U397, MLA144, ACT IV, MOLT4, DA-1, JURKAT, WEHI, K-562, COS, RAJI, NIH 3T3, HL-60, MLA144, NAMAIWA, NEURO, etc.). 2A, CHO, PerC.6, YB2 / O, etc., or heterologous myelomas, fusion products thereof, or any cells or fusion cells derived therefrom, or any other suitable cell line known in the art (see, e.g., www.atcc.org, accessed Nov. 26, 2007, www.lifetech.com., etc.), antibody producing cells, such as, but not limited to, isolated or cloned spleen, peripheral blood, lymph, tonsils, or other immune or B cell containing cells, or constant or variable heavy or light chain expression cells, The variant or framework or CDR sequences are produced by fusing with any other cell that expresses the variant or framework or CDR sequences as endogenous or heterologous nucleic acid, recombinant or endogenous, viral, bacterial, algal, prokaryotic, amphibian, insect, reptile, fish, mammalian, rodent, equine, ovine, caprine, ovine, primate, eukaryotic, genomic DNA, cDNA, rDNA, mitochondrial DNA or RNA, chloroplast DNA or RNA, hnRNA, mRNA, tRNA, single-stranded, double-stranded or triple-stranded, hybridized, or the like, or any combination thereof, as endogenous or heterologous nucleic acid. Antibody producing cells can also be obtained from the peripheral blood, or preferably the spleen or lymph nodes, of humans or other suitable animals immunized with the antigen of interest. Any other suitable host cell can also be used to express heterologous or endogenous nucleic acid encoding the antibody, specified fragment or variant thereof, of the present invention.The fused cells (hybridomas) or recombinant cells can be isolated using selective culture conditions or other appropriate known methods and cloned by limiting dilution or cell sorting, or other known methods.

[0201] In one embodiment, the antibodies described herein can be produced using the multiple antigen peptide (MAP) system. The MAP system utilizes a peptidyl core of three or seven radially branched lysine residues onto which antigenic peptides of interest can be constructed using standard solid-phase chemistry. The lysine core results in MAPs carrying approximately 4-8 copies of peptide epitopes, depending on the inner core, which generally accounts for less than 10% of the total molecular weight. The MAP system does not require a carrier protein for conjugation. High molar ratios and dense packing of multiple copies of antigenic epitopes within MAPs have been shown to generate strong immunogenic responses. This method is described in U.S. Patent No. 5,229,490, which is incorporated herein by reference in its entirety.

[0202] Other suitable methods for producing or isolating antibodies of the required specificity can be used, including but not limited to peptide or protein libraries (e.g., but not limited to, bacteriophage, ribosomal, oligonucleotide, RNA, cDNA, etc., display libraries; e.g., Cambridge Antibody The present invention includes the selection of recombinant antibodies using methods known in the art, including those available from a variety of commercial vendors, such as BioSystems Technologies (Cambridgeshire, UK), MorphoSys (Martinsreid / Planegg, Del.), Biovation (Aberdeen, Scotland), and BioInvent (Lund, Sweden). See U.S. Patent Nos. 4,704,692; 5,723,323; 5,763,192; 5,814,476; 5,817,483; 5,824,514; and 5,976,862. An alternative method relies on immunization of transgenic animals (e.g., SCID mice, Nguyen et al. (1977) Microbiol. Immunol. 41:901-907 (1997); Sandhu et al. (1996) Crit. Rev. Biotechnol. 16:95-118; Eren et al. (1998) Immunol. 93:154-161) that are capable of generating a repertoire of human antibodies as known in the art and / or described herein.Such techniques include, but are not limited to, ribosome display (Hanes et al. (1997) Proc. Natl. Acad. Sci. USA 94:4937-4942; Hanes et al. (1998) Proc. Natl. Acad. Sci. USA 95:14130-14135); single cell antibody production techniques (e.g., the selected lymphocyte antibody method ("SLAM") (U.S. Patent No. 5,627,052; Wen et al. (1987) J. Immunol. 17:887-892; Babcook et al., Proc. Natl. Acad. Sci. USA (1996) 93:7843-7848); gel microdroplets and flow cytometry (Powell et al. (1990) Biotechnol. 8:333-337; One Cell Systems, (Cambridge, Mass).; Gray et al. (1995) J. Imm. Meth. 182:155-163; and Kenny et al. (1995) Bio. Technol. 13:787-790); B cell selection (Steenbakkers et al. (1994) Molec. Biol. Reports 19:125-134).

[0203] Antibody derivatives of the invention can also be prepared by delivering a polynucleotide encoding an antibody of the invention to a suitable host to provide a transgenic animal or mammal, such as a goat, cow, horse, sheep, etc., which produces such antibodies in its milk. These methods are known in the art and are described in U.S. Patent Nos. 5,827,690; 5,849,992; 4,873,316; 5,849,992; 5,994,616; 5,565,362; and 5,304,489.

[0204] The term "antibody derivatives" includes post-translational modifications to the linear polypeptide sequence of an antibody or fragment. For example, U.S. Patent No. 6,602,684 B1 describes methods for producing modified glycoforms of antibodies, including whole antibody molecules, antibody fragments, or fusion proteins, that contain a region equivalent to the Fc region of an immunoglobulin and have enhanced Fc-mediated cytotoxicity, and the glycoproteins so produced.

[0205] Antibody derivatives can also be prepared by providing transgenic plants and cultured plant cells (such as, but not limited to, tobacco, corn, and duckweed) that deliver polynucleotides of the invention to produce such antibodies, specified portions or variants in plant parts or cells cultured therefrom. For example, Cramer et al. (1999) Curr. Top. Microbol. Immunol. 240:95-118 and references cited therein describe the generation of transgenic tobacco leaves expressing large amounts of recombinant proteins, for example using inducible promoters. Transgenic corn has been used to express mammalian proteins at commercial production levels with biological activity equivalent to those produced in other recombinant systems or purified from natural sources. See, for example, Hood et al. (1999) Adv. Exp. Med. Biol. 464:127-147 and references cited therein. Antibody derivatives, including antibody fragments such as single chain antibodies (scFv), have also been produced in large quantities from transgenic plant seeds, including tobacco species and potato tubers. See, for example, Conrad et al. (1998) Plant Mol. Biol., 38:101-109 and references cited therein. Thus, the antibodies of the present invention can also be produced using transgenic plants according to known methods.

[0206] Antibody derivatives can also be produced by the addition of exogenous sequences to, for example, alter immunogenicity or to decrease, enhance or alter binding affinity, association rate, dissociation rate, avidity, specificity, half-life, or any other suitable characteristic. Generally, some or all of the non-human or human CDR sequences are maintained, while the non-human sequences of the variable and constant regions are replaced with human or other amino acids.

[0207] Generally, CDR residues are directly and most substantially involved in influencing antigen binding.The humanization or engineering of the antibody of the present invention can be carried out by any known method, such as, but not limited to, those described in U.S. Patent Nos. 5,723,323; 5,976,862; 5,824,514; 5,817,483; 5,814,476; 5,763,192; 5,723,323; 5,766,886; 5,714,352; 6,204,023; 6,180,370; 5,693,762; 5,530,101; 5,585,089; 5,225,539; and 4,816,567.

[0208] Techniques for producing partially or fully human antibodies are known in the art, and any such technique may be used. According to one embodiment, fully human antibody sequences are produced in transgenic mice engineered to express human heavy and light chain antibody genes. Several strains of such transgenic mice have been produced that can produce different classes of antibodies. B cells from transgenic mice producing the desired antibodies can be fused to generate hybridoma cell lines that continuously produce the desired antibodies. (See, e.g., Russell et al. (2000) Infection and Immunity, April 2000: 1820-1826; Gallo et al. (2000) European J. of Immun., 30: 534-540; Green (1999) J. of Immun. Methods, 231: 11-23; Yang et al. (1999A) J. of Leukocyte Biology, 66: 401-410; Yang (1999B) Cancer Research, 59(6): 1236-1243; Jakobovits. (1998) Advanced Drug Delivery Reviews, 31: 33-42; Green and Jakobovits (1998) J. Exp. Med., 188(3):483-495; Jakobovits (1998) Exp. Opin. Invest. Drugs, 7(4):607-614; Tsuda et al. (1997) Genomics, 42:413-421; Sherman-Gold (1997) Genetic Engineering News, 17(14); Mendez et al. (1997) Nature Genetics, 15:146-156; Jakobovits (1996) Weir's Handbook of Experimental Immunology, The Integrated Immune System, IV, 194.1-194.7; Jakobovits (1995) Current Opinion in Biotechnology, 6:561-566; Mendez et al. (1995) Genomics, 26:294-307; Jakobovits (1994) Current (See, e.g., Biology, 4(8):761-763; Arbones et al. (1994) Immunity, 1(4):247-260; Jakobovits (1993) Nature, 362(6417):255-258; Jakobovits et al. (1993) Proc. Natl. Acad. Sci. USA, 90(6):2551-2555; and U.S. Patent No. 6,075,181.) The antibodies of the present invention can also be modified to produce chimeric antibodies, which are antibodies in which the various domains of the heavy and light chains of the antibody are encoded by DNA from multiple species. See, for example, U.S. Patent No. 4,816,567.

[0209] Alternatively, the antibodies of the present invention can be modified to create veneered antibodies. A veneered antibody is one in which the outer amino acid residues of an antibody of one species are appropriately replaced or "veneered" with those of a second species to render the antibody of the first species non-immunogenic in the second species, thus reducing the immunogenicity of the antibody. Since the antigenicity of a protein depends primarily on the nature of its surface, the immunogenicity of the antibody can be reduced by replacing exposed residues that are different from those normally found in the antibodies of another mammalian species. This judicious replacement of the outer residues should have little or no effect on the inner domains or inter-domain contacts. Thus, the ligand binding properties should not be affected as a result of the changes limited to the variable region framework residues. This process is called "veneered" because it only changes the outer surface or epidermis of the antibody, leaving the supporting residues undisturbed.

[0210] The "veneering" procedure utilizes available sequence data of human antibody variable domains compiled by Kabat et al. (1987) Sequences of Proteins of Immunological Interest, 4th ed., Bethesda, Md., National Institutes of Health, updates to this database, as well as other accessible U.S. and foreign databases (both nucleic acid and protein). Non-limiting examples of methods used to generate veneered antibodies include those described in EP 519596; U.S. Patent No. 6,797,492; and Padlan et al. (1991) Mol. Immunol. 28(4-5):489-498.

[0211] The term "antibody derivative" also includes "bispecific antibodies," which are small antibody fragments with two antigen-binding sites, the fragments comprising a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain. (See, e.g., EP 404,097; WO 93 / 11161; and Hollinger et al., (1993) Proc. Natl. Acad. Sci. USA, 90:6444-6448.) By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with complementary domains on another chain to form two antigen-binding sites. (See also U.S. Pat. No. 6,632,926 to Chen et al., which discloses antibody mutants in which one or more amino acids have been inserted into the hypervariable region of the parent antibody and which have a binding affinity for a target antigen that is at least about two-fold stronger than the binding affinity of the parent antibody for the antigen.) The term "antibody derivatives" further includes "linear antibodies." Procedures for making linear antibodies are known in the art and are described in Zapata et al. (1995) Protein Eng., 8(10):1057-1062. Briefly, these antibodies comprise a pair of tandem Fd segments (V H -C H 1-VH-C H 1). Linear antibodies can be bispecific or monospecific.

[0212] The antibodies of the invention can be recovered and purified from recombinant cell culture by known methods, including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography, and lectin chromatography. High performance liquid chromatography ("HPLC") can also be used for purification.

[0213] Antibodies of the present invention include natural purified products, the products of chemical synthetic procedures, and, as discussed above, products produced by recombinant techniques from, for example, yeast, higher plant, insect and mammalian cells, or eukaryotic hosts including prokaryotic cells.

[0214] If the monoclonal antibody being tested binds to a protein or polypeptide, the antibody being tested and the antibody provided by the hybridoma of the present invention are equivalent. It is also possible, without undue experimentation, to determine whether an antibody has the same specificity as the monoclonal antibody of the present invention by determining whether the antibody being tested prevents the binding of the monoclonal antibody of the present invention to the protein or polypeptide with which the monoclonal antibody is normally reactive. If the antibody being tested competes with the monoclonal antibody of the present invention, as indicated by a decrease in binding by the monoclonal antibody of the present invention, it is likely that the two antibodies bind to the same or closely related epitopes. Alternatively, the monoclonal antibody of the present invention can be pre-incubated with the protein with which it is normally reactive to determine whether the ability of the monoclonal antibody being tested to bind to the antigen is inhibited. If the monoclonal antibody being tested is inhibited, it probably has the same or closely related epitope specificity as the monoclonal antibody of the present invention.

[0215] The term "antibody" is also intended to include antibodies of all isotypes. A particular isotype of monoclonal antibody can be prepared directly by selection from the initial fusion or can be prepared secondarily by sib selection techniques to isolate class-switched variants using the procedures described in Steplewski et al. (1985) Proc. Natl. Acad. Sci. USA 82:8653 or Spira et al. (1984) J. Immunol. Methods 74:307 from parent hybridomas secreting monoclonal antibodies of different isotypes.

[0216] Isolation of other hybridomas secreting monoclonal antibodies having the specificity of the monoclonal antibodies of the present invention can also be accomplished by one skilled in the art by producing anti-idiotypic antibodies. Herlyn et al. (1986) Science 232:100. Anti-idiotypic antibodies are antibodies which recognize unique determinants present on the monoclonal antibody produced by the hybridoma of interest.

[0217] Idiotypic identity between the monoclonal antibodies of two hybridomas demonstrates that the two monoclonal antibodies are identical in their recognition of the same epitopic determinant. Therefore, by using antibodies against epitopic determinants on a monoclonal antibody, it is possible to identify other hybridomas expressing monoclonal antibodies of the same epitopic specificity.

[0218] It is also possible to use anti-idiotype technology to produce monoclonal antibodies that mimic epitopes. For example, anti-idiotype monoclonal antibodies made against a first monoclonal antibody have a binding domain in the hypervariable region that is the mirror image of the epitope bound by the first monoclonal antibody. Therefore, in this case, anti-idiotype monoclonal antibodies can be used for immunization to produce these antibodies.

[0219] In some aspects of the present invention, it is useful to detectably or therapeutically label the antibody. Suitable labels are described above. Methods for conjugating antibodies with these agents are known in the art. For illustrative purposes only, the antibody can be labeled with a detectable moiety such as a radioactive atom, a chromophore, a fluorophore, etc. Such labeled antibodies can be used in diagnostic techniques, in vivo or in isolated test samples.

[0220] Coupling of an antibody to a low molecular weight hapten can increase the sensitivity of the antibody in an assay. The hapten can then be specifically detected by a second reaction. For example, it is common to use haptens such as biotin, which reacts with avidin, or dinitrophenol, pyridoxal, and fluorescein, which can react with specific anti-hapten antibodies. See Harlow and Lane (1988) supra.

[0221] Antibodies can be labeled with detectable moieties, such as radioactive atoms, chromophores, fluorophores, etc. Such labeled antibodies can be used in diagnostic techniques, in vivo or in isolated test samples. Antibodies can also be conjugated to pharmaceuticals, such as, for example, chemotherapeutic drugs or toxins. These can be linked to cytokines, ligands, or other antibodies. Suitable agents for coupling to antibodies to achieve antitumor effects include cytokines, such as interleukin 2 (IL-2) and tumor necrosis factor (TNF); photosensitizers for use in photodynamic therapy, including aluminum (III) phthalocyanine tetrasulfonate, hematoporphyrin, and phthalocyanine; iodine-131 ( 131 I), Yttrium-90( 90 Y), Bismuth-212( 212 Bi), Bismuth-213( 213 Bi), Technetium-99m ( 99m Tc), rhenium-186( 186 Re), and rhenium-188 ( 188radionuclides, such as Re; antibiotics, such as doxorubicin, adriamycin, daunorubicin, methotrexate, daunomycin, neocarzinostatin, and carboplatin; bacterial, plant, and other toxins, such as diphtheria toxin, Pseudomonas aeruginosa exotoxin A, Staphylococcus aeruginosa enterotoxin A, abrin-A toxin, ricin A (deglycosylated ricin A and native ricin A), TGF-α toxin, cytotoxin from the Chinese cobra (naja naja atra), and gelonin (a plant toxin); restrictocin (a ribosome-inactivating protein produced by Aspergillus restrictus), saporin (Saponaria officinalis), and RNase; tyrosine kinase inhibitors; ly207702 (a difluorinated purine nucleoside); liposomes containing anticystants (e.g., antisense oligonucleotides, plasmids encoding toxins, methotrexate, etc.); and other antibodies and antibody fragments such as F(ab).

[0222] The antibody of the present invention can also be bound to many different carriers.Thus, the present invention also provides a composition containing the antibody and another active or inactive substance.Examples of well-known carriers include glass, polystyrene, polypropylene, polyethylene, dextran, nylon, amylase, natural and modified cellulose, polyacrylamide, agarose and magnetite.The nature of the carrier can be either soluble or insoluble for the purpose of the present invention.Those skilled in the art will know other suitable carriers for binding monoclonal antibodies, or can ascertain them using routine experimentation. IV.Treatment The present invention relates to a therapeutic method for preventing or reducing bleeding in subjects undergoing anticoagulant therapy. It is contemplated that the antidotes or derivatives of the present invention may be short-term drugs for use in elective or emergency situations that can safely and specifically neutralize the conventional anticoagulant properties of fXa inhibitors without causing adverse hemodynamic side effects or exacerbation of the proliferative vascular response to injury.

[0223] In one embodiment, a therapeutically effective amount of an antidote exhibits a high therapeutic index. Therapeutic index is the dose ratio between toxic and therapeutic effects, and is the LD 50 and ED 50 It can be expressed as the ratio between LD 50 is the dose that is lethal to 50% of the population, and ED 50 LD is the dose that is therapeutically effective in 50% of the population. 50 and ED 50 is determined by standard pharmaceutical procedures in animal cell cultures or experimental animals. The antidote or derivative of the present invention may be administered once or several times when neutralization of the effect of fXa inhibitors present in the plasma of a subject is required. Preferably, the antidote of the present invention is sufficient when administered in a single dose.

[0224] It is contemplated that the typical dose of the antidote of the present invention will depend on the actual clinical situation and the inhibitor concentration in plasma. In in vitro assays, such as clinical coagulation assays such as thrombin generation, aPTT, PT and ACT, a therapeutically effective amount of the antidote is predicted to produce a correction of ex vivo coagulation activity of 10% or more. In vitro assays have shown that an antidote / inhibitor ratio higher than 1.0 shows a reversal effect. The maximum plasma concentration of the antidote is predicted to be in the micromolar range, possibly between 10 micromolar or less.

[0225] In clinical situations, one of the criteria in determining the effectiveness of an antidote is that it produces some change in the actual measurement of bleeding. In clinical trials, classification of major bleeding includes fatal bleeding, bleeding into vital organs (intracranial, intraocular, retroperitoneal, spinal cord, pericardium), any bleeding requiring reoperation or a new therapeutic procedure (e.g., aspiration of an operated knee, chest tube insertion for hemothorax, endoscopic electrocoagulation, etc.), or a bleeding index of ≥ 2.0 if associated with overt bleeding. The bleeding index is defined as the number of units of packed red blood cells or whole blood transfused + the hemoglobin value before the onset of bleeding - the hemoglobin value (grams per deciliter) after the bleeding has stabilized.

[0226] Another criterion for the effectiveness of an antidote in a clinical setting is the reduction of clinically significant non-massive bleeding. This classification of bleeding includes bleeding that is not massive but requires more clinical attention than usual, including nosebleeds that are persistent or recurrent and substantial or do not stop without treatment; rectal or urinary tract bleeding that does not rise to a level that requires a therapeutic procedure (e.g., new insertion of a Foley catheter or cystoscopy), substantial hematomas at injection sites or other locations that occur spontaneously or with minor trauma; substantial blood loss; bleeding that requires unplanned blood transfusion. As used herein, "substantial blood loss" refers to an amount of blood loss that is greater than that normally associated with a surgical procedure. Substantial blood loss results in swelling that is not sufficient to require drainage and is therefore managed conservatively.

[0227] In one embodiment, the derivatives of the invention have a plasma circulating half-life sufficient to substantially neutralize fXa inhibitors present in plasma. Active fXa has essentially no circulating half-life in humans, since it is effectively inhibited by ATIII, TFPI and other plasma inhibitors (Fuchs, HE and Pizzo, SV, J. Clin. Invest., 1983, 72:2041-2049). Inactive fXa has been shown to have a circulating half-life of 2-3 hours in humans. In a baboon model, the half-life of fXa whose active site is blocked by DEGR ([5-(dimethylamino) 1-naphthalenesulfonyl]-glutamyl glycylarginyl chloromethyl ketone) was about 10 hours or 2 hours, respectively, as determined by isotope or enzyme-linked immunosorbent assay (Taylor, FB et al., Blood, 1991, 78(2):364-368).

[0228] It may be desirable to extend the half-life of the antidote fXa derivative to 24-48 hours. It is contemplated that the plasma half-life of the antidote will be increased by conjugating or appending one or more of the following moieties: a) polyethylene glycol; b) an acyl group; c) liposomes and encapsulants; d) carrier proteins; e) artificial phospholipid membranes; f) immunoglobulins; and g) Nanoparticles.

[0229] The conjugation site may not be limited to a particular chain or residue, so long as the conjugation does not mask the inhibitor binding site of the antidote. The antidotes described herein may be administered in combination with any one or more of the compounds described above.

[0230] In general, the administered antibody has a much longer half-life than circulating blood coagulation proteins. A complex consisting of Gla-domain-deleted fXa and an antibody bound to the non-active site of fXa can be used as an antidote with an extended circulating half-life. The formation of a complex between fXa and an antibody targeting the non-active site may reduce the interaction of Gla-domain-deleted fXa with macromolecular substrates and inhibitors such as prothrombin and antithrombin III, while leaving the active site cleft undisturbed so that the complex can act as an antidote binding to active site-specific small molecule inhibitors. The formation of α-2-macroglobulin-fXa complexes may also be useful as an antidote for small molecule inhibitors of fXa.

[0231] The effectiveness of the antidote in reversing the anticoagulant activity of fXa inhibitors and its procoagulant activity can be determined by those skilled in the art in in vitro assays and animal models. Examples of in vitro assays are clinical coagulation assays such as thrombin generation, aPTT, PT and ACT. It is contemplated that the antidote of the present invention can produce a correction of ex vivo coagulation activity of 10% or more. For example, several in vivo animal models of bleeding time and / or blood loss in rodents such as mice, primates such as dogs and monkeys can be used to measure effectiveness. V. Pharmaceutical Compositions The present invention further provides a composition comprising a fXa derivative and a pharma- ceutically acceptable carrier.

[0232] "Pharmaceutically acceptable carrier" refers to any diluent, excipient, or carrier that can be used in the composition of the present invention. Pharmaceutically acceptable carriers include ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human serum albumin, buffer substances such as phosphates, glycine, sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, electrolytes such as water, salts or protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulosic substances, polyethylene glycol, sodium carboxymethylcellulose, polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers, polyethylene glycol, and wool fat. Suitable pharmaceutical carriers are described in Remington's Pharmaceutical Sciences, Mack Publishing Company, a standard reference textbook in the field. They are preferably selected with respect to the intended form of administration, i.e., oral tablets, capsules, elixirs, syrups, etc., and are not inconsistent with conventional pharmaceutical practice.

[0233] The pharmaceutical compositions of the present invention can be manufactured by methods well known in the art, such as conventional granulation, mixing, dissolving, encapsulation, lyophilization, or emulsification processes, among others. The compositions can be produced in a variety of forms, including granules, precipitates, or particles, powders, including freeze-dried, rotary-dried, or spray-dried powders, amorphous powders, injectables, emulsions, elixirs, suspensions, or solutions. The formulations can optionally contain stabilizers, pH adjusters, surfactants, bioavailability modifiers, and combinations thereof.

[0234] Pharmaceutical formulations may be prepared as liquid suspensions or solutions using sterile liquids such as oil, water, alcohol, and combinations thereof. Pharmaceutically suitable surfactants, suspending agents, or emulsifying agents may be added for oral or parenteral administration. Suspensions may include oils such as peanut oil, sesame oil, cottonseed oil, corn oil, and olive oil. Suspension preparations may also contain esters of fatty acids such as ethyl oleate, isopropyl myristate, fatty acid glycerides, and acetylated fatty acid glycerides. Suspension formulations may include alcohols such as ethanol, isopropyl alcohol, hexadecyl alcohol, glycerol, and propylene glycol. Ethers such as poly(ethylene glycol), petroleum hydrocarbons such as mineral oil and petrolatum, and water may also be used in suspension formulations.

[0235] The compositions of this invention are formulated for pharmaceutical administration to a mammal, preferably a human being. Such pharmaceutical compositions of this invention may be administered in a variety of ways, preferably parenterally.

[0236] It is contemplated that the antidote of the present invention can or may be administered to the systemic circulation by parenteral administration in order to quickly reverse the anticoagulant activity of fXa inhibitors present in the plasma of patients during emergency situations. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intrahepatic, intralesional and intracranial injection or infusion techniques. However, if the neutralizing fXa inhibitor has a long plasma half-life, a sustained infusion or sustained release formulation may be required to bind to the fXa inhibitor and thereby release active fXa before the fXa inhibitor is cleared from the body.

[0237] Sterile injectable forms of the compositions of the present invention may be aqueous or oleaginous suspensions. These suspensions may be formulated using suitable dispersing or wetting agents and suspending agents according to techniques known in the art. Sterile injectable preparations may also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example as a solution in 1,3-butanediol. Acceptable vehicles and solvents that may be used are, among others, water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally used as solvents or suspending media. For this purpose, any bland, fixed oil may be used, including synthetic mono- or di-glycerides. Fatty acids such as oleic acid and its glyceride derivatives are useful in the preparation of injectables, as are natural pharma-ceutically acceptable oils such as olive oil or castor oil, particularly in their polyoxyethylated forms. These oil solutions or suspensions may also contain long-chain alcohol diluents or dispersants, such as carboxymethylcellulose or similar dispersants commonly used in the formulation of pharma- ceutical acceptable dosage forms, including emulsions and suspensions. Other commonly used surfactants, such as Tween, Span and other emulsifiers or bioavailability enhancers commonly used in the manufacture of pharma- ceutical acceptable solid, liquid, or other dosage forms, may also be used for formulation purposes. The compounds may be formulated for parenteral administration by injection, such as by bolus injection or continuous infusion. The unit dosage form for injection may be in ampoules or in multi-dose containers.

[0238] In addition to the above-mentioned dosage forms, pharma- ceutically acceptable excipients and carriers and dosage forms are generally known to those skilled in the art and are included in the present invention.It should be understood that the specific dose and treatment regimen for any particular patient will depend on a variety of factors, including the activity of the specific antidote used, the patient's age, weight, general health, sex and diet, renal and hepatic function, time of administration, excretion rate, drug combination, the judgment of the treating physician or veterinarian, and the severity of the particular disease being treated. VI. Kit The present invention further provides a kit or package. In some embodiments, the kit of the present invention comprises (a) a first container containing a fXa inhibitor that is routinely administered to treat thrombosis, and (b) a second container containing an antidote of the present invention for use when there is an excess of fXa inhibitor in (a) or when normal hemostasis needs to be restored to stop or prevent bleeding. In other embodiments, the kit further comprises a label that explains when the two agents in (a) and (b) should be used.

[0239] The first and second containers can be bottles, jars, vials, flasks, syringes, tubes, bags, or any other containers used in the manufacture, storage, or distribution of pharmaceutical products. The package insert can be a label, tag, marker, etc. that lists information about the pharmaceutical composition of the kit. The information listed is usually determined by a regulatory agency that governs the geographic area in which the pharmaceutical composition is sold, such as the U.S. Food and Drug Administration. Preferably, the package insert specifically lists the indications for which the pharmaceutical composition is approved. The package insert can be made of any material that allows a person to read the information contained therein or thereon. Preferably, the package insert is a printable material, such as paper, paper cardboard with adhesive backing, foil, or plastic, on which the desired information is printed or applied. EXAMPLES

[0240] The present invention will be further understood with reference to the following examples, which are intended to be purely illustrative of the present invention. The present invention is not limited in scope by the illustrated embodiments, which are intended only as illustrations of single aspects of the invention. Any method that is functionally equivalent is within the scope of the present invention. In addition to those described herein, various modifications of the present invention will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are within the scope of the appended claims.

[0241] Unless otherwise stated, all temperatures are degrees Celsius. Also, in these examples and elsewhere, the following abbreviations have the following meanings:

[0242] [ka] Example 1 Preparation of des-Gla anhydro-fXa by chymotrypsin digestion Des-Gla anhydro-fXa was prepared by incubating anhydro-fXa, in which dehydroalanine replaces the active site serine, with chymotrypsin in 0.05 M Tris-HCl, 0.1 M NaCl, pH 7.5 for 60 min at 22°C according to the procedure of Morita, T. et al., J. Bio. Chem., 1986, 261(9):4015-4023. In a typical experimental set-up, 0.5 milligrams per milliliter (mg / mL) of anhydro-fXa was incubated with 5 units per milliliter (U / mL) of α-chymotrypsin-agarose beads with gentle agitation. At the end of the reaction, the α-chymotrypsin-agarose beads were removed by centrifugation or filtration. This was then incubated with excess amounts of the inhibitors 4-amidino-phenyl-methane-sulfonyl fluoride (APMSF), tosyl-L-lysine chloromethyl ketone (TLCK), and tosyl-L-phenylalanine chloromethyl ketone (TPCK) to quench any residual fXa activity or any chymotrypsin activity that may have leached from the beads. Gla-domain fragments and inhibitors were removed from the final product des-Gla anhydro-fXa by Amicon ultracentrifugal filtration devices (YM10 membranes) or by conventional dialysis. Concentration or buffer exchange was also achieved simultaneously, if necessary. Gla-domain-containing anhydro-fXa was prepared according to the procedure reported by Nogami et al., J. Biol. Chem., 1999, 274(43):31000-7. α-Chymotrypsin-agarose beads were purchased from Sigma, and specific activity (U / mL) was based on manufacturer's data for the specific lot number used.

[0243] Chymotrypsin digestion of active fXa can be performed according to the above procedure without APMSF. The clotting activity of active fXa was determined before chymotrypsin digestion and after 15, 30 and 60 minutes of chymotrypsin digestion according to the procedure described in Example 3 below. Figure 7 shows that clotting activity was completely lost after 30 minutes of chymotrypsin digestion. To ensure complete removal of the Gla domain, the incubation time was extended to 60 minutes.

[0244] Example 2 Thrombin generation assay in platelet-poor plasma (PPP) or platelet-rich plasma (PRP) In this example, human platelet-poor or platelet-rich plasma samples were prepared from healthy donor blood drawn into 0.32% citrate. PRP and PPP were prepared by centrifuging anticoagulated blood at approximately 100× gravity or 1000× gravity, respectively, for 20 minutes at room temperature. 75–100 microliters (uL) of plasma were mixed with CaCl2 and Z-Gly-Gly-Arg-aminomethylcoumarin (Z-GGR-AMC, a fluorogenic substrate for thrombin). Tissue factor (Innovin, Dade Behring) was added to initiate thrombin generation. In a typical experiment, the reaction mixture contained 15 millimolar (mM) Ca 2+ , 100 micromolar (μM) Z-GGR-AMC, and 0.1 nanomolar (nM) tissue factor (TF) (Innovin). Thrombin formation was measured using a fluorometric plate reader (Molecular The plasma was continuously monitored by a 35-μL flow cytometer (Fiberglass-Tuned Devices) and relative fluorescence units (RFU) were measured. Inhibitors and antidotes, when present, were pre-incubated with plasma for 20 min at room temperature before initiating thrombin generation.

[0245] The results of various experiments using this assay can be found in Figures 4, 6, and 9.

[0246] Example 3 Clotting Prolongation Assay Two coagulation assay formats were used to test the effect of factor Xa inhibitors and antidotes on coagulation prolongation. In the first format, multiple samples were measured simultaneously using 96-well plates. In the second assay format, aPTT was measured with a conventional coagulation instrument (MLA Electra800 automated coagulation timer).

[0247] For the 96-well plate format method, human platelet-poor or platelet-rich plasma was prepared similarly to the procedure in Example 2. 75–100 μL of plasma was recalcified with CaCl2, incubated at 37°C for 3 min, and clot formation was initiated by adding tissue factor (Innovin, Dade Behring) or aPTT reagent (Actin FS, Dade Behring). The change in OD405 was continuously monitored by a plate reader (Molecular Devices). Clotting time was defined as the time (in seconds) at which the half-maximal change in absorbance (OD405 nm) was reached. Factor Xa inhibitors and antidotes, if present, were preincubated with plasma for 20 min at room temperature before initiating the reaction.

[0248] When active fXa was tested for its clotting activity as shown in Figure 7, 75-100 uL of fX-deficient plasma (George King Bio-Medical, Inc.) was recalcified with CaCl2 and incubated at 37°C for 3 min, and the fXa product after chymotrypsin digestion was added to the plasma to initiate clot formation. The change in OD405 was continuously monitored by a plate reader as described above.

[0249] In Figure 13, the effect of 400 nM betrixaban on aPTT prolongation of normal human plasma and the reversal of the inhibitory effect of betrixaban by the antidote des-Gla anhydro-fXa were measured with an MLA Electra800 automated clotting timer. 100 μL of pooled human plasma was mixed with 400 nM betrixaban and various concentrations of the antidote. To measure clotting time, aPTT reagent (Actin FS, Dade Behring) and CaCl2 were added according to the manufacturer's instructions.

[0250] The results of further experiments using this assay can be found in FIGS.

[0251] Example 4 Reversal of betrixaban-induced inhibition of fXa by des-Gla anhydro-fXa To measure the inhibition of fXa activity by betrixaban and the reversal of its inhibitory effect, purified active fXa, various concentrations of betrixaban and the antidote des-Gla anhydro-fXa were incubated in 20 mM Tris, 150 mM NaCl, 5 mM CaCl, 1 mM HCl, 10 mM NaCl, 1 ... 2+ , and 0.1% bovine serum albumin (BSA). After incubation at room temperature for 20 min, 100 μM Spectrozyme-fXa (factor Xa chromogenic substrate, Chromogenix) was added to the mixture, and the rate of cleavage of the substrate was continuously monitored by a plate reader at 405 nanometers (nm) for 5 min. In Figure 5, the chromogenic activity was normalized to active fXa in the absence of any inhibitor. The initial rate of product formation was analyzed by nonlinear regression as a function of inhibitor and antidote concentrations to estimate the affinity of betrixaban for the antidote (Figure 8).

[0252] The effect of the antidote des-Gla anhydro-fXa on thrombin activity toward the chromogenic substrate S2288 (200 μM) was measured with and without the specific small molecule IIa inhibitor argatroban as before. As expected, the antidote (538 nM) has no effect on the amidolytic activity of IIa (5 nM) or its inhibition by 50 nM argatroban.

[0253] Example 5 Preparation of fXa with decarboxylated γ-carboxyglutamic acid residues fXa derivatives with decarboxylated γ-carboxyglutamic acid residues can be prepared by treating fXa protein, for example, according to the procedure reported by Bajaj et al., J. Biol. Chem., 1982, 257(7):3726-3731. 2-5 mg of purified or recombinant fXa in 2 mL of 0.1 molar ammonium bicarbonate, pH 8.0, was lyophilized. The resulting powder was sealed under a vacuum of <20 μM and heated at 110° C. for various times to obtain decarboxylated fXa.

[0254] Example 6 Preparation of recombinant des-Gla fXa-S379A The fXa derivatives may be produced by recombinant DNA methods using one of the following procedures based on the fX cDNA (SEQ ID NO: 2) for expressing fX (SEQ ID NO: 1, 3) or fXa derivatives (SEQ ID NO: 4, 5, 9, and 11) in a suitable host organism following general procedures of mutagenesis and molecular biology.

[0255] Recombinant fX and fX derivatives can be expressed, for example, in human embryonic kidney cells HEK293, based on the procedures described in Larson, PJ et al., Biochem., 1998, 37:5029-5038, and Camire, RM et al., Biochem., 2000, 39, 14322-14329. Recombinant fX can be activated to rfXa by the factor X activator Russell's Viper Venom (RVV). Based on the procedures described in Example 1, rfXa can be further processed to des-Gla anhydro-fXa.

[0256] Recombinant fX-S379A in which the active site serine residue has been replaced by alanine (S195A in chymotrypsin numbering), preferably the active fXa mutant rfXa-S379A, can be expressed, for example, in Chinese hamster ovary (CHO) cells based on the procedures described by Sinha et al., Protein Expression and Purif., 1992, 3:518-524; Wolf, DL et al., J. Biol. Chem., 1991, 266(21):13726-13730.

[0257] Des-Gla fXa-S379A may be prepared by chymotrypsin digestion of fXa-S379A according to the procedure described in Example 1.

[0258] More preferably, des-Gla fXa-S379A may be directly expressed using deletion of the Gla-domain fragment by mutagenesis procedures, following previous procedures. For example, recombinant protein expression may be used to express des-Gla(1-39)-fXa-S379A following deletion of Gla-domain fragment 1-39 of SEQ ID NO:3; des-Gla(1-44)-fXa-S379A, the equivalent of SEQ ID NO:10 in which dehydro-alanine is replaced by alanine; and des-Gla(1-45)-fXa-S379A in which the entire Gla-domain has been deleted (SEQ ID NO:11).

[0259] Further truncations in the EGF1 or EGF1+EGF2 domains (Figure 2) can also be made to express des(1-84)-fXa-S379A or des(1-128)-fXa-S379A derivatives.

[0260] Example 7 Expression of recombinant fXa mutants in CHO cells In this example, we describe a recombinant protein expression construct and a cell line for directly expressing the Gla-domainless fXa-S379A (S195A in chymotrypsin numbering) mutant. The recombinant antidote does not require the activation or chemical modification steps required to produce the pd-antidote and has comparable affinity for plasma-derived proteins in the in vitro assays described herein.

[0261] In this example, the fXa mutant (SEQ ID NO: 13, Table 12a) was expressed directly in CHO cells (see FIG. 14 for expression vector) and functional protein was purified from conditioned medium as described below. The functional activity of the recombinant antidote (r-antidote) was tested in vitro and in animal models (Example 8).

[0262] Using PCR, the cDNA sequence of fX (SEQ ID NO:2) was mutated in three regions. The first mutation was the deletion of amino acids 6-39 in the Gla-domain of FX (SEQ ID NO:3, Figure 3). The second mutation was the replacement of amino acids 143-194 of the activation peptide sequence with -RKR-. This resulted in a -RKRRKR- linker connecting the light and heavy chains. After secretion, this linker is removed in CHO, resulting in a two-chain fXa molecule. The third mutation was the mutation of the active site residue S379 to an Ala residue.

[0263] The polypeptide produced by the cDNA described immediately above (SEQ ID NO: 16) is set forth in Table 12 (SEQ ID NO: 12). The alignment of the cDNA to the polypeptide is shown in Table 20. The resulting two-chain fXa molecule after secretion is the light chain fragment set forth in Table 12b (SEQ ID NO: 14) and the heavy chain fragment set forth in Table 12c (SEQ ID NO: 15).

[0264] The first 1 to 5 amino acids in the fX sequence are reserved and used to connect the fXa mutant polypeptide to the prepropeptide of fX (SEQ ID NO:1, FIG. 1 ) to ensure proper processing of the prepropeptide in the fXa mutant.

[0265] The DNA sequences encoding the above fXa mutant polypeptides were sequenced and inserted into an expression vector shown in Figure 14. The polynucleotide of the expression vector is shown in SEQ ID NO: 18. The plasmid DNA was linearized and transfected into CHO dhfr(-) cells. The cells were selected using tetrahydrofolate (HT)-deficient medium and methotrexate (MTX). Stable clones were screened for high protein expression using the fX ELISA kit (Enzyme Research Laboratories, Cat. No. FX-EIA). The FXa mutant proteins were expressed in serum-free and conditioned medium, harvested, and processed for purification.

[0266] The target protein in the medium can be isolated by ion exchange chromatography and then purified by a single step affinity chromatography (such as anti-fXa antibodies coupled to a matrix) or a combination of several chromatographic steps such as a hydrophobic matrix. Affinity purification can include chromatographic materials that selectively bind to the fXa active site cleft, such as benzamidine-sepharose or soybean trypsin inhibitor-agarose (STI-agarose).

[0267] FIG. 15 shows monoclonal antibodies (Enzyme Western blot of affinity (STI-agarose, Sigma catalog #T0637) purified fXa mutant using a FX-EIA from Sigma Research Laboratories. After reduction of the disulfide bond connecting the light and heavy chains, r-antidote shows the predicted heavy chain band similar to plasma-derived fXa in the Western blot. Deletion of amino acids 6-39 in the Gla-domain of the fXa mutant results in a lower molecular weight band of the light chain of r-antidote compared to plasma-derived fXa. Molecular weight markers can also be seen in the blot.

[0268] Example 8 In vivo mouse model The pharmacokinetic and pharmacodynamic (PK-PD) profile of betrixaban in male C57Bl / 6 mice with or without antidote was studied. A single oral dose of betrixaban was dosed at 0, 15, 25, and 75 mg / kg in the control groups. 15 mg / kg was used for the antidote treatment groups. A single intravenous (IV) injection of antidote (300ug / 200μL) or vehicle (normal saline, 200μL) was administered 5 minutes prior to the 1.5 hour time point.

[0269] At 1.5, 2.0, and 4.0 hours after oral administration of betrixaban, mice were anesthetized with ketamine cocktail (SC) and exsanguinated by cardiac puncture. Blood samples (0.5 mL) were obtained in 50 μL of trisodium citrate. Whole blood INR was measured using Hemochron Jr. cartridges (International Technidyne Corporation) according to the manufacturer's instructions. Mouse platelet-poor plasma was prepared by centrifugation for plasma concentration determination of betrixaban and antidote (ELISA).

[0270] For recombinant antidote (r-antidote) experiments, control groups of mice were orally dosed with 0, 15, 25, and 75 mg / kg betrixaban. 15 mg / kg was used for the antidote (300 μg / 200 μL) treatment group. Samples were taken 1.5 hours after oral administration of betrixaban (5 minutes after antidote injection).

[0271] As shown in Figures 16 and 17 and Tables 13 and 14, a single injection (300 μg, IV) of plasma-derived antidote (pd-antidote) or recombinant fXa mutant (r-antidote) in mice after administration of betrixaban (15 mg / kg, PO) effectively captured the inhibitor in vivo. The PK-PD correlation of whole blood INR and antidote plasma concentration (Tables 13-14) showed a >50% reduction in functional betrixaban based on INR measurements, justifying effective neutralization of fXa inhibitors with antidote by multiple injections or other regimes. It is contemplated that these results demonstrate that the fXa derivatives of the present invention have the potential to act as universal antidotes to reverse the anticoagulant effects of fXa inhibitors in patients undergoing bleeding or other medical emergencies.

[0272] FIG. 22 shows a mouse study with a single IV injection (1 s.) or two injections (2 s.) of r-Antidote after oral administration of betrixaban (15 mg / kg) (n=5 mice / group, 312 ug / 200 ul of r-Antidote). In the single injection group, mouse blood samples were taken 1 hour after oral administration of betrixaban. Vehicle (Control_1) or r-Antidote (1 s.) was administered 5 minutes before the 1 hour time point. In the double injection group, vehicle or r-Antidote was injected at 55 minutes and repeated 115 minutes after oral administration of betrixaban. Mouse blood samples were taken at 2 hours for mice treated with vehicle (Control_2) and r-Antidote (2 s.). The INR measured in mouse plasma as a function of antidote / betrixaban ratio after a single or two injections of antidote is shown in FIG. 22B.

[0273] Example 9 In vitro reversal of rivaroxaban and apixaban by antidotes As expected, the antidote contemplated by the present invention was also able to bind and neutralize other active site-specific fXa inhibitors. Tables 15 and 16 show the in vitro correction of inhibition by betrixaban, rivaroxaban and apixaban by pd-antidote and r-antidote. Purified fXa (3.0 nM), inhibitor (7.5 nM) and various concentrations of antidote were incubated for 10 minutes at 22°C in a buffer containing 20 mM Tris, 150 mM NaCl, 0.1% BSA, pH 7.4. fXa activity was assayed as in Example 4.

[0274] As shown in Table 15, the pd-antidote at 204 nM produced at least 60% correction of the inhibitory effect of the inhibitors tested, while in Table 16, the r-antidote (186 nM) achieved >95% correction of inhibition for betrixaban and rivaroxaban and >70% reversal of apixaban.

[0275] Example 10 In vitro reversal of betrixaban by r-antidote In Table 17, the effect of recombinant antidote protein on the reversal of anticoagulation by betrixaban was tested in a human plasma clotting assay. The effect of 300nM and 400nM betrixaban on the prolongation of plasma aPTT and the reversal of the inhibitory effect was measured by an MLA Electra800 automated clotting timer. 100μL of pooled citrate-anticoagulated human plasma was mixed with 300nM or 400nM betrixaban and various concentrations of antidote. aPTT reagent (Actin FS, Dade Behring) and CaCl2 were added according to the manufacturer's instructions.

[0276] Example 11 In vitro reversal of low molecular weight heparin ("LMWH") by r-antidote In Figure 18, the effect of r-Antidote to reverse the inhibitory effect of the LMWH enoxaparin (Sanofi-Aventis) was tested by turbidity changes in human plasma. Enoxaparin (0-1.25 U / mL) was incubated with or without 508 nM r-Antidote for 20 min at 22°C. Turbidity changes were measured according to the procedure described in Example 3. 508 nM r-Antidote substantially corrected (>75%) the inhibitory effect of 0.3125-1.25 U / mL enoxaparin.

[0277] In Figure 19, the effect of r-antidote on the reversal of anticoagulation by low molecular weight heparin (LMWH enoxaparin, Sanofi-Aventis) was tested in a human plasma clotting assay. The effect of 1 anti-Xa unit / mL of LMWH on the prolongation of plasma aPTT and the reversal of the inhibitory effect was measured by an MLA Electra800 automated clotting timer. 100 μL of pooled citrate-anticoagulated human plasma was mixed with enoxaparin and various concentrations of antidote. Before measuring the clotting time, aPTT reagent (Actin FS, Dade Behring) and CaCl2 were added according to the manufacturer's instructions. The addition of 1.14 μM of recombinant antidote resulted in a 52% correction of the anticoagulation produced by 1 unit / mL of enoxaparin.

[0278] Example 12 In vitro reversal of rivaroxaban by r-antidote In FIG. 20, the effect of recombinant antidote protein on the reversal of anticoagulation by a small molecule factor Xa inhibitor (rivaroxaban, Bay59-7939) was tested in a human plasma clotting assay. As reported by Perzborn et al., J. Thromb. Haemost., vol. 3:514-521, 2005, measurement of prothrombin time is an accurate method to evaluate rivaroxaban anticoagulant effect. The effect of 1 μM rivaroxaban on prothrombin time (PT) prolongation and reversal of inhibitory effect of pooled human plasma was measured by MLA Electra800 automated coagulation timer. 100 μL of pooled citrate-anticoagulated human plasma was mixed with rivaroxaban and various concentrations of antidote. Prior to measuring clotting time, rabbit brain thromboplastin C Plus reagent (Dade Behring) was added to the plasma samples according to the manufacturer's instructions. Addition of 1.9 μM of the recombinant antidote resulted in 100% correction of the anticoagulation produced by 1 μM rivaroxaban.

[0279] (Example 13) In vitro reversal of apixaban by r-antidote In Table 18, the effect of recombinant antidote protein on the reversal of anticoagulation by apixaban was tested in a human plasma clotting assay. As reported by Pinto et al., J. Med. Chem., vol. 55(22):5339-5356, 2007, measurement of prothrombin time (PT) is an accurate method to evaluate the ex vivo anticoagulant effect of apixaban. The effect of 1 μM and 1.5 μM apixaban on the prothrombin time (PT) prolongation and reversal of inhibitory effects of pooled human plasma was measured by an MLA Electra800 automated clotting timer. 100 μL of pooled citrate-anticoagulated human plasma was mixed with apixaban and various concentrations of antidote. Before measuring clotting time, rabbit brain thromboplastin C Plus reagent (Dade Behring) was added to the plasma samples according to the manufacturer's instructions. Addition of 1.9 μM recombinant antidote resulted in 97% correction of the anticoagulation produced by 1.5 μM apixaban.

[0280] Example 14 In vitro inhibition of argatroban by des-Gla anhydro-fXa To measure the inhibition of thrombin activity by argatroban and the reversal of its inhibitory effect, purified human thrombin (5 nM), argatroban (50 nM) and various concentrations of the antidote des-Gla anhydrofXa were added to a buffer containing 20 mM Tris, 0.15 M NaCl, 5 mM calcium chloride, 0.1% bovine serum albumin, pH 7.4. After incubation at room temperature for 20 minutes, the amidolytic substrate S2288 (200 uM) was added to the mixture and the cleavage rate of the p-nitroanilide substrate was monitored by absorbance at 405 nm. The results are presented in Figure 12.

[0281] While the invention has been described in conjunction with the above embodiments, it should be understood that the foregoing description and examples are intended to be illustrative and not limiting of the scope of the invention. Other aspects, advantages and modifications within the scope of the invention will be apparent to those skilled in the art to which the invention pertains.

[0282] [Table 1]

[0283] [Table 2]

[0284] [Table 3]

[0285] [Table 4]

[0286] [Table 5]

[0287]

Table 6

[0288]

Table 7

[0289]

Table 8

[0290]

Table 9

[0291]

Table 10

[0292]

Table 11

[0293]

Table 12

[0294]

Table 12a

[0295]

Table 12b

[0296]

Table 12c

[0297]

Table 13

[0298]

Table 14

[0299]

Table 15

[0300]

Table 16

[0301]

Table 17

[0302]

Table 18

[0303]

Table 19

[0304]

Table 20

Table 20-2

Claims

[Claim 1] An antibody as described in the specification.

Images