Antibodies for binding to plasminogen

Abstract

The present invention relates to antigen-binding proteins comprising an antigen-binding domain that binds to plasminogen, wherein the antigen-binding protein reduces the activation of plasminogen to plasmin, pharmaceutical compositions comprising the same, and methods and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to antigen-binding proteins and related fragments thereof for binding to plasminogen, the production of said antigen-binding proteins and fragments, and the use of said antibodies and fragments for the detection and treatment of various disease states.

[0002] Related Applications This application claims priority to Australian Provisional Patent Application No. AU 2019904052, the contents of which are incorporated herein by reference in their entirety. [Background technology]

[0003] Plasminogen (PLG) is the inactive zymogen form of plasmin, a serine protease with broad specificity for target substrates including fibrin, fibrinogen, complement components 3 and 5 (C3 and C5), vitronectin, osteocalcin, factors V, VIII, and X, and some collagenases. Thus, PLG and PLM together are involved in a variety of important physiological and pathological processes, including fibrinolysis and hemostasis, extracellular matrix degradation, cell migration, embryonic development, tissue remodeling, inflammation, wound healing, angiogenesis, and tissue invasion.

[0004] PLG is primarily synthesized in the liver, but is also synthesized in major organs and tissues. Therefore, PLG is present in significant amounts in plasma and many extravascular fluids. Under physiological conditions, PLG is converted to its active form, plasmin (PLM), by cleavage in the activation loop. Activation, which can be mediated by urokinase plasminogen activator (uPA) or tissue plasminogen activator (tPA), or various other proteases, converts single-chain PLG (amino acid residues 20–810) into PLM, which consists of a heavy chain A (residues 20–580) and a light chain B (residues 581–810) linked by disulfide bonds. Heavy chain A contains five kringle domains (which mediate substrate binding via lysine-binding regions), and light chain B corresponds to the serine protease domain. A fragment consisting of the first four kringle domains has been named angiostatin and is a novel angiogenesis inhibitor.

[0005] The plasminogen / plasmin system has been implicated in a variety of physiological and pathological processes, including fibrinolysis, tissue remodeling, cell migration, inflammation, and tumor invasion and metastasis. Genetic defects in plasminogen are a risk factor for the development of thromboembolic diseases.

[0006] There is a need for compositions and methods for modulating the plasmin system for the treatment and / or prevention of various disease states.

[0007] The reference to any prior art herein is not an admission or suggestion that this prior art forms part of common general knowledge in any jurisdiction, or that this prior art would be reasonably expected to be understood, considered relevant, and / or could be combined with other pieces of prior art by a person skilled in the art. Summary of the Invention [Means for solving the problem]

[0008] The present invention provides an antigen binding protein comprising an antigen binding domain that binds to plasminogen, wherein the antigen binding protein reduces the activation of plasminogen.

[0009] In any aspect, the antigen binding proteins of the present invention may reduce the activation of plasminogen by any one or more plasminogen activators. The plasminogen activator may comprise any one of the amino acids Arg 561 -Val 562 The plasminogen activator is any enzyme capable of cleaving the bond (numbering is based on human plasminogen). Exemplary plasminogen activators are plasminogen-cleaving serine proteases, including the coagulation proteins factor IX, factor X, and prothrombin (factor II), protein C, chymotrypsin and trypsin, various leukocyte elastases, streptokinase (SK), staphylokinase, urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and plasmin. Preferably, the plasminogen activator is selected from the group consisting of streptokinase (SK), urokinase plasminogen activator (uPA), and tissue plasminogen activator (tPA). Preferably, the reduction in SK-, uPA-, and / or tPA-mediated activation of plasminogen is in the presence of one or more ligands or substrates of plasminogen / plasmin. Preferably, the cofactor is selected from the group consisting of ε-aminocaproic acid (EACA) or other lysine analogues, fibrinogen (Fg) and fibrin (Fn). Methods for measuring plasminogen activation are known in the art and can be used to determine the reduction in plasminogen activation by the antigen binding proteins of the invention. Exemplary methods are described herein. The reduction in plasminogen activation can be measured by measuring the decrease in the amount of active plasmin formed.

[0010] In any embodiment, the antigen binding proteins of the present invention do not detectably or significantly inhibit the activity of plasmin. In another embodiment, the antigen binding proteins of the present invention do not detectably bind to the active site (i.e., catalytic triad) of plasmin. Binding of the antigen binding protein to plasmin may be determined by any of the methods described herein.

[0011] In any embodiment, the antigen binding proteins of the present invention bind to the kringle domain of plasminogen, preferably kringle domain 5. In any embodiment, the antigen binding proteins of the present invention bind to the kringle domain and serine protease domain of plasminogen. In any embodiment, the antigen binding proteins of the present invention bind to the kringle domain, activation loop and serine protease domain of plasminogen. In any embodiment, the antigen binding proteins of the present invention bind only to a region of plasminogen that includes the activation loop and kringle domain (preferably kringle 5) of plasminogen, but not the serine protease domain. In other words, the antigen binding proteins of the present invention do not bind to a region of plasminogen that does not include the activation loop. In any embodiment, the antigen binding proteins of the present invention bind to the serine protease domain of plasminogen, but do not bind to the kringle domain of plasminogen. In another embodiment, the antigen binding proteins of the present invention also interact with the serine protease domain.

[0012] In any embodiment, the antigen binding protein of the present invention binds to a region of plasminogen that contains the activation loop of plasminogen. Preferably, the region that contains the activation loop is Arg of plasminogen. 493 From His 569 or comprising an amino acid sequence between Lys 468 From His 569 and more preferably, in this case the activation loop comprises the sequence between Ala of plasminogen according to the numbering set forth in SEQ ID NO: 65. 543 From Arg 582Preferably, the antigen binding protein binds to a peptide comprising the sequence shown in SEQ ID NO: 66, or a fragment thereof. Preferably, the antigen binding protein of the present invention binds to a peptide comprising the sequence shown in SEQ ID NO: 66, or a fragment thereof. Preferably, the antigen binding protein of the present invention binds to a peptide comprising the sequence shown in SEQ ID NO: 66, or a fragment thereof. 561 and Val 562 Binds to one or more of:

[0013] In any embodiment, the antigen binding protein of the present invention has a nucleotide sequence at position Arg 493 , Ser 495 , Ile 496 , Asp 516 , Gly 517 , Asp 518 , Val 519 , Tyr 525 , and Tyr 533 or a position corresponding thereto. Preferably, the antigen binding proteins of the invention bind to one or more residues in the kringle 5 domain of plasminogen at or a position corresponding thereto as shown in Table 2. Preferably, the residues of the antigen binding proteins of the invention that bind to one or more residues in the kringle 5 domain of plasminogen are those defined in Table 2 or are amino acid residues at positions corresponding to those shown in Table 2.

[0014] In any embodiment, the antigen binding protein of the present invention has a nucleotide sequence at position Lys 468 , Arg 493 , Ser 495 , Ile 496 , Asp 516 , Gly 517 , and Tyr 525or a position equivalent thereto. Preferably, the antigen binding proteins of the invention bind to one or more residues in the kringle 5 domain of plasminogen at or a position equivalent thereto as shown in Table 3. Preferably, the residues of the antigen binding proteins of the invention that bind to one or more residues in the kringle 5 domain of plasminogen are as defined in Table 3 or are amino acid residues at positions equivalent thereto as shown in Table 3.

[0015] In any embodiment, the antigen binding protein of the present invention has a nucleotide sequence at position Glu 554 , Lys 556 , Lys 557 , His 569 , and Asp 751 or a position equivalent thereto. Preferably, antigen binding proteins of the invention bind to one or more residues in the serine protease domain of plasminogen at or a position equivalent thereto as shown in Table 2. Preferably, the residues of antigen binding proteins of the invention that bind to one or more residues in the serine protease domain of plasminogen are as defined in Table 2 or are amino acid residues at positions equivalent thereto as shown in Table 2.

[0016] In any embodiment, the antigen binding protein of the present invention has a nucleotide sequence at position Glu 554 , Lys 556 , Lys 557 , Arg 561 , and His 569or a position equivalent thereto. Preferably, antigen binding proteins of the invention bind to one or more residues in the serine protease domain of plasminogen at or a position equivalent thereto as shown in Table 3. Preferably, the residues of antigen binding proteins of the invention that bind to one or more residues in the serine protease domain of plasminogen are as defined in Table 3 or are amino acid residues at positions equivalent thereto as shown in Table 3.

[0017] In any embodiment, the antigen binding protein of the present invention has a nucleotide sequence at position Asp 676 , Arg 677 , Arg 712 , Glu 714 , and Asn 769 or a position equivalent thereto. Preferably, antigen binding proteins of the invention bind to one or more residues in the serine protease domain of plasminogen at or a position equivalent thereto as shown in Table 4. Preferably, the residues of antigen binding proteins of the invention that bind to one or more residues in the serine protease domain of plasminogen are as defined in Table 4 or are amino acid residues at positions equivalent thereto as shown in Table 4.

[0018] The present invention also provides antigen binding proteins (eg, antibodies) that have the same or substantially the same amino acids at positions relative to, or equivalent to, the residues designated for the G05 antibody in Tables 2 or 3.

[0019] The present invention also provides antigen binding proteins (eg, antibodies) that have the same or substantially the same amino acids at positions relative to, or equivalent to, the residues designated for the G11 antibody in Table 4.

[0020] The present invention also provides antigen binding proteins that bind or specifically bind to plasminogen, wherein said antigen binding proteins competitively inhibit the binding of the G05 or G11 antibody (i.e. comprising the following: a VH comprising the sequence set forth in SEQ ID NO:8 and a VL having the sequence set forth in SEQ ID NO:7; or a VH comprising the sequence set forth in SEQ ID NO:40 and a VL having the sequence set forth in SEQ ID NO:39) to plasminogen.

[0021] In any aspect of the present invention, the interaction between one residue of an antigen-binding protein of the present invention and one residue of plasminogen can be determined by X-ray crystal structure analysis and contact distance analysis of 0 to 3.9 Å (inclusive).

[0022] The present invention also provides an antigen binding protein that binds to the same epitope on plasminogen as an antibody comprising a VH domain having the amino acid sequence set forth in SEQ ID NO:8, wherein the antigen binding protein reduces or inhibits activation of plasminogen.

[0023] The present invention also provides an antigen-binding protein that binds to the same epitope on plasminogen as an antibody comprising a VH domain having the amino acid sequence set forth in SEQ ID NO: 8 and a VL domain having the amino acid sequence set forth in SEQ ID NO: 7, wherein the antigen-binding protein reduces or inhibits plasminogen activation. In one embodiment, the epitope is defined by X-ray crystallography. Preferably, the epitope is defined by X-ray crystallography and contact distance analysis of 0 to 3.9 Å (inclusive). Preferably, the antigen-binding protein has a contact distance of approximately 1000 to 1300 Å. 2 The surface area of ​​the plasminogen of SEQ ID NO:65 is covered.

[0024] The present invention also provides an antigen-binding protein that binds to the same epitope on plasminogen as an antibody comprising a VH domain having the amino acid sequence set forth in SEQ ID NO: 40 and a VL domain having the amino acid sequence set forth in SEQ ID NO: 39, wherein the antigen-binding protein reduces or inhibits plasminogen activation. In one embodiment, the epitope is defined by X-ray crystallography. Preferably, the epitope is defined by X-ray crystallography and contact distance analysis of 0 to 3.9 Å (inclusive).

[0025] In any embodiment, the antigen binding proteins of the invention bind to plasminogen with a K of less than 15 nM, less than 10 nM, or about 8 nM or less. D Preferably, K D is determined using any of the assays described herein, for example, surface plasmon resonance (SPR).

[0026] In any embodiment, the antigen binding protein of the invention binds to plasminogen and produces approximately 1 x 10 4 Super, about 1×10 5 More than or about 4 x 10 5 More than k a (M -1 s -1 ) can be presented.

[0027] In any embodiment, the antigen binding protein of the invention binds to plasminogen and produces approximately 1 x 10 -3 Less than 5 × 10 -3 Less than or 2 x 10 -3 Less than or about 10 x 10 -4 The following k d (s -1 ) can be presented. Preferably, k d is about 7 x 10 -4 , or any value described herein.

[0028] In any aspect of the invention, the antigen binding protein of the invention has an IC of less than 500 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 80 nM, 60 nM or 50 nM. 50 and can inhibit streptokinase-, tPA-, or uPA-mediated activation of plasminogen. Preferably, IC 50 is about 18 nM or any value described herein, as measured in solution in the presence of EACA. 50 is about 290 nM, or any value described herein, measured in the presence of fibrin.

[0029] Antigen binding proteins of the present invention may bind to a peptide derived from SEQ ID NO: 65. For example, antigen binding proteins of the present invention may bind to a peptide consisting of 4, 5, 7, 8, 9, 10 or more consecutive amino acid residues of the sequence of SEQ ID NO: 65. In some embodiments, antigen binding proteins of the present invention bind to a peptide comprising, consisting essentially of, or consisting of residues 554-569 of SEQ ID NO: 65. In some embodiments, antigen binding proteins of the present invention bind to a peptide comprising, consisting essentially of, or consisting of residues 493-533 of SEQ ID NO: 65. In some embodiments, antigen binding proteins of the present invention bind to a peptide comprising, consisting essentially of, or consisting of residues 468-525 of SEQ ID NO: 65. In some embodiments, antigen binding proteins of the present invention bind to a peptide comprising, consisting essentially of, or consisting of residues 554-569 of SEQ ID NO: 65. In some embodiments, antigen binding proteins of the present invention bind to a peptide comprising, consisting essentially of, or consisting of residues 554-569 of SEQ ID NO: 65. In some embodiments, antigen binding proteins of the invention bind to a peptide comprising, consisting essentially of, or consisting of residues 493-533 of SEQ ID NO: 65 and bind to a peptide comprising, consisting essentially of, or consisting of residues 554-569 of SEQ ID NO: 65. In some embodiments, antigen binding proteins of the invention bind to a peptide comprising, consisting essentially of, or consisting of residues 468-525 of SEQ ID NO: 65 and bind to a peptide comprising, consisting essentially of, or consisting of residues 554-569 of SEQ ID NO: 65. In yet another embodiment, antigen binding proteins of the invention bind to a peptide comprising, consisting essentially of, or consisting of residues 468-525 of SEQ ID NO: 65 and bind to a peptide comprising, consisting essentially of, or consisting of residues 554-751 of SEQ ID NO: 65.

[0030] In a preferred embodiment, the antigen binding proteins of the present invention do not bind to a region of plasminogen or plasmin that contains only the serine protease domain, in other words, the antigen binding proteins of the present invention are proteins that bind to a region of plasminogen that contains the activation loop that lies between the region known as kringle 5 and the serine protease domain.

[0031] In another embodiment, the antigen binding proteins of the invention do not detectably bind to a region of plasminogen that contains the catalytic triad of the serpin protease domain. Thus, in a specific embodiment, the antigen binding proteins of the invention comprise or consist of the amino acid sequence 570 to 741 of SEQ ID NO: 65, preferably the amino acid sequence His 603 From Ala 741 It does not bind to peptides that contain or consist of the amino acid sequence

[0032] In any embodiment, the antigen binding protein does not significantly reduce the activity of plasmin. Preferably, the antigen binding protein does not result in a significant reduction in the activity of plasmin as measured using any assay described herein capable of measuring plasmin activity.

[0033] In any embodiment, the antigen binding protein does not significantly reduce the activity of any one or more of tPA, thrombin, trypsin, factor Xa (FXa), and plasma kallikrein.

[0034] In certain embodiments, the CDRs of an antigen binding protein can be determined using the IMGT domain gap numbering system.

[0035] The present invention provides an antigen binding protein for binding to plasminogen, the antigen binding protein comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a where: FR1, FR2, FR3 and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are each a framework region; CDR1a, CDR2a and CDR3a are each a complementarity determining region; The sequences of any of the framework regions or complementarity determining regions are as described herein.

[0036] The present invention provides an antigen binding protein for binding to plasminogen, the antigen binding protein comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a where: FR1, FR2, FR3 and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are each a framework region; CDR1a, CDR2a and CDR3a are each a complementarity determining region; The sequence of any of the above complementarity determining regions has the amino acid sequence shown in Table 1 below. Preferably, the framework regions also have the amino acid sequence shown in Table 1 below, containing amino acid mutations at specific residues, which can be determined by aligning the various framework regions from each antibody. The present invention also encompasses cases where CDR1, CDR2, and CDR3 are sequences from VH and CDR1a, CDR2a, and CDR3a are sequences from VL, or where CDR1, CDR2, and CDR3 are sequences from VL and CDR1a, CDR2a, and CDR3a are sequences from VH.

[0037] In one embodiment, the antigen binding protein is FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Includes.

[0038] As defined herein, a linker may be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0039] The present invention provides a compound comprising (in N to C-terminal or C to N-terminal order): SEQ ID NOs: 7 and 8: or SEQ ID NOs: 39 and 40 The present invention provides an antigen binding protein comprising, consisting essentially of, or consisting of the amino acid sequence of:

[0040] The present invention also provides an antigen-binding protein comprising an antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to plasminogen, and wherein the antigen-binding domain comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:4, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:5, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:6; (ii) a VH comprising a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO:8; (iii) a VL comprising a CDR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:1, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:2, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:3; (iv) a VL having a sequence at least about 95% identical to the sequence set forth in SEQ ID NO:7; (v) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO: 4, a CDR2 having the sequence set forth in SEQ ID NO: 5, and a CDR3 having the sequence set forth in SEQ ID NO: 6; (vi) a VH having the sequence set forth in SEQ ID NO: 8; (vii) a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 1, a CDR2 having the sequence set forth in SEQ ID NO: 2, and a CDR3 having the sequence set forth in SEQ ID NO: 3; (viii) VL having the sequence set forth in SEQ ID NO: 7; (ix) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO: 4, a CDR2 having the sequence set forth in SEQ ID NO: 5, and a CDR3 having the sequence set forth in SEQ ID NO: 6; and a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 1, a CDR2 having the sequence set forth in SEQ ID NO: 2, and a CDR3 having the sequence set forth in SEQ ID NO: 3; (x) A VH having the sequence set forth in SEQ ID NO: 8, and a VL having the sequence set forth in SEQ ID NO: 7.

[0041] In any embodiment of the invention, the antigen binding domain further comprises at least one of the following: (i) a VH comprising a framework region (FR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 21; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 22; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 23; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 24; (ii) a VL comprising: an FR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 17; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 18; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 19; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 20; (iii) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 21, an FR2 having the sequence set forth in SEQ ID NO: 22, an FR3 having the sequence set forth in SEQ ID NO: 23, and an FR4 having the sequence set forth in SEQ ID NO: 24; (iv) a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 17, an FR2 having the sequence set forth in SEQ ID NO: 18, an FR3 having the sequence set forth in SEQ ID NO: 19, and an FR4 having the sequence set forth in SEQ ID NO: 20; or (v) A VH comprising an FR1 having the sequence set forth in SEQ ID NO: 21, an FR2 having the sequence set forth in SEQ ID NO: 22, an FR3 having the sequence set forth in SEQ ID NO: 23, and an FR4 having the sequence set forth in SEQ ID NO: 24; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 17, an FR2 having the sequence set forth in SEQ ID NO: 18, an FR3 having the sequence set forth in SEQ ID NO: 19, and an FR4 having the sequence set forth in SEQ ID NO: 20.

[0042] The present invention also provides an antigen-binding protein comprising an antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to plasminogen, and wherein the antigen-binding domain comprises at least one of the following: (i) a VH comprising a complementarity determining region (CDR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 36, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 37, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 38; (ii) a VH comprising a sequence at least about 95%, or 96%, or at least 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 40; (iii) a VL comprising a CDR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 33, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 34, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 35; (iv) a VL having a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 39; (v) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO: 36, a CDR2 having the sequence set forth in SEQ ID NO: 37, and a CDR3 having the sequence set forth in SEQ ID NO: 38; (vi) a VH having the sequence set forth in SEQ ID NO: 40; (vii) a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 33, a CDR2 having the sequence set forth in SEQ ID NO: 34, and a CDR3 having the sequence set forth in SEQ ID NO: 35; (viii) a VL having the sequence set forth in SEQ ID NO: 39; (ix) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO: 36, a CDR2 having the sequence set forth in SEQ ID NO: 37, and a CDR3 having the sequence set forth in SEQ ID NO: 38; and a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 33, a CDR2 having the sequence set forth in SEQ ID NO: 34, and a CDR3 having the sequence set forth in SEQ ID NO: 35; or (x) A VH having the sequence set forth in SEQ ID NO: 40 and a VL having the sequence set forth in SEQ ID NO: 39.

[0043] In any embodiment of the invention, the antigen binding domain further comprises at least one of the following: (i) a VH comprising a framework region (FR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 53; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 54; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 55; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 56; (ii) a VL comprising: an FR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:49; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:50; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:51; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:52; (iii) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 53, an FR2 having the sequence set forth in SEQ ID NO: 54, an FR3 having the sequence set forth in SEQ ID NO: 55, and an FR4 having the sequence set forth in SEQ ID NO: 56; (iv) a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 49, an FR2 having the sequence set forth in SEQ ID NO: 50, an FR3 having the sequence set forth in SEQ ID NO: 51, and an FR4 having the sequence set forth in SEQ ID NO: 52; (v) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 53, an FR2 having the sequence set forth in SEQ ID NO: 54, an FR3 having the sequence set forth in SEQ ID NO: 55, and an FR4 having the sequence set forth in SEQ ID NO: 56; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 49, an FR2 having the sequence set forth in SEQ ID NO: 50, an FR3 having the sequence set forth in SEQ ID NO: 51, and an FR4 having the sequence set forth in SEQ ID NO: 52.

[0044] As described herein, an antigen binding protein may be any of the following: (i) single-chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) one of (i) or (ii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (iv) one of (i) or (ii) linked to a protein that binds to immune effector cells. It may be in the form of:

[0045] Additionally, as described herein, the antigen binding protein may be: (i) Diabodies; (ii) triabodies; (iii) tetrabodies; (iv)Fab; (v) F(ab'): (vi) Fv; (vii) bispecific or other forms of multispecific antibodies; (viii) one of (i)-(vii) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (viv) one of (i) to (vii) linked to a protein that binds to immune effector cells; It may be in the form of:

[0046] The antigen-binding protein described above may also be referred to as the antigen-binding domain of an antibody.

[0047] Preferably, the antigen-binding proteins described herein are antibodies or antigen-binding fragments thereof. Typically, the antigen-binding proteins are antibodies, for example, monoclonal antibodies. They may be in the form of recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half antibodies, bispecific antibodies, trispecific antibodies, or multispecific antibodies). Antibodies may further comprise chemical modifications, such as conjugation with an active drug or radiolabel, or an agent to improve solubility, or other chemical modifications described herein.

[0048] As described herein, the antigen binding protein may be a variable domain.

[0049] The present invention also provides a plasminogen antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region is selected from the group consisting of: CDR L1 set forth in SEQ ID NO: 1, CDR L2 set forth in SEQ ID NO: 2, and CDR L3 set forth in SEQ ID NO: 3 including; and The heavy chain variable region comprises: CDR H1 set forth in SEQ ID NO: 4, CDR H2 set forth in SEQ ID NO: 5, and CDR H3 set forth in SEQ ID NO: 6 Includes.

[0050] In any embodiment of the invention, the plasminogen antibody comprises a light chain variable region having the sequence of SEQ ID NO:7.

[0051] In any embodiment of the invention, the plasminogen antibody comprises a heavy chain variable region having the sequence of SEQ ID NO:8.

[0052] In any embodiment of the present invention, the plasminogen antibody comprises a light chain variable region having FR L1 set forth in SEQ ID NO: 17, FR L2 set forth in SEQ ID NO: 18, FR L3 set forth in SEQ ID NO: 19, and FR L4 set forth in SEQ ID NO: 20.

[0053] In any embodiment of the invention, the plasminogen antibody comprises a heavy chain variable region having FR H1 set forth in SEQ ID NO: 21, FR H2 set forth in SEQ ID NO: 22, FR H3 set forth in SEQ ID NO: 23, and FR H4 set forth in SEQ ID NO: 24.

[0054] The present invention also provides a plasminogen antibody comprising a light chain variable region and a heavy chain variable region, wherein the light chain variable region is selected from the group consisting of: CDR L1 set forth in SEQ ID NO: 33, CDR L2 set forth in SEQ ID NO: 34, and CDR L3 set forth in SEQ ID NO: 35 including; and The heavy chain variable region comprises: CDR H1 set forth in SEQ ID NO: 36, CDR H2 set forth in SEQ ID NO: 37, and CDR H3 set forth in SEQ ID NO: 38 Includes.

[0055] In any embodiment of the invention, the plasminogen antibody comprises a light chain variable region having the sequence of SEQ ID NO:39.

[0056] In any embodiment of the invention, the plasminogen antibody comprises a heavy chain variable region having the sequence of SEQ ID NO:40.

[0057] In any aspect of the invention, the plasminogen antibody comprises a light chain variable region having FR L1 set forth in SEQ ID NO: 49, FR L2 set forth in SEQ ID NO: 50, FR L3 set forth in SEQ ID NO: 51, and FR L4 set forth in SEQ ID NO: 52.

[0058] In any embodiment of the invention, the plasminogen antibody comprises a light chain variable region having FR H1 set forth in SEQ ID NO: 53, FR H2 set forth in SEQ ID NO: 54, FR H3 set forth in SEQ ID NO: 55, and FR H4 set forth in SEQ ID NO: 56.

[0059] In any aspect or embodiment, the antibody is a naked antibody, specifically, the antibody is in unconjugated form and has not been configured to form a conjugate.

[0060] In certain embodiments, the complementarity determining region sequences (CDRs) of the antigen binding proteins of the invention can be defined according to the IMGT numbering system.

[0061] Reference herein to a protein or antibody that "binds" plasminogen gives literal support to the protein or antibody "specifically binds to" or "specifically binds to" plasminogen.

[0062] The present invention also provides an antigen-binding domain or antigen-binding fragment of the above antibody.

[0063] The present invention also provides fusion proteins comprising an antigen binding protein, immunoglobulin variable domain, antibody, dab (single domain antibody), di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single chain antibody molecule, or multispecific antibody described herein.

[0064] The present invention also provides conjugates in the form of antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies or fusion proteins as described herein, conjugated to a label or a cytotoxic agent.

[0065] The present invention also provides antibodies for binding to the antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies, fusion proteins, or conjugates described herein.

[0066] The present invention also provides nucleic acids encoding the antigen binding proteins, immunoglobulin variable domains, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies, fusion proteins, or conjugates described herein.

[0067] In one example, such a nucleic acid is included in an expression construct in which the nucleic acid is operably linked to a promoter. Such an expression construct can be a vector, for example, a plasmid.

[0068] In the example of the invention relating to a single polypeptide chain antigen binding protein, the expression construct may include a promoter linked to the nucleic acid encoding the polypeptide chain.

[0069] In examples involving multiple polypeptide chains forming an antigen binding protein, the expression construct comprises a nucleic acid operably linked to a promoter, e.g., encoding a polypeptide comprising a VH, and a nucleic acid operably linked to a promoter, e.g., encoding a polypeptide comprising a VL.

[0070] In another example, the expression construct may comprise, for example, the following components operably linked in 5'-3' order: (i) Promoter (ii) a nucleic acid encoding a first polypeptide; (iii) an internal ribosome entry site; and (iv) a nucleic acid encoding a second polypeptide is a bicistronic expression construct comprising wherein the first polypeptide comprises a VH and the second polypeptide comprises a VL, or vice versa.

[0071] The present invention also contemplates separate expression constructs, one encoding a first polypeptide comprising a VH and the other encoding a second polypeptide comprising a VL. For example, the present invention also contemplates (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH, operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL, operably linked to a promoter; A composition comprising:

[0072] The present invention provides cells comprising the vectors or nucleic acids described herein. Preferably, the cells are isolated, substantially purified, or recombinant. In one example, the cells contain an expression construct of the invention or: (i) a first expression construct comprising a nucleic acid encoding a polypeptide comprising a VH, operably linked to a promoter; and (ii) a second expression construct comprising a nucleic acid encoding a polypeptide comprising a VL, operably linked to a promoter; Including, The first and second polypeptides now associate to form the antigen binding protein of the invention.

[0073] Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0074] The present invention also provides a peptide comprising, consisting essentially of, or consisting of residues 554-569 of SEQ ID NO:65, which binds to a peptide comprising, consisting essentially of, or consisting of residues 493-533, and which binds to a peptide comprising, consisting essentially of, or consisting of residues 468-525 of SEQ ID NO:65.

[0075] The present invention also provides pharmaceutical compositions comprising the antigen binding proteins described herein, or comprising the CDR and / or FR sequences, or immunoglobulin variable domains described herein, antibodies, dabs (single domain antibodies), di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, or multispecific antibodies, fusion proteins, or conjugates, and a pharmaceutically acceptable carrier, diluent or excipient.

[0076] The present invention also provides diagnostic compositions comprising the antigen binding proteins described herein, or comprising the CDR and / or FR sequences, or antigen binding sites described herein, immunoglobulin variable domains, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, or multispecific antibodies, fusion proteins or conjugates, a diluent and optionally a label.

[0077] The present invention also provides kits or articles of manufacture comprising the antigen binding proteins described herein, or comprising the CDR and / or FR sequences or immunoglobulin variable domains described herein, antibodies, dabs, di-scFvs, scFvs, Fabs, Fab's, F(ab')2, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single chain antibody molecules, or multispecific antibodies, fusion proteins or conjugates.

[0078] The antigen-binding proteins described herein may comprise a human constant region, e.g., an IgG constant region, such as an IgG1, IgG2, IgG3, or IgG4 constant region, or a mixture thereof. In the case of antibodies or proteins comprising a VH and a VL, the VH may be linked to a heavy chain constant region, and the VL may be linked to a light chain constant region.

[0079] In one example, the antigen binding protein described herein comprises the constant region of an IgG4 antibody or a stabilized constant region of an IgG4 antibody. In one example, the protein or antibody comprises an IgG4 constant region with a proline at position 241 (according to the Kabat numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991)).

[0080] In one example, an antigen binding protein described herein, or a composition of an antigen binding protein described herein, comprises a heavy chain constant region, which comprises a stabilized heavy chain constant region comprising a mixture of sequences that completely, partially, or completely lack a C-terminal lysine residue.

[0081] In one example, the antigen binding protein comprises a VH disclosed herein linked or fused to an IgG4 constant region or a stabilized IgG4 constant region (e.g., as described above), and the VL linked or fused to a κ light chain constant region.

[0082] It is understood that the functional properties of the antigen binding proteins of the present invention apply mutatis mutandis to the antibodies of the present invention.

[0083] The antigen binding proteins described herein can be purified, substantially purified, isolated, and / or recombinant.

[0084] The antigen binding protein of the invention may be part of the supernatant harvested from the culture medium in which a hybridoma expressing the antigen binding protein of the invention has been grown.

[0085] The present invention also provides a method of restoring hemostasis or inhibiting plasminogen activation in a subject that has suffered a trauma or has suffered a hemorrhage or is bleeding (e.g. due to surgery, trauma or after childbirth), which method comprises administering to the subject an antigen binding protein of the invention, thereby restoring hemostasis or inhibiting plasminogen activation in the subject.

[0086] The present invention provides a method of inhibiting fibrinolysis in a subject in need thereof, the method comprising administering to the subject an antigen binding protein of the invention, thereby inhibiting fibrinolysis in the subject. As used herein, a subject in need of inhibition of fibrinolysis may include a subject who has suffered a trauma (e.g., a car accident, fall or other severe injury), has undergone surgery, is experiencing bleeding due to childbirth, or for any other reason requiring inhibition of fibrinolysis.

[0087] The present invention also provides a method of treating or preventing, or reducing the severity or progression of, a bacterial infection in a subject, which method comprises administering to the subject an antigen binding protein of the invention, thereby treating or preventing, or reducing the severity or progression of the bacterial infection in the subject. In this regard, the antigen binding protein may also be used to prevent recurrence of the infection, which is considered to be preventing the infection.

[0088] The present invention also provides a method of treating a condition associated with or caused by a bacterial infection in a subject, the method comprising administering to the subject an effective amount of an antigen binding protein of the invention, thereby treating the condition associated with or caused by a bacterial infection in the subject. The condition associated with or caused by a bacterial infection may be any of the conditions described herein. In any embodiment, the bacterial infection may be either chronic or acute.

[0089] The present invention also provides a method of reducing the severity of a bacterial infection in a subject, the method comprising administering to the subject an antigen binding protein of the invention, thereby reducing the severity of the bacterial infection in the subject.

[0090] Furthermore, the present invention also provides a method of treating or preventing cancer in a subject, which method comprises administering to the subject an antigen binding protein of the invention, thereby treating or preventing cancer in the subject. As used herein, methods of treating cancer include methods of inhibiting, preventing, or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer.

[0091] In any of the treatment methods of the invention, the method may comprise the administration of any combination of antigen binding proteins of the invention. In a specific embodiment, the combination comprises a first antigen binding protein comprising a light chain variable region comprising the sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the sequence of SEQ ID NO: 40; and a second antigen binding protein having a light chain variable region comprising the sequence of SEQ ID NO: 7; and a heavy chain variable region comprising the sequence of SEQ ID NO: 8.

[0092] The present invention also provides the use of a plasminogen binding antigen binding protein of the invention in the manufacture of a medicament for restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered trauma or who is in need of restoring hemostasis or inhibiting plasminogen activation after surgery or childbirth.

[0093] The present invention also provides the use of a plasminogen binding antigen binding protein of the present invention in the manufacture of a medicament for restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered a traumatic injury.

[0094] The present invention also provides the use of an antigen binding protein of the invention in the manufacture of a medicament for inhibiting fibrinolysis in a subject in need thereof. As used herein, a subject in need of inhibiting fibrinolysis may include a subject who has suffered a trauma (e.g. a car accident, fall or other severe injury), has undergone surgery, is experiencing bleeding due to childbirth, or for any other reason requiring the inhibition of fibrinolysis.

[0095] The present invention also provides the use of a plasminogen-binding antigen-binding protein of the present invention in the manufacture of a medicament for the treatment or prevention of a bacterial infection.

[0096] The present invention also provides the use of a plasminogen binding antigen binding protein of the present invention in the manufacture of a medicament for the treatment, prevention or lessening of the severity of any condition or disease caused by or associated with a bacterial infection.

[0097] Furthermore, the present invention provides the use of an antigen binding protein of the present invention in the manufacture of a medicament for the treatment or prevention of cancer in a subject, which medicament may also be intended to inhibit, prevent or minimise the spread or progression of cancer, including metastasis of cancer.

[0098] In any use of the invention, said use comprises the combination of any of the antigen binding proteins of the invention in the manufacture of a medicament, preferably wherein said combination comprises a first protein having a light chain variable region comprising the sequence of SEQ ID NO: 39 and a heavy chain variable region comprising the sequence of SEQ ID NO: 40; and a second protein having a light chain variable region comprising the sequence of SEQ ID NO: 7; and a heavy chain variable region comprising the sequence of SEQ ID NO: 8.

[0099] The present invention also provides a pharmaceutical composition comprising a plasminogen binding antigen binding protein of the present invention and a pharmaceutically acceptable excipient.

[0100] The pharmaceutical composition is preferably intended for the uses described herein. Accordingly, the present invention provides a pharmaceutical composition comprising a plasminogen binding antigen binding protein of the present invention for use in restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered trauma or who is in need of restoring hemostasis or inhibiting plasminogen activation after surgery or childbirth.

[0101] The present invention also provides a pharmaceutical composition comprising a plasminogen binding antigen binding protein of the present invention for use in restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered a traumatic injury.

[0102] The present invention provides pharmaceutical compositions comprising an antigen binding protein of the invention for use in inhibiting fibrinolysis in a subject in need thereof. As used herein, a subject in need of inhibition of fibrinolysis may include a subject who has suffered a trauma (e.g., a car accident, fall or other severe injury), has undergone surgery, is experiencing bleeding due to childbirth, or for any other reason requiring inhibition of fibrinolysis.

[0103] The present invention also provides a pharmaceutical composition comprising a plasminogen binding antigen binding protein of the present invention for use in the treatment or prevention of a bacterial infection.

[0104] The present invention also provides a pharmaceutical composition comprising a plasminogen binding antigen binding protein of the present invention for use in the treatment, prevention or lessening of the severity of any condition or disease caused by or associated with a bacterial infection.

[0105] Furthermore, the present invention provides pharmaceutical compositions comprising the plasminogen binding antigen binding proteins of the present invention for use in treating or preventing cancer in a subject. The pharmaceutical compositions may also be aimed at inhibiting, preventing, or minimizing the spread or progression of cancer, including cancer metastasis.

[0106] In any of the aforementioned methods or uses of the invention, the bacterial infection may be caused by spore-forming or non-spore-forming bacteria. Where the infection is caused by spore-forming bacteria, the infection may be characterised by the bacteria in a vegetative state or in the spore form. It will be appreciated that upon targeting the spore form of the bacteria, the antigen binding protein of the invention may thereby act to prevent or reduce the likelihood of re-infection with the bacteria.

[0107] In any embodiment of the present invention, the bacterium is a gram-positive bacterium, preferably a gram-positive cocci. In any embodiment, the gram-positive bacterium belongs to the order Bacillales, Clostridiales or Lactobacillales.

[0108] In any embodiment of the present invention, the bacterium belongs to the family Streptococcaceae. Preferably, the bacterium belongs to the genus Streptococcus. More preferably, the bacterium is a Group A Streptococcus (GAS), preferably Streptococcus pyogenes. In certain embodiments, the infection may be an infection caused by a bacterium selected from the group consisting of Streptococcus pyogenes, Streptococcus dysgalactiae, Streptococcus pneumonia, Streptococcus agalactiae, Streptococcus canis, Streptococcus equisimilis, Streptococcus mutans, and Streptococcus suis.

[0109] In any aspect of the invention, the bacterium belongs to the family Bacillaceae, preferably the genus Bacillus, more preferably in which case the infection is caused by bacteria of the species Bacillus anthracis and Bacillus cereus.

[0110] In any embodiment of the present invention, the bacterium belongs to the family Staphylococcusaceae. Preferably, the bacterium belongs to the genus Staphylococcus. More preferably, the infection may be caused by a bacterium selected from the group consisting of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), and vancomycin-resistant Staphylococcus aureus (VRSA). In a specific embodiment, the bacterium is Staphylococcus epidermidis.

[0111] In any embodiment of the present invention, the bacterium belongs to the family Peptostreptococcaceae. Preferably, the bacterium belongs to the genus Clostridioides. More preferably, the bacterial infection is an infection caused by a bacterium selected from the group consisting of Clostridium difficile (also known as Clostridioides difficile).

[0112] In some embodiments, the bacterium belongs to the family Clostridiaceae. The bacterium may belong to the genus Clostridium, optionally where the bacterial infection is an infection by any one of Clostridium botulinum, Clostridium tetani, Clostridium perfringens, and Clostridium sordellii.

[0113] In some embodiments, the bacterium belongs to the family Porphyromonadaceae, including Porphyromonas gingivalis.

[0114] In any aspect of the invention, the bacterium is Gram-negative and optionally belongs to the order Enterobacteriales, Campylobacterales or Bacteroidales.

[0115] In any embodiment of the invention, the bacterium belongs to the family Yersiniaceae, preferably to the genus Yersinia, more preferably in this case the infection is caused by bacteria of the species Yersinia pestis, Yersinia enterocolitica.

[0116] In any embodiment of the invention, the bacterium belongs to the family Helicobacteraceae, preferably the genus Helicobacter, and more preferably the infection is caused by bacteria of the species Helicobacter pylori.

[0117] Thus, the methods, uses or pharmaceutical compositions of the present invention are useful for treating, preventing or reducing the severity of any disease caused by or associated with the bacteria set forth herein. For example, the methods, uses or pharmaceutical compositions of the invention may be aimed at treating, preventing or reducing the severity of any disease / infection caused by or associated with Group A Streptococcus (GAS), Staphylococcus or Bacillus species, including but not limited to pharyngitis, tonsillitis, scarlet fever, cellulitis, erysipelas, rheumatic fever, skin and soft tissue infections, endocarditis, osteoarticular infections, implant infections, post-streptococcal glomerulonephritis, necrotizing fasciitis, myonecrosis, subperiosteal abscess, necrotizing pneumonia, pyomyositis, mediastinitis, myocardial, perirenal, hepatic and pancreatic abscesses, septic thrombophlebitis, and severe eye infections such as endophthalmitis and lymphangitis.

[0118] The methods, uses or pharmaceutical compositions of the invention may also be aimed at treating, preventing or reducing the severity of any disease / infection caused by or associated with Clostroides difficile (Clostridium difficile), including gastrointestinal infections, mild diarrhea and enteritis.

[0119] The methods, uses or pharmaceutical compositions of the invention may also be aimed at treating, preventing or reducing the severity of any disease / infection caused by or associated with P. gingivalis difficile, including periodontal disease and gum inflammation.

[0120] The methods, uses or pharmaceutical compositions of the invention may also be aimed at treating, preventing or reducing the severity of any disease / infection caused by or associated with Yersinia species, including plague (pneumonic, septicemic or bubonic) and yersiniosis.

[0121] The methods, uses or pharmaceutical compositions of the present invention may also be aimed at treating, preventing or reducing the severity of any disease / infection caused by or associated with Helicobacter, including acute or chronic gastritis, gastric ulcers, atrophy of the stomach wall, duodenal ulcers and susceptibility to or risk of gastric cancer.

[0122] In any embodiment of the present invention, the method or use for treating or preventing a bacterial infection includes a method or use for inhibiting or minimizing the extent of bacterial invasion of host tissues / organs.

[0123] The methods and uses of the present invention can be applied to the treatment or prevention of cancer, examples of which include blood cancer, epithelial cancer, liver cancer, pancreatic cancer, gastric cancer, osteosarcoma, endometrial cancer and ovarian cancer.

[0124] The present invention further provides nucleic acid molecules encoding the antigen binding proteins of the present invention, or functional fragments or derivatives thereof.

[0125] The present invention also provides cells comprising the vectors or nucleic acid molecules described herein.

[0126] The present invention also provides animals or tissues derived from the cells described herein.

[0127] In another aspect, the present invention provides a kit or article of manufacture comprising an antigen binding protein of the invention or a pharmaceutical composition described herein.

[0128] In other embodiments, there is provided a kit for use in the therapeutic or prophylactic applications described herein, the kit comprising: a container holding an antigen-binding protein or pharmaceutical composition of the invention; - A label or package insert containing instructions for use Includes.

[0129] As used herein, unless the context requires otherwise, the term "comprise" and conjugations of this term (e.g., "comprising," "comprises," and "comprised") do not exclude additional additives, components, integers, or steps.

[0130] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which: [Brief explanation of the drawings]

[0131] [Figure 1] Binding of G05 to plasminogen and plasmin. G05 was immobilized on a NiHc chip; the binding kinetics of (A) plasminogen and (B) plasmin were measured by multi-cycle surface plasmon resonance (SPR). The dotted lines represent a 1:1 Langmuir model fit to the experimental data. (C) Table showing the kinetic constants (ka and kd) and affinity constant (KD). [Figure 2] Binding of G11 to plasminogen and plasmin. G11 was immobilized on a NiHc chip; the binding kinetics of (A) plasminogen and (B) plasmin were measured by multi-cycle surface plasmon resonance (SPR). The dotted lines represent a 1:1 Langmuir model fit to the experimental data. (C) Table showing the kinetic constants (ka and kd) and affinity constant (KD). [Figure 3]Inhibition of tPA-mediated plasminogen activation by G05 (A) and G11 (B) in solution in the presence of EACA. [Figure 4] Inhibition of tPA-mediated plasminogen activation by G05 (A) and G11 (B) in the presence of fibrin. [Figure 5] G05 does not inhibit plasmin activity; G11 significantly inhibits plasmin activity. Plasmin activity measured in the presence of G05 or G11 (0-2000 nM); 5 nM plasmin. [Figure 6] Inhibition of plasminogen binding to streptokinase by G05. (A) 10 nM plasminogen R561,S741A was passed over streptokinase immobilized on a CM4 chip in the presence of G05 or naive chicken Ab, i.e., gAb (0–500 nM). G05 showed inhibition, especially at high concentrations. The control naive chicken Ab showed no inhibition of Pig binding to SK. (B) Percentage of SK binding in the presence of 500 nM Ab, normalized to the no-antibody control. [Figure 7] Inhibition of streptokinase-mediated plasminogen activation by G05. G05 inhibits plasminogen activation by SK, as early addition of antibody at t=0 and 10 min completely abolished plasmin generation. [Figure 8] Binding of G05 and G11 to recombinant serine protease domains. (A) Size-exclusion chromatography showing binding of G05 to recombinant kringle 5-serine protease domain (K5SP). A higher molecular weight complex (K5SP + G05, solid line) was observed compared to K5SP alone (dashed line) or G05 alone (dotted line). (B) Size-exclusion chromatography showing binding of G11 to recombinant serine protease domain (SP). Size-exclusion chromatography shows a higher molecular weight complex (SP + G11, solid line) compared to SP alone (dashed line) or G11 alone (dotted line). [Figure 9]Crystal structure of G05 and G11 bound to the serine protease domain of plasminogen. (A) The crystal structure of the KR5-SP / G05 binary complex shows that G05 binds to the plasminogen activation loop. KR5 and SP are indicated, and the stick-shaped activation loop, G05 light chain (LC), and heavy chain (HC) are labeled. The top image shows the binary complex, and the bottom image shows key residues (labeled and numbered) involved in the intramolecular interactions described above. Dashed lines are used to indicate polar interactions. (B) The crystal structure of the SP / G11 binary complex reveals that G11 forms polar interactions with the serine protease domain of plasminogen / plasmin, thereby distorting the catalytic pocket and surrounding loops and preventing plasmin production and activity. SP is indicated, and the G11 light chain (LC) and heavy chain (HC) are labeled. The top image shows the binary complex, and the bottom image shows key residues (labeled and numbered) involved in the intramolecular interactions described above. Dashed lines are used to indicate polar interactions. [Figure 10] G05 inhibition of plasminogen binding to Group A Streptococcus (GAS) as measured by flow cytometry. Binding of fluorescently labeled plasminogen to GAS is inhibited by G05 compared to gAb and control. Also shown is the inhibition of plasminogen binding to GAS by tranexamic acid (TXA). Bar graphs show median fluorescence intensity plotted for each scFv and control. [Figure 11] G05 inhibits plasminogen binding to Group A Streptococcus (GAS) as measured by SDS-PAGE. Binding of fluorescently labeled plasminogen to GAS was measured by fluorescent scanning of (A) SDS-PAGE gels, which show total plasminogen added to the assay (total), bound, and unbound, as well as washes collected during the assay. (B) The plasminogen band intensity in (A) was normalized to the HBS control. Of all samples, only G05 showed significant inhibition of Plg binding to GAS cells. [Figure 12] G05 inhibits plasminogen activation by Group A Streptococcus (GAS) (enzyme assay). (Top) Plasminogen bound to G05 was incubated with GAS. The plasmin produced was measured with a fluorogenic substrate. Plasminogen activation by GAS is presumably mediated by streptokinase (SK). (Bottom) Progress curves of plasminogen activation in the presence and absence of recombinant SK. [Figure 13]hPLG is recruited to the CDI-injured intestine, exacerbating tissue damage and disease and aiding in spore dissemination to infected hosts. (A) Representative PAS / Alcian blue-stained cecal sections from hPLG transgenic or hPLG-injected WT mice uninfected or infected with C. difficile M7404WT or M7404 toxin mutant (AB-). Brackets ([) indicate crypt hyperplasia, arrowheads (▲) indicate epithelial damage, and asterisks (*) indicate edema and inflammation. Scale bars represent 200 μm. (B) Western blot of standard amounts of hPLG from the cecum of hPLG transgenic C. difficile-infected mice versus uninfected mice. Purified hPLG is included as a positive control (lane 1). Shown are hPLG extracted from the ceca of uninfected hPLG (lane 2) and C. difficile-infected hPLG mice (lane 3), and hPLG-injected uninfected (lane 4), M7404 toxin mutant (AB-) (lane 5), or M7404-infected C57BL / 6 mice (lane 6). Units (100 and 75) refer to the molecular weight (kDa) of the protein standard. (C) Stool consistency, (D) cage appearance, and (E) physiological appearance. (F) Survival time for hPLG transgenic (n = 20) and C57BL / 6 (n = 13) C. difficile M7404-infected mice, and uninfected controls for hPLG (n = 7) and C57BL / 6 (n = 11) mice. (G, H) Toxin levels in feces from C. difficile-infected hPLG transgenic or C57BL / 6 mice assessed using HT29 cells (TcdA) (G) and Vero cells (TcdB). (H) No statistically significant differences in toxin levels were observed. (I) C. difficile spore counts in the kidney, spleen, and thymus of C. difficile-infected hPLG transgenic or C57BL / 6 mice. (J) Inflammation in hPLG transgenic-infected and uninfected mice.(K) Survival times for mPLG KO (n = 18) and C57BL / 6 (n = 18) C. difficile M7404-infected mice and for uninfected controls: mPLG KO (n = 5) and C57BL / 6 (n = 6) mice. Statistical analysis was determined using one-way ANOVA with Tukey's multiple comparisons (**P < 0.005; ***P < 0.0005; ****P < 0.0001) (C, D, E, J) or Mann-Whitney test (*P < 0.05; **P < 0.001; ***P < 0.0001) (F, G, H, I, K). [Figure 14] hPLG alters host inflammatory responses and tissue integrity during CDI. (A) Cytokine levels using a 22-plex cytokine array and TIMP-1 ELISA are shown for tissue lysates from the cecum of either PBS-injected (white bars) or hPLG-injected (black bars) C. difficile M7404-infected mice. The multiplication factor represents the cytokine levels present in the intestinal tissue of infected mice normalized to the levels obtained from the respective uninfected mouse group. Only cytokine levels that differed between hPLG- and PBS-injected and infected mice are shown. (B) The abundance (Log10) of the same cytokines that differed between infected hPLG-injected (orange squares) and infected PBS-injected (black circles) mice are shown for the cecum of each uninfected mouse group. Note that cytokine levels in uninfected mice were examined to confirm that changes in cytokine expression were not due to the presence of hPLG alone. Cytokine levels in hPLG-injected but uninfected mice were slightly (but not significantly) lower than in PBS-injected uninfected mice, but cytokine levels in both uninfected groups were lower compared to those in infected mice. Statistical analysis was determined using the Mann-Whitney test (*P<0.05; **P<0.001; ***P<0.0001). [Figure 15]Human, equine, and porcine PLG bind to C. difficile spores, but mouse PLG does not. (A) Western blot of C. difficile M7404 vegetative cells using anti-human PLG Ab. Samples show vegetative cells incubated in the absence (V) or presence (VP) of hPLG. (B) Representative Biacore sensor curves showing the interaction between human, equine, and porcine PLG and M7404 spores (black squares, purple circles, and orange triangles, respectively), and the lack of interaction between mouse PLG and spores (blue diamonds). Data were fitted to a one-site specific binding model. (C) Binding of hPLG to C. difficile spores isolated from strains from various locations and origins (M7404 Canadian human isolate (E1); R20291 UK human isolate (E2); JGS6133 US animal isolate (A-US); A135 Australian animal isolate (A-AU); DLL3109 Australian human isolate (E-AU); VPI10463 US human reference isolate (R); and CD37 US nontoxigenic isolate (NT)) detected with an anti-human PLG Ab. For (A) and (C), samples representing spores incubated in the absence (S) or presence (SP) of hPLG are shown. Purified hPLG (positive control) protein (P) and supernatant (W) washed five times with binding buffer to remove all unbound PLG are also included as controls. The units (100 and 75) refer to the molecular weight (kDa) of the protein standard. (D-F) Stimulated emission depletion (STED) microscopy of in vitro-derived M7404 spores stained with anti-spore antibodies (green) conjugated to hPLG (red). (G-I) STED microscopy of human-derived fecal C. difficile spores stained with anti-spore antibodies (green) conjugated to hPLG (red). (J-L) STED microscopy of mouse-derived fecal M7404 spores. Anti-spore antibody staining is shown in green; hPLG staining is shown in red. Merged STED images of in vitro-derived spores, human fecal-derived spores, and mouse fecal-derived spores are shown in F, I, and L, respectively. [Figure 16]Plasmin binding to C. difficile spores remodels the spore surface and increases germination efficiency. TEM cross-sections of (A) untreated or (B) plasmin-bound M7404 spores. Boxed regions from (A) and (B) are shown in (C) and (D), respectively. (E) The average exosporium length (nm) was measured from untreated and plasmin-bound M7404 spores (n = 40 spores / group, 5 measurements per spore). Statistical analysis was determined using the Mann-Whitney U test (P < 0.0001). (F) Germination rates of untreated (orange squares) or plasmin-bound (blue triangles) M7404 spores in the presence of 50 μM sodium taurocholate. Untreated spores (black squares) incubated in the absence of sodium taurocholate were included as a negative germination control. Data represent the ratio of OD600 at each time point (OD600(t)) to OD600 at time point 0 (OD600(t0)), measured over time (minutes). Statistical analysis was determined using a two-way one-way ANOVA with Tukey's multiple comparisons (P < 0.01 for untreated spores versus plasmin-bound spores in the presence of germinant). [Figure 17] Administration of anti-plasminogen antibody reduces disease severity, delays onset, and increases survival in mice infected with C. difficile. (A) Cage appearance, (B) stool consistency, (C) physiological appearance, and (D) survival curves of hPLG-injected and C. difficile M7404-infected mice IP injected with either HEPES alone, G05 antibody, or naive chicken antibody. Statistical analysis was determined using the Mann-Whitney test; *P<0.05. [Figure 18] Cross-reactivity of G05 and G11 antibodies. G05 antibody does not significantly inhibit related serine proteases, including plasmin. G11 antibody does not significantly inhibit related serine proteases, except for plasmin. [Figure 19]Administration of the G05 antibody reduces bleeding. Bleeding volume (measured at OD550nm) was measured from wild-type C56BL / 6 (WT) mice and mice with a homozygous loss-of-function mutation in the gene encoding plasminogen (Plg- / -) in tail bleeding experiments. All mice received 200 μg of human plasminogen and 50 μg of either gAb (negative control) or G05. The amount of bleeding in mice receiving the G05 antibody was significantly reduced compared to mice receiving the naive antibody. [Figure 20] G11 inhibits whole blood clot lysis in the presence of red blood cells and platelets. The time required to achieve 50% whole blood clot lysis (IC50) by G11, α2AP, and aprotinin is shown. G11 is approximately 2-fold more effective than α2AP and approximately 2.5-fold more effective than aprotinin in inhibiting whole blood clot lysis. DETAILED DESCRIPTION OF THE INVENTION

[0132] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention.

[0133] Further aspects of the invention and further embodiments of the aspects described in the preceding paragraphs will become apparent from the following description, given by way of example and with reference to the accompanying drawings, in which:

[0134] Reference will now be made in detail to specific embodiments of the invention. While the invention will be described in conjunction with the embodiments, it will be understood that it is not intended to limit the invention to those embodiments. Rather, the invention is intended to cover all alternatives, modifications, and equivalents which may be included within the scope of the present invention as defined by the claims.

[0135] The present inventors have developed antigen binding proteins, eg, antibodies, that bind to plasminogen and inhibit or reduce its activation.

[0136] By virtue of their ability to bind to and inhibit or reduce the activation of plasminogen, and as described in more detail herein, the antigen binding proteins of the invention are useful in treating, preventing or slowing the progression of conditions or diseases mediated by plasminogen activation to plasmin, for example, they are useful in inhibiting fibrinolysis and promoting hemostasis following trauma or surgery, bleeding after childbirth, or any other situation in which inhibition of plasminogen activation is required.

[0137] The antigen binding proteins of the invention also have utility for treating or preventing bacterial infections in which bacterial pathogens recruit the plasmin system to promote invasion of host tissues. In particular, the antigen binding proteins of the invention are useful for treating or preventing infections caused by Streptococcus spp., Staphylococcus spp., Yersinia pestis, Helicobacter pylori, E. coli, Salmonella spp., and P. gingivalis.

[0138] The plasmin system may also be used by invasive tumors to promote angiogenesis and metastasis. Thus, the antigen binding proteins of the invention also have the ability to inhibit or reduce one or more aspects of inflammatory, tumor growth and metastatic activity.

[0139] Overview Throughout this specification, unless specifically stated otherwise or the context requires otherwise, reference to a single step, composition, group of steps, or group of compositions shall be understood to include one and a plurality (i.e., one or more) of those steps, compositions, group of steps, or group of compositions. Thus, as used herein, the singular forms "a," "an," and "the" include plural aspects, and vice versa, unless the context clearly indicates otherwise. For example, reference to "a" includes one as well as two or more; reference to "an" includes one as well as two or more; reference to "the" includes one as well as two or more, etc.

[0140] Those skilled in the art will understand that the present invention is susceptible to variations and modifications other than those specifically described. It is to be understood that the present invention includes all such variations and modifications. The present invention also includes all of the steps, features, compositions, and compounds mentioned or shown in this specification, individually or collectively, as well as any combination of any two or more of said steps or features.

[0141] One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in the practice of the present invention, and is in no way intended to be limited to the methods and materials described.

[0142] All patents and publications referenced herein are incorporated by reference in their entirety.

[0143] The present invention is not to be limited in scope by the specific examples described herein, which are intended as illustrations only. Functionally equivalent products, compositions and methods are clearly within the scope of the invention.

[0144] Any example or embodiment of the present invention herein shall be understood to apply mutatis mutandis to any other example or embodiment of the present invention, unless expressly stated otherwise.

[0145] Unless otherwise defined, all technical and scientific terms used herein shall be understood to have the same meaning as commonly understood by one of ordinary skill in the art (e.g., in cell culture, molecular genetics, immunology, immunohistochemistry, protein chemistry, and biochemistry).

[0146] Unless otherwise indicated, the recombinant protein, cell culture, and immunological techniques utilized in this disclosure are standard procedures, well known to those skilled in the art. Such techniques are described in J. Perbal, A Practical Guide to Molecular Cloning, John Wiley and Sons (1984), J. Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989), T.A. Brown (ed.), Essential Molecular Biology: A Practical Approach, Volumes 1 and 2, IRL Press (1991), D.M.G. Lover and B.D.H.Means (eds.), DNA Cloning: A Practical Approach, Volumes 1-4, IRL Press (1995 and 1996), and F.M.A.usubel et al. (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current revisions), Ed. Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988), and J.E. Coligan et al. al. (eds.) Current Protocols in Immunology, John Wiley & Sons (including all current editions), and other sources.

[0147] The descriptions and definitions of variable regions and portions thereof, immunoglobulins, antibodies and fragments thereof herein may be further clarified by the descriptions in Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991; Bork et al., J. Mol. Biol. 242, 309-320, 1994; Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al. Nature 342, 877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273, 927-948, 1997.

[0148] The term "and / or", e.g., "X and / or Y", shall be understood to mean either "X and Y" or "X or Y", and shall be understood to give clear support for both meanings or for either meaning.

[0149] As used herein, the term "derived from" shall be understood to indicate that the specified integer may be obtained from a particular source, but is not necessarily obtained directly from that source.

[0150] For example, references herein to ranges of residues are understood to include the endpoints, e.g., a reference to "a region comprising amino acids 56-65" is understood in an inclusive manner, i.e., this region includes the sequence of amino acids numbered 56, 57, 58, 59, 60, 61, 62, 63, 64, and 65 in a specified sequence.

[0151] Selected Definitions Plasminogen is the inactive precursor of plasmin, the major fibrolytic enzyme in mammals. Plasmin also plays an important role in cell migration, tissue remodeling, and bacterial invasion. Plasmin is a serine protease that preferentially cleaves Lys-Xaa and Arg-Xaa bonds with higher selectivity than trypsin. Plasminogen activators, such as tissue plasminogen activator (tPA) or urokinase, cleave Arg-Xaa bonds. 561 -Val 562 It cleaves human plasminogen molecules at the bond to produce active plasmin. The resulting two plasmin chains are held together by two interchain disulfide bridges. The light chain (25 kDa) carries the catalytic center (containing the catalytic triad) and shares sequence similarity with trypsin and other serine proteases. The heavy chain (60 kDa) is composed of five highly similar triple-loop structures (called kringles). Some of the kringles contain lysine-binding sites, which mediate the plasminogen / plasmin interaction with fibrin. Plasmin belongs to the peptidase family Si.

[0152] Plasminogen (or Plg) is a seven-domain glycoprotein containing a Pap or pan-apple domain, five kringle domains (KR1-KR5), and a serine protease (SP) domain. Plg circulates in plasma in a closed, activation-resistant conformation. Upon localization at target sites, plasminogen binds to surface lysine / arginine residues on targets (including fibrin clots and cell surface receptors). Binding occurs via a lysine-binding site (LBS) on the kringle domain, an event that triggers the rearrangement of plasminogen to the open conformation. Upon conversion from the closed to the open conformation, the activation loop between the KR5 and SP domains becomes exposed and is cleaved by plasminogen activators (such as tissue plasminogen activator or urokinase plasminogen activator) to form the enzymatically active form, plasmin (Plm). The plasminogen activation system is tightly regulated by host serine protease inhibitors: plasminogen activator inhibitors 1 and 2 (PAI-1 and PAI-2). Active plasmin released from targets is typically cleared from the circulation by the specific inhibitor α-2-antiplasmin or the housekeeping enzyme α-2-macroglobulin.

[0153] The term "plasminogen" as used herein includes any of plasminogen variants, including Glu-plasminogen (Glu-Plg), Lys-plasminogen (Lys-Plg), and mini-, midi-, and micro-plasminogen. Lys-plasminogen is an N-cleaved form of Glu-Plg formed by cleavage of Glu-plasminogen by plasmin. Compared to Glu-Plg, Lys-plasminogen exhibits a higher affinity for fibrin and is more easily activated by uPA and tPA. Midi-plasminogen contains kringle domains 4 and 5 of plasminogen and a light chain (serine protease domain). It is formed by cleavage of kringle domains 1 to 3 from Glu-plasminogen. Mini-plasminogen (also known as 442Val-Plg or neoplasminogen) is produced by the action of elastase on residue 442 (located within kringle domain 4) of Glu-plasminogen. Mini-plasminogen therefore contains kringle domain 4, kringle domain 5, and the serine protease domain of plasminogen. Micro-plasminogen is composed of the zymogen domain of plasminogen with a peptide and a section connecting several residues of kringle 5 attached to the N-terminus. It is produced by the action of plasmin on plasminogen. Micro-plasminogen (or micro-Plg) therefore contains the light chain (serine protease domain) of plasminogen but does not contain the kringle domain. (See, e.g., Shi et al. (1980) J. Biol. Chem. 263:17071-5.) Like plasminogen, microplasminogen is activated by tPA and urokinase to form a proteolytically active molecule. Human microplasmin has a molecular weight of approximately 29 kDa and has a lower affinity for fibrin compared to plasmin.

[0154] For nomenclature purposes only, but not by way of limitation, an exemplary amino acid sequence of human plasminogen ("glu-Plg") is set forth in SEQ ID NO:65. The sequence containing the hPlg activation loop is set forth in SEQ ID NO:66.

[0155] As used herein, plasminogen refers to a molecule that has at least one biochemical or biophysical activity of plasminogen that can be distinguished from that of plasmin.

[0156] The phrases "inhibit plasminogen activation" or "reduce plasminogen activation" mean that the antigen binding protein of the present invention inhibits or reduces the conversion of plasminogen to plasmin. Furthermore, activity is measured using a suitable in vitro, cellular, or in vivo assay, and activity is blocked or reduced by at least 1%, 5%, 10%, 25%, 50%, 60%, 70%, 80%, or 90% or more compared to plasminogen activation in the same assay under the same conditions but without the antigen binding protein. Preferably, plasminogen activation is mediated or induced by any one or more plasminogen activators. The plasminogen activator may be any one of the following: Arg 561 -Val 562The plasminogen-cleaving serine protease is any enzyme capable of cleaving the bond (numbering is based on human plasminogen). Exemplary plasminogen-cleaving serine proteases, and therefore plasminogen activators, include the clotting proteins factor IX, factor X, and prothrombin (factor II), protein C, chymotrypsin and trypsin, various leukocyte elastases, streptokinase (SK), urokinase (uPA), and tissue plasminogen activator (tPA), and plasmin. Preferably, the plasminogen activator is selected from the group consisting of streptokinase (SK), urokinase (uPA), and tissue plasminogen activator (tPA). Preferably, the inhibition or reduction of plasminogen activation is carried out in the presence of one or more cofactors. Preferably, the cofactor is selected from the group consisting of epsilon-aminocaproic acid (EACA), fibrinogen (Fg), and fibrin (Fn).

[0157] The term "isolated protein" or "isolated polypeptide" refers to a protein or polypeptide that, by virtue of its origin or source from which it is derived, is not associated with naturally associated components that accompany it in its native state; and is substantially free of other proteins from the same source. A protein may be rendered substantially free of naturally associated components by isolation using protein purification techniques known in the art, or may be substantially purified by such isolation. "Substantially purified" means that the protein is substantially free of contaminants, e.g., at least about 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% removed.

[0158] The term "recombinant" shall be understood to mean a product of artificial genetic recombination. Thus, with respect to a recombinant protein comprising an antibody antigen-binding domain, this term does not encompass naturally occurring antibodies within a subject's body that are the product of natural recombination that occurs during B-cell maturation. However, if such an antibody is isolated, it is considered to be an isolated protein comprising an antibody antigen-binding domain. Similarly, if a nucleic acid encoding a protein is isolated and expressed using recombinant means, the resulting protein is a recombinant protein comprising an antibody antigen-binding domain. Recombinant protein also encompasses proteins expressed by artificial recombinant means when it is present within a cell, tissue, or subject, e.g., where it is expressed.

[0159] The term "protein" is understood to include a single polypeptide chain, i.e., a series of consecutive amino acids linked by peptide bonds, or a series of polypeptide chains covalently or non-covalently bonded to one another (i.e., a polypeptide complex). For example, a series of polypeptide chains can be covalently bonded using suitable chemical bonds or disulfide bonds. Examples of non-covalent bonds include hydrogen bonds, ionic bonds, van der Waals forces, and hydrophobic interactions. A protein may also include one or more unnatural amino acids.

[0160] It will be understood from the previous paragraph that the term "polypeptide" or "polypeptide chain" means a series of consecutive amino acids linked by peptide bonds.

[0161] As used herein, the term "antigen-binding domain" shall be understood to mean a region of an antibody capable of specifically binding to an antigen, i.e., an Fv comprising a VH or VL or both a VH and a VL. The antigen-binding domain need not be in the context of a complete antibody, for example, it may be isolated (e.g., a domain antibody) or in another form, such as, for example, an scFv, as described herein.

[0162] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or several closely related antigens (e.g., plasminogen) via an antigen-binding domain contained in an Fv. This term includes four-chain antibodies (e.g., two light chains and two heavy chains), recombinant or modified antibodies (e.g., chimeric antibodies, humanized antibodies, human antibodies, CDR-grafted antibodies, primatized antibodies, de-immunized antibodies, synhumanized antibodies, half antibodies, bispecific antibodies).

[0163] Antibodies generally contain a constant domain, which can be organized into a constant region, constant fragment, or crystallizable fragment (Fc). Exemplary forms of antibodies contain a four-chain structure as their basic unit. Full-length antibodies contain two covalently bonded heavy chains (approximately 50-70 kDa) and two light chains (approximately 23 kDa each). The light chains generally contain a variable region (if present) and a constant domain, and in mammals, they are either kappa or lambda light chains. The heavy chains generally contain a variable region and one or two constant domains connected by a hinge region to additional constant domains. Mammalian heavy chains are of one of the following types: α, δ, ε, γ, or μ. Each light chain is also covalently bonded to one of the heavy chains. For example, two heavy chains and heavy and light chains are bound by interchain disulfide bonds and non-covalent interactions. The number of interchain disulfide bonds can vary depending on the type of antibody. Each chain has an N-terminal variable region (VH or VL, each approximately 110 amino acids long) and one or more constant domains at the C-terminus. The light chain constant domain (CL, approximately 110 amino acids long) is aligned and disulfide-bonded to the first constant domain of the heavy chain (CH1, 330-440 amino acids long). The light chain variable region is aligned with the heavy chain variable region. An antibody heavy chain can include two or more additional CH domains (CH2, CH3, etc.) and can include a hinge region between the CH1 and CH2 constant domains. Antibodies can be of any type (e.g., IgG, IgE, IgM, IgD, IgA, and IgY), class (e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2), or subclass. In one example, the antibody is a murine (mouse or rat) antibody or a primate (e.g., human) antibody. In one example, the antibody heavy chain lacks a C-terminal lysine residue. In one example, the antibody is a humanized, synthetic humanized, chimeric, CDR-grafted or deimmunized antibody.

[0164] The terms "full-length antibody," "intact antibody," or "complete antibody" are used interchangeably to refer to an antibody in its substantially intact form, as opposed to an antigen-binding fragment thereof. Specifically, complete antibodies include those having heavy and light chains, including the Fc region. The constant domains may be wild-type sequence constant domains (e.g., human wild-type sequence constant domains) or amino acid sequence variants thereof.

[0165] As used herein, "variable region" refers to the portion of the light chain and / or heavy chain of an antibody defined herein that is capable of specifically binding to an antigen, and includes the amino acid sequences of the complementarity-determining regions (CDRs); i.e., CDR1, CDR2, and CDR3, and framework regions (FRs). For example, a variable region includes three CDRs together with three or four FRs (e.g., FR1, FR2, FR3, and optionally FR4). VH refers to the variable region of the heavy chain. VL refers to the variable region of the light chain.

[0166] As used herein, the term "complementarity determining region" (synonyms are CDRs; i.e., CDR1, CDR2, and CDR3) refers to the amino acid residues in an antibody variable region whose presence is primarily responsible for specific antigen binding. Each variable region domain (VH or VL) typically has three CDRs, identified as CDR1, CDR2, and CDR3. The CDRs of VH are also referred to herein as CDR H1, CDR H2, and CDR H3, respectively, where CDR H1 corresponds to CDR1 of VH, CDR H2 corresponds to CDR2 of VH, and CDR H3 corresponds to CDR3 of VH. Similarly, the CDRs of a VL are referred to herein as CDR L1, CDR L2, and CDR L3, respectively, where CDR L1 corresponds to CDR 1 of a VL, CDR L2 corresponds to CDR 2 of a VL, and CDR L3 corresponds to CDR 3 of a VL. In one example, the amino acid positions assigned to CDRs and FRs are defined according to the Kabat Sequences of Proteins of Immunological Interest, National Institutes of Health, Bethesda, Md., 1987 and 1991 (also referred to herein as the "Kabat numbering system"). In another example, the amino acid positions assigned to CDRs and FRs are defined according to the Enhanced Chothia Numbering Scheme (http: / / www.bioinfo.org.uk / mdex.html).The present invention is not limited to FRs and CDRs defined by the Kabat numbering system, but encompasses all numbering systems, including the standard numbering system, or the numbering system of Chothia and Lesk J. Mol. Biol. 196:901-917, 1987; Chothia et al., Nature 342:877-883, 1989; and / or Al-Lazikani et al., J. Mol. Biol. 273:927-948, 1997; Honnegher and Pluekthun J. Mol. Biol. 309:657-670, 2001; or the IMGT system described in Giudicelli et al., Nucleic Acids Res. 25:206-211, 1997. In one example, the CDRs are defined according to the Kabat numbering system. Optionally, the heavy chain CDR2 according to the Kabat numbering system does not include the five C-terminal amino acids listed herein, or any one or more of those amino acids are substituted with another naturally occurring amino acid. In this regard, Padlan et al., FASEB J., 9:133-139, 1995, established that the five C-terminal amino acids of heavy chain CDR2 are generally not involved in antigen binding.

[0167] "Framework regions" (FRs) are variable region residues other than the CDR residues. The FRs of VH are also referred to herein as FR H1, FR H2, FR H3, and FR H4, respectively, where FR H1 corresponds to FR 1 of VH, FR H2 corresponds to FR 2 of VH, FR H3 corresponds to FR 3 of VH, and FR H4 corresponds to FR 4 of VH. Similarly, the FRs of VL are also referred to herein as FR L1, FR L2, FR L3, and FR L4, respectively, where FR L1 corresponds to FR 1 of VL, FR L2 corresponds to FR 2 of VL, FR L3 corresponds to FR 3 of VL, and FR L4 corresponds to FR 4 of VL.

[0168] As used herein, the term "Fv" shall be understood to mean any protein, whether composed of multiple polypeptides or a single polypeptide, in which a VL and a VH associate to form a complex having an antigen-binding domain, i.e., capable of specifically binding to an antigen. The VH and VL forming the antigen-binding domain may be in a single polypeptide chain or in different polypeptide chains. Furthermore, an Fv of the present invention (as well as any protein of the present invention) may have multiple antigen-binding domains that may or may not bind to the same antigen. This term shall be understood to encompass fragments derived directly from antibodies as well as proteins corresponding to such fragments produced using recombinant means. In some examples, the VH is not linked to the heavy chain constant domain (CH)1 and / or the VL is not linked to the light chain constant domain (CL). Exemplary Fv-comprising polypeptides or proteins include Fab fragments, Fab' fragments, F(ab') fragments, scFvs, diabodies, triabodies, tetrabodies, or higher order complexes, or any of the above linked to a constant region or domain thereof, e.g., a CH2 or CH3 domain, e.g., a minibody. A "Fab fragment" consists of a monovalent antigen-binding fragment of an immunoglobulin and can be produced by digesting a whole antibody with the enzyme papain to yield a fragment consisting of an intact light chain and a portion of the heavy chain, or can be produced using recombinant means. An "Fab' fragment" of an antibody can be obtained by treating a whole antibody with pepsin, followed by reduction, to yield a molecule consisting of an intact light chain and a portion of the heavy chain including the VH and a single constant domain. Two Fab' fragments are obtained for each antibody treated in this manner. Fab' fragments can also be produced by recombinant means. An "F(ab')2 fragment" of an antibody consists of a dimer of two Fab' fragments linked by two disulfide bonds and can be obtained by treating a whole antibody molecule with the enzyme pepsin without subsequent reduction. A "Fab2" fragment is a recombinant fragment containing two Fab fragments linked, for example, using a leucine zipper or CH3 domain.A "single-chain Fv" or "scFv" is a recombinant molecule comprising the variable region fragment (Fv) of an antibody in which the variable region of the light chain and the variable region of the heavy chain are covalently linked by a suitable flexible polypeptide linker.

[0169] As used herein, the term "bind" in reference to the interaction of an antigen-binding protein, or antigen-binding domain thereof, with an antigen means that the interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, an antibody recognizes and binds to a specific protein structure, rather than protein in general. If an antibody binds to epitope "A," the presence of a molecule containing epitope "A" (or free, unlabeled "A") will reduce the amount of labeled "A" bound to the antibody in a reaction involving labeled "A" and protein.

[0170] As used herein, the terms "specifically binds" or "binds specifically" shall be understood to mean that an antigen-binding protein of the invention reacts or associates with a particular antigen or cell expressing it at a higher frequency, more rapidly, for a longer period of time, and / or with higher affinity than with another antigen or cell. For example, the antigen-binding protein binds to plasminogen (e.g., human plasminogen) with substantially higher affinity (e.g., 1.5-fold, 2-fold, 5-fold, 10-fold, 20-fold, 40-fold, 60-fold, 80-fold to 100-fold, 150-fold, or 200-fold) than it binds to other related molecules, such as other serine proteases. In one example of the invention, the antigen-binding protein "specifically binds" to plasminogen (preferably human) with at least 1.5-fold or 2-fold or more higher affinity (e.g., 5-fold, 10-fold, 20-fold, 50-fold, 100-fold, or 200-fold) than it binds to plasmin. Generally, although not necessarily, reference to binding refers to specific binding, and each term shall be understood to provide clear support for the other term.

[0171] As used herein, the term "does not detectably bind" shall be understood to mean that the antigen-binding protein, e.g., antibody, binds to the candidate antigen at a level less than 10%, or 8%, or 6%, or 5% above background. Background may be the level of binding signal detected in the absence of protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control antigen. The level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen-binding protein is immobilized and contacted with the antigen.

[0172] As used herein, the term "does not significantly bind" shall be understood to mean that the level of binding of an antigen binding protein of the invention to a polypeptide is not statistically significantly higher than the background, e.g., the level of binding signal detected in the absence of the antigen binding protein and / or in the presence of a negative control protein (e.g., an isotype control antibody), and / or the level of binding detected in the presence of a negative control polypeptide. The level of binding is detected using a biosensor assay (e.g., Biacore) in which the antigen binding protein is immobilized and contacted with the antigen.

[0173] As used herein, the term "epitope" (synonymously "antigenic determinant") is understood to mean a region of plasminogen that is bound by an antigen-binding protein, including the antigen-binding domain of an antibody. Unless otherwise defined, the term is not necessarily limited to the specific residues or structures contacted by the antigen-binding protein. For example, the term includes the region spanning the amino acids contacted by the antigen-binding protein, as well as 5-10 (or more), or 2-5, or 1-3 amino acids outside this region. In some instances, an epitope includes a series of discontinuous amino acids that are positioned near each other when the antigen-binding protein is folded, i.e., a "conformational epitope." Those skilled in the art will also recognize that the term "epitope" is not limited to peptides or polypeptides. For example, the term "epitope" includes chemically active surface groups such as sugar, phosphoryl, or sulfonyl side chains, and, in certain instances, may have specific three-dimensional structural features and / or specific charge characteristics.

[0174] As used herein, the term "disease" refers to a disruption or interference with normal function and includes any disease or disorder, without being limited to any particular condition.

[0175] As used herein, the terms "preventing," "prevent" or "prevention" include administering an antigen binding protein of the invention, thereby stopping or preventing the onset of at least one symptom of a disease. The term also encompasses treatment of a subject in remission to prevent or prevent recurrence.

[0176] As used herein, the terms "treating," "treat," or "treatment" include administering an antigen binding protein described herein, thereby reducing or eliminating at least one symptom of a particular disease or disorder.

[0177] As used herein, the term "subject" shall be understood to mean any animal, including humans, e.g., mammals. Exemplary subjects include, but are not limited to, humans and non-human primates. For example, the subject is a human.

[0178] antibody In one example, the antigen binding protein or plasminogen binding protein described herein according to any embodiment is an antibody.

[0179] Methods for producing antibodies are known in the art and / or are described in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, plasminogen (e.g., human plasminogen) or a region thereof (e.g., the extracellular region) or an immunogenic fragment or epitope thereof, or a cell expressing and presenting it (i.e., the immunogen), optionally formulated with any suitable or desired carrier, adjuvant, or pharmaceutically acceptable excipient, is administered to a non-human animal, e.g., a mouse, chicken, rat, rabbit, guinea pig, dog, horse, cow, goat, or pig. The immunogen may be administered intranasally, intramuscularly, subcutaneously, intravenously, intradermally, intraperitoneally, or by other known routes.

[0180] The production of polyclonal antibodies can be monitored by sampling the blood of the immunized animal at various time points after immunization. One or more further immunizations can be given as needed to obtain a desired antibody titer. The process of boosting and titering is repeated until a suitable titer is obtained. When a desired level of immunogenicity is obtained, the immunized animal is bled and the serum is isolated and stored, and / or the animal is used to generate monoclonal antibodies (mAbs).

[0181] Monoclonal antibodies are one exemplary form of antibody contemplated by the present invention. The term "monoclonal antibody" or "mAb" refers to a homogeneous antibody population capable of binding to the same antigen, e.g., the same epitope within the antigen. The term is not limited regarding the source of the antibody or the method by which it is made.

[0182] For the production of mAbs, any one of several well-known techniques can be used, such as the procedures exemplified in US Pat. No. 4,196,265, mentioned above, or Harlow and Lane (1988).

[0183] For example, a suitable animal is immunized with an immunogen under conditions sufficient to stimulate antibody-producing cells. Rodents such as rabbits, mice, and rats are exemplary animals. For example, mice that do not express murine antibodies but have been genetically modified to express human antibodies can also be used to produce the antibodies of the present invention (e.g., as described in WO 2002 / 066630).

[0184] Following immunization, somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), are selected for use in the mAb generation protocol. These cells are obtained from biopsies of the spleen, tonsils, or lymph nodes, or from a peripheral blood sample. The B cells from the immunized animal are then fused with an immunogen with immortal myeloma cells, generally derived from the same species as the animal immunized.

[0185] The hybrids are amplified by culture in selective media containing drugs that block de novo synthesis of nucleotides in tissue culture media. Exemplary drugs are aminopterin, methotrexate, and azaserine.

[0186] The amplified hybridomas are subjected to functional selection for antibody specificity and / or titer, for example, by flow cytometry and / or immunohistochemistry and / or immunoassays (e.g., radioimmunoassays, enzyme immunoassays, cytotoxicity assays, plaque assays, dot immunoassays, etc.).

[0187] Alternatively, ABL-MYC technology (NeoClone, Madison WI 53713, USA) is used to generate MAb-secreting cell lines (eg, as described in Largaespada et al., J. Immunol. Methods. 197:85-95, 1996).

[0188] Antibodies can also be produced or isolated by screening display libraries, e.g., phage display libraries, as described, for example, in U.S. Patent No. 6,300,064 and / or U.S. Patent No. 5,885,793. For example, the present inventors have isolated fully human antibodies from phage display libraries.

[0189] The antibody of the invention can be a synthetic antibody, for example, the antibody is a chimeric antibody, a humanized antibody, a human antibody, a synthetic humanized antibody, a primatized antibody or a deimmunized antibody.

[0190] Proteins containing antibody-binding domains Single Domain Antibodies In some instances, a protein of the invention is or comprises a single domain antibody (used interchangeably with the terms "domain antibody" or "dAb"). A single domain antibody is a single polypeptide chain that comprises all or a portion of the heavy chain variable region of an antibody. In particular instances, a single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, MA; see, e.g., U.S. Pat. No. 6,248,516).

[0191] Diabodies, triabodies, and tetrabodies In some instances, the proteins of the invention are or comprise diabodies, triabodies, tetrabodies or higher order protein complexes such as those described in WO 98 / 044001 and / or WO 94 / 007921.

[0192] For example, a diabody is a protein that includes two associated polypeptide chains, each of which has the structure V L -XV H or V H -XV L where V L is the antibody light chain variable region, and V H is an antibody heavy chain variable region, and X is a V in a single polypeptide chain. H and V L A linker that contains insufficient residues to allow the V of one polypeptide chain to associate (or form an Fv) or is absent and H V of another polypeptide chain L to form an antigen-binding domain, i.e., an Fv molecule capable of specifically binding to one or more antigens. L and V H may be the same in each polypeptide chain, or V L and V H can be different in each polypeptide chain, thereby forming a bispecific diabody (ie, comprising two Fvs with different specificities).

[0193] Single chain Fv (scFv) Those skilled in the art will appreciate that an scFv is a V polypeptide chain consisting of a single polypeptide chain. H and V L The V domain, as well as the scFv, form the desired structure for antigen binding (i.e., the V domain of a single polypeptide chain). H and V L V allows V to associate with each other to form Fv H and V LIt will be appreciated that the present invention also includes a polypeptide linker between the scFv and the nucleotides (Gly4Ser)3, for example, a linker comprising more than 12 amino acid residues, with (Gly4Ser)3 being one of the more preferred linkers for scFv.

[0194] The present invention also contemplates disulfide-stabilized Fvs (or diFvs or dsFvs), in which a single cysteine ​​residue is present in the V H FR and V L The cysteine ​​residues are introduced into the FR of the Fv and linked by disulfide bonds to generate a stable Fv.

[0195] Alternatively, or in addition, the invention encompasses dimeric scFvs, i.e., proteins comprising two scFv molecules non-covalently or covalently linked, for example, by a leucine zipper domain (e.g., derived from Fos or Jun), or the two scFvs are linked by a peptide linker of sufficient length to allow both scFvs to form and bind to antigen, as described, for example, in U.S. Patent Application Publication No. 20060263367.

[0196] Heavy chain antibodies Heavy chain antibodies are structurally distinct from many other forms of antibodies in that they contain heavy chains but no light chains. Thus, these antibodies are also called "heavy chain-only antibodies." Heavy chain antibodies are found, for example, in camelids and cartilaginous fish (also called IgNARs).

[0197] The heavy chain variable region (V) present in conventional four-chain antibodies H domain) and the light chain variable region (called the "V domain") present in conventional four-chain antibodies. L The variable regions present in native heavy chain antibodies are generally referred to as "V domains" in camelid antibodies to distinguish them from the "V domains" HH domain" and in IgNARs, V-NARs.

[0198] A general description of camelid-derived heavy chain antibodies and their variable regions and methods for their production and / or isolation and / or use can be found in, inter alia, the following references: WO 94 / 04678, WO 97 / 49805 and WO 97 / 49805.

[0199] A general description of cartilaginous fish-derived heavy chain antibodies and their variable regions, and methods for their production and / or isolation and / or use, can be found, inter alia, in WO 2005 / 118629.

[0200] Other antibodies and proteins containing their antigen-binding domains The present invention also provides (i) "key and hole" bispecific proteins as described in U.S. Pat. No. 5,731,168; (ii) heteroconjugate proteins (e.g., as described in U.S. Pat. No. 4,676,980); (iii) heteroconjugate proteins produced using chemical cross-linkers (e.g., as described in U.S. Pat. No. 4,676,980); and (iv) Fab3 (e.g., as described in EP19930302894) Other antibodies and proteins comprising their antigen-binding domains are contemplated, such as:

[0201] Mutations to proteins The present invention also provides antigen binding proteins, or nucleic acids encoding same, which have at least 80% identity to the sequences disclosed herein, hi one example, an antigen binding protein or nucleic acid of the invention comprises a sequence that is at least about 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence disclosed herein.

[0202] Alternatively, or in addition, the antigen binding protein may be a V H or V Lor 97%, or 98%, or 99% identical to the CDRs of (e.g., three CDRs).

[0203] In another example, a nucleic acid of the invention comprises a sequence that is at least about 80%, or 85%, or 90%, or 95%, or 97%, or 98%, or 99% identical to a sequence that encodes an antigen binding protein having a function as described herein according to any of the Examples. The invention also encompasses nucleic acids that encode antigen binding proteins of the invention that differ from the sequences exemplified herein due to the degeneracy of the genetic code.

[0204] The percent identity of nucleic acids or polypeptides is determined by GAP (Needleman and Wunsch. Mol. Biol. 48, 443-453, 1970) analysis (GCG program) with a gap creation penalty of 5 and a gap extension penalty of 0.3. The query sequence is at least 50 residues long, and the GAP analysis aligns the two sequences over a region of at least 50 residues. For example, the query sequence is at least 100 residues long, and the GAP analysis aligns the two sequences over a region of at least 100 residues. For example, the two sequences are aligned over their entire lengths.

[0205] The present invention also contemplates nucleic acids that hybridize to the nucleic acids encoding the antigen-binding sites described herein under stringent hybridization conditions. "Moderate stringency" is defined herein as hybridization and / or washing in 2x SSC buffer, 0.1% (w / v) SDS, at a temperature ranging from 45°C to 65°C, or equivalent conditions. "High stringency" is defined herein as hybridization and / or washing in 0.1x SSC buffer, 0.1% (w / v) SDS, or at a lower salt concentration, and at a temperature of at least 65°C, or equivalent conditions. References herein to a particular level of stringency encompass equivalent conditions using washing / hybridization solutions other than SSC, as known to those skilled in the art. For example, methods for calculating the temperature at which the strands of a double-stranded nucleic acid dissociate (also known as the melting temperature, or Tm) are known in the art. A temperature similar to (e.g., within 5°C or within 10°C) or equal to the Tm of the nucleic acid is considered high stringency. Moderate stringency should be considered to be within 10°C-20°C or 10°C-15°C of the calculated Tm of the nucleic acid.

[0206] The present invention also contemplates mutant forms of the antigen binding proteins of the present invention which contain one or more conservative amino acid substitutions compared to the sequences described herein. In some examples, the antigen binding protein contains no more than 10 conservative amino acid substitutions, for example, 9 or 8 or 7 or 6 or 5 or 4 or 3 or 2 or 1. A "conservative amino acid substitution" is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain and / or hydropathicity and / or hydrophilicity.

[0207] Families of amino acid residues with similar side chains have been defined in the art, including basic side chains (e.g., lysine, arginine, histidine), acidic side chains (e.g., aspartic acid, glutamic acid), uncharged polar side chains (e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine), nonpolar side chains (e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan), β-branched side chains (e.g., threonine, valine, isoleucine), and aromatic side chains (e.g., tyrosine, phenylalanine, tryptophan, histidine). The hydropathic index is described, for example, in Kyte and Doolittle J. Mol. Biol., 157:105-132, 1982, and the hydrophilicity index is described, for example, in U.S. Pat. No. 4,554,101.

[0208] The present invention also contemplates non-conservative amino acid mutations. For example, substitution of a charged amino acid with another charged amino acid, and substitution with a neutral or positively charged amino acid, are of particular interest. In some examples, the antigen-binding protein contains 10 or fewer non-conservative amino acid substitutions, such as 9, 8, 7, 6, 5, 4, 3, 2, or 1.

[0209] In one example, the mutations occur within the FRs of the antigen-binding domain of the antigen-binding protein of the present invention, hi another example, the mutations occur within the CDRs of the antigen-binding protein of the present invention.

[0210] Exemplary methods for producing mutant forms of antigen binding proteins include: Mutagenesis of DNA (Thie et al., Methods Mol. Biol. 525:309-322, 2009) or RNA (Kopsidas et al., Immunol. Lett. 107:163-168, 2006; Kopsidas et al. BMC Biotechnology, 7:18, 2007; and WO 1999 / 058661); Introduction of a nucleic acid encoding a polypeptide into a mutagenized cell, e.g., XL-1Red, XL-mutS, and XL-mutS-Kanr bacterial cells (Stratagene); DNA shuffling (e.g., as disclosed in Stemmer, Nature 370:389-91, 1994); and Site-directed mutagenesis (as described, for example, in Dieffenbach (ed.) and Dveksler (ed.) (PCR Primer: A Laboratory Manual, Cold Spring Harbor Laboratories, NY, 1995)).

[0211] Exemplary methods for determining the biological activity of mutant antigen binding proteins of the invention will be apparent to those of skill in the art and / or are described herein, e.g., for antigen binding. For example, methods for determining antigen binding, competitive inhibition of binding, affinity, association, dissociation, and therapeutic effect are described herein.

[0212] constant region The present invention encompasses antigen binding proteins and / or antibodies described herein comprising an antibody constant region, including an antigen-binding fragment of an antibody fused to Fc.

[0213] The sequences of constant regions useful for producing the proteins of the invention can be obtained from several different sources. In some instances, the constant region of the protein, or a portion thereof, is derived from a human antibody. The constant region, or a portion thereof, can be derived from any antibody class, including IgM, IgG, IgD, IgA, and IgE, as well as any antibody isotype, including IgG1, IgG2, IgG3, and IgG4. In one example, the constant region is a human isotype IgG4 or stabilized IgG4 constant region.

[0214] In one example, the Fc region of the constant region has a reduced ability to induce effector function, for example, compared to a native or wild-type human IgG1 or IgG3 Fc region. In one example, the effector function is antibody-dependent cell-mediated cytotoxicity (ADCC) and / or antibody-dependent cell-mediated phagocytosis (ADCP) and / or complement-dependent cytotoxicity (CDC). Methods for assessing the level of effector function of an Fc region containing protein are known in the art and / or described herein.

[0215] In one example, the Fc region is an IgG4 Fc region (i.e., an IgG4 constant region), e.g., a human IgG4 Fc region. The sequences of suitable IgG4 Fc regions will be apparent to those of skill in the art and / or available in publicly accessible databases (e.g., available from the National Center for Biotechnology Information).

[0216] In one example, the constant region is a stabilized IgG4 constant region. The term "stabilized IgG4 constant region" will be understood to mean an IgG4 constant region that has been modified to reduce the tendency to undergo Fab arm exchange or the formation of half antibodies. "Fab arm exchange" refers to a type of protein modification of human IgG4, in which an IgG4 heavy chain and associated light chain (half molecule) are exchanged with a heavy chain-light chain pair from another IgG4 molecule. Thus, an IgG4 molecule can acquire two different Fab arms that recognize two different antigens (resulting in a bispecific molecule). Fab arm exchange occurs naturally in vivo and can be induced in vitro with purified blood cells or a reducing agent such as reduced glutathione. "Half antibodies" form when an IgG4 antibody dissociates to form two molecules, each containing a single heavy chain and a single light chain.

[0217] In one example, the stabilized IgG4 constant region contains a proline at position 241 of the hinge region according to the Kabat system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 1987 and / or 1991). This position corresponds to position 228 of the hinge region according to the EU numbering system (Kabat et al., Sequences of Proteins of Immunological Interest Washington DC United States Department of Health and Human Services, 2001 and Edelman et al., Proc. Natl. Acad. USA, 63, 78-85, 1969). In the case of human IgG4, this residue is generally serine. After the replacement of the proline with serine, the IgG4 hinge region contains the sequence CPPC. In this regard, those skilled in the art will recognize that a "hinge region" is a proline-rich portion of an antibody heavy chain constant region that connects the Fc and Fab regions and confers flexibility to the two Fab arms of an antibody. The hinge region contains cysteine ​​residues that participate in inter-heavy chain disulfide bonds. It is generally defined as the stretch from Glu226 to Pro243 in human IgG1 according to the Kabat numbering system. Hinge regions of other IgG isotypes can be aligned with the IgG1 sequence by placing the first and last cysteine ​​residues that form inter-heavy chain disulfide (SS) bonds at the same positions (see, for example, WO 2010 / 080538).

[0218] Another example of a stabilized IgG4 antibody is an antibody in which the arginine at position 409 (according to the EU numbering system) of the heavy chain constant region of human IgG4 is substituted with lysine, threonine, methionine, or leucine (e.g., as described in WO 2006 / 033386). Additionally or alternatively, the Fc region of the constant region comprises a residue selected from the group consisting of alanine, valine, glycine, isoleucine, and leucine at the position corresponding to 405 (according to the EU numbering system). Optionally, the hinge region comprises a proline at position 241 (i.e., the CPPC sequence) (as described above).

[0219] In another example, the Fc region is a region modified to have reduced effector function, i.e., a "non-immunostimulatory Fc region." For example, the Fc region is an IgG1 Fc region containing substitutions at one or more positions selected from the group consisting of 268, 309, 330, and 331. In another example, the Fc region is an IgG1 Fc region containing one or more of the following mutations: E233P, L234V, L235A, and a deletion of G236, and / or one or more of the following mutations: A327G, A330S, and P331S (Armour et al., Eur J Immunol. 29:2613-2624, 1999; Shields et al., J Biol Chem. 276(9):6591-604, 2001). Further examples of non-immunostimulatory Fc regions are described, for example, in Dall'Acqua et al., J Immunol. 177:1129-1138 2006; and / or Hezareh J Virol; 75:12161-12168, 2001).

[0220] In another example, the Fc region may comprise at least one C, e.g., from an IgG4 antibody. H 2 domain and at least one C from an IgG1 antibody HA chimeric Fc region comprising three domains, wherein the Fc region comprises a substitution at one or more amino acid positions selected from the group consisting of 240, 262, 264, 266, 297, 299, 307, 309, 323, 399, 409, and 427 (EU numbering) (e.g., as described in WO 2010 / 085682). Exemplary substitutions include 240F, 262L, 264T, 266F, 297Q, 299A, 299K, 307P, 309K, 309M, 309P, 323F, 399S, and 427F.

[0221] Further modifications The present invention also contemplates further modifications to antibodies or antigen binding proteins comprising the Fc region or constant region.

[0222] For example, the antibody contains one or more amino acid substitutions that increase the half-life of the protein. For example, the antibody contains an Fc region containing one or more amino acid substitutions that increase the affinity of the Fc region for neonatal Fc region (FcRn). For example, the Fc region has increased affinity for FcRn at lower pH, e.g., about pH 6.0, thereby promoting Fc / FcRn binding in endosomes. In one example, the Fc region has increased affinity for FcRn at about pH 6 compared to its affinity at about pH 7.4, thereby promoting re-release of Fc into the blood after cellular recycling. These amino acid substitutions are useful for extending the half-life of proteins by reducing clearance from the blood.

[0223] Exemplary amino acid substitutions include T250Q and / or M428L or T252A, T254S and T266F or M252Y, S254T and T256E or H433K and N434F according to the EU numbering system. Additional or alternative amino acid substitutions are described, for example, in U.S. Patent Application Publication No. 20070135620 or U.S. Patent No. 7,083,784.

[0224] Protein production In one example, an antigen binding protein described herein according to any of the Examples is produced by culturing a hybridoma under conditions sufficient to produce the protein, e.g., as described herein and / or known in the art.

[0225] Recombinant expression In another example, the antigen binding proteins described herein according to any of the Examples are recombinant.

[0226] In the case of a recombinant protein, the nucleic acid encoding it can be cloned into an expression construct or vector, which is then transfected into host cells that do not naturally produce the protein, such as Escherichia coli (E. coli) cells, yeast cells, insect cells, or mammalian cells, such as simian COS cells, Chinese hamster ovary (CHO) cells, human embryonic kidney (HEK) cells, or myeloma cells. Exemplary cells used to express proteins are CHO cells, myeloma cells, or HEK cells. Molecular cloning techniques to achieve these goals are known in the art and are described, for example, in Ausubel et al., (eds.), Current Protocols in Molecular Biology, Greene Pub. Associates and Wiley-Interscience (1988, including all current editions) or Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press (1989). A wide variety of cloning and in vitro amplification methods are suitable for constructing recombinant nucleic acids. Methods for producing recombinant antibodies are also known in the art, see, for example, US Pat. No. 4,816,567 or US Pat. No. 5,530,101.

[0227] After isolation, the nucleic acid is operably linked and inserted into a promoter or expression vector in an expression construct for further cloning (amplification of the DNA) or for expression in a cell-free system or in a cell.

[0228] As used herein, the term "promoter" should be interpreted in its broadest sense and includes the transcriptional regulatory sequences of a genomic gene, including the TATA box or initiation element, required for accurate transcription initiation, with or without additional regulatory elements (e.g., upstream activating sequences, transcription factor binding sites, enhancers, and silencers) that alter expression of the nucleic acid, e.g., in response to developmental and / or external stimuli, or in a tissue-specific manner. In this context, the term "promoter" is also used to refer to a recombinant, synthetic, or fusion nucleic acid, or derivative thereof, that confers, activates, or enhances expression of a nucleic acid to which it is operably linked. Exemplary promoters may contain additional copies of one or more specific regulatory elements to further enhance expression and / or alter the spatial and / or temporal expression of the nucleic acid.

[0229] As used herein, the term "operably linked" means positioning a promoter relative to a nucleic acid such that expression of the nucleic acid is controlled by the promoter.

[0230] Many vectors for expression in cells are available. Vector components generally include, but are not limited to, one or more of the following: a signal sequence, a protein-coding sequence (e.g., obtained from the information provided herein), an enhancer element, a promoter, and a transcription termination sequence. Those skilled in the art will recognize sequences suitable for protein expression. Exemplary signal sequences include prokaryotic secretion signals (e.g., pelB, alkaline phosphatase, penicillinase, Ipp, or heat-stable enterotoxin II), yeast secretion signals (e.g., invertase leader, α-factor leader, or acid phosphatase leader), or mammalian secretion signals (e.g., herpes simplex gD signal).

[0231] Exemplary promoters active in mammalian cells include the cytomegalovirus immediate-early promoter (CMV-IE), the human elongation factor 1-alpha promoter (EF1), small nuclear RNA promoters (U1a and U1b), the alpha-myosin heavy chain promoter, the simian virus 40 promoter (SV40), the Rous sarcoma virus promoter (RSV), the adenovirus major late promoter, the β-actin promoter; hybrid regulatory elements comprising the CMV enhancer / β-actin promoter or an immunoglobulin promoter or an active fragment thereof. Examples of useful mammalian host cell lines are the SV40-transformed monkey kidney CV1 cell line (COS-7, ATCC CRL 1651); the human embryonic kidney cell line (293 cells or 293 cells subcloned for growth in suspension culture); baby hamster kidney cells (BHK, ATCC CCL 10); or Chinese hamster ovary cells (CHO).

[0232] Exemplary promoters suitable for expression in yeast cells, such as yeast cells selected from the group including, for example, Pichia pastoris, Saccharomyces cerevisiae, and S. pombe, include, but are not limited to, the ADH1 promoter, the GAL1 promoter, the GAL4 promoter, the CUP1 promoter, the PHO5 promoter, the nmt promoter, the RPR1 promoter, or the TEF1 promoter.

[0233] Means for introducing isolated nucleic acids or expression constructs containing the same into cells for expression are known to those of skill in the art. The technique used for a given cell depends on known, established techniques. Means for introducing recombinant DNA into cells include, inter alia, microinjection, DEAE-dextran-mediated transfection, liposome-mediated transfection using, for example, lipofectamine (Gibco, MD, USA) and / or cellfectin (Gibco, MD, USA), PEG-mediated DNA uptake, electroporation, and microparticle bombardment using, for example, DNA-coated tungsten or gold particles (Agracetus Inc., WI, USA).

[0234] Host cells used to produce proteins may be cultured in a variety of media, depending on the type of cell used. Commercially available media such as Ham's F10 (Sigma), Minimal Essential Medium ((MEM), (Sigma), RPMI-1640 (Sigma), and Dulbecco's Modified Eagle's Medium ((DMEM), Sigma) are suitable for culturing mammalian cells. Media for culturing the other cell types described herein are known in the art.

[0235] Protein isolation Methods for isolating proteins are known in the art and / or described herein.

[0236] If the antigen-binding protein is secreted into the culture medium, the supernatant from such an expression system is first concentrated using a commercially available protein concentration filter, e.g., an Amicon or Millipore Pellicon ultrafiltration unit. A protease inhibitor such as PMSF may be included in any of the above steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of incidental contaminants. Alternatively, or in addition, the supernatant may be filtered and / or separated from the cells expressing the protein, e.g., using continuous centrifugation.

[0237] Antigen-binding proteins prepared from cells can be purified using, for example, ion exchange, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, affinity chromatography (e.g., Protein A affinity chromatography or Protein G chromatography), or any combination of the above. These methods are known in the art and are described, for example, in WO 99 / 57134 or in Ed Harlow and David Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988).

[0238] Those skilled in the art will also recognize that proteins can be modified to include tags for ease of purification or detection, such as a polyhistidine tag, e.g., a hexahistidine tag, or an influenza virus hemagglutinin (HA) tag, or a simian virus 5 (V5) tag, or a FLAG tag, or a glutathione S-transferase (GST) tag. The resulting protein is then purified using methods known in the art, such as affinity purification. For example, a protein containing a hexa-His tag is purified by contacting a sample containing the protein with nickel-nitrilotriacetic acid (Ni-NTA) immobilized on a solid or semi-solid support, which specifically binds to the hexa-His tag, washing the sample to remove unbound proteins, and then eluting the bound protein. Alternatively, or in addition, a ligand or antibody that binds to the tag is used in the affinity purification method.

[0239] Assaying the activity of antigen-binding proteins Binding to plasminogen and its mutants It will be clear to those skilled in the art from the disclosure herein that the antigen-binding proteins of the present invention bind to plasminogen / plasmin. Methods for assessing protein binding are known in the art and are described, for example, in Scopes (Protein purification: principles and practice, Third Edition, Springer Verlag, 1994). Such methods generally involve immobilizing the antigen-binding protein and contacting it with labeled antigen (plasminogen). After washing to remove non-specifically bound proteins, the amount of label and, consequently, bound antigen are detected. Of course, the antigen-binding protein may be labeled and the antigen may be immobilized. Panning-type assays may also be used. Alternatively, or in addition, surface plasmon resonance assays may be used.

[0240] Optionally, the dissociation constant (Kd), association constant (Ka) and / or affinity constant (K) of the immobilized antigen binding protein for plasminogen or an epitope thereof are measured. D ) is determined. The "Kd" or "Ka" or "K" of the plasminogen binding protein is D "Kd" is measured, in one example, by a radioactively or fluorescently labeled plasminogen ligand binding assay. In the case of "Kd," the assay equilibrates the antigen-binding protein with a minimal concentration of labeled plasminogen or its epitope in the presence of a titration series of unlabeled plasminogen. After washing to remove unbound plasminogen or its epitope, the amount of label, which is an indicator of the protein's Kd, is determined.

[0241] In another example, Kd, ​​Ka or K D is measured using a surface plasmon resonance assay, for example, using a BIAcore surface plasmon resonance (BIAcore, Inc., Piscataway, NJ) with immobilized plasminogen or plasmin or a region thereof or an immobilized antigen binding protein.

[0242] Measurement of inhibitory activity The antigen binding proteins of the present invention are preferably capable of inhibiting plasminogen activation and / or plasmin activity.

[0243] A variety of assays are known in the art to assess the ability of a protein to inhibit or reduce the activation of plasminogen to plasmin.

[0244] In one example, the antigen binding protein inhibits activators of plasminogen (plasminogen activators) from binding to plasminogen. Preferably, the antigen binding protein of the present invention binds to plasminogen and prevents binding and / or cleavage of plasminogen in the activation loop, preferably in this case by binding to the Arg 561 -Val 562 Cleavage at the nucleotide sequence is prevented, reduced or inhibited.

[0245] Preferably, the antigen binding proteins of the present invention inhibit or block the activation of plasminogen by any known plasminogen activator, including, but not limited to, streptokinase (SK), tissue-plasminogen activator (tPA), or urokinase-plasminogen activator (uPA). Plasminogen activators whose activity is inhibited by the binding of an antigen binding protein of the present invention to plasminogen may be plasminogen activators produced by the host organism (e.g., in humans) or may be secreted or produced by bacterial pathogens and activate plasminogen in the host. Thus, activation of plasminogen by either host or pathogen plasminogen activators can be inhibited or blocked by the antigen binding proteins of the present invention.

[0246] The antigen-binding proteins of the present invention may also inhibit the binding of plasminogen to pathogens, including inhibiting or preventing the binding of plasminogen to bacteria. Inhibition may also relate to the binding of plasminogen to bacterial spores or vegetative forms of bacteria. Inhibition of the binding of plasminogen to pathogens preferably inhibits the activation of plasminogen to plasmin.

[0247] Exemplary methods for measuring inhibition of plasminogen activation, inhibition of binding of plasminogen activators to plasminogen, and inhibition of binding of plasminogen to pathogens are described in Examples 2, 6, and 7.

[0248] Measurement of inhibitory activity The antigen binding proteins of the invention are also capable of inhibiting plasmin activity, including inhibiting plasmin-mediated clot lysis at a level that is comparable to or significantly greater than physiological inhibitors of plasmin activity or pathological inhibitors of plasmin activity.

[0249] A variety of assays are known in the art for assessing the ability of a protein to inhibit or reduce plasmin activity.

[0250] In one example, the antigen binding protein inhibits the proteolysis of any substrate by plasmin. Preferably, the antigen binding protein of the present invention binds to plasmin and prevents binding and / or cleavage of the plasmin substrate by the serine protease domain. Preferably, the antigen binding protein of the present invention binds to or sterically shields the catalytic triad of plasmin, thereby preventing the catalytic triad from cleaving the plasmin substrate, where the catalytic triad comprises residues His603, Asp646 and Ala / Ser741 of SEQ ID NO: 65 (corresponding to His57, Asp102 and Ser195 using chymotrypsin numbering). Thus, in a preferred embodiment, the antigen binding protein of the present invention is an anti-catalytic antigen binding protein.

[0251] Preferably, the antigen binding proteins of the invention inhibit or block cleavage of any of plasmin's known substrates, including but not limited to: fibrin, fibrinogen, factors V, VIII and X, protease-activated receptor I, fibronectin, thrombospondin, laminin, von Willebrand factor, vitronectin, pro-brain-derived neurotrophic factor, cofactors C3 and C5, tenascin, osteocalcin, CUB domain-containing protein 1 and other proteases such as collagenase.

[0252] Exemplary methods for measuring inhibition of plasmin activity are described, eg, in Examples 2 and 10.

[0253] The antigen binding proteins of the invention are also useful in applications requiring the detection of plasmin and / or plasminogen in biological samples, for example, they may be useful for diagnostic applications, including where the proteins are used in histology and ELISA, and similar applications where binding of the antigen binding protein to a target protein can provide useful diagnostic information.

[0254] Conditions being treated The antigen binding proteins of the present invention are useful in the treatment of conditions requiring the inhibition of the conversion of plasminogen to plasmin (ie, inhibition of plasminogen activation) and / or inhibition of plasmin activity.

[0255] Active plasmin has a very broad range of target substrate specificity. Thus, plasmin targets include fibrin, fibrinogen, complement component 3, complement component 5, vitronectin, osteocalcin, factors V, VIII, and X, protease-activated receptors, damage-induced aggregation proteins, and some collagenases. Plasmin also targets the important plasminogen activators tPa and uPa, creating a positive feedback loop.

[0256] Plasmin is a highly efficient enzyme, and uninhibited plasmin can rapidly deplete all circulating fibrinogen stores, causing a systemic hemorrhagic state within minutes. Therefore, under normal physiological conditions, active plasmin is only present at the surface of target sites such as fibrin clots or on cell surfaces.

[0257] In severe trauma patients, coagulation abnormalities are often observed during the acute phase of trauma. Traumatic coagulation abnormalities are coagulation disorders caused by the trauma itself. The pathophysiology of traumatic coagulation abnormalities consists of coagulation activation, hyperfibrinolysis, and consumptive coagulopathy. These pathophysiological mechanisms are characteristic of DIC with a fibrinolytic phenotype.

[0258] Fibrinolytic dysregulation is a key mechanism in traumatic coagulation disorders. It is a poorly understood process ranging from excessive degradation (hyperfibrinolysis) to blockade of fibrinolysis. Both hyperfibrinolysis and blockade are associated with excess mortality and post-traumatic organ failure. The pathophysiology is thought to be related to endothelial injury and hypoperfusion, and several molecular markers that play specific roles have been identified. This pathology is mediated, in part, by excessive upregulation of profibrinolytic tPA in the absence of a concomitant increase in antifibrinolytic PAI-1, resulting in excessive activation of plasminogen to plasmin. Therefore, in the specific case of trauma, excessive plasminogen activation needs to be inhibited, which can be achieved using the antigen-binding proteins of the present invention.

[0259] Furthermore, several pathologies characterized by excessive plasminogen activation include those characterized by elevated levels of plasma plasminogen activators that are physically and immunologically related to those in human tissues and vascular endothelium. Thrombotic or hemorrhagic disorders due to defective fibrinolysis can also result from congenital or acquired defects in the fibrinolytic system.

[0260] The antigen binding proteins of the present invention are useful for minimizing or reducing hemorrhage or bleeding after surgery, injury, or in individuals with clotting factor disorders. Use of the antigen binding proteins in these situations inhibits plasmin-mediated fibrinolysis or clot lysis, thereby reducing blood loss and reducing or minimizing the need for blood transfusions. Blood transfusions are associated with a high risk of incompatibility, allergic reactions, multiple organ dysfunction, and infection, leading to increased morbidity and mortality.

[0261] The antigen binding proteins of the invention may also be used to prevent bleeding in other conditions such as hemophilia, menorrhagia, von Willebrand syndrome and thrombolysis-induced bleeding.

[0262] The antigen binding proteins of the invention are useful for inhibiting fibrinolysis in several clinical conditions, including reducing bleeding in patients who have undergone cardiac surgery, orthopedic surgery, neurosurgery, liver transplantation, vascular surgery, thoracic surgery, gynecological surgery, or in patients with end-stage renal disease, peripartum bleeding, gastrointestinal bleeding, trauma, traumatic brain injury, intracerebral hemorrhage and subarachnoid hemorrhage. In other words, the antigen binding proteins of the invention are useful for inhibiting plasmin in individuals with a hyperfibrinolytic state.

[0263] Thus, the antigen binding proteins of the present invention are useful for inhibiting fibrinolysis in a wide variety of scenarios where inhibition of plasminogen activation and / or inhibition of plasmin activity is required.

[0264] Other conditions requiring inhibition of plasminogen activation (and / or plasmin activity) include conditions characterized by excessive bleeding (hemorrhage), including after childbirth and after surgery (such as transplant, chest, cardiac and orthopedic surgery).

[0265] The antigen binding proteins of the present invention are useful in the treatment or prevention of any condition associated with or resulting from the presence or increased levels of a bacterial pathogen.

[0266] Invasive pathogens mediate various stages of disease through diverse mechanisms, including the mammalian plasminogen / plasmin system. A vast number of pathogens express plasmin(ogen) receptors, which immobilize plasmin(ogen) on bacterial surfaces, enhancing plasminogen activation by mammalian plasminogen activators. Bacteria also affect the secretion of plasminogen activators and their inhibitors from mammalian cells. For example, the prokaryotic plasminogen activators streptokinase and staphylokinase form complexes with plasmin(ogen), thus enhancing plasminogen activation. The Pla surface protease of Yersinia pestis is functionally similar to mammalian activators and converts plasminogen to plasmin by limited proteolysis. In essence, plasminogen receptors and activators utilize host-derived systems to transform bacteria into proteolytic organisms.

[0267] Some exemplary bacterial infections that can be treated / prevented by the antigen binding proteins of the invention are described in detail below. However, it will be understood that the use of the antigen binding proteins of the invention is not limited to the bacterial infections described. More specifically, it will be understood that the antigen binding proteins of the invention have general application in treating / reducing the invasion of bacterial pathogens that secrete plasminogen activators in order to invade host tissues.

[0268] In Gram-negative bacteria, the filamentous surface appendages, fimbriae and flagella, form the major group of plasminogen receptors. In Gram-positive bacteria, surface-bound enzyme molecules and M-protein-related structures have been identified as plasminogen receptors, and the former receptor type is also present on mammalian cells. Consequently, plasmin produced by or activated by Haemophilus influenzae, Salmonella typhimurium, Streptococcus pneumoniae, Y. pestis, and Borrelia burgdorferi has been shown to degrade mammalian extracellular matrix. In some cases, plasminogen activation has been shown to enhance bacterial translocation in vitro through reconstituted basement membranes or epithelial cell monolayers.

[0269] Streptococcus pyogenes, or Group A Streptococcus (GAS), is a facultative, Gram-positive cocci that grows in chains and causes a variety of infections in humans, including pharyngitis, tonsillitis, scarlet fever, cellulitis, erysipelas, rheumatic fever, poststreptococcal glomerulonephritis, necrotizing fasciitis, myonecrosis, and lymphangitis.

[0270] In particular, streptokinase (SK), secreted by Streptococcus species, promotes infection by overcoming immune defenses and activating circulating plasminogen. Streptokinase has been reported to directly activate the closed conformation of plasminogen by enveloping its serine protease domain; once bound, the N-terminus of streptokinase inserts into the activation loop of the protease domain to generate active plasmin. Antigen-binding proteins of the present invention have been demonstrated to inhibit streptokinase from binding to plasminogen, thereby inhibiting the activation of plasminogen by streptokinase. A similar (although slightly different) mechanism of plasminogen activation is also understood to occur upon binding of staphylokinase, an important virulence factor secreted by Staphylococcus species. Thus, the antigen binding proteins of the invention are particularly useful for treating, minimizing the severity of or slowing the progression of infections caused by Gram-positive bacteria such as Streptococcus spp., Staphylococcus spp. and Enterococcus spp.

[0271] The antigen binding proteins of the invention are also useful in the treatment of infections caused by Gram-negative bacteria such as Helicobacter pylori, Yersinia pestis, Salmonella spp., Escherichia spp., Campylobacter spp., and Shigella spp., and the spirochete Borrelia (the causative agent of Lyme disease).

[0272] As used herein, any treatment of a bacterial infection includes reducing the severity of the symptoms or signs of a microbial infection or preventing the progression or worsening of the infection. It will be understood that such treatment, reduction, or prevention includes reducing the number of bacteria or preventing the spread of bacteria to other sites within the host (i.e., reducing the invasion of the pathogen).

[0273] In particular, the present inventors have demonstrated that promotion of sporulation and virulence by Clostridium difficile is mediated by plasminogen activation. Specifically, the present inventors demonstrate that plasminogen specifically binds to C. difficile spores, allowing active plasmin to degrade their surface and facilitate rapid germination. More specifically, the binding and activation of human plasminogen directly alters the structural and functional properties of C. difficile spores, leading to faster germination in the presence of physiologically relevant host germinative factors. This contributes to earlier disease onset and more severe disease outcomes. The antigen-binding proteins of the present invention have been demonstrated to be particularly useful in reducing the severity of infections caused by Clostridium difficile (also known as Clostroides difficile, CDI).

[0274] Plasminogen activation has been reported to be associated with the promotion of angiogenesis, including in the case of cancer growth / metastasis. Furthermore, inhibitors of plasminogen activation (e.g., PAI-1) are useful in inhibiting cancer-associated angiogenesis.

[0275] Thus, the antigen binding proteins of the invention are also useful for treating cancer in a subject, or for delaying the progression or metastasis of cancer. Exemplary cancers include cystic and solid tumors, bone and soft tissue tumors, including tumors in the anal tissue, bile duct, bladder, blood cells, intestine, brain, breast, carcinoid, cervix, eye, esophagus, head and neck, kidney, larynx, leukemia, liver, lung, lymph nodes, lymphoma, melanoma, mesothelioma, myeloma, ovary, pancreas, penis, prostate, skin (e.g., squamous cell carcinoma), sarcoma, stomach, testes, thyroid, vagina, and vulva. Soft tissue tumors include benign schwannoma monosomy, desmoid tumor, lipoblastoma, lipoma, uterine leiomyoma, clear cell sarcoma, dermatofibrosarcoma protuberans, Ewing's sarcoma, extraskeletal myxoid chondrosarcoma, myxoid liposarcoma, alveolar rhabdomyosarcoma, and synovial sarcoma. Specific bone tumors include non-ossifying fibroma, unicameral bone cyst, enchondroma, aneurysmal bone cyst, osteoblastoma, chondroblastoma, chondromyxoid fibroma, osteogenic fibroma and adamantinoma, giant cell tumor of bone, fibrous dysplasia of bone, Ewing's sarcoma, eosinophilic granuloma, osteosarcoma, chondroma, chondrosarcoma, malignant fibrous histiocytoma, and metastatic carcinoma. Leukemias include acute lymphoblastic, acute myeloblastic, chronic lymphoblastic, and chronic myeloma leukemia.

[0276] Other examples include breast tumors, colon tumors, adenocarcinoma, mesothelioma, bladder tumors, prostate tumors, germ cell tumors, hepatocellular carcinoma / bile duct tumors, carcinoma, neuroendocrine tumors, pituitary tumors, small round cell tumors, squamous cell carcinoma, melanoma, atypical fibroxanthoma, seminoma, nonseminoma, interstitial Leydig cell tumors, Sertoli cell tumors, skin tumors, kidney tumors, testicular tumors, brain tumors, ovarian tumors, stomach tumors, oral tumors, bladder tumors, bone tumors, cervical tumors, esophageal tumors, laryngeal tumors, liver tumors, lung tumors, vaginal tumors, and Wilms' tumor.

[0277] As used herein, "subject" refers to an animal, such as a mammal or avian species, including humans, monkeys, horses, cows, sheep, goats, dogs, and cats.

[0278] The subject may have a bacterial infection, be exposed to infectious bacteria, be at risk of developing a bacterial infection, or be at a higher risk of developing a bacterial infection than the general population.Examples of subjects at a higher risk of developing a bacterial infection include patients who are being treated for a bacterial infection and whose normal intestinal microflora is disrupted by antimicrobial therapy, patients with immune dysfunction (e.g., immunoglobulin deficiency, splenic insufficiency, splenectomy, HIV infection, leukocyte dysfunction, hemoglobinopathy), elderly people, people with certain malignant diseases (e.g., multiple myeloma, chronic lymphocytic leukemia, lymphoma), people with high occupational risk (e.g., firefighters, waterworks, sanitation workers, police, medical and public service workers, such as laboratory workers, hospital workers), people in closed groups (e.g., prisons, military, nursing homes), and other people with immunodeficiencies that may increase susceptibility to bacterial infection.

[0279] Bacterial infections generally refer to: (1) higher levels of bacteria in samples taken from subjects compared with uninfected control samples; (2) a high proportion of one or more species of bacteria in samples taken from the subject compared with the total level of bacteria in uninfected control samples; (3) a higher proportion of bacteria relative to one or more other bacterial species in the sample collected from the subject when compared to an uninfected control sample; (4) The presence of a bacterium in a sample compared to an uninfected control sample when the bacterium is below the limit of detection in the uninfected control.

[0280] Subjects can be diagnosed as having bacterial infection by any method described herein or known in the art.Biological samples such as body fluid samples (for example, blood) or tissue samples or scrapings.Then, sample is prepared (by various methods), and then cultured on various agar plates containing synthetic media to classify microorganisms.Real-time PCR is another method that can identify bacteria in sample.

[0281] An acute infection refers to an infection in a subject that requires prompt treatment (generally within 15 to 60 minutes) or the infection may progress and endanger the subject's life. Such acute infections may occur in infants or immunocompromised subjects as described herein.

[0282] In one aspect, the terms "infection" and "bacterial infection" refer to an infection caused by gram-positive bacteria, also referred to as a "gram-positive infection."

[0283] Gram-positive bacteria refer to bacteria that stain blue or purple with Gram staining, including, for example, Clostroides difficile (Clostridium difficile), Streptococcus pyrogenes, Staphylococcus aureus, Lactobacillus species, Bifidobacteria, Scardovia wiggsiae, and Bacillus anthracis. Gram-positive bacteria are characterized by a thick peptidoglycan layer around the cell membrane and the absence of an outer membrane around the cell membrane. Gram-positive bacteria are not limited to gram-positive cocci or gram-positive bacilli.

[0284] The antigen-binding proteins of the invention are also useful in reducing the severity of or preventing toxic shock. Symptoms of toxic shock or toxic shock syndrome (TSS) can vary depending on the underlying cause. TSS, caused by infection with the bacterium Staphylococcus aureus, typically manifests in otherwise healthy subjects through signs and symptoms such as low blood pressure, high fever accompanied by malaise and confusion, which can rapidly progress to stupor, coma, and multiple organ failure. A characteristic rash, often seen early in the disease process, resembles a sunburn and can affect any area of ​​the body, including the lips, inside the mouth, eyes, palms, and soles of the feet. In patients who survive the initial stage of infection, the rash desquamates or peels after 10 to 14 days.

[0285] In contrast, TSS, or TSLS, caused by the bacterium Streptococcus pyogenes typically occurs in people with a pre-existing skin infection caused by the same bacterium. These subjects often experience severe pain at the site of the skin infection, followed by a rapid progression of the symptoms described above for TSS. In contrast to TSS caused by Staphylococcus, streptococcal TSS is rarely accompanied by a sunburn-like rash.

[0286] In the case of Staphylococcal Toxic Shock Syndrome, the diagnosis is made strictly based on the CDC criteria established in 2011, as follows: 1. Body temperature >38.9°C (102.02°F) 2. Systolic blood pressure < 90mmHg 3. Diffuse macular erythroderma 4. Desquamation (especially from the palms and soles) 1 to 2 weeks after the onset of symptoms 5. Involvement of three or more of the following organ systems: Gastrointestinal (vomiting, diarrhea) Muscle: Severe muscle pain or creatine phosphokinase levels at least twice the upper limit of normal Mucosal congestion (vagina, oral cavity, conjunctiva) Renal failure (serum creatinine >2x normal) Hepatitis (bilirubin, AST, or ALT >2x normal) ·Low platelet count (platelet count <100,000 / mm 3 ) Central nervous system involvement (confusion without focal neurological symptoms) 6. Negative results for: Blood, throat, and CSF cultures for other bacteria (other than S. aureus); · Serologically negative for rickettsial infections, leptospirosis, and measles. Cases are classified as confirmed or probable based on: Confirmed case: all six of the above criteria are met (unless the patient dies before desquamation occurs); Possible example: Five of the six criteria above are met.

[0287] In certain embodiments, the terms "infection" and "bacterial infection" refer to an infection caused by gram-negative bacteria. The infection may be due to Enterobacteriaceae. An infection caused by "Enterobacteriaceae" refers to any of the gram-positive members of this family of bacteria, including, but not limited to, Salmonella spp., Escherichia coli, Yersinia pestis, Klebsiella spp., Shigella spp., Proteus spp., Enterobacter spp., Serratia spp., and Citrobacter spp. Infections can also be caused by other Gram-negative bacteria, including Helicobacteraceae, such as Helicobacter pylori, or Campylobacteraceae, such as Campylobacter jejuni.

[0288] Exemplary bacteria which may cause infections and for which the present invention has particular application in the treatment, prevention or prevention are described below. Also described are examples of pathologies associated with or resulting from bacterial infections which comprise or consist of different types of bacteria. It will be understood that these examples do not limit the use of the antigen binding proteins of the present invention and that the antigen binding proteins of the present invention are useful in a variety of pathologies associated with bacteria which use plasminogen activation to promote pathogenesis.

[0289] Escherichia coli (E. coli) is a Gram-negative bacterium that is part of the normal flora of the gastrointestinal tract. There are hundreds of strains of E. coli, most of which are harmless and inhabit the gastrointestinal tract of healthy humans and animals. Currently, four classes of enterotoxigenic E. coli ("EEC group") are recognized, which cause gastroenteritis in humans. Among these are enteropathogenic (EPEC) strains and those whose mechanism of virulence is related to the excretion of typical E. coli enterotoxins. These E. coli strains can cause a variety of diseases, including those associated with gastrointestinal and urinary tract infections, sepsis, pneumonia, and meningitis. Antibiotics are not effective against some strains and do not necessarily prevent recurrence of infection.

[0290] For example, the E. coli strain O157:H7 is estimated to be responsible for 10,000 to 20,000 cases annually in the United States (Federal Centers for Disease Control and Prevention). Hemorrhagic colitis is the name for the acute illness caused by the E. coli strain O157:H7. Preschool children and the elderly are at highest risk for serious complications.

[0291] Exemplary sequences of enterotoxigenic E. coli strains include GenBank Accession Nos. AB011549, X97542, AF074613, Y11275, and AJ007716.

[0292] Salmonella typhimurium is a Gram-negative bacterium that causes a variety of clinical conditions ranging from localized gastrointestinal infection, gastroenteritis (diarrhea, abdominal cramps, and fever), to the severe systemic disease typhoid fever (including typhoid fever). Salmonella infections also result in substantial livestock losses.

[0293] Unique to Gram-negative bacilli, the cell wall of Salmonella species contains complex lipopolysaccharide (LPS) structures that are released upon cell lysis and can function as endotoxins, contributing to the virulence of the organism.

[0294] Contaminated food is the primary mode of transmission of nontyphoidal Salmonella infections, with Salmonella remaining viable in undercooked meat and animal products. The most common animal sources are chickens, turkeys, pigs, and cattle; as well as a variety of other domestic and wild animals. The epidemiology of typhoid fever and other typhoid fevers caused by Salmonella species is associated with water contaminated with human feces.

[0295] Pseudomonas species are motile, gram-negative bacilli that are resistant to most antibiotics and are a major cause of hospital-acquired infections. Infections are most common in immunocompromised subjects, burn patients, ventilator-assisted subjects, subjects with indwelling catheters, IV anesthesia users, and subjects with chronic lung disease (e.g., cystic fibrosis). Although infections are rare in healthy subjects, they can occur at many sites and lead to urinary tract infections, sepsis, pneumonia, pharyngitis, and a variety of other disorders, with frequent treatment failure and significant mortality.

[0296] Pseudomonas aeruginosa is a Gram-negative, aerobic, rod-shaped bacterium with unipolar motility. An opportunistic human pathogen, P. aeruginosa is also an opportunistic plant pathogen. Like other Pseudomonads, P. aeruginosa secretes a variety of pigments. Definitive clinical identification of P. aeruginosa may involve determining the production of both pyocyanin and fluorescence, as well as the organism's ability to grow at 42°C. P. aeruginosa can also grow in diesel and jet fuel, which is why it is also known as a hydrocarbon-utilizing organism (or "HUM bug") and causes microbial corrosion.

[0297] Vibrio cholerae is a gram-negative bacillus that infects humans, causing cholera, a disease spread by poor sanitation and contributing to contaminated water supplies. Vibrio cholerae can localize in the human small intestine, where it produces a toxin that interferes with ion transport across the mucosa, causing diarrhea and dehydration. Subjects infected with Vibrio cholerae require intravenous or oral rehydration with solutions containing electrolytes. The disease is generally self-limited; however, death can occur from dehydration and loss of essential electrolytes. Antibiotics such as tetracycline have been demonstrated to shorten the duration of the disease, and an oral vaccine is currently in development.

[0298] Neisseria gonorrhea is a gram-negative cocci that causes the sexually transmitted disease gonorrhea. Because its surface antigens can vary, Neisseria gonorrhea prevents the development of immunity against reinfection. Approximately 750,000 cases of gonorrhea are reported annually in the United States, with an estimated 750,000 more cases annually, the majority of which occur in teenagers and young adolescents. Ampicillin, amoxicillin, or some types of penicillin were previously recommended for the treatment of gonorrhea. However, due to the increasing incidence of penicillin-resistant gonorrhea, newer antibiotics administered by injection, such as ceftriaxone or spectinomycin, are now used to treat most gonorrhea infections.

[0299] Staphylococcus aureus is a gram-positive coccus that is usually localized in the human nose and occasionally found on the skin. Staphylococcus can cause bloodstream infections, pneumonia, and surgical site infections in hospital settings (i.e., nosocomial infections). Staphylococcus aureus can cause severe food poisoning, and many strains grow in food and produce exotoxins. Staphylococcus resistant to common antibiotics, such as vancomycin, has emerged in the United States and abroad as a major public health challenge in both community and hospital settings. Recently, vancomycin-resistant Staphylococcus aureus isolates have also been identified in Japan.

[0300] Mycobacterium tuberculosis is a gram-positive bacterium that is the causative agent of tuberculosis, a serious and sometimes fatal disease. Tuberculosis is an increasing global disease and the leading cause of death from a single infectious disease (currently 3 million deaths per year). Although it can affect multiple organs in the human body, including the brain, kidneys, and bones, tuberculosis most commonly affects the lungs.

[0301] In the United States, approximately 10 million individuals are infected with Mycobacterium tuberculosis, as indicated by a positive skin test, with 26,000 new cases of active disease each year. The increase in tuberculosis (TB) cases is associated with HIV / AIDS, homelessness, drug abuse, and the importation of people with active infection. Because no single drug can destroy all TB bacteria, current treatment programs for drug-susceptible TB involve taking two or four drugs (e.g., isoniazid, rifampin, pyrazinamide, ethambutol, or streptomycin) over a 6- to 9-month period. Additionally, drug-resistant and multidrug-resistant strains of Mycobacterium tuberculosis are increasingly being observed.

[0302] Helicobacter pylori (H. pylori) is a microaerophilic, Gram-negative, slow-growing, flagellated, spiral- or S-shaped organism that infects the stomach lining. H. pylori is a human gastric pathogen associated with chronic superficial gastritis, peptic ulcer disease, and chronic atrophic gastritis, which can lead to gastric adenocarcinoma. H. pylori is one of the most common chronic bacterial infections in humans, affecting over 90% of patients with active gastritis. Current treatments include triple therapy using bismuth, metronidazole, and either tetracycline or amoxicillin, which eradicates H. pylori in most cases. Problems associated with triple therapy include patient compatibility, side effects, and metronidazole resistance. Promising alternative regimens to dual therapy are amoxicillin and metronidazole or omeprazole and amoxicillin.

[0303] Streptococcus pneumoniae is a gram-positive cocci that is one of the most common causes of bacterial pneumonia, as well as middle ear infections (otitis media) and meningitis. Each year in the United States, pneumococcal infections result in approximately 50,000 cases of bacteremia, 3,000 cases of meningitis, 100,000 to 135,000 hospitalizations, and 7 million cases of otitis media. Pneumococcal infections are responsible for an estimated 40,000 deaths annually in the United States. Children under the age of 2, adults over the age of 65, and people of all ages with underlying medical conditions, such as congestive heart disease, diabetes, emphysema, liver disease, sickle cell anemia, and HIV, as well as those living in special environments, such as nursing homes and long-term care facilities, are at higher risk of infection.

[0304] Drug-resistant S. pneumoniae strains have become common in the United States, and many penicillin-resistant pneumococci are also resistant to other antimicrobial drugs, such as erythromycin or trimethoprim-sulfamethoxazole.

[0305] Treponema pallidum is the spirochete that causes syphilis. T. pallidum is the causative agent of syphilis, yaws, and nonvenereal syphilis, or pinta, exclusively. Treponema pallidum cannot grow in vitro and replicates in the absence of mammalian cells. Initial infection results in ulcers at the site of infection; however, the bacteria migrate throughout the body, damaging many organs over time. In later stages, untreated syphilis, although not contagious, can cause serious cardiac abnormalities, mental impairment, blindness, other neurological disorders, and death.

[0306] Haemophilus influenzae (H. influenzae) is a family of Gram-negative bacteria; six types are known, and most H. influenzae-related illnesses are caused by type B, or "HIB." Until the development of a vaccine for H. influenzae, H. influenzae was a common cause of otitis media, sinus infections, and bronchitis, the most common cause of meningitis, and a major cause of pneumonia, septic arthritis (joint infections), cellulitis (soft tissue infections), and pericarditis (infection of the pericardium). H. influenzae type B bacteria are widespread in humans, typically living in the throat and nasal passages without causing disease. Unvaccinated children under the age of five are at risk for H. influenzae infection. Meningitis and other serious infections caused by H. influenzae infection can cause brain damage or death.

[0307] Shigella dysenteriae (Shigella dys.) is a gram-negative bacillus that causes dysentery. In the colon, the bacterium invades and divides within mucosal cells, triggering a significant inflammatory response. Shigella infection can cause severe diarrhea, which can lead to dehydration and be dangerous for children, the elderly, or those with chronic illnesses. Shigella dys. produces potent cytotoxic, enterotoxic, and neurotoxic toxins (Shiga toxins) that act as inhibitors of protein synthesis. Although resistance to antibiotics such as ampicillin and TMP-SMX has developed, treatment with newer, more expensive antibiotics such as ciprofloxacin, norfloxacin, and enoxacin remains effective.

[0308] Listeria is a gram-positive, motile bacterium present in human and animal feces. L. monocytogenes causes diseases such as listeriosis, meningoencephalitis, and meningitis. This organism is one of the leading causes of death from foodborne pathogens, particularly in pregnant women, newborns, the elderly, and immunocompromised subjects. It is present in environments such as decaying plant matter, sewage, water, and soil and can survive extremes of temperature and salinity, making it a highly dangerous foodborne pathogen, especially in unheated foods. The bacterium can spread from the intestinal infection site to the central nervous system and feto-placental unit. Infection can result in meningitis, gastroenteritis, and septicemia. In cattle and sheep, Listeria infection can cause encephalitis and spontaneous abortion.

[0309] Proteus mirabilis is an intestinal, Gram-negative commensal organism, distantly related to Escherichia coli (E. coli). It normally colonizes the human urethra and is an opportunistic pathogen that is a major cause of urinary tract infections in catheterized subjects. P. mirabilis possesses two special characteristics: 1) extremely fast motility, which manifests as chemotaxis on culture plates; and 2) it produces urease, which confers the ability to degrade urea and survive within the genitourinary tract.

[0310] Yersinia pestis is the causative agent of plague (bubonic and pulmonary), a devastating disease that has caused millions of deaths worldwide. The organism can be transmitted from rats to humans through the bite of infected fleas or from person to person through the air during an epidemic. Yersinia pestis is an extremely pathogenic organism, requiring only small numbers to cause disease, which, if left untreated, is often fatal. The organism is enteroinvasive and can survive and multiply in macrophages before being disseminated systemically within the host.

[0311] Bacillus anthracis, also known as anthrax, is contracted by humans when they come into contact with contaminated animals. Anthrax is not transmitted through person-to-person contact. Three forms of the disease represent sites of infection, including cutaneous (skin), pulmonary (lungs), and intestinal. Pulmonary and intestinal infections are often fatal if left untreated. Spores are taken up by macrophages and internalized into phagolysosomes (membrane compartments), where germination begins. Lysis of infected macrophages releases the bacteria into the bloodstream, where they rapidly multiply and disseminate throughout the circulatory and lymphatic systems, a process that can lead to septic shock, respiratory distress, and organ failure. Spores of this pathogen have also been used as a terrorist weapon.

[0312] Burkholderia mallei is a gram-negative aerobic bacterium that causes glanders, an infection primarily affecting horses, mules, and donkeys. It is rarely associated with human infections and is more commonly found in livestock animals. The organism is similar to B. pseudomallei and is distinguished by its nonmotile nature. This pathogen is host-adapted and does not reside in the environment outside of its host. Glanders is often fatal unless treated with antibiotics, and transmission can occur via the air or, more commonly, through contact with infected animals. Acute-onset pneumonia, bacteremia (bloodborne transmission of the organism), pustules, and death are common outcomes during infection. The mechanism of virulence is not fully understood but requires a type III secretion system similar to the one from Salmonella typhimurium. There is no vaccine against this potentially dangerous organism, which is considered a potential bioterrorism agent. The genome of this organism carries a large number of insertion sequences (below) compared to the closely related B. pseudomallei, as well as a large number of simple sequence repeats that may function in antigenic variation of cell surface proteins.

[0313] Burkholderia pseudomallei is a Gram-negative bacterium that causes melioidosis in humans and animals. Melioidosis is a disease found in certain regions of Asia, Thailand, and Australia. B. pseudomallei is typically a soil-dwelling organism recovered from rice paddies and humid tropical soils; however, as an opportunistic pathogen, it can cause disease in susceptible subjects, such as those with diabetes. The organism can reside intracellularly, causing pneumonia and bacteremia (dissemination of bacteria through the bloodstream). The incubation period can be extremely long, sometimes taking decades from infection to disease. Treatment can require the use of antibiotics for several months, although relapses are common. Intracellular spread may occur through the induction of actin polymerization at one pole of the cell, allowing movement through the cytoplasm and from cell to cell. This organism carries several small sequence repeats, which may promote antigenic variation similar to that observed in the B. mallei genome.

[0314] Burkholderia cepacia is a Gram-negative bacterium consisting of at least seven distinct subspecies, including Burkholderia multivorans, Burkholderia vietnamiensis, Burkholderia stabilis, Burkholderia cenocepacia, and Burkholderia ambifaria. B. cepacia is an important human pathogen, causing pneumonia, most often in people with underlying lung diseases (e.g., cystic fibrosis) or immune disorders (e.g., chronic granulomatous disease). B. cepacia is typically found in water and soil and can survive for long periods in moist environments. Person-to-person transmission has been documented; as a result, many hospitals, clinics, and detention facilities for cystic fibrosis patients have implemented strict isolation measures regarding B. cepacia. Patients with this bacterium are often treated in separate areas from those without the bacterium to limit transmission. This is because B. cepacia infection can cause a rapid decline in lung function, leading to death. Diagnosis of B. cepacia involves isolating the bacterium from a saliva culture. Treatment is challenging because B. cepacia is naturally resistant to many common antibiotics, including aminoglycosides (e.g., tobramycin) and polymyxin B. Treatment typically involves multiple antibiotics, including ceftazidime, doxycycline, piperacillin, chloramphenicol, and co-trimoxazole.

[0315] Francisella tularensis was first recognized in the early 20th century by Edward Francis in Tulare County, California, as the causative agent of a plague-like disease affecting squirrels. The organism is now named after him. The disease, called tularemia, has been described throughout history. This organism is a potential bioterrorism agent because it can be transmitted to humans from infected ticks or deer flies, by infected meat, or via aerosols. It is aquatic and can be found living within protozoa, similar to those observed with Legionella. It has a high infectivity rate and can invade and rapidly multiply in phagocytic and non-phagocytic cells. Once inside macrophages, the organism can evade the phagosome and survive in the cytoplasm.

[0316] Bacterial infections may also be caused by Porphyromonas gingivalis, a gram-negative, anaerobic bacterium implicated in the pathogenesis of periodontal disease and found in the upper gastrointestinal tract, respiratory system, and colon. P. gingivalis pathogenesis has also been shown to involve mobilization of plasminogen activators.

[0317] The present invention is also useful for veterinary applications. A healthy microflora in the gastrointestinal tract of livestock is crucial to their health and the production of corresponding related foods. As in humans, the gastrointestinal tract of a healthy animal contains a wide variety of bacteria (i.e., E. coli, Pseudomonas aeruginosa, and Salmonella species), which live in ecological equilibrium with one another. This equilibrium can be disturbed by dietary changes, stress, or in response to antibiotic or other therapeutic treatment, causing bacterial diseases in the animal, commonly caused by Salmonella, Campylobacter, Enterococci, Tularemia, and E. coli. Bacterial diseases in such animals often require therapeutic intervention, which has treatment costs as well as well-being, and which are often associated with reduced productivity.

[0318] The terms "treat," "treating," or "treatment," as used herein, refer to the administration of one or more of the compositions or pharmaceutically active ingredients described herein, with or without another pharmaceutically active or inactive ingredient, to (i) alleviate or reduce any of the bacterial infection or one or more symptoms of a bacterial infection, or (ii) slow the progression of the bacterial infection or one or more symptoms of a bacterial infection, or (iii) reduce the severity of the bacterial infection or one or more symptoms of a bacterial infection, or (iv) inhibit the clinical symptoms of a bacterial infection, or (v) inhibit the manifestation of an adverse symptom of a bacterial infection. Furthermore, the terms "treating" or "treatment" can include one or more of the following: ameliorating symptoms of a bacterial infection in a subject, blocking or ameliorating the recurrence of symptoms of a bacterial infection in a subject, reducing the severity and / or frequency of symptoms of a bacterial infection in a subject, arresting, reducing, or inhibiting the growth of vegetative forms of bacteria in a subject, inhibiting bacterial sporulation in a subject, inhibiting the activation of bacterial spores in a subject, inhibiting the germination of bacterial spores in a subject, and inhibiting the post-germination growth of bacterial spores in a subject. Treating refers to about 1% to about 100% improvement, blocking, reduction, decrease, or inhibition relative to a subject not administered an antigen binding protein or composition of the invention. Preferably, the amelioration, blocking, reduction, decrease, or inhibition is 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% relative to a subject not administered an antigen binding protein or composition of the invention.

[0319] Successful treatment may generally refer to an improvement in any of the symptoms associated with or resulting from a gram-positive or gram-negative bacterial infection, and may refer to, for example, an improvement in any of the following: fever, inflammation, swelling, vomiting, fatigue, cramps, cough, snoring, respiratory illness, diarrhea, meningitis, headache, joint pain, body aches, blisters, rash, nausea, chills, dizziness, drowsiness, insomnia, vomiting, dermatitis, excessive mucus production (e.g., in the eyes, gastrointestinal tract, sinuses, or respiratory system), ulcers, gastrointestinal discomfort, skin loss, hair loss, necrosis, and organ dysfunction. Improvement in any of these symptoms or any of the bacterial infections or conditions described herein can be readily assessed according to standard methods and techniques known in the art. The population of subjects treated by the disease method includes subjects suffering from an undesirable condition or disease, as well as subjects at risk of developing such a condition or disease.

[0320] As used herein, the terms "inhibit", "inhibiting" and "inhibition" have their ordinary and accustomed meanings and include one or more of the following: inhibition of bacterial growth or function, inhibition of the growth of vegetative forms of bacteria, inhibition of the function of vegetative forms of bacteria, inhibition of bacterial growth, inhibition of bacterial sporulation, inhibition of bacterial spore activation, inhibition of bacterial spore germination, and inhibition of post-germination growth of bacterial spores. Such inhibition is about 1% to about 100% inhibition of a particular activity relative to the activity in a subject to which an antigen binding protein or composition of the invention has not been administered. Preferably, inhibition is 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5% or 1% inhibition of said activity relative to a subject to which an antigen binding protein or composition of the invention has not been administered. As used herein, "spore" refers to both the commonly used terms "spore" and "endospore."

[0321] As used herein, the terms "preventing" or "prevention" have their ordinary and accustomed meaning and may include one or more of the following: preventing bacterial colonization in a subject, preventing an increase in the growth of a bacterial population in a subject, preventing the activation, germination or post-germination growth of bacterial spores in a subject, preventing bacterial sporulation in a subject, preventing the onset of disease caused by bacteria in a subject, and preventing symptoms of disease caused by bacteria in a subject. As used herein, prevention lasts for at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 25, 30, 35, 40 or more days after administration of an antigen binding protein or composition of the invention.

[0322] As used herein, the term "prophylaxis" includes inhibiting bacterial growth or the development of a progressive infection in a subject, wherein the prevention lasts for at least about 0.5, 1, 2, 3, 4, 5, 6, 7, 10, 12, 15, 20, 25, 30, 35, 40 days or more after administration of an antigen binding protein or composition of the invention. Inhibiting growth or the development of a progressive infection means that the severity of the infection in a subject is reduced by about 1% to about 100% relative to a subject not administered an antigen binding protein or composition of the invention. Preferably, the reduction in severity of infection is a 100%, 99%, 98%, 97%, 96%, 95%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1% reduction in severity. The severity of the infection may be based on, among other factors, the amount of bacteria present in the subject, the period for which the bacteria can be detected in the subject, and / or the severity of the symptoms of the bacterial infection.

[0323] composition In some examples, the antigen binding proteins described herein may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically, rectally, nasally, buccally, vaginally, via an implanted reservoir, as a dosage formulation containing a conventional non-toxic pharmaceutically acceptable carrier, or by any other convenient dosage form. The term "parenteral" as used herein includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intraventricular, intrasternal, and intracranial injection or infusion techniques.

[0324] Methods for preparing antigen-binding proteins into suitable forms (e.g., pharmaceutical compositions) for administration to a subject are known in the art and include, for example, those described in Remington's Pharmaceutical Sciences (18th ed., Mack Publishing Co., Easton, Pa., 1990) and US Pharmacopeia: National Formulary (Mack Publishing Company, Easton, Pa., 1984).

[0325] The pharmaceutical compositions of the present invention are particularly useful for parenteral administration, e.g., intravenous administration or administration into the cavity or lumen of an organ or joint. Compositions for administration generally comprise a solution of the antigen-binding protein dissolved in a pharmaceutically acceptable carrier, e.g., an aqueous carrier. A variety of aqueous carriers, such as buffered saline, can be used. The compositions may contain pharmaceutically acceptable auxiliary substances required to mimic physiological conditions, such as pH adjusting and buffering agents, toxicity adjusting agents, etc., e.g., sodium acetate, sodium chloride, potassium chloride, calcium chloride, sodium lactate, etc. The concentration of the antigen-binding protein of the present invention in these formulations can vary widely and is selected primarily based on fluid volume, viscosity, body weight, etc., depending on the particular administration method selected and the patient's needs. Exemplary carriers include water, saline, Ringer's solution, dextrose solution, and 5% human serum albumin. Non-aqueous vehicles such as mixed oils and ethyl oleate can also be used. Liposomes can also be used as carriers. The vehicle may contain minor amounts of additives that enhance isotonicity and chemical stability, such as buffers and preservatives.

[0326] The antigen binding proteins of the invention can be formulated for local or topical administration, for example, topical application to the skin or tissue in need of treatment. Formulations for topical application typically include a topical vehicle in combination with the active agent, with or without other optional ingredients. Pharmaceutical compositions of the invention may be in the form of a spray, cream, gel, lotion, etc. for topical administration.

[0327] Suitable topical vehicle and additional components are known in the art, and it will be clear that vehicle selection can depend on specific physical form and delivery method.Topical vehicle includes organic solvents, for example, alcohol (for example, ethanol, isopropyl alcohol or glycerin), glycols such as butylene, isoprene or propylene glycol, aliphatic alcohols such as lanolin, the mixture of water and organic solvents, and the mixture of organic solvents such as alcohol and glycerin, lipid-based materials such as fatty acids, acylglycerols (including oils such as mineral oil, and natural or synthetic fats), phosphoglycerides, sphingolipids and waxes, protein-based materials such as collagen and gelatin, silicone-based materials (both non-volatile and volatile), and hydrocarbon-based materials such as microsponges and polymer matrices.

[0328] The composition may further contain one or more ingredients configured to improve the stability or effectiveness of the applied formulation, such as stabilizers, suspending agents, emulsifiers, viscosity adjusters, gelling agents, preservatives, antioxidants, skin permeation enhancers, moisturizers, and sustained-release materials. Examples of such ingredients are described in Martindale-The Extra Pharmacopoeia (Pharmaceutical Press, London 1993) and Martin (ed.), Remington's Pharmaceutical Sciences. The formulation includes microcapsules, such as hydroxymethylcellulose or gelatin microcapsules, liposomes, albumin microspheres, microemulsions, nanoparticles, or nanocapsules.

[0329] Topical formulations may be prepared in a variety of physical forms, including, for example, solids, pastes, creams, foams, lotions, gels, powders, aqueous liquids, emulsions, sprays, and skin patches. The physical appearance and viscosity of these forms can be determined by the presence and amount of emulsifiers and viscosity modifiers present in the formulation. Solids are generally rigid and non-pourable and are commonly formulated into bars or sticks or specific shapes. Solids may be opaque or transparent and may optionally contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product. Creams and lotions are often similar to each other, differing primarily in their viscosity. Both lotions and creams may be opaque, translucent, or transparent and often contain emulsifiers, solvents, and viscosity modifiers, as well as moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product. Gels can be prepared in a variety of viscosities, from strong or thick to weak or thin. These formulations, similar to lotion and cream formulations, may contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product. Liquids are thinner than creams, lotions, or gels and often do not contain emulsifiers. Liquid topical formulations often contain solvents, emulsifiers, moisturizers, emollients, fragrances, dyes / colorants, preservatives, and other active ingredients that increase or enhance the efficacy of the final product.

[0330] Emulsifiers used in topical formulations include, but are not limited to, ionic emulsifiers, nonionic emulsifiers such as cetearyl alcohol, polyoxyethylene oleyl ether, PEG-40 stearate, ceteareth-12, ceteareth-20, ceteareth-30, ceteareth alcohol, PEG-100 stearate, and glyceryl stearate. Suitable viscosity modifiers include, but are not limited to, protective colloids or nonionic gums, such as hydroxyethylcellulose, xanthan gum, magnesium aluminum silicate, silica, microcrystalline wax, beeswax, paraffin, and cetyl palmitate. Gel compositions can be formed by adding gelling agents such as chitosan, methylcellulose, ethylcellulose, polyvinyl alcohol, polyquaterniums, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, carbomer, or ammonium glycyrrhizinate. Suitable surfactants include, but are not limited to, nonionic, amphoteric, ionic, and cationic surfactants, such as one or more of dimethicone copolyol, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, lauramide DEA, cocamide DEA, and cocamide MEA, oleyl betaine, cocamidopropyl phosphatidyl PG-dimonium chloride, and ammonium laureth sulfate, which may be used in topical formulations.

[0331] Preservatives include, but are not limited to, antibacterial agents such as methylparaben, propylparaben, sorbic acid, benzoic acid, and formaldehyde, as well as physical stabilizers and antioxidants such as vitamin E, sodium ascorbate / ascorbic acid, and propyl gallate. Suitable moisturizers include, but are not limited to, lactic acid and other hydroxy acids and their salts, glycerin, propylene glycol, and butylene glycol. Suitable emollients include lanolin alcohol, lanolin, lanolin derivatives, cholesterol, petrolatum, isostearyl neopentanoate, and mineral oil. Suitable fragrances and colorants include, but are not limited to, FD&C Red No. 40 and FD&C Yellow No. 5. Other suitable additional ingredients that may be included in the topical formulation include, but are not limited to, abrasives, moisture absorbents, anti-caking agents, anti-foaming agents, anti-static agents, astringents (e.g., witch hazel), alcohols and herbal extracts such as chamomile extract, binders / excipients, buffers, chelating agents, film formers, conditioning agents, propellants, opacifying agents, pH adjusters, and protectants.

[0332] Typical delivery methods for topical compositions include application with the fingers, application with a physical applicator such as a cloth, tissue, cotton swab, stick, or brush, spraying, including misting, aerosol, or foam spraying, dripping, dusting, dipping, and rinsing. Additionally, controlled release vehicles can be used, and the compositions can be formulated for transdermal administration (e.g., as a transdermal patch).

[0333] Once formulated, the antigen binding proteins of the present invention are administered in a manner compatible with the dosage formulation, and in such amount as will be therapeutically / prophylactically effective. The formulations are easily administered in a variety of dosage forms, such as the types of injectable solutions described above, although other pharmaceutically acceptable forms are also contemplated, such as tablets, pills, capsules, or other solid forms for oral administration, suppositories, pessaries, nasal solutions or sprays, aerosols, inhalants, liposomal forms, and the like. Pharmaceutical "sustained-release" capsules or compositions may also be used. Sustained-release formulations are generally designed to provide a constant drug level over an extended period of time, and may be used to deliver the antigen binding proteins of the present invention.

[0334] WO 2002 / 080967 describes compositions and methods for administering aerosolized compositions comprising antibodies, for example for the treatment of asthma, which are also suitable for administering the antigen binding proteins of the present invention.

[0335] Dosage and timing of administration Suitable dosages of antigen-binding proteins of the present invention will vary depending on the particular antigen-binding protein, the disease being treated, and / or the subject being treated. Determining suitable dosages is within the capabilities of a skilled physician, and can be done, for example, by starting with a suboptimal dosage and gradually modifying the dosage to determine an optimal or useful dosage. Alternatively, data from cell culture assays or animal studies can be used to determine appropriate dosages for treatment / prophylaxis, where a suitable dose is determined to be the ED of the active compound with little or no toxicity. 50 The therapeutically / prophylactically effective dose can be estimated initially from cell culture assays. The dose can be determined based on the IC50 / IC100 / IC200 / IC100 / IC200 / IC100 / IC200 / IC100 / IC200 / IC100 / IC200 / IC100 / IC200 / IC200 / IC200 / IC200 / IC200 / IC300 / IC400 / IC500 / IC600 / IC700 / IC800 / IC9 ... 50The compound may be formulated in animal models to achieve a circulating plasma concentration range that includes the compound's potency (i.e., the concentration or amount of compound that achieves a half-maximal inhibition of symptoms). Such information can be used to more accurately determine useful doses in humans. Levels in plasma can be measured, for example, by high performance liquid chromatography.

[0336] In some instances, the methods of the present invention comprise administering a prophylactically or therapeutically effective amount of a protein described herein.

[0337] The term "therapeutically effective amount" is an amount that, when administered to a subject in need of treatment, improves the subject's prognosis and / or condition and / or reduces or inhibits one or more symptoms of a clinical condition described herein to a level below that observed or recognized as a clinical diagnostic or clinical feature of that condition. The amount administered to a subject will depend on the specific characteristics of the disease being treated, the type and stage of the disease being treated, the method of administration, and characteristics of the subject, such as overall health, other diseases, age, sex, genotype, and weight. One of skill in the art will be able to determine appropriate dosages depending on these and other factors. Thus, this term should not be construed to limit the invention to a particular amount, e.g., weight or amount of protein; rather, the invention encompasses any amount of antigen-binding protein sufficient to achieve a desired result in a subject.

[0338] As used herein, the term "prophylactically effective amount" shall be understood to mean an amount of protein sufficient to prevent or inhibit or delay the onset of one or more detectable symptoms of a clinical condition. One of skill in the art will recognize that such an amount will vary depending, for example, on the particular antigen binding protein administered and / or the particular subject and / or the type or severity or level of disease and / or predisposition to the disease (genetic or otherwise). Thus, this term should not be construed to limit the invention to a particular amount, e.g., weight or amount of antigen binding protein; rather, the invention encompasses any amount of antigen binding protein sufficient to achieve a predetermined result in a subject.

[0339] kit The present invention further includes kits comprising one or more of the following: (i) an antigen-binding protein of the invention or an expression construct encoding same; (ii) a cell of the invention; (iii) a conjugate of the present invention; or (iii) The pharmaceutical composition of the present invention.

[0340] In the case of a kit for detecting plasminogen, the kit may further comprise, for example, a detection means linked to the antigen-binding protein of the present invention.

[0341] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharmaceutically acceptable carrier.

[0342] Optionally, the kits of the invention are packaged with instructions for use of the methods described herein according to any of the Examples.

[0343] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings, all of which constitute various alternative aspects of the invention.

[0344] Table 1

[0345] Table 2

[0346] Table 3

[0347] Table 4

[0348] Table 5

[0349] Table 6

[0350] Table 7

[0351] Table 8

[0352] Table 9

[0353] Table 10

[0354] Table 11

[0355] [Table 12] [Example]

[0356] Example 1: Generation of G05 and G11 antibodies Antibodies for binding to plasminogen were obtained by raising antibody responses to full-length plasminogen in chickens. Antibody variable heavy and light chains (VH and VL) were amplified from cDNA from chicken spleen and linked via a flexible linker to generate an scFv library.

[0357] Selection was performed by screening plasminogen-binding antibodies using Biacore. SK-mediated plasminogen activation and fibrinolysis assays were used to identify antibodies that prevented or inhibited the activation of plasminogen to plasmin.

[0358] Example 2: Characterization of G05 and G11 antibodies for binding to the plasminogen activation loop SPR assay Chicken antibodies were immobilized on a Series S CM4 (GE Healthcare) chip by amine coupling. Plasminogen or plasmin binding to the antibodies of the present invention was analyzed at concentrations ranging from 0.39 nM up to 50 nM using a Biacore T200 (GE Healthcare) in a buffer consisting of 10 mM HEPES pH 7.4, 150 mM NaCl, 3 mM EDTA, and 0.05% Tween 20 in the presence of a protease inhibitor cocktail. Plasminogen / plasmin was injected at 30 μl / min for 180 seconds of association followed by 600 seconds of dissociation. At the end of each cycle, the sensor chip was regenerated with 10 mM glycine pH 1.8 before the next injection, a minimum of six cycles. To obtain kinetic parameters, the sensorgrams were fitted with a Langmuir 1:1 binding model using Biacore T200 evaluation software (GE Healthcare).

[0359] Binding of plasminogen or plasmin on a CM5 chip The results of the binding experiments (Figures 1 and 2) show that the antigen-binding proteins of the present invention bind to both plasminogen and plasmin, but Plasminogen compared to Plasmin The affinity for β-glucan is approximately 10 times higher.

[0360] Inhibition of tPA-mediated plasminogen activation in solution and on fibrin. In solution: 20 nM plasminogen was mixed with various G05 and G11 concentrations (0-200 nM) in the presence of 20 mM EACA for 30 minutes at room temperature. After incubation, plasminogen activation by 4 nM tPA was measured using a fluorogenic substrate in a Fluostar Omega plate reader (BMG Labtech) at excitation and emission wavelengths of 355 nm and 460 nm, respectively. Progress curves were fitted to a nonlinear exponential equation using GraphPad Prism 6: Y = Y0*exp(rate of activation*X) (wherein, when X=0, Y0 is the Y value) The rate of activation was plotted against the corresponding G05 or G11 concentration to obtain the inhibition curve, which was fitted using the inhibitor vs. response model in GraphPad Prism 6: Y=Min + (Max-Min) / (1+((X HillSlope ) / (I C 50 HillSlope ))) where Top and Bottom are the plateaus of the fluorescence readings and HillSlope is a measure of the steepness of the curve. 50 The IC value, i.e., the concentration of G05 that inhibits 50% of tPA-mediated plasminogen activation in solution, is 17.29 ± 2.73 nM (Figure 3A). 50 The value is 20 nM (FIG. 3B).

[0361] On fibrin: Plasminogen activation was measured on the surface of preformed fibrin clots prepared by mixing 3 mg / ml fibrinogen (Banksia Scientific); 1 U bovine thrombin (Jomar Life Research); and 10 nM tPA (Boehringer Ingelheim) for 2 hours at 37°C. 100 nM plasminogen mixed with concentrations (0-2 μM) of G05 or G11 was added to the surface of the clot. Plasmin activity was monitored using 200 μM fluorogenic substrate (H-Ala-Phe-Lys-AMC, Bachem) as previously described. The rate and IC of plasminogen activation were calculated. 50 was calculated as previously described. The IC obtained for the inhibition of tPA-mediated plasminogen activation on fibrin by G05 50 The IC value obtained for the inhibition of tPA-mediated plasminogen activation on fibrin by G11 is 292 nM (Figure 4A). 50 The value is 47 nM (FIG. 4B).

[0362] No inhibition of plasmin: Plasmin (Haematologic Technologies) activity was measured in the presence of 200 μM fluorogenic substrate (H-Ala-Phe-Lys-AMC, Bachem) using a Fluostar Omega plate reader (BMG Labtech) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. Progress curves were obtained for G05 and A01 (a non-inhibitory plasminogen antibody) at a 10:1 antibody:plasmin ratio. G05 exhibited a slight agonistic effect on Plam activity compared to A01. Plasmin was measured at 20 nM and antibody G05 (0–200 nM) at 37°C using a Fluostar Omega plate reader (BMG Labtech) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. A weak G05 concentration-dependent agonistic effect was observed (Figure 5).

[0363] Inhibition of plasminogen-streptokinase binding by G05. The effect of G05 on the binding of plasminogen to streptokinase was examined using a Biacore T200 (GE Healthcare). 10 nM plasminogen was passed across streptokinase immobilized on a CM4 (GE Healthcare) chip in the presence of 0, 62.5, 125, 250, and 500 nM G05 or naive chicken antibody (gAb). G05 showed inhibition at 125 nM and above. Naive chicken antibody gAb (control) showed no inhibition. Percentage of SK binding in the presence of 500 nM G05 and gAb normalized to the no-antibody control.

[0364] The results, shown in Figure 6, indicate that G05 partially competes with streptokinase for plasminogen binding.

[0365] Inhibition of SK-mediated plasminogen activation Plasminogen activation by streptokinase was used to evaluate the potency of the antibodies of the present invention. 50 nM plasminogen was activated with 5 nM recombinant streptokinase at 37°C. The progress of plasminogen activation was monitored using 200 μM plasmin fluorogenic substrate H-Ala-Phe-Lys-AMC (Bachem) in a Fluostar Omega plate reader (BMG Labtech) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. Individually, 0.5 μM antibody was added at specific time points during the process (t = 0, t = 60 min, and t = 160 min). As a negative control, HEPES-buffered saline was added instead of antibody.

[0366] The results in Figure 7 show that the G05 antibody effectively inhibits streptokinase-mediated plasminogen activation when added at t = 0 and 10, and partially at t = 60 minutes.

[0367] Example 3: Binding of recombinant serine protease domains to G05 and G11 The binding of recombinant kringle 5-serine protease domain (KR5-SP) to G05 was evaluated. G05 binds to KR5-SP and forms a stable binary complex with it, which can be co-purified by size-exclusion chromatography. A Superdex 200 16 / 60 column (GE Healthcare) was used, and the buffer was HEPES-buffered saline. As shown in Figure 8A, the co-complex eluted as a single peak at 58.8 ml. As a reference, KR5-SP and G05 antibodies eluted as single peaks at 66.7 ml and 74.6 ml, respectively.

[0368] G11 binds to the serine protease domain (SP) and forms a stable binary complex with it, which can be co-purified by size-exclusion chromatography. A Superdex 75 16 / 60 column (GE Healthcare) was used, and the buffer was HEPES-buffered saline. As shown in Figure 8B, the co-complex eluted as a single peak at 58.0 ml. For reference, SP and G11 both eluted at 82 ml and 66.2 ml, respectively.

[0369] Example 4: Crystal structure of G05 bound to a single recombinant kringle 5-serine protease domain The purified G05 / KR5-SP complex was crystallized at 10 mg / ml in the presence of 0.1 M MES pH 6.5 and 10% (w / v) PEG 20,000 at 20°C. Crystals were flash-cooled in liquid N2 in the presence of 20% (w / v) glycerol. A 2.0 Å dataset was collected at the Australian Synchrotron MX2 beamline using an EIGER X 16M pixel detector (Dectris Ltd, Switzerland, also known as the ACRF detector). The crystal structure was solved by molecular replacement using the program PHASER (CCP4) with the KR5 and SP domains from the structures of plasminogen (PDB ID 4DUR) and chicken single-chain fragment variable (PDB ID 4P48) as search models. Multiple rounds of modeling with COOT and refinement with PHENIX were performed. The final model shown in Figure 9A was generated using PyMOL (www.pymol.org / ).

[0370] The G05-KR5-SP complex structure reveals that the antibody forms a polar interaction with the activation loop of plasminogen, thereby interfering with plasmin production by preventing plasminogen activator from cleaving the activation loop.

[0371] Example 5: Crystal structure of G11 bound to a single recombinant serine protease domain The purified G11 / SP complex was crystallized at 15 mg / ml in the presence of 0.1 M MES pH 6.0, 0.2 M lithium sulfate, and 20% (w / v) PEG 4,000 at 20°C. Crystals were flash-cooled in liquid N2 in 20% (w / v) glycerol. A 2.5 Å dataset was collected at the Australian Synchrotron MX2 beamline using an EIGER X 16M pixel detector (Dectris Ltd, Switzerland, also known as the ACRF detector). The crystal structure was solved using the program PHASER (CCP4) by molecular replacement, using the SP domains from the structures of plasminogen (PDB ID 4DUR) and chicken single-chain fragment variable (PDB ID 4P48) as search models. Multiple rounds of modeling with COOT and refinement with PHENIX were performed. The final model (Figure 9B) was generated using PyMOL (www.pymol.org / ).

[0372] The G11-SP complex structure reveals that the antibody forms polar interactions with the serine protease domain of plasminogen / plasmin, thereby disrupting plasmin production and activity by distorting the catalytic site and surrounding loops, meaning that the enzyme is catalytically impaired.

[0373] Example 6: Inhibition of GAS-plasminogen binding To determine binding to GAS, an inactivatable mutant of plasminogen was used in the following experiments.

[0374] By flow cytometry GAS cells were grown overnight in Todd-Hewitt (TH) broth from a glycerol stock stub in a shaker incubator at 37°C. On the day of the experiment, fresh TH broth was added to an OD of 0.1. 600 Cultures were sub-inoculated and grown in a shaker incubator as before until an OD of 0.35. 600Early logarithmic phase GAS cells (in) were washed twice with phosphate-buffered saline (PBS) supplemented with 2% plasminogen-deficient fetal calf serum (FCS) and then resuspended in an equivalent volume. Alexa-Fluor 647-labeled recombinant plasminogen (22.2 nM) was incubated with 250 μl of washed GAS cells (in) at 4°C for 30 minutes. Cells were then washed and immediately analyzed by flow cytometry. To ensure a homogenous cell population was analyzed, single cells were gated to exclude larger entities (presumably streptococcal chains). Following this, viable cell populations were selected using Syto-9 nucleic acid dye (ThermoFisher).

[0375] The results shown in Figure 10 demonstrate that at 2.22 μM, G05 inhibited the binding of GAS to recombinant plasminogen by approximately 40%. As a control, 25 mM tranexamic acid was used, which reduced the binding of recombinant plasminogen by approximately 80%. Naive chicken antibody gAb showed no inhibition.

[0376] By SDS-PAGE 1.0 OD 600GAS cultures were grown as described above, except that 1 ml of culture was used for each condition. Cells were washed twice and resuspended in 250 μl of PBS supplemented with 2% plasminogen-deficient FCS. 0.2 μM recombinant plasminogen was incubated with GAS along with 7.5 μM G05, gAb, 13.5 mM TXA, or a buffer-only control. After 30 minutes of incubation at 4°C, the supernatant (unbound fraction) was collected. Cells were then washed twice as described above, and the second wash was retained for SDS-PAGE. Bound plasminogen was eluted with 50 μl of 500 mM TXA, in which the cells were resuspended and spun down again. The resulting supernatant was collected as the "bound fraction." The unbound, washed, and bound fractions were separated on a 12% SDS-PAGE gel along with 0.2 μM (total) recombinant plasminogen, followed by fluorescence scanning under a Cy5 filter using a Typhoon Gel Imaging System (Amersham). Band intensities were determined using standard 2D gel analysis with ImageQuant TL software (GE Healthcare).

[0377] The results, shown in Figure 11, demonstrate a 70% reduction in the binding of recombinant plasminogen to GAS by G05 compared to the gAb and HEPES-buffered saline controls. Interestingly, the positive control, TXA, showed no inhibition. This result suggests that TXA, a plasmin inhibitor, does not inhibit the binding of plasminogen to GAS.

[0378] Example 7: Inhibition of Plasminogen Activation Against Group A Streptococcus (GAS) (Enzyme Assay) 1.0 OD 600GAS cultures were grown as described above, except for the following: 1 ml of culture was used for each sample. Cells were washed twice and resuspended in 250 μl of PBS supplemented with 2% plasminogen-deficient FCS. 80 nM plasminogen and 8 μM G05 / gAb or 13.5 mM TXA were mixed for 15 minutes at room temperature and then incubated with washed GAS cells for 1 hour at room temperature. After the 1-hour incubation, cells were washed twice and finally resuspended in 50 μl of PBS supplemented with 2% plasminogen-deficient FCS. 2 nM recombinant SK was added to half of the test samples to determine whether SK needed to be replaced. Ten microliters of resuspended cells were added to 100 μl of a reaction mixture buffered with 25 mM Tris, 150 mM NaCl, 0.05% Tween 20 pH 7.4, and 200 μM fluorogenic substrate H-Ala-Phe-Lys-AMC (Bachem). Plasmin activity was measured at 37°C using a Fluostar Omega plate reader (BMG Labtech) at excitation and emission wavelengths of 355 nm and 460 nm, respectively.

[0379] The results, shown in Figure 12, demonstrate that G05 inhibits plasminogen activation in the presence and absence of recombinant SK. As expected, gAb has no effect. TXA dissociates plasminogen from the cell surface, contributing to the increased activity observed with the addition of SK (solution activation of Plg). The inset in Figure 12 shows that recombinant SK ultimately activates Plg in solution in the absence of GAS cells.

[0380] Example 8: hPLG is recruited to the CDI-injured intestine and exacerbates tissue damage and disease, and anti-plasminogen antibodies reduce disease severity, delay disease onset, and increase survival in mice. Clostridium difficile (now known as Clostridioides difficile) infects the gastrointestinal tract, causing severe tissue damage. A key driver of C. difficile infection (CDI) is the bacterium's ability to form an inactive yet highly robust spore form, which allows the bacterium to survive in hostile environments. Spores initiate and transmit disease and contribute to disease recurrence, an event that occurs in up to 30% of patients. In the colon, spores germinate into vegetative cells, which colonize the intestinal tract and produce up to three toxins: the large clostridial toxins TcdA and TcdB and the C. difficile transferase (CDT) toxin. These molecules act directly to damage colonic tissue by their action on the actin cytoskeleton of host cells, rendering the tissue permeable. C. difficile vegetative cells also produce spores, which can be found in up to 1 x 10 per gram of feces from infected patients. 7 It is found in spores; these spores transmit the disease and prolong the infection cycle.

[0381] Plasmin, the active form of the liver-produced zymogen plasminogen, is a serine protease that plays an important role in fibrinolysis, cell migration, and tissue remodeling. Several pathogens, such as Group A Streptococcus and Bacillus anthracis, have been shown to utilize the fibrinolytic system by recruiting and activating plasminogen to the microbial surface. Due to its ability to degrade host cells, microbe-bound plasmin significantly enhances bacterial invasion of the host, thereby increasing disease severity during infection.

[0382] Under normal conditions, plasmin is present in low abundance in the gastrointestinal tract. However, due to its role in wound healing, it is hypothesized that the plasminogen system may be recruited to sites of infection, where it may exacerbate or inhibit disease progression. To test this idea, mice injected with or transgenic for human plasminogen (hPLG) were infected with a toxigenic wild-type (WT) strain of C. difficile (M7404) or a mutant strain (DLL3121) that no longer produces the major toxins, TcdA and TcdB. WT-infected mice exhibited epithelial damage, edema, and inflammation compared with uninfected mice (Figure 13A). hPLG levels in the cecum of WT-infected mice were significantly elevated compared with all controls (Figure 13B). In addition, slightly elevated levels of hPLG were detected in DLL3121-infected mice compared with uninfected animals (Figure 13B). These results suggest that CDI and tissue damage induce hPLG influx into gastrointestinal tissues at the site of infection. Although the actions of the major toxins TcdA and TcdB appear to be the primary drivers of hPLG migration to the site of infection, other bacterial factors, such as C. difficile dual toxins (CDTs) or other virulence factors, are also sufficient to mediate partial hPLG influx. Collectively, these results suggest that C. difficile infection and tissue damage induce hPLG influx into gastrointestinal tissues at the site of infection.

[0383] To determine whether hPLG tissue infusion affected infection outcomes, we compared disease severity in wild-type C57BL / 6 mice with that observed in transgenic mice expressing hPLG (both infected with toxigenic C. difficile). Disease was significantly worse in the presence of hPLG, with hPLG-expressing mice exhibiting earlier disease onset as indicated by stool consistency scores (Figure 13C), increased contamination of nesting material (Figure 13D), and impaired physiological status (Figure 13E). The severity of disease symptoms led to a significant decrease in survival time, with hPLG-transgenic mice requiring euthanasia 24 h earlier than WT mice (Figure 13F). To confirm that the worsening disease was not due to differences in colonization or toxin production between the mouse groups, feces were collected and analyzed 24 h postinfection. TcdA and TcdB production was comparable between the two groups of mice (Fig. 13G,H), with no discernible difference in the number of spores shed by the mice, with all mice receiving 5 × 10 6 ~1×10 7 Spores / g (feces) excreted.

[0384] We next examined whether leakage of intestinal contents and dissemination of luminal contents to distal organs occurred in infected animals. It was reasonable to assume that such an outcome could explain the exacerbated disease observed in hPLG mice. Therefore, 24 h after infection, we harvested the thymus, spleen, and kidneys and examined them for the presence of spores. Spores were detected in the kidneys, spleen, and thymus of hPLG mice but not in C57BL / 6 control mice, except for very small numbers in the thymus (Fig. 13I). Examination of cecal tissue harvested at 24 h after infection revealed significantly more inflammation evident in hPLG mice compared with C57BL / 6 mice (Fig. 13J). These findings were supported by experiments performed with hPLG-injected mice, which also showed accelerated disease symptoms. Thus, the exacerbated disease observed in hPLG-expressing or -injected mice was reflected in the cecal pathology of these mice, suggesting that hPLG exacerbates intestinal inflammation during toxigenic C. difficile infection, leading to severe disease outcomes. The presence of hPLG in infected tissue also altered the host inflammatory profile, with significantly increased pro- and anti-inflammatory cytokine production detected in infected hPLG mice compared with infected PBS control mice (Figure 14A). Quantitative proteomic analysis of infected versus uninfected cecal tissues from PBS- and hPLG-injected mice supported these findings (data not shown). Taken together, these data suggest that C. difficile can recruit hPLG during the course of infection and that the presence of this protease increases inflammation and disease severity, in turn promoting translocation of C. difficile spores to extraintestinal organs, a previously unreported finding.

[0385] Because all mice used in these infection experiments produce mouse plasminogen (mPLG) in addition to hPLG, we investigated the role of mPLG during infection by examining disease outcomes after toxigenic C. difficile infection in genetically modified mice that no longer express mPLG (mPLG KO) versus unmodified C57BL / 6 (WT) mice. No differences in disease progression or survival were observed between the two groups (Figure 13K), suggesting that mPLG does not contribute to disease in this infection model.

[0386] Because several different invasive pathogens, including bacteria and parasites, recruit plasminogen to their surface to allow host invasion and subvert the host immune response, we investigated whether C. difficile possesses the same ability. In the case of B. anthracis, it is the vegetative form of the pathogen that normally binds plasminogen, but the spore form was found to bind plasminogen, albeit without consequences for disease severity or virulence. Unexpectedly, hPLG did not bind to C. difficile vegetative cells (Figure 15A), but instead was found to strongly bind to spores (Figures 15A and C), regardless of the bacterial strain's origin (human or animal), geographic location, or toxin-producing status. Furthermore, immunofluorescence (IF) and STED super-resolution microscopy confirmed that hPLG (Alexa488 for IF and Alexa647 for STED) binds to spores produced under laboratory culture conditions (Figure 15D–F). Importantly, spores from C. difficile-infected patients (Figure 15G–I) or mice (Figure 15J–L) were also found to have hPLG on their surface, and STED images showed that in all cases, hPLG was bound in clusters around the spore surface (Figure 15E, H, K). This result suggests that spores are naturally coated with hPLG under the conditions of an infected host.

[0387] Surface plasmon resonance revealed that hPLG binds to C. difficile spores with high affinity (KD of 13.4 nM ± 3.2 nM, K of 55,520 (± 6953) M / s). on and 7.3 x 10 -4 (±1.1×10 -4 )1 / s K off ) (Fig. 3B). Importantly, no concentration-dependent interaction was detected between spores and mPLG (Fig. 15B). These data support the finding that no difference in virulence was observed between WT and mouse plasminogen-deficient mice (Fig. 13K). C. difficile spores also bound to porcine and equine PLG, albeit with weaker affinity than human PLG (Fig. 15B), which may suggest that these proteins also contribute to disease exacerbation in these hosts.

[0388] The conversion of PLG to its most active form, plasmin, is tightly regulated. hPLG is converted to plasmin by host factors, such as urokinase plasminogen activator (uPA) and tissue plasminogen activator (tPA). Therefore, PLG activation assays were performed on spore-bound forms. Unbound or spore-bound hPLG were incubated with human or mouse uPA (huPA and muPA) and human tPA (htPA). Both mouse and human factors activated hPLG to plasmin, whether unbound or spore-bound, suggesting that spores possess plasmin on their surface and can transport this active protease to any site they occupy, either within or outside the intestinal tract. This idea is supported by proteomic analysis, which showed increased abundance of a mouse variant of the plasminogen activation cascade only in infected hPLG mice (data not shown), indicating that hPLG is activated and functional in the gastrointestinal tract of these animals.

[0389] Because plasmin has broad substrate specificity, to determine the functional consequences of plasminogen activation in the context of C. difficile spores, we examined spore structure to determine whether it was modified, particularly at the surface. Surprisingly, transmission electron microscopy demonstrated that hPLG bound to spores and activated them with huPA, reducing the thickness of the exosporium, or spore surface layer, by half, from an average of 73.9 nm for untreated spores to 34.6 nm (Figure 16A–E). Because germinants must pass through the exosporium layer to enable activation of spore germination, the thickness and density of this layer may affect the efficiency of the germination process. Therefore, germination assays performed in the presence of taurocholate, a known host-derived bile acid germinant, showed that plasmin-bound spores germinated faster than untreated spores (Figure 16F). These results demonstrate that hPLG binding and activation directly alter the structural and functional properties of C. difficile spores, leading to faster germination in the presence of physiologically relevant host germinants, which may contribute to the earlier disease onset and severe disease outcomes observed in the previously described hPLG-mouse model. We also examined other functional consequences of spore-hPLG binding and activation on host interactions, revealing that plasmin-bound C. difficile spores cleave C3b and the extracellular matrix (ECM) protein, fibronectin, suggesting that spore-hPLG binding and activation to plasmin alters spore-host interactions. Plasmin-mediated modification of the surface of bacterial cells (in this case, spores) has not previously been demonstrated, providing a novel understanding of how pathogens are modified to exploit the proteolytic activity of host proteins.

[0390] Collectively, these findings suggest that the presence of hPLG thus far appears to contribute to disease progression in a multifactorial manner by inducing immune responses, degrading host tissues, and facilitating extraintestinal spore dissemination, the latter of which may be associated with disease recurrence.

[0391] Building on these findings, we investigated whether therapeutic targeting of hPLG could provide an effective treatment for CDI. Previous studies have shown that it is difficult to specifically inhibit the plasmin system using small-molecule plasmin inhibitors that cross-react with other key proteases in the coagulation system. To overcome these challenges, antibodies specifically targeting the plasmin / plasmin system were developed. Two antibodies were tested. One antibody, G05, was found to effectively inhibit the production of active plasmin. The other antibody was found to effectively inhibit plasmin activity by binding to the catalytic triad of plasmin. The X-ray crystal structure of the scFv fragment of G05 in complex with a fragment of plasminogen (KR5-SP) (referred to as KR5-SP, Figure 9) was determined. The G05 / KR5-SP complex structure revealed that the antibody specifically interacts with the activation loop of hPLG, thereby preventing activators such as tPA and uPA from cleaving and activating the zymogen.

[0392] C57BL / 6 mice injected with hPLG and infected with C. difficile were intraperitoneally injected with HEPES-buffered saline (HBS) alone, or with naive antibody or antibody G05 as a control. Compared to the control group, the G05-treated group had lower scores for both stool consistency (Figure 17B) and nesting material contamination (Figure 17A) detected 24 hours after infection, and disease onset was less rapid. Physiological characteristics between the G05-treated and control groups also trended toward reduced disease severity 24 hours after infection (Figure 17C). Mouse survival beyond this time point was also significant, with G05-treated mice surviving 24 hours longer than control mice (Figure 17D). These results demonstrate that administration of the plasmin production-inhibiting antibody G05 (i.e., an antibody that inhibits the activation of plasminogen to plasmin) protects against severe disease and prolongs the survival of infected mice.

[0393] These data indicate that dysregulation and increase in the amount of hPLG in the gastrointestinal tract after toxin injury alters the dynamics of C. difficile infection, allowing us to propose a new model of CDI progression. In this model, toxin-mediated intestinal injury recruits hPLG to the damaged area. The abnormal localization and activation of hPLG further promotes tissue damage through an inflammatory response that increases immune cell influx and host protein degradation. Once infection is established, hPLG can be recruited to both ingested spores and newly produced spores in the intestine. Plasmin induces rapid spore germination and increases the number of toxin-producing vegetative cells, further exacerbating host tissue damage and increasing disease severity. Plasmin-bound spores also have greater invasive potential and may disseminate systemically within the host, thereby increasing the propensity for disease recurrence. In support of this model, the antibody G05, which inhibits plasminogen activation, successfully reduced disease severity. Because several gastrointestinal pathogens mediate disease through toxin-producing effectors that compromise tissue integrity, and because many other gastrointestinal disorders cause similar leaky gut effects, these results have far-reaching implications for understanding the mechanisms underlying intestinal disease and raise the possibility that inhibition of human plasminogen or plasmin may have broad therapeutic utility.

[0394] Example 9: Administration of antibodies that inhibit plasminogen activation is useful in reducing bleeding Wild-type C56BL / 6 (WT) mice and mice carrying a double mutation in the gene encoding plasminogen (Plg knockout, Plg- / -) were subjected to tail bleeding experiments.

[0395] Each mouse was intravenously injected with 200 μg of human plasminogen. Five minutes later, 50 μg of antibody (gAb or G05) was administered intravenously. Tail bleeding experiments were performed by immersing the cut tail in warm saline for 20 minutes. The amount of bleeding was measured by collecting red blood cells, which were then suspended in a fixed amount of lysis buffer; OD was determined for each animal. 550 was recorded.

[0396] The data (shown in Figure 19) show that the amount of bleeding in mice receiving the G05 antibody was significantly reduced compared to mice receiving the naive (gAb) antibody. These results indicate that antibodies that inhibit plasminogen activation are useful in inhibiting bleeding in a model of traumatic hemorrhage.

[0397] Example 10: G05 and G11 inhibit the lysis of synthetic and whole blood clots Synthetic fibrin clots were formed by mixing 3 mg / ml fibrinogen (Banksia Scientific), 1 U bovine thrombin (Jomar Life Research), and 10 nM tPA (Boehringer Ingelheim) for 2 h at 37°C. Fibrinolysis was initiated by adding 45 nM plasminogen mixed with 0-3 μM G05; 0-2 μM G11; 0-90 nM α2AP; or 0-6.25 mM TXA to the surface of the clot.

[0398] Fibrinolysis was monitored using a Nephelometer (BMG) for up to 10 hours at 37°C. The time required to achieve 50% clot lysis was defined as IC 50 Used for calculations. The combination of G05 and G11 significantly inhibited clot lysis, approximately 4-6 times more than either antibody used alone.

[0399] Whole blood clots were prepared from human blood collected from healthy donors. Halo-shaped clots were generated by mixing 15% of a mixture containing recombinant tissue factor and 67 mM CaCl2 in HBS supplemented with synthetic phospholipids (Dade Innovin, Siemens Germany) with whole blood at a 1:4 ratio. The plates were sealed and incubated at 37°C for 60 minutes before use.

[0400] Clot lysis was induced by the addition of up to 7 nM tPA, as well as antibodies or Plm inhibitors at the following concentrations: 0-312.5 nM B10; 0-1000 nM α2AP; and 0-1000 nM aprotinin. Clot lysis was observed as an increase in turbidity and was monitored using a plate reader as OD6. 10 Monitored in nM.

[0401] At high concentrations (e.g., up to 1,000 nM), only α2AP and aprotinin partially inhibited clot lysis; TXA at ​​concentrations up to 100 μM delayed clot lysis, and complete inhibition of clot lysis was observed at 300 μM or higher (data not shown).

[0402] The time taken to achieve 50% clot lysis was determined as IC 50 The IC obtained for G11 was used for calculations. 50 The value was approximately twice that of α2AP and approximately 2.5 times that of aprotinin (FIG. 20).

[0403] Thus, in a clot lysis assay that closely resembles a biological system, antibody G11 is more effective at inhibiting plasmin-induced lysis than α2AP, a physiological inhibitor of plasmin, and more effective than aprotinin, a pharmacological inhibitor of plasmin.

[0404] It will be understood that the invention disclosed and defined herein extends to all alternative combinations of two or more of the individual features described or apparent from the text or drawings, all of these different combinations constituting various alternative aspects of the invention. Further aspects of the present invention are described below: [Section 1] 1. An antigen binding protein comprising an antigen binding domain that binds to plasminogen, wherein said antigen binding protein reduces activation of plasminogen. [Section 2] Item 10. The antigen-binding protein of Item 1, wherein the antigen-binding protein reduces or inhibits activation of the plasminogen by any one or more plasminogen activators. [Section 3] The plasminogen activator is a plasminogen activator that binds to Arg of plasminogen. 561 -Val 562 3. The antigen-binding protein of clause 2, wherein the plasminogen activator is any enzyme capable of cleaving the bond (numbering according to human plasminogen), optionally wherein the plasminogen activator is a plasminogen-cleaving serine protease selected from the group consisting of the coagulation proteins factor IX, factor X, and prothrombin (factor II), protein C, chymotrypsin and trypsin, various leukocyte elastases, streptokinase (SK), staphylokinase, urokinase plasminogen activator (uPA), tissue plasminogen activator (tPA), and plasmin. [Section 4] Item 4. The antigen-binding protein according to any one of Items 1 to 3, wherein the antigen-binding protein binds to an activation loop of plasminogen. [Section 5] Item 5. The antigen-binding protein according to any one of Items 1 to 4, which binds to a Kringle domain, preferably Kringle domain 5, of plasminogen. [Section 6] Item 6. The antigen-binding protein according to any one of Items 1 to 5, wherein the antigen-binding protein also binds to the kringle domain 5 and serine protease domain of plasminogen. [Section 7] The antigen-binding protein binds to Arg of plasminogen 493 From His 569 and preferably the protein binds to a region of plasminogen comprising or consisting of an amino acid sequence between Lys 468 From His 569and more preferably, the activation loop binds to a region comprising or consisting of an amino acid sequence between Ala of plasminogen according to the numbering set forth in SEQ ID NO: 65. 543 From Arg 582 Item 7. The antigen-binding protein of any one of Items 1 to 6, comprising the sequence: [Section 8] Item 8. The antigen-binding protein of any one of Items 1 to 7, wherein the antigen-binding protein binds to a peptide comprising, consisting of, or consisting essentially of the sequence shown in SEQ ID NO: 66, or a fragment thereof. [Section 9] 10. The antigen binding protein of claim 1, wherein the antigen binding protein has a sequence at position Arg 493 , Ser 495 , Ile 496 , Asp 516 , Gly 517 , Asp 518 , Val 519 , Tyr 525 , and Tyr 533 or a residue in the kringle 5 domain of plasminogen at a position shown in Table 2, or a residue in the kringle 5 domain of plasminogen at a position corresponding thereto; preferably, the antigen-binding protein binds to a residue in the kringle 5 domain of plasminogen at a position shown in Table 2, or a residue in the kringle 5 domain of plasminogen at a position corresponding thereto. [Section 10] 10. The antigen binding protein, comprising: 554 , Lys 556 , Lys 557 , His 569 , and Asp 751 or a position corresponding thereto, and preferably, the antigen-binding protein of the invention binds to one or more residues in the serine protease domain of plasminogen at the positions shown in Table 2, or a position corresponding thereto. [Section 11] 10. The antigen binding protein of claim 1, wherein the antigen binding protein has a nucleotide at position Lys 468 , Arg 493 , Ser495 , Ile 496 , Asp 516 , Gly 517 , and Tyr 525 or a residue in the kringle 5 domain of plasminogen at a position shown in Table 3, or a residue in the kringle 5 domain of plasminogen at a position corresponding thereto; preferably, the antigen-binding protein of the present invention binds to a residue in the kringle 5 domain of plasminogen at a position shown in Table 3, or a residue in the kringle 5 domain of plasminogen at a position corresponding thereto. [Section 12] 10. The antigen binding protein, comprising: 554 , Lys 556 , Lys 557 , Asp 561 , and His 569 The antigen-binding protein of any one of Items 1 to 8 or Item 11, which binds to one or more residues in the serine protease domain of plasminogen at positions shown in Table 3, or at positions corresponding thereto; preferably, the antigen-binding protein of the invention binds to one or more residues in the serine protease domain of plasminogen at positions shown in Table 3, or at positions corresponding thereto. [Section 13] Item 13. The antigen-binding protein according to any one of Items 1 to 12, wherein the interaction between one residue of the antigen-binding protein and one residue of plasminogen is elucidated by X-ray crystal structure analysis and contact distance analysis of 0 to 3.9 Å (inclusive). [Section 14] Item 14. The antigen-binding protein according to any one of Items 1 to 13, wherein the antigen-binding protein binds to the same epitope on plasminogen as an antibody comprising a VH domain having the amino acid sequence shown in SEQ ID NO: 8 and a VL domain having the amino acid sequence shown in SEQ ID NO: 7, and the antigen-binding protein reduces or inhibits activation of the plasminogen. [Section 15] 10. The antigen binding protein of claim 1, wherein the antigen binding protein is 676 , Arg 677 , Arg 712 , Glu 714 , and Asn 769or a residue in the serine protease domain of plasminogen at a position shown in Table 4, or a position corresponding thereto; preferably, the antigen-binding protein of the invention binds to one or more residues in the serine protease domain of plasminogen at a position shown in Table 4, or a position corresponding thereto. [Section 16] The antigen-binding protein according to any one of Items 1 to 3, 14, or 15, wherein the antigen-binding protein binds to the same epitope on plasminogen as an antibody comprising a VH domain having the amino acid sequence shown in SEQ ID NO: 40 and a VL domain having the amino acid sequence shown in SEQ ID NO: 39, and the antigen-binding protein reduces or inhibits activation of plasminogen. [Section 17] The antigen binding protein binds to plasminogen and has a K of less than 15 nM, less than 10 nM, or about 8 nM or less. D Item 17. The antigen-binding protein of any one of Items 1 to 16, which exhibits the following: [Section 18] The antigen-binding protein binds to plasminogen and produces approximately 1×10 4 Super, about 1×10 4 More than or about 1 x 10 5 or more or about 4 x 10 5 More than k a (M -1 s -1 Item 18. The antigen-binding protein of any one of Items 1 to 17, which exhibits the following formula: [Section 19] The antigen-binding protein binds to plasminogen and produces approximately 1×10 -3 Less than 5 × 10 -3 Less than or 2 x 10 -3 The following k d (s -1 ) or the k d is about 7 x 10 -4 Item 19. The antigen-binding protein of any one of Items 1 to 18, which is: [Section 20] the antigen binding protein has an IC50 of less than 500 nM, 300 nM, 250 nM, 200 nM, 150 nM, 100 nM, 80 nM, 60 nM or 50 nM, preferably about 40 nM 50 Item 20. The antigen-binding protein of any one of Items 1 to 19, which inhibits streptokinase-, tPA-, or uPA-mediated activation of plasminogen. [Section 21] 21. The antigen-binding protein of any one of Aspects 1 to 20, wherein the antigen-binding protein binds to a peptide derived from the amino acid sequence set forth in SEQ ID NO: 65, and optionally the antigen-binding protein binds to a peptide consisting of 4, 5, 7, 8, 9, 10 or more consecutive amino acid residues of the sequence of SEQ ID NO: 65. [Section 22] The antigen binding protein is selected from the group consisting of: binds to a peptide comprising, consisting essentially of, or consisting of residues 554 to 569 of SEQ ID NO: 65; binds to a peptide comprising, consisting essentially of, or consisting of residues 493 to 533 of SEQ ID NO: 65; binds to a peptide comprising, consisting essentially of, or consisting of residues 468 to 525 of SEQ ID NO: 65; binds to a peptide comprising, consisting essentially of, or consisting of residues 554 to 569 of SEQ ID NO: 65; binds to a peptide comprising, consisting essentially of, or consisting of residues 554 to 569 of SEQ ID NO: 65; binds to a peptide comprising, consisting essentially of, or consisting of residues 493 to 533 of SEQ ID NO: 65 and binds to a peptide comprising, consisting essentially of, or consisting of residues 554 to 569 of SEQ ID NO: 65; 22. The antigen-binding protein of any one of items 1 to 21, which binds to a peptide comprising, consisting essentially of, or consisting of residues 468 to 525 of SEQ ID NO: 65, and binds to a peptide comprising, consisting essentially of, or consisting of residues 554 to 569 of SEQ ID NO: 65. [Section 23] Item 23. The antigen-binding protein of any one of Items 1 to 22, wherein the antigen-binding protein does not bind to a region of plasminogen or plasmin that includes only the serine protease domain, and preferably the antigen-binding protein does not bind to the catalytic triad of plasminogen or plasmin and / or does not significantly reduce the activity of plasmin. [Section 24] Item 24. The antigen-binding protein of any one of Items 1 to 23, wherein the antigen-binding protein does not significantly reduce the activity of any one or more of tPA, thrombin, trypsin, factor Xa (FXa), and plasma kallikrein. [Section 25] The antigen binding protein is selected from the group consisting of: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, FR1, FR2, FR3 and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are each a framework region; CDR1a, CDR2a and CDR3a are each a complementarity determining region; Item 25. The antigen-binding protein that binds to plasminogen according to any one of Items 1 to 24, wherein any of the sequences of the complementarity determining regions has an amino acid sequence shown in Table 1, and preferably the framework regions also have an amino acid sequence shown in Table 1 below, which contains amino acid mutations at specific residues that can be determined by aligning various framework regions derived from each antibody. [Section 26] 26. The antigen-binding protein of claim 25, wherein CDR1, CDR2, and CDR3 are sequences from the VH and CDR1a, CDR2a, and CDR3a are sequences from the VL, or wherein CDR1, CDR2, and CDR3 are sequences from the VL and CDR1a, CDR2a, and CDR3a are sequences from the VH. [Section 27] 27. The antigen-binding protein of clause 25 or 26, wherein FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4- and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked by a linker, optionally the linker being a chemical compound, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues. [Section 28] (in N to C-terminal or C to N-terminal order) SEQ ID NOs: 7 and 8: or SEQ ID NOs: 39 and 40 28. The antigen-binding protein of any one of items 1 to 27, comprising, consisting essentially of, or consisting of the amino acid sequence: [Section 29] Item 29. The antigen-binding protein of any one of Items 1 to 28, comprising an antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to plasminogen, and the antigen-binding domain is selected from the group consisting of: (i) a VH comprising a complementarity determining region (CDR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:4, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:5, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:6; (ii) a VH comprising a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO:8; (iii) a VL comprising a CDR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:1, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:2, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:3; (iv) a VL having a sequence at least about 95% identical to the sequence set forth in SEQ ID NO:7; (v) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO: 4, a CDR2 having the sequence set forth in SEQ ID NO: 5, and a CDR3 having the sequence set forth in SEQ ID NO: 6; (vi) a VH having the sequence set forth in SEQ ID NO: 8; (vii) a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 1, a CDR2 having the sequence set forth in SEQ ID NO: 2, and a CDR3 having the sequence set forth in SEQ ID NO: 3; (viii) VL having the sequence set forth in SEQ ID NO: 7; (ix) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO:4, a CDR2 having the sequence set forth in SEQ ID NO:5, and a CDR3 having the sequence set forth in SEQ ID NO:6; and a VL comprising a CDR1 having the sequence set forth in SEQ ID NO:1, a CDR2 having the sequence set forth in SEQ ID NO:2, and a CDR3 having the sequence set forth in SEQ ID NO:3; or (x) a VH having the sequence set forth in SEQ ID NO: 8 and a VL having the sequence set forth in SEQ ID NO: 7 An antigen-binding protein comprising at least one of: [Section 30] the antigen-binding domain comprising: (i) a VH comprising a framework region (FR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 21; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 22; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 23; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 24; (ii) a VL comprising: an FR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 17; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 18; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 19; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 20; (iii) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 21, an FR2 having the sequence set forth in SEQ ID NO: 22, an FR3 having the sequence set forth in SEQ ID NO: 23, and an FR4 having the sequence set forth in SEQ ID NO: 24; (iv) a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 17, an FR2 having the sequence set forth in SEQ ID NO: 18, an FR3 having the sequence set forth in SEQ ID NO: 19, and an FR4 having the sequence set forth in SEQ ID NO: 20; or (v) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 21, an FR2 having the sequence set forth in SEQ ID NO: 22, an FR3 having the sequence set forth in SEQ ID NO: 23, and an FR4 having the sequence set forth in SEQ ID NO: 24; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 17, an FR2 having the sequence set forth in SEQ ID NO: 18, an FR3 having the sequence set forth in SEQ ID NO: 19, and an FR4 having the sequence set forth in SEQ ID NO: 20. 30. The antigen-binding protein of paragraph 29, further comprising at least one of: [Section 31] Item 29. The antigen-binding protein of any one of Items 1 to 28, comprising an antigen-binding domain of an antibody, wherein the antigen-binding domain binds to or specifically binds to plasminogen, and the antigen-binding domain is selected from the group consisting of: (i) a VH comprising a complementarity determining region (CDR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 36, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 37, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 38; (ii) a VH having a sequence at least about 95%, or 96%, or 97%, or 98%, or 99% identical to the sequence set forth in SEQ ID NO: 40; (iii) a VL comprising a CDR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 33, a CDR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 34, and a CDR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 35; (iv) a VL having a sequence at least about 95% identical to the sequence set forth in SEQ ID NO: 39; (v) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO: 36, a CDR2 having the sequence set forth in SEQ ID NO: 37, and a CDR3 having the sequence set forth in SEQ ID NO: 38; (vi) a VH having the sequence set forth in SEQ ID NO: 40; (vii) a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 33, a CDR2 having the sequence set forth in SEQ ID NO: 34, and a CDR3 having the sequence set forth in SEQ ID NO: 35; (viii) a VL having the sequence set forth in SEQ ID NO: 39; (ix) a VH comprising a CDR1 having the sequence set forth in SEQ ID NO: 36, a CDR2 having the sequence set forth in SEQ ID NO: 37, and a CDR3 having the sequence set forth in SEQ ID NO: 38; and a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 33, a CDR2 having the sequence set forth in SEQ ID NO: 34, and a CDR3 having the sequence set forth in SEQ ID NO: 35; or (x) a VH having the sequence set forth in SEQ ID NO: 40 and a VL having the sequence set forth in SEQ ID NO: 39 An antigen-binding protein comprising at least one of: [Section 32] the antigen-binding domain comprising: (i) a VH comprising a framework region (FR) 1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 53; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 54; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 55; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 56; (ii) a VL comprising: an FR1 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:49; an FR2 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:50; an FR3 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:51; and an FR4 having a sequence at least about 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:52; (iii) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 53, an FR2 having the sequence set forth in SEQ ID NO: 54, an FR3 having the sequence set forth in SEQ ID NO: 55, and an FR4 having the sequence set forth in SEQ ID NO: 56; (iv) a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 49, an FR2 having the sequence set forth in SEQ ID NO: 50, an FR3 having the sequence set forth in SEQ ID NO: 51, and an FR4 having the sequence set forth in SEQ ID NO: 52; (v) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 53, an FR2 having the sequence set forth in SEQ ID NO: 54, an FR3 having the sequence set forth in SEQ ID NO: 55, and an FR4 having the sequence set forth in SEQ ID NO: 56; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 49, an FR2 having the sequence set forth in SEQ ID NO: 50, an FR3 having the sequence set forth in SEQ ID NO: 51, and an FR4 having the sequence set forth in SEQ ID NO: 52. 32. The antigen-binding protein of claim 31, further comprising at least one of: [Section 33] The antigen binding protein is selected from the group consisting of: (i) single-chain Fv fragment (scFv); (ii) dimeric scFv (di-scFv); (iii) one of (i) or (ii) linked to the constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (iv) one of (i) or (ii) linked to a protein that binds to immune effector cells. Item 33. The antigen-binding protein according to any one of Items 1 to 32, which is in the form of: [Section 34] The antigen binding protein is selected from the group consisting of: (i) Diabodies; (ii) triabodies; (iii) tetrabodies; (iv)Fab; (v) F(ab'): (vi) Fv; (vii) bispecific antibodies; (viii) one of (i)-(vii) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; or (viv) one of (i) to (vii) linked to a protein that binds to immune effector cells; Item 34. The antigen-binding protein according to any one of Items 1 to 33, which is in the form of: [Section 35] Item 35. The antigen-binding protein according to any one of Items 1 to 34, wherein the antigen-binding protein is an antibody or an antigen-binding fragment thereof, and preferably the antigen-binding protein is a monoclonal antibody. [Section 36] Item 36. The antigen-binding protein according to any one of Items 1 to 35, wherein the antigen-binding protein is a variable domain. [Section 37] the protein is a plasminogen-binding antibody, the antibody comprising a light chain variable region and a heavy chain variable region; The light chain variable region comprises: CDR L1 set forth in SEQ ID NO: 1, CDR L2 set forth in SEQ ID NO: 2, and CDR L3 set forth in SEQ ID NO: 3 Includes; The heavy chain variable region comprises: CDR H1 set forth in SEQ ID NO: 4, CDR H2 set forth in SEQ ID NO: 5, and CDR H3 set forth in SEQ ID NO: 6 Item 37. The antigen-binding protein of any one of Items 1 to 36, comprising: [Section 38] 38. The antigen binding protein of clause 37, wherein the antibody comprises a light chain variable region having FR L1 set forth in SEQ ID NO: 17, FR L2 set forth in SEQ ID NO: 18, FR L3 set forth in SEQ ID NO: 19, and FR L4 set forth in SEQ ID NO: 20. [Section 39] 39. The antigen-binding protein of paragraph 37 or 38, wherein the antibody comprises a heavy chain variable region having FR H1 set forth in SEQ ID NO: 21, FR H2 set forth in SEQ ID NO: 22, FR H3 set forth in SEQ ID NO: 23, and FR H4 set forth in SEQ ID NO: 24. [Section 40] 38. The antigen binding protein of clause 37, wherein the antibody comprises a light chain variable region having the sequence of SEQ ID NO:7. [Section 41] 41. The antigen-binding protein of clause 37 or 40, wherein the antibody comprises a heavy chain variable region having the sequence of SEQ ID NO:8. [Section 42] the protein is a plasminogen-binding antibody, the antibody comprising a light chain variable region and a heavy chain variable region; The light chain variable region comprises: CDR L1 set forth in SEQ ID NO: 33, CDR L2 set forth in SEQ ID NO: 34, and CDR L3 set forth in SEQ ID NO: 35 Includes; The heavy chain variable region comprises: CDR H1 set forth in SEQ ID NO: 36, CDR H2 set forth in SEQ ID NO: 37, and CDR H3 set forth in SEQ ID NO: 38 Item 37. The antigen-binding protein of any one of Items 1 to 36, comprising: [Section 43] 43. The antigen binding protein of clause 42, wherein the antibody comprises a light chain variable region having FR L1 set forth in SEQ ID NO: 49, FR L2 set forth in SEQ ID NO: 50, FR L3 set forth in SEQ ID NO: 51, and FR L4 set forth in SEQ ID NO: 52. [Section 44] 44. The antigen-binding protein of paragraph 42 or 43, wherein the antibody comprises a heavy chain variable region having FR H1 set forth in SEQ ID NO: 53, FR H2 set forth in SEQ ID NO: 54, FR H3 set forth in SEQ ID NO: 55, and FR H4 set forth in SEQ ID NO: 56. [Section 45] 43. The antigen binding protein of clause 42, wherein the antibody comprises a light chain variable region having the sequence of SEQ ID NO:39. [Section 46] 46. ​​The antigen binding protein of clause 42 or 45, wherein the antibody comprises a heavy chain variable region having the sequence of SEQ ID NO: 40. [Section 47] Item 47. The antigen-binding protein according to any one of Items 1 to 46, wherein the antigen-binding protein is a naked antibody. [Section 48] A fusion protein comprising the antigen-binding protein, immunoglobulin variable domain, antibody, dab (single domain antibody), di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody according to any one of Items 1 to 46. [Section 49] A conjugate in the form of an antigen-binding protein, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody or fusion protein according to any one of Items 1 to 46 or 48, conjugated to a label or a cytotoxic agent. [Section 50] A nucleic acid encoding the antigen-binding protein, immunoglobulin variable domain, antibody, dab, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody, fusion protein, or conjugate according to any one of Items 1 to 49. [Section 51] A vector comprising the nucleic acid of item 50. [Section 52] A pharmaceutical composition comprising the antigen-binding protein of any one of Items 1 to 47, the fusion protein of Item 48, or the conjugate of Item 49, and a pharmaceutically acceptable carrier, diluent, or excipient. [Section 53] A method of restoring hemostasis in a subject that has suffered a trauma, suffered a hemorrhage, or is bleeding, comprising administering to a subject in need thereof the antigen-binding protein of any one of items 1 to 48, thereby restoring hemostasis in said subject. [Section 54] 49. A method of inhibiting fibrinolysis in a subject in need thereof, comprising administering to a subject in need thereof the antigen-binding protein of any one of paragraphs 1 to 48, thereby inhibiting fibrinolysis in said subject, optionally wherein said subject has suffered trauma or has bleeding due to surgery or childbirth. [Section 55] A method of treating a bacterial infection in a subject, comprising administering to the subject the antigen-binding protein of any one of items 1 to 48, thereby treating the bacterial infection in the subject. [Section 56] A method of treating a condition associated with or resulting from a bacterial infection in a subject, said method comprising administering to said subject an effective amount of an antigen binding protein of the invention, thereby treating said condition associated with or resulting from a bacterial infection in said subject. [Section 57] A method for reducing the severity of a bacterial infection in a subject, comprising administering to the subject the antigen-binding protein of any one of Items 1 to 48, thereby reducing the severity of the bacterial infection in the subject. [Section 58] A method for treating or preventing cancer in a subject, the method comprising administering to the subject the antigen-binding protein of any one of items 1 to 48, thereby treating or preventing cancer in the subject. [Section 59] The method comprises inhibiting, preventing, or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer. [Section 60] Item 49. Use of the plasminogen-binding antigen-binding protein according to any one of Items 1 to 48 in the manufacture of a medicament for restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered trauma, or who needs to restore hemostasis or inhibit plasminogen activation after surgery or childbirth. [Section 61] Item 49. Use of the plasminogen-binding antigen-binding protein according to any one of Items 1 to 48 in the manufacture of a medicament for the treatment or prevention of a bacterial infection. [Section 62] Item 49. Use of the antigen-binding protein of any one of Items 1 to 48 in the manufacture of a medicament for the treatment, prevention, or reduction of severity of any condition or disease caused by or associated with a bacterial infection. [Section 63] 50. Use of the antigen-binding protein of any one of paragraphs 1 to 48 in the manufacture of a medicament for treating or preventing cancer in a subject, optionally wherein the medicament is intended to inhibit, prevent or minimize the spread or progression of cancer, including metastasis of cancer. [Section 64] Item 53. The pharmaceutical composition according to Item 52, for use in restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered trauma or who needs to restore hemostasis or inhibit plasminogen activation after surgery or childbirth. [Section 65] Item 53. The pharmaceutical composition according to Item 52, for use in treating or preventing a bacterial infection. [Section 66] Item 53. The pharmaceutical composition according to Item 52, for use in treating, preventing or reducing the severity of any condition or disease caused by or associated with a bacterial infection. [Section 67] 53. The pharmaceutical composition of claim 52 for use in treating or preventing cancer in a subject, optionally wherein the composition is intended to inhibit, prevent, or minimize the spread or progression of cancer, including metastasis of cancer. [Section 68] Item 57. The method of item 55 or 56, the use of item 61 or 62, or the composition of item 65 or 66, wherein the bacterial infection is chronic or acute. [Section 69] 69. The method, use or composition of paragraph 68, wherein the infection is a bacterial infection. [Section 70] 70. The method, use or composition of paragraph 69, wherein the bacterial infection is caused by spore-forming bacteria, and optionally the infection may be characterized by bacteria in a vegetative state or in spore form. [Section 71] 70. The method, use or composition of clause 69, wherein the bacterial infection is caused by a bacterium that is gram-positive, preferably a gram-positive cocci. [Section 72] Item 72. The method, use or composition according to Item 71, wherein the bacterium belongs to the family Streptococcaceae, preferably the bacterium belongs to the genus Streptococcus, more preferably the bacterium is a Group A Streptococcus (GAS), including Streptococcus pyogenes. [Section 73] 72. The method, use or composition of Clause 71, wherein the bacterium belongs to the family Staphylococcaceae, preferably the bacterium belongs to the genus Staphylococcus, and more preferably the infection is an infection caused by a bacterium selected from the group consisting of Staphylococcus aureus, including Methicillin-resistant Staphylococcus aureus (MRSA), Vancomycin-intermediate Staphylococcus aureus (VISA), and Vancomycin-resistant Staphylococcus aureus (VRSA). [Section 74] 72. The method, use or composition of clause 71, wherein the bacterium belongs to the family Peptostreptococcaceae, preferably the bacterium belongs to the genus Clostridioides. [Section 75] 72. The method, use or composition according to paragraph 71, wherein the bacterial infection is an infection caused by Clostridium difficile (also known as Clostridioides difficile). [Section 76] 70. The method, use or composition of clause 69, wherein the infection is caused by a gram-negative bacterium, preferably from the order Enterobacteriales. [Section 77] 77. The method, use or composition of clause 76, wherein the bacterium is selected from the group consisting of Yersinia pestis, Yersinia enterocolitica, Helicobacter pylori, E. coli, Salmonella spp., Pseudomonas spp., Shigella spp.

Claims

1. An antibody or antigen-binding fragment thereof comprising an antigen-binding domain that binds to plasminogen, wherein the antigen-binding domain is (i) a VH comprising a complementarity determining region (CDR) 1 having the sequence set forth in SEQ ID NO: 36, a CDR2 having the sequence set forth in SEQ ID NO: 37, and a CDR3 having the sequence set forth in SEQ ID NO: 38; and (ii) a VL comprising a CDR1 having the sequence set forth in SEQ ID NO: 33, a CDR2 having the sequence set forth in SEQ ID NO: 34, and a CDR3 having the sequence set forth in SEQ ID NO: 35; An antibody or antigen-binding fragment thereof.

2. the antibody or antigen-binding fragment: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, FR1, FR2, FR3 and FR4 are each a framework region; CDR1, CDR2 and CDR3 are each a complementarity determining region; FR1a, FR2a, FR3a and FR4a are each a framework region; CDR1a, CDR2a, and CDR3a are each a complementarity determining region; wherein CDR1, CDR2, and CDR3 are sequences from the VH, and CDR1a, CDR2a, and CDR3a are sequences from the VL, or CDR1, CDR2, and CDR3 are sequences from the VL, and CDR1a, CDR2a, and CDR3a are sequences from the VH; FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4 and FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a are linked directly or via a linker; The antibody or antigen-binding fragment thereof according to claim 1.

3. The antibody or antigen-binding fragment thereof of claim 2, wherein the linker is a chemical compound, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

4. 4. The antibody or antigen-binding fragment thereof according to any one of claims 1 to 3, wherein the antigen-binding domain is: (i) a VH having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 40; (ii) a VL having a sequence at least 95% identical to the sequence set forth in SEQ ID NO: 39; (iii) a VH having the sequence set forth in SEQ ID NO: 40; (iv) a VL having the sequence set forth in SEQ ID NO: 39; or (v) VH having the sequence set forth in SEQ ID NO: 40 and VL having the sequence set forth in SEQ ID NO: 39 An antibody or antigen-binding fragment thereof comprising at least one of:

5. The antigen-binding domain comprises: (i) a VH comprising a framework region (FR) 1 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 53; an FR2 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 54; an FR3 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 55; and an FR4 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO: 56; (ii) a VL comprising an FR1 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:49, an FR2 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:50, an FR3 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:51, and an FR4 having a sequence at least 80%, at least 85%, at least 90%, at least 92%, at least 95%, at least 97%, or at least 99% identical to the sequence set forth in SEQ ID NO:52; (iii) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 53, an FR2 having the sequence set forth in SEQ ID NO: 54, an FR3 having the sequence set forth in SEQ ID NO: 55, and an FR4 having the sequence set forth in SEQ ID NO: 56; (iv) a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 49, an FR2 having the sequence set forth in SEQ ID NO: 50, an FR3 having the sequence set forth in SEQ ID NO: 51, and an FR4 having the sequence set forth in SEQ ID NO: 52; or (v) a VH comprising an FR1 having the sequence set forth in SEQ ID NO: 53, an FR2 having the sequence set forth in SEQ ID NO: 54, an FR3 having the sequence set forth in SEQ ID NO: 55, and an FR4 having the sequence set forth in SEQ ID NO: 56; and a VL comprising an FR1 having the sequence set forth in SEQ ID NO: 49, an FR2 having the sequence set forth in SEQ ID NO: 50, an FR3 having the sequence set forth in SEQ ID NO: 51, and an FR4 having the sequence set forth in SEQ ID NO:

52. further comprising at least one of: The antibody or antigen-binding fragment thereof according to claim 4.

6. the antibody or antigen-binding fragment thereof (i) single chain Fv fragments (scFv); (ii) dimeric scFv (di-scFv); (iii) one of (i) or (ii) linked to a constant region, Fc or heavy chain constant domain (CH)2 and / or CH3, of an antibody; (iv) one of (i) or (ii) linked to a protein that binds to an immune effector cell. (v) diabodies; (vi) triabodies; (vii) tetrabodies; (viii) Fab; (ix)F(ab')2: (x) Fv; (xi) bispecific antibodies; (xii) one of (v)-(xi) linked to a constant region of an antibody, Fc or heavy chain constant domain (CH)2 and / or CH3; or (xiii) A fusion protein comprising any one of the antibodies or antigen-binding fragments thereof, immunoglobulin variable domains, antibodies, di-scFvs, scFvs, Fabs, Fab's, F(ab')2s, Fv fragments, diabodies, triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, or multispecific antibodies of (i) to (xii). The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, in the form:

7. 7. A conjugate in the form of an antibody or antigen-binding fragment thereof, immunoglobulin variable domain, antibody, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody or fusion protein according to any one of claims 1 to 6, conjugated to a label or a cytotoxic agent.

8. A nucleic acid encoding the antibody or antigen-binding fragment thereof, immunoglobulin variable domain, antibody, di-scFv, scFv, Fab, Fab', F(ab')2, Fv fragment, diabody, triabody, tetrabody, linear antibody, single-chain antibody molecule, or multispecific antibody, or fusion protein of any one of claims 1 to 6.

9. The nucleic acid of claim 8 , wherein the nucleic acid is in the form of a vector.

10. A pharmaceutical composition comprising an antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, or a conjugate according to claim 7, and a pharmaceutically acceptable carrier, diluent or excipient.

11. 11. The antibody or antigen-binding fragment thereof of any one of claims 1 to 6, the conjugate of claim 7, or the pharmaceutical composition of claim 10, a) an antibody or antigen-binding fragment thereof, conjugate, or pharmaceutical composition for use in restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered a traumatic injury or who is in need of restoring hemostasis or inhibiting plasminogen activation after surgery or childbirth; b) an antibody or antigen-binding fragment thereof, conjugate or pharmaceutical composition for use in the treatment or prevention of a bacterial infection; c) an antibody or antigen-binding fragment thereof, conjugate, or pharmaceutical composition for use in the treatment, prevention, or reduction of the severity of any condition or disease caused by or associated with a bacterial infection; or d) An antibody or antigen-binding fragment thereof, conjugate, or pharmaceutical composition for use in treating or preventing cancer in a subject, wherein said composition is intended to inhibit, prevent, or minimize the spread or progression of cancer, including metastasis of cancer.

12. a) the bacterial infection is caused by spore-forming bacteria; b) the bacterial infection is caused by a gram-positive bacterium, or c) the bacterial infection is caused by gram-negative bacteria; The antibody or antigen-binding fragment thereof, conjugate or pharmaceutical composition of claim 11.

13. - Spore-forming bacterial infections are caused by bacteria in the vegetative state or in the spore form, - Gram-positive bacterial infections are caused by gram-positive cocci, - the gram-negative bacterial infection is caused by a bacterium selected from the group consisting of Yersinia pestis, Yersinia enterocolitica, Helicobacter pylori, E. coli, Salmonella spp., Pseudomonas spp., Shigella spp., - the bacterial infection is caused by bacteria belonging to the Streptococcus family (Streptococcus pyogenes), - the bacterial infection is caused by bacteria belonging to the genus Streptococcus, - the bacterial infection is caused by Group A Streptococcus (GAS) bacteria, including Streptococcus pyogenes; - the bacterial infection is caused by bacteria belonging to the family Staphylococcus aureus, - the bacterial infection is caused by bacteria belonging to the genus Staphylococcus, the bacterial infection is caused by a bacterium selected from the group consisting of Staphylococcus aureus, including methicillin-resistant Staphylococcus aureus (MRSA), vancomycin-intermediate Staphylococcus aureus (VISA), and vancomycin-resistant Staphylococcus aureus (VRSA); - the bacterial infection is caused by bacteria belonging to the Peptostreptococcus family, - the bacterial infection is caused by bacteria belonging to the genus Clostridioides, or - the bacterial infection is caused by the bacterium Clostridium difficile (also known as Clostridioides difficile), 13. The antibody or antigen-binding fragment thereof, conjugate or pharmaceutical composition of claim 12.

14. Use of the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6, the conjugate according to claim 7 or the pharmaceutical composition according to claim 10, a) Use in the manufacture of a medicament for restoring hemostasis or inhibiting excessive plasminogen activation in a subject who has suffered trauma or who needs to do so after surgery or childbirth; b) Use in the manufacture of a medicament for the treatment or prevention of a bacterial infection and / or for the treatment, prevention or reduction of the severity of any condition or disease caused by or associated with a bacterial infection, wherein the bacterial infection is chronic or acute; or c) Use in the manufacture of a medicament for the treatment or prevention of cancer in a subject, wherein the medicament is intended to inhibit, prevent or minimize the spread or progression of cancer, including metastasis of cancer.

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