Antibodies for binding to plasmin

Abstract

To provide an antigen-binding protein comprising an antigen-binding domain that binds to plasmin. Also provided are compositions comprising the antigen binding proteins, as well as uses thereof and methods of treatment comprising the same.SOLUTION: To provide antigen-binding proteins or antigen-binding fragments thereof that specifically bind to the serine protease domain of plasmin and inhibit or reduce plasmin activity via interaction with the catalytic site of the protein.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to antigen-binding proteins and related fragments thereof for binding to plasmin, 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 2019904049, 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 plasmin, wherein the antigen binding protein reduces the activity of plasmin.

[0009] Preferably, the plasmin is human plasmin.

[0010] In any embodiment of the invention, the antigen binding protein of the invention binds to the serine protease domain of plasmin.

[0011] Preferably, the antigen binding proteins of the present invention bind to the catalytic site of the plasmin protease domain. 603 , Asp 646 and Ala / Ser 741 (Numbering is according to human plasmin as set forth in SEQ ID NO: 33). Preferably, the antigen binding protein binds to a peptide comprising the sequence set forth in SEQ ID NO: 34, or a fragment thereof.

[0012] In any embodiment of the invention, the antigen binding protein of the invention may comprise the amino acid sequence of Arg 637 , Leu 638 , Leu 640 , Pro 642 , Arg 644 , Lys 645 , Gln 721 , Trp 783 , and Asn 791 or a position equivalent thereto. Preferably, the antigen binding proteins of the invention bind to one or more residues in the serine protease domain of plasmin at or a position equivalent thereto as shown in Table 2. More preferably, the residue(s) of the antigen binding protein that bind to one or more residues in the serine protease domain of plasmin are one or more of the amino acid residues defined in Table 2.

[0013] The present invention also provides antigen binding proteins (eg, antibodies) having the same amino acids at positions relative to, or equivalent to, the residues designated for the B10 antibody in Table 2.

[0014] In any aspect of the invention, the antigen binding proteins of the invention may also bind to plasminogen, however, as plasminogen is the inactive form of a zymogen, it will be understood that the antigen binding proteins of the invention do not inhibit the activation of plasminogen, but rather the activity of plasmin-activated plasminogen.

[0015] In certain embodiments of the invention, the antigen binding proteins of the invention inhibit the binding of the plasminogen activator, ie, streptokinase, to plasminogen and the activation of plasminogen by streptokinase.

[0016] 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 plasmin can be determined by X-ray crystal structure analysis and contact distance analysis of 0 to 3.9 Å (inclusive).

[0017] The present invention also provides an antigen-binding protein that binds to the same epitope on plasmin 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 the activity of plasmin. 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 925 Å or less. 2 Covers ±5% of the surface area of ​​plasmin with SEQ ID NO:33.

[0018] In any embodiment of the invention, the antigen binding protein of the invention binds to plasmin and produces approximately 1 x 10 4 Super, about 5×10 4 Super, about 1×105 Over or about 5 x 10 5 k equal to or greater than or any value described herein a (M -1 s -1 Preferably, the antigen binding proteins of the present invention can bind to plasmin and exhibit approximately 8 x 10 5 The value of k a (M -1 s -1 ) is presented.

[0019] In any embodiment of the invention, the antigen binding protein of the invention binds to plasmin and produces approximately 1 x 10 -3 Less than or about 5 x 10 -4 Less than k d (s -1 Preferably, the antigen binding proteins of the present invention bind to plasmin and exhibit a concentration of about 4.5 x 10 -4 ±0.2 or any value of k described herein d (s -1 ) is presented.

[0020] In any aspect of the invention, the antigen binding protein of the invention binds to plasmin with a K of less than 2 mM, less than 100 μM, less than about 100 nM, or less than about 500 pM. D Preferably, K D is determined using any of the assays described herein, for example, surface plasmon resonance (SPR), such as multi-cycle SPR.

[0021] Antigen binding proteins of the present invention may bind to a peptide derived from SEQ ID NO: 33. 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: 33. More preferably, 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: 34. In some embodiments, antigen binding proteins of the present invention bind to a peptide comprising, consisting essentially of, or consisting of residues 637 to 791 of the sequence of SEQ ID NO: 33. More preferably, antigen binding proteins bind to a peptide comprising at least residues His according to the sequence of plasmin set forth in SEQ ID NO: 33. 603 , Asp 646 and Ala / Ser 741 Combine with.

[0022] In any aspect of the invention, the antigen binding protein does not significantly reduce or inhibit the activity of any one or more of tPA, thrombin, trypsin, factor Xa (FXa) and plasma kallikrein.

[0023] In some embodiments, the antigen binding proteins of the invention do not specifically bind to the serine proteases trypsin, thrombin, activated protein C, kallikrein, neutrophil elastase, or combinations thereof.

[0024] The present invention provides an antigen binding protein for binding to plasmin, 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.

[0025] The present invention provides an antigen binding protein for binding to plasmin, 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.

[0026] The present invention also provides an antigen binding protein that binds to or specifically binds to plasmin, wherein the antigen binding protein competitively inhibits the binding of the B10 antibody (i.e. comprising 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) to plasmin.

[0027] The present invention provides an antigen binding protein for binding to plasmin, the antigen binding protein comprising: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-Linker-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.

[0028] As defined herein, a linker may be a chemical entity, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

[0029] The present invention provides antigen binding proteins comprising, consisting essentially of, or consisting of (in N to C-terminal or C to N-terminal order) the amino acid sequences of SEQ ID NOs: 7 and 8.

[0030] 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 plasmin, 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.

[0031] 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.

[0032] 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:

[0033] 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:

[0034] The antigen-binding protein described above may also be referred to as the antigen-binding domain of an antibody.

[0035] 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.

[0036] As described herein, the antigen binding protein may be a variable domain.

[0037] The present invention also provides an anti-plasmin 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 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:

[0038] In any embodiment of the invention, the anti-plasmin antibody comprises a light chain variable region having the sequence of SEQ ID NO:7.

[0039] In any embodiment of the invention, the anti-plasmin antibody comprises a heavy chain variable region having the sequence of SEQ ID NO:8.

[0040] In any embodiment of the invention, the anti-plasmin 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.

[0041] In any embodiment of the invention, the anti-plasmin 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.

[0042] In any embodiment of the invention, the antibody is a naked antibody, specifically, the antibody is in unconjugated form and has not been configured to form a conjugate.

[0043] 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.

[0044] Reference herein to a protein or antibody that "binds" to plasmin gives literal support to the protein or antibody that "specifically binds to" or "specifically binds to" plasmin.

[0045] The present invention also provides an antigen-binding domain or antigen-binding fragment of the above antibody.

[0046] 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.

[0047] 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.

[0048] 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 (e.g. bispecific or triabodies), fusion proteins, or conjugates described herein.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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.

[0053] 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.

[0054] 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:

[0055] 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.

[0056] Examples of cells of the invention include bacterial cells, yeast cells, insect cells or mammalian cells.

[0057] 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.

[0058] 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.

[0059] 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.

[0060] 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.

[0061] 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)).

[0062] 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.

[0063] 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.

[0064] 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.

[0065] The antigen binding proteins described herein can be purified, substantially purified, isolated, and / or recombinant.

[0066] 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.

[0067] The present invention provides a method for inhibiting plasmin activity in a subject in need thereof, comprising administering a plasmin-inhibiting antigen-binding protein of the present invention to the subject, thereby inhibiting plasmin activity in the subject. The subject in need may include a subject suffering from hemophilia, menorrhagia, von Willebrand syndrome, or thrombolysis-induced bleeding.

[0068] Preferably, the inhibition of plasmin activity comprises the inhibition of plasmin cleavage of one or more substrates of plasmin selected from the group consisting of 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.

[0069] 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.

[0070] The present invention also provides a method of restoring hemostasis or inhibiting plasmin activity 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 plasmin activity in the subject.

[0071] The present invention also provides a method of treating or preventing a streptococcal infection in a subject, which method comprises administering to the subject an antigen binding protein of the invention, thereby treating or preventing the streptococcal 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.

[0072] 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.

[0073] The present invention also provides a method of treating a condition associated with or caused by a streptococcal 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 streptococcal infection in the subject. The condition associated with or caused by a streptococcal infection may be any of the conditions described herein. In any aspect of the invention, the streptococcal infection may be either chronic or acute.

[0074] The present invention also provides a method of reducing the severity of a Streptococcal infection in a subject, the method comprising administering to the subject an antigen binding protein of the invention, thereby reducing the severity of the Streptococcal infection in the subject.

[0075] 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.

[0076] The invention provides use of an antigen binding protein of the invention in the manufacture of a medicament for inhibiting plasmin activity in a subject in need thereof, including inhibiting plasmin activity in a subject suffering from hemophilia, menorrhagia, von Willebrand syndrome or thrombolysis-induced bleeding.

[0077] The present invention also provides the use of an antigen binding protein of the invention in the manufacture of a medicament for restoring haemostasis or inhibiting excessive plasmin activity in a subject who has suffered a traumatic injury or who needs to do so after surgery or childbirth.

[0078] The present invention also provides the use of an antigen binding protein of the invention in the manufacture of a medicament for restoring haemostasis or inhibiting excessive plasmin activity in a subject who has suffered a traumatic injury.

[0079] 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.

[0080] The present invention also provides the use of an antigen binding protein of the invention in the manufacture of a medicament for the treatment or prevention of a streptococcal infection.

[0081] The present invention also provides the use of an antigen binding protein of the 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 Streptococcal infection.

[0082] 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.

[0083] The present invention also provides a pharmaceutical composition comprising an antigen-binding protein of the present invention and a pharmaceutically acceptable excipient.

[0084] The pharmaceutical composition is preferably intended for the uses described herein. Accordingly, the present invention provides a pharmaceutical composition comprising an antigen binding protein of the invention for use in inhibiting plasmin activity in a subject in need thereof, including inhibiting plasmin activity in a subject suffering from hemophilia, menorrhagia, von Willebrand syndrome or thrombolysis-induced bleeding.

[0085] The present invention also provides pharmaceutical compositions for use in restoring hemostasis or inhibiting excessive plasmin activity in a subject who has suffered a traumatic injury or who needs to do so after surgery or childbirth.

[0086] The present invention also provides a pharmaceutical composition comprising an antigen binding protein of the invention for use in restoring hemostasis or inhibiting excessive plasmin activity in a subject who has suffered a traumatic injury.

[0087] The present invention also provides a pharmaceutical composition comprising an antigen binding protein of the invention for use in the treatment or prevention of a streptococcal infection.

[0088] The present invention also provides a pharmaceutical composition comprising an 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 Streptococcal infection.

[0089] Furthermore, the present invention provides pharmaceutical compositions comprising the 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.

[0090] In any embodiment of the present invention, the streptococcal infection is caused by a bacterium belonging 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 of the present invention, the infection can be caused by a bacterium selected from the group consisting of Streptococcus pyogenes, Streptococcus dysgalactiae, and Streptococcus pneumonia.

[0091] Therefore, the method, use, or pharmaceutical composition of the present invention is useful for treating, preventing, or reducing the severity of any disease caused by or associated with the bacteria described herein. For example, the method, use, or pharmaceutical composition of the present invention may be intended to treat, prevent, or reduce the severity of any disease / infection caused by or associated with Group A Streptococcus (GAS), 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.

[0092] 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.

[0093] The present invention further provides nucleic acid molecules encoding the antigen binding proteins of the present invention, or functional fragments or derivatives thereof.

[0094] The present invention also provides cells comprising the vectors or nucleic acid molecules described herein.

[0095] The present invention also provides animals or tissues derived from the cells described herein.

[0096] 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.

[0097] In another aspect of the invention, 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:

[0098] 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.

[0099] 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]

[0100] [Figure 1]Binding of B10 to plasminogen and plasmin. B10 was immobilized on a NiHc chip; the binding kinetics of B10 to (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) Summary table showing the kinetic constants (ka and kd) and affinity constant (KD). [Figure 2] Dose-dependent inhibition of tPA-mediated plasminogen activation by B10. B10 inhibits the activity of plasmin produced by tPA-mediated plasminogen activation in the presence of EACA in solution (A) or on a fibrin clot (B). [Figure 3] Inhibition of SK-mediated plasminogen activation by B10. B10, when added to the reaction (at a 20-fold molar excess) at any time during the assay, immediately inhibits plasmin activity. This result indicates that B10 inhibits plasmin activity. [Figure 4] Effect of B10 on the enzymatic activity of plasmin and other serine proteases. (A) Progress curves of plasmin activity in the presence of B10 or a non-inhibitory plasminogen-binding antibody (A01). Plasmin activity was undetectable in the presence of B10. (B) Plasmin activity was assayed in the presence of B10 (0–100 nM), with an IC50 of 24.3 ± 1.4 nM; complete inhibition was recorded at a 1:10 ratio of plasmin and B10. (C) With the exception of plasmin (*), B10 does not inhibit the enzymatic activity of serine proteases, including human activated kallikrein (HPKa), protein C, factor IIa, thrombin, factor Xa, uPA, and tPA. Enzyme activity in the presence of a naive antibody gAb is also shown. [Figure 5]Inhibition of plasminogen binding to streptokinase by B10. (A) 10 nM plasminogen was passed over streptokinase immobilized on a CM4 chip in the presence of B10 or naive chicken Ab, i.e., gAb (0–500 nM). B10 showed inhibition at concentrations between 62.5 and 500 nM. The control naive chicken Ab, gAb, showed no inhibition of Plg binding to SK. (B) Percentage of plasminogen binding to SK in the presence of 500 nM Ab, normalized to the no-antibody control. [Figure 6] Binding of B10 to a single recombinant serine protease domain. Size exclusion chromatography shows a higher molecular weight complex (SP+B10, solid line) compared to SP alone (dashed line) or B10 alone (dotted line). [Figure 7] Crystal structure of B10 bound to the serine protease domain (SP) of plasmin. The crystal structure of the SP / B10 binary complex shows B10 bound to the plasmin catalytic triad. SP is shown, the catalytic triad is shown as stick figures and labeled, and the B10 light chain (LC) and heavy chain (HC) are labeled. Above is a representation of the binary complex, and below are the key residues (labeled and numbered) involved in intramolecular interactions and labeled as described above. Dashed lines are used to indicate polar interactions. [Figure 8] B10 inhibits plasmin production by Group A Streptococcus (GAS) (enzyme assay). (Top) Plasminogen bound to B10 was incubated with GAS. The activity of the produced plasmin was measured with a fluorogenic substrate. In this case, plasmin produced by GAS is presumably mediated by streptokinase (SK). (Bottom) Progress curve of plasmin activity in the presence or absence of recombinant SK. [Figure 9]B10 inhibits fibrinolysis of synthetic clots. Fibrinolysis was measured on preformed synthetic fibrin clots prepared by mixing 3 mg / ml fibrinogen, 1 U of bovine thrombin, and 10 nM tPA for 2 hours at 37°C. 45 nM plasminogen mixed with 0-90 M B10, α2AP, or 0-6.25 mM TXA was added to the surface of the clot. Fibrinolysis was monitored for up to 10 hours at 37°C using a nephelometer. The time required to achieve 50% clot lysis was used for IC50 calculations. The IC50 values ​​obtained for B10 were comparable to those for α2AP and aprotinin. [Figure 10] B10 inhibits the lysis of whole blood clots in the presence of red blood cells and platelets. The percentage of clot lysis (compared to negative and positive controls) is plotted as a function of inhibitor concentration at the time point when complete lysis is achieved in the positive control (30-40 minutes) and plotted using nonlinear regression to calculate the IC50. B10 is approximately 8 times more effective than α2AP and 9 times more effective than aprotinin in inhibiting whole blood clot lysis. DETAILED DESCRIPTION OF THE INVENTION

[0101] 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.

[0102] 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:

[0103] 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.

[0104] Hemorrhage, or bleeding, is a serious or fatal complication of surgery, injury, or clotting factor disorders. Antifibrinolytic agents that inhibit plasmin-mediated fibrinolysis or clot lysis may reduce blood loss, emergency reoperation, morbidity, and mortality during severe bleeding.

[0105] Uninhibited plasmin dissolves blood clots (fibrinolysis) and degrades clotting factors (fibrinogen, factor V, factor VIII), impairing blood clotting and thereby increasing the risk of bleeding. In addition, uninhibited plasmin activates neutrophils and macrophages, increasing chemotaxis and oxidative stress, as well as promoting the release of proinflammatory cytokines and matrix metalloproteinases.

[0106] Since their first use over 50 years ago, small molecule plasmin inhibitors have been proven to reduce bleeding and related complications. Currently available plasmin inhibitors are typically in the form of small molecules that block the enzyme active site or interfere with the interaction of plasmin with a substrate.

[0107] The lysine analogs, epsilon-aminocaproic acid (EACA) and tranexamic acid (TX), mimic lysine residues and interact with the lysine-binding site on plasmin kringles, blocking their interaction with fibrin. Due to their molecular size and mechanism of action, lysine analogs have low potency and are not very specific; they accumulate in kidney disease and cross the blood-brain barrier and placenta. Lysine analogs inhibit plasminogen activation and fibrinolysis by preventing plasminogen and tPA from binding to fibrin. By the same mechanism, lysine analogs can actually increase plasmin activity by blocking the kringle interaction with α2-antiplasmin and increasing plasminogen activation by tPA or uPA in solution. Lysine analogs also interfere with plasminogen-plasmin interactions with cellular receptors, blocking the interaction of tissue factor with plasminogen. The biological effects of the interaction of lysine analogs with other kringle-containing proteins (tPA, (pro)thrombin, hepatocyte growth factor, uPA, and the apoprotein of lipoprotein(a)) are not fully understood. Because lysine analogs cross the placenta and the blood-brain barrier, they can induce epileptic seizures in cardiac surgery patients and increase the incidence of cerebral infarction in patients with subarachnoid hemorrhage.

[0108] Major clinical trials have aimed to understand the efficacy of small molecule plasminogen activator inhibitors in inhibiting fibrinolysis and restoring hemostasis during trauma. These studies have found that small molecule plasmin inhibitors are limited by nonspecific mechanisms of action, off-target effects, low potency, and lack of efficacy against certain types of bleeding. In particular, the CRASH-2 and MATTER trials examined the efficacy of the lysine analog tranexamic acid (TXA) and found that administration of TXA to critically ill patients within 3 hours of injury significantly reduced mortality. Importantly, these studies also revealed that the survival benefit of TXA decreased by 10% for every 15-minute delay in administration, with no benefit observed after 3 hours. This is thought to be due to changes in the in vivo coagulation and fibrinolytic proteome, which lead to increased uPA-mediated plasminogen activation to plasmin. Thus, plasmin generated in the plasma increases the likelihood of nonspecific fibrinolysis due to fibrinogen degradation and α2-antiplasmin depletion. Therefore, there is also a need for plasmin active site inhibitors for use in clinical situations where inhibition of plasminogen activation is no longer useful.

[0109] Fibrinolysis inhibitors with greater specificity and potency are needed, particularly in patients with severe, life-threatening bleeding, such as cerebral hemorrhage, where existing therapies are ineffective and can be harmful, in part due to off-target effects. Creating highly specific catalytic inhibitors of plasmin is challenging because its enzymatic active site shares significant homology with other trypsin-like serine proteases. For example, the most commonly used plasmin active site inhibitor is aprotinin. However, this molecule is a nonspecific inhibitor of many other serine proteases, including trypsin, thrombin, activated protein C, kallikrein, neutrophil elastase, and other proteases. Aprotinin is no longer available in the United States due to safety concerns associated with increased mortality. Therefore, there is a need for plasmin-specific inhibitors with minimal inhibitory activity against other serine proteases.

[0110] The present inventors have developed antigen binding proteins, eg, antibodies, that bind to plasmin and inhibit or reduce its catalytic activity.

[0111] The antigen binding proteins of the invention have been demonstrated to specifically bind to the serine protease domain of plasmin and inhibit plasmin activity through interaction with the catalytic site of the protein.

[0112] Advantageously, the antigen binding proteins of the present invention do not inhibit the activity of other serine proteases such as tPA, thrombin, trypsin, factor Xa and plasma kallikrein.

[0113] By virtue of their ability to bind to and inhibit or reduce the activity of plasmin, 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 the plasmin and fibrinolytic system, for example, they are useful in promoting hemostasis of bleeding following trauma, surgery or childbirth.

[0114] The antigen binding proteins of the invention also have utility in treating or preventing bacterial infections in which bacteria utilise streptokinase or related enzymes to recruit the plasmin system to facilitate invasion of host tissues, in particular the antigen binding proteins of the invention are useful in treating or preventing infections caused by Streptococcus species.

[0115] 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.

[0116] 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.

[0117] 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.

[0118] 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.

[0119] All patents and publications referenced herein are incorporated by reference in their entirety.

[0120] 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.

[0121] 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.

[0122] 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).

[0123] 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.

[0124] 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.

[0125] 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.

[0126] 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.

[0127] 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.

[0128] Selected Definitions Plasmin 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. Plasmin is the active form of the zymogen plasminogen. Plasminogen is activated to plasmin by the proteolytic action of various activators on plasminogen. Plasminogen activators, such as tissue plasminogen activator (tPA) or urokinase (uPa), activate the Arg-Xaa bond. 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.

[0129] Plasmin (or Plm) is a seven-domain glycoprotein containing a Pap or pan-apple domain, five kringle domains (KR1-KR5), and a serine protease (SP) domain. Its inactive form, plasminogen (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 reorganization of plasminogen into 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.

[0130] 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.

[0131] 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: 33. The sequence containing the hPlm serine protease domain is set forth in SEQ ID NO: 34, where the catalytic triad is underlined and shown in bold.

[0132] As used herein, the term plasmin refers to a molecule that has at least one biochemical or biophysical activity of plasmin, which biochemical or biophysical activity can be distinguished from that of plasminogen.

[0133] The phrases "inhibit plasmin activity" or "reduce plasmin activity" mean that the antigen binding protein of the present invention inhibits or reduces the enzymatic activity of 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 plasmin activity in the same assay under the same conditions but without the antigen binding protein. Preferably, plasmin activity is measured after activation of plasminogen with any one or more plasminogen activators. The plasminogen activator may be selected from the group consisting of Arg, 561 -Val 562 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.

[0134] 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.

[0135] 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.

[0136] 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.

[0137] 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.

[0138] 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.

[0139] For the purposes of this disclosure, the term "antibody" includes proteins capable of specifically binding to one or several closely related antigens (e.g., plasmin) via an antigen-binding domain contained in an Fv. The 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). Antibodies may further include chemical modifications, e.g., conjugation with an active agent or radiolabel, or an agent to improve solubility, e.g., pegylation of an antibody or antigen-binding protein thereof described herein.

[0140] 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.

[0141] 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.

[0142] 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.

[0143] 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.

[0144] "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.

[0145] 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.

[0146] As used herein, the term "bind" in reference to the interaction of an antigen-binding site or its antigen-binding domain with an antigen means that this interaction is dependent on the presence of a specific structure (e.g., an antigenic determinant or epitope) on the antigen. For example, antibodies recognize and bind to specific protein structures, rather than proteins 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.

[0147] 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 a higher affinity than with another antigen or cell. For example, the antigen-binding protein binds to plasmin (e.g., human plasmin) with substantially higher affinity (e.g., 1.5-fold, or 2-fold, or 5-fold, or 10-fold, or 20-fold, or 40-fold, or 60-fold, or 80-fold to 100-fold, or 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 plasmin (preferably human) with at least 1.5-fold or 2-fold or more higher affinity (e.g., 5-fold, or 10-fold, or 20-fold, or 50-fold, or 100-fold, or 200-fold) than it binds to related serine proteases. 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.

[0148] 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.

[0149] 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.

[0150] As used herein, the term "epitope" (synonymously "antigenic determinant") shall be understood to mean the region of plasmin to which an antigen-binding protein, including the antigen-binding domain of an antibody, binds. 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 folds, 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.

[0151] 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.

[0152] 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.

[0153] 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.

[0154] 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.

[0155] antibody In one example, the antigen binding protein or plasmin binding protein described herein according to any embodiment is an antibody.

[0156] Methods for producing antibodies are known in the art and / or described in Harlow and Lane (eds.), Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, (1988). Generally, in such methods, plasmin (e.g., human plasmin) 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.

[0157] 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).

[0158] 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.

[0159] For the production of mAbs, any one of several known techniques can be used, such as the procedures exemplified in the above-mentioned US Pat. No. 4,196,265 or Harlow and Lane (1988).

[0160] 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).

[0161] 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.

[0162] 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.

[0163] 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.).

[0164] 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).

[0165] 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.

[0166] 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.

[0167] 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).

[0168] 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.

[0169] 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).

[0170] 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.

[0171] 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.

[0172] 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.

[0173] 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).

[0174] 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.

[0175] 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.

[0176] 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.

[0177] 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:

[0178] 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.

[0179] 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).

[0180] 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.

[0181] 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.

[0182] The present invention also contemplates nucleic acids that hybridize to nucleic acids encoding the antigen-binding proteins 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.

[0183] 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.

[0184] 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.

[0185] 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.

[0186] 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.

[0187] 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)).

[0188] 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.

[0189] 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.

[0190] 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.

[0191] 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.

[0192] 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).

[0193] 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.

[0194] 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).

[0195] 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).

[0196] 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).

[0197] 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.

[0198] Further modifications The present invention also contemplates further modifications to antibodies or antigen binding proteins comprising the Fc region or constant region.

[0199] 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.

[0200] 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.

[0201] 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.

[0202] Recombinant expression In another example, the antigen binding proteins described herein according to any of the Examples are recombinant.

[0203] 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.

[0204] 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.

[0205] 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.

[0206] 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.

[0207] 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).

[0208] 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).

[0209] 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.

[0210] 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).

[0211] 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.

[0212] Protein isolation Methods for isolating proteins are known in the art and / or described herein.

[0213] 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.

[0214] 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).

[0215] 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.

[0216] Assaying the activity of antigen-binding proteins Binding to plasmin 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 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 site and contacting it with labeled antigen (plasmin). 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.

[0217] Optionally, the dissociation constant (Kd), association constant (Ka) and / or affinity constant (K) of the immobilized antigen binding protein for plasmin or an epitope thereof are measured.D ) is determined. The "Kd" or "Ka" or "K" of the plasmin binding protein is D "Kd" is measured, in one example, by a radioactively or fluorescently labeled plasmin ligand binding assay. In the case of "Kd," the assay equilibrates the antigen-binding protein with a minimal concentration of labeled plasmin or its epitope in the presence of a titration series of unlabeled plasmin. After washing to remove unbound plasmin or its epitope, the amount of label, which is an indicator of the protein's Kd, is determined.

[0218] 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 plasmin or a region thereof or an immobilized antigen binding protein.

[0219] Measurement of inhibitory activity The antigen binding proteins of the present invention are preferably capable of inhibiting plasmin activity. Advantageously, the antigen binding proteins of the present invention inhibit plasmin-mediated clot lysis at levels similar to or significantly greater than physiological inhibitors of plasmin activity or pathological inhibitors of plasmin activity.

[0220] A variety of assays are known in the art for assessing the ability of a protein to inhibit or reduce plasmin activity.

[0221] 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: 33 (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.

[0222] 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.

[0223] The antigen binding proteins of the present invention may inhibit the binding of streptokinase to plasmin or may inhibit the binding of pathogen-derived proteins that bind to plasmin by a mechanism similar to streptokinase. More specifically, streptokinase, secreted by Streptococcus pyogenes, is known to encapsulate the serine protease domain of plasminogen / plasmin and, once bound, activates plasminogen to form plasmin. The antigen binding proteins of the present invention advantageously inhibit the binding of streptokinase to the serine protease domain of plasmin, thereby inhibiting plasmin activity mediated by plasminogen activation by streptokinase.

[0224] Exemplary methods for measuring inhibition of plasmin activity are described, eg, in Examples 2, 5, and 6.

[0225] 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.

[0226] Conditions being treated 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.

[0227] 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.

[0228] 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.

[0229] Thus, the antigen binding proteins of the present invention are useful for inhibiting fibrinolysis in a wide variety of scenarios where inhibition of plasmin activity is required.

[0230] The antigen binding sites of the invention are also useful in the treatment or prevention of any condition associated with or caused by the presence of or increased levels of bacteria that mediate their pathogenesis via streptokinase and related enzymes.

[0231] 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.

[0232] Thus, the antigen binding proteins of the invention are useful for inhibiting or preventing skin hyperpigmentation or inflammation, impetigo, pharyngitis, tonsillitis, scarlet fever, cellulitis, erysipelas, rheumatic fever, post-streptococcal glomerulonephritis, necrotizing fasciitis, myonecrosis and lymphangitis caused by Streptococcus pyogenes, Streptococcus dysgalactiae or Streptococcus pneumoniae.

[0233] The antigen binding proteins of the invention are also useful in inhibiting tumor metastasis, which is also known to recruit the plasmin system. Examples of cancers that can be treated with the antigen binding proteins of the invention include cystic and solid tumors, bone and soft tissue tumors, including tumors of 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.

[0234] 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.

[0235] composition In some examples, the antigen binding proteins described herein may be administered orally, parenterally, by inhalation spray, adsorption, absorption, topically (including as a spray or lotion), rectally, nasally, buccally, vaginally, intracerebroventricularly, via an implanted reservoir, as a dosage formulation containing a conventional non-toxic pharmaceutically acceptable carrier, or by any other convenient dosage form. As used herein, the term "parenteral" includes subcutaneous, intravenous, intramuscular, intraperitoneal, intrathecal, intracerebroventricular, intrasternal, and intracranial injection or infusion techniques.

[0236] 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).

[0237] 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.

[0238] 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.

[0239] 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.

[0240] 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.

[0241] 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.

[0242] 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.

[0243] 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.

[0244] 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).

[0245] 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 drops or sprays, aerosols, inhalants, liposomal forms, gels, creams, sprays, 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.

[0246] 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.

[0247] 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.

[0248] In some instances, the methods of the present invention comprise administering a prophylactically or therapeutically effective amount of a protein described herein.

[0249] The timing of administration can be determined based on the clinical condition of the subject receiving the antigen binding protein of the present invention. For example, in situations where the subject has suffered a trauma, it will be appreciated that it will be beneficial to administer the antigen binding protein of the present invention as soon as possible after the trauma in order to attenuate hyperfibrinolysis in the subject.

[0250] 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.

[0251] 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.

[0252] 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.

[0253] In the case of a kit for detecting plasmin, the kit may further comprise, for example, a detection means linked to the antigen-binding protein of the invention.

[0254] In the case of a kit for therapeutic / prophylactic use, the kit may further comprise a pharmaceutically acceptable carrier.

[0255] Optionally, the kits of the invention are packaged with instructions for use of the methods described herein according to any of the Examples.

[0256] 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.

[0257] [Table 1]

[0258] [Table 2]

[0259] [Table 3]

[0260] [Table 4]

[0261] [Table 5]

[0262] [Table 6] [Example]

[0263] Example 1: Generation of B10 antibody Antibodies for binding to plasmin were obtained by raising antibody responses to full-length plasminogen in chickens. Antibody variable heavy and light chains (VH and VL) were amplified from chicken cDNA by PCR and linked together via a flexible linker to generate an scFv library.

[0264] Selection was performed by screening plasminogen-binding antibodies using ELISA and Biacore. SK-mediated plasminogen activation and fibrinolysis assays were performed to identify antibodies that prevented or inhibited the activation of plasminogen to plasmin or the inhibition of plasmin activity.

[0265] Example 2: Characterization of antibody B10 for binding to the plasmin active site 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).

[0266] The results of the binding experiments (Figure 1) show that the antigen-binding proteins of the present invention bind to both plasminogen and plasmin, but with a slightly higher affinity for plasminogen compared to plasmin.

[0267] B10 inhibits the activity of plasmin formed by tPA-mediated plasminogen activation

[0268] In solution: 20 nM plasminogen was mixed with various B10 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 B10 concentration to obtain the inhibition curve, which was fitted with 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 value, ie, the concentration of B10 that inhibits 50% of tPA-mediated plasminogen activation in solution, is 21.51±2.28 nM.

[0269] 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 of B10 (0-2 μM) 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 50 The value was 86±11.6 nM. The results are shown in FIG.

[0270] B10 inhibits the activity of plasmin formed by SK-mediated activation of plasminogen.

[0271] 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.

[0272] The results in Figure 3 show that B10 antibody immediately inhibits plasmin activity regardless of when it is added to the reaction at any point during the course of the assay.

[0273] Effect of B10 on the activity of plasmin and other plasma proteases 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 for B10 and A01 (non-inhibitory plasminogen antibodies) at a 10:1 antibody:plasmin ratio. B10 showed complete inhibition of Plm activity compared to A01. Plasmin activity (20 nM) in the presence of antibody B10 (0–200 nM) was measured at 37°C using a Fluostar Omega plate reader (BMG Labtech) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. IC 50 was 24.3±1.4nM.

[0274] To examine cross-reactivity with other plasma serine proteases, 250 nM B10 was mixed with 10 nM of human plasma proteases: plasma kallikrein (Molecular Innovations), factor Xa (Molecular Innovations), factor XIIa (Enzyme Research Laboratories), protein C (Molecular Innovations), thrombin (Molecular Innovations), uPA (Abbokinase), tPA (Actilyse), and plasmin. Thrombin, factor Xa, tPA, and plasmin activities were measured using the chromogenic substrate T2943 (Sigma-Aldrich) at 405 nm and 37°C using a Fluostar Omega plate reader (BMG Labtech). Plasma kallikrein, factor XIIa, and protein C activities were measured using the chromogenic substrate S2032 (Chromogenix). uPA activity was measured at 37°C using a fluorogenic substrate (Spectrofluor) on a Fluostar Omega plate reader (BMG Labtech) with excitation and emission wavelengths of 355 nm and 460 nm, respectively. Also shown is the activity of the enzyme in the presence of naive antibody gAb. Here, enzyme activity was normalized to a HEPES-buffered saline control in which the antibody was replaced with buffer. As shown in Figure 4C, B10 has no effect on activity against the tested plasma proteases other than Plm.

[0275] Inhibition of SK binding to plasminogen by B10 The effect of B10 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 B10 or naive chicken antibody (gAb). B10 showed inhibition at all concentrations tested, from 62.5 to 500 nM. Naive chicken antibody gAb (control) showed no inhibition. Figure 5B shows the percentage of SK binding to plasminogen (normalized to the no-antibody control) in the presence of 500 nM G05 and gAb.

[0276] The results, shown in Figure 5, indicate that B10 inhibits the binding of streptokinase to plasminogen by approximately 33% compared to the control antibody.

[0277] Example 3: Binding of B10 to the serine protease domain B10 binds to the kringle 5-serine protease domain (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 6, the co-complex eluted as a single peak at 58.5 ml. For reference, the KR5-SP and B10 antibodies eluted at 67.2 ml and 73.3 ml, respectively, also as single peaks.

[0278] Example 4: Crystal structure of B10 bound to a single recombinant kringle 5-serine protease domain The purified complex was crystallized at 10 mg / ml in a buffer containing 25 mM HEPES and 150 mM NaCl at 20°C in the presence of 0.1 M MES pH 6.5 and 0.2 M (NH4)2SO4, 20% (w / v) PEG8K. Crystals were flash-cooled in liquid N2 in the presence of 20% (v / v) glycerol. A 2.7 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 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, created using PyMOL (www.pymol.org / ), is shown in Figure 7.

[0279] The B10-SP complex structure reveals that binding of B10 to the catalytic domain prevents the formation of a functional catalytic site, thereby preventing plasmin binding / substrate cleavage.

[0280] Example 5: Effect of B10 on plasmin activity in the presence of GAS 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 B10 / gAb or 13.5 mM TXA were mixed at room temperature for 15 minutes 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. 10 μl of resuspended cells was added to a 100 μl 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.

[0281] The results shown in Figure 8 demonstrate that B10 reduces plasmin activity generated by GAS by approximately 60% compared to the control antibody and HEPES-buffered saline controls. The bottom panel shows that recombinant SK (2 nM) activates Plg in solution in the absence of GAS cells.

[0282] Example 6: B10 inhibits 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 hours at 37°C. Fibrinolysis was initiated by adding 45 nM plasminogen mixed with 0–90 nM B10, 0–90 nM α2AP, or 0–6.25 mM TXA to the surface of the clot.

[0283] 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 The IC obtained for B10 was used for calculations. 50 The values ​​were comparable to those of α2AP and aprotinin (FIG. 9) and significantly lower than that of TXA (approximately 50-fold).

[0284] Whole blood clots were prepared from human blood collected from healthy donors. Halo-shaped clots were generated by mixing 15% of a mixture containing synthetic phospholipids, recombinant tissue factor (Dade Innovin, Siemens Germany), and 67 mM CaCl2 in HBS at a 1:4 ratio with whole blood. The plate was sealed and incubated at 37°C for 60 minutes before use.

[0285] 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.

[0286] 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). Antibody B10 completely inhibited clot lysis at concentrations of 250 nM or higher.

[0287] The time taken to achieve 50% clot lysis was determined as IC 50 The IC obtained for B10 was used for calculations. 50 The values ​​were approximately 8-fold higher than α2AP and 9.5-fold higher than aprotinin (FIG. 10), indicating that B10 was significantly more effective at inhibiting whole blood clot lysis. B10 was over 180-fold more effective than TXA (not shown).

[0288] Thus, in a clot lysis assay that closely resembles a biological system, antibody B10 is significantly more effective at inhibiting plasmin-induced lysis than α2AP, a physiological inhibitor of plasmin. Furthermore, antibody B10 is significantly more effective at inhibiting plasmin-induced lysis than the existing pharmacological agents TXA and aprotinin.

[0289] 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.

Claims

1. 1. An antigen-binding protein comprising an antigen-binding domain that binds to the serine protease domain of plasmin, wherein said antigen-binding protein specifically inhibits the activity of plasmin.

2. 2. The antigen-binding protein of claim 1, wherein the antigen-binding domain binds to the catalytic site of the serine protease domain of plasmin.

3. 3. The antigen-binding protein of claim 1 or 2, wherein the antigen-binding domain binds to a peptide comprising the sequence: SEQ ID NO: 34, or a fragment thereof.

4. The antigen binding protein comprises the Arg ６３７ , Leu ６３８ , Leu ６４０ , Pro ６４２ , Arg ６４４ , Lys ６４５ , Gln ７２１ , Trp ７８３ , and Asn ７９１ 4. The antigen-binding protein of any one of claims 1 to 3, which binds to one or more residues in the serine protease domain of plasmin at, or a position corresponding thereto, or which binds to one or more residues in the serine protease domain of plasmin at, or a position corresponding thereto, as shown in Table 2.

5. 5. The antigen-binding protein of any one of claims 1 to 4, wherein the protein binds to human plasmin.

6. 6. The antigen-binding protein of any one of claims 1 to 5, wherein the protein inhibits the binding of plasmin to streptokinase.

7. 7. The antigen binding protein of any one of claims 1 to 6, wherein the protein does not significantly bind to, or significantly reduce or inhibit the activity of, a serine protease selected from the list consisting of: tissue plasminogen activator (tPA), urokinase plasminogen activator (uPA), thrombin, trypsin, Factor Xa, plasma kallikrein, human activated kallikrein (HPKa), protein C, and Factor XIIa.

8. 8. The antigen binding protein of any one of claims 1 to 7, wherein the antigen binding protein inhibits or reduces plasmin-mediated cleavage of one or more of the following plasmin substrates selected from: 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 collagenase.

9. 9. The antigen-binding protein of any one of claims 1 to 8, wherein interactions between residues of said antigen-binding protein and residues of plasmin are revealed by X-ray crystal structure analysis and contact distance analysis of 0 to 3.9 Å (inclusive).

10. 10. The antigen-binding protein of any one of claims 1 to 9, wherein the antigen-binding protein binds to the same epitope on plasmin 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 the activity of the plasmin.

11. 11. The antigen binding protein of claim 10, wherein the epitope is defined by X-ray crystallography, and optionally, the epitope is defined by contact distance analysis of 0 to 3.9 Å, inclusive.

12. The antigen-binding protein binds to plasmin and produces approximately 1 x 10 ４ Super, about 5 x 10 ４ Super, about 1×10 ５ Over or about 8 x 10 ５ Above k ａ (M －１ s －１ ), and preferably the antigen binding protein binds to plasmin and exhibits approximately 8×10 ５ No.K ａ (M －１ s －１ 12. The antigen-binding protein of claim 1, wherein the antigen-binding protein exhibits the following structure:

13. The antigen-binding protein binds to plasmin and produces approximately 1 x 10 －３ less than, or about 5 x 10 －４ Less than k ｄ (s －１ ), and preferably, the antigen binding of the present invention binds to plasmin and exhibits approximately 4.5 x 10 －４ No.K ｄ (s －１ 13. The antigen-binding protein of any one of claims 1 to 12, which exhibits the formula:

14. The antigen binding protein binds to plasmin and has a K of less than 2 mM, less than 100 μM, less than about 100 nM, or less than about 500 pM. Ｄ and presenting the K Ｄ The antigen-binding protein of any one of claims 1 to 13, wherein is determined using surface plasmon resonance (SPR).

15. 15. The antigen binding protein of any one of claims 1 to 14, wherein the antigen binding protein binds to a peptide taken from SEQ ID NO: 33, optionally wherein 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:

33.

16. 16. The antigen-binding protein of claim 15, wherein 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:

34.

17. 17. The antigen binding protein of any one of claims 1 to 16, wherein the antigen binding protein binds to a peptide comprising, consisting essentially of, or consisting of residues 637 to 791 of SEQ ID NO:

33.

18. 18. The antigen-binding protein of claim 17, wherein the antigen-binding protein binds to at least residues His603, Asp646 and Ala / Ser741 according to the sequence of plasmin set forth in SEQ ID NO:

33.

19. 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; the sequences of any of the framework regions or complementarity determining regions are as described herein, Optionally, the sequence of any of the complementarity determining regions has an amino acid sequence shown in Table 1.

20. 19. The antigen binding protein of any one of claims 1 to 18, wherein the antigen binding protein comprises, consists essentially of, or consists of the amino acid sequence of SEQ ID NOs: 7 and 8 (in N to C-terminal or C to N-terminal order).

21. The antigen-binding domain comprises: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4-linker-FR1a-CDR1a-FR2a-CDR2a-FR3a-CDR3a-FR4a Including, The antigen-binding domain comprises: (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 21. The antigen-binding protein of any one of claims 1 to 20, comprising at least one of:

22. The protein is: (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.

22. The antigen binding protein of claim 21, further comprising at least one of:

23. The antigen-binding protein is selected from the group consisting of: (i) single chain Fv fragments (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 an immune effector cell.

23. The antigen-binding protein of any one of claims 1 to 22, in the form:

24. The antigen-binding protein is selected from the group consisting of: (i) diabodies; (ii) triabodies; (iii) tetrabodies; (iv) Fab; (v)F(ab')2: (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.

23. The antigen-binding protein of any one of claims 1 to 22, in the form:

25. 25. The antigen-binding protein of any one of claims 1 to 24, wherein the antigen-binding protein is an antibody or antigen-binding fragment thereof, preferably wherein the binding protein is a monoclonal antibody or variable domain.

26. 26. The antigen-binding protein of any one of claims 21 to 25, wherein the linker is a chemical compound, one or more amino acids, or a disulfide bond formed between two cysteine ​​residues.

27. An anti-plasmin 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 Including; 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 Antiplasmin antibodies, including:

28. 28. The anti-plasmin antibody of claim 27, wherein the antibody comprises a light chain variable region having the sequence of SEQ ID NO:

7.

29. 29. The anti-plasmin antibody of claim 27 or 28, wherein the antibody comprises a heavy chain variable region having the sequence of SEQ ID NO:

8.

30. The anti-plasmin antibody of any one of claims 27 to 29, 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.

31. The anti-plasmin antibody of any one of claims 27 to 30, 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.

32. 32. The antigen-binding protein or antiplasmin antibody of any one of claims 1 to 31, wherein the protein or antibody is a naked antibody.

33. A fusion protein comprising the antigen-binding protein or antibody of any one of claims 1 to 32.

34. A conjugate in the form of an antigen-binding protein or antibody according to any one of claims 1 to 32 conjugated to a label or a cytotoxic drug.

35. A nucleic acid encoding the antigen-binding protein, antibody, fusion protein or conjugate of any one of claims 1 to 34.

36. A vector comprising the nucleic acid of claim 35.

37. 35. A method of inhibiting plasmin activity in a subject in need thereof, comprising administering to the subject the antigen binding protein, antibody, fusion protein, or conjugate of any one of claims 1 to 34, thereby inhibiting plasmin activity in the subject.

38. 35. A method of inhibiting fibrinolysis in a subject in need thereof, comprising administering to the subject the antigen binding protein, antibody, fusion protein, or conjugate of any one of claims 1 to 34, thereby inhibiting fibrinolysis in the subject.

39. 39. The method of claim 37 or 38, wherein the method is for inhibiting plasmin activity in a subject suffering from hemophilia, menorrhagia, von Willebrand syndrome or thrombolysis-induced bleeding, or for restoring hemostasis in a subject who has suffered a trauma or is bleeding due to surgery or childbirth.

40. 35. A method of treating or preventing a streptococcal infection in a subject, said method comprising administering to said subject an antigen binding protein, antibody, fusion protein or conjugate of any one of claims 1 to 34, thereby treating or preventing a streptococcal infection in said subject.

41. 41. The method of claim 40, wherein the method comprises reducing the severity of an infection or treating a condition associated with or resulting from a Streptococcal infection in the subject.

42. 35. A method of treating, inhibiting, preventing or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer, in a subject, said method comprising administering to a subject in need thereof the antigen binding protein, antibody, fusion protein or conjugate of any one of claims 1 to 34, thereby treating, inhibiting, preventing or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer, in said subject.

43. 35. Use of the antigen binding protein, antibody, fusion protein or conjugate of any one of claims 1 to 34 in the manufacture of a medicament for inhibiting plasmin activity, or in a subject in need thereof.

44. 35. Use of an antigen binding protein, antibody, fusion protein or conjugate according to any one of claims 1 to 34 in the manufacture of a medicament for inhibiting fibrinolysis in a subject in need thereof.

45. 45. The use of claim 43 or 44, wherein the medicament is intended for inhibiting plasmin activity in a subject suffering from hemophilia, menorrhagia, von Willebrand syndrome or thrombolysis-induced bleeding, or for restoring hemostasis in a subject who has suffered a trauma or is bleeding due to surgery or childbirth.

46. 35. Use of an antigen-binding protein, antibody, fusion protein or conjugate according to any one of claims 1 to 34 in the manufacture of a medicament for the treatment or prevention of streptococcal infections.

47. 35. Use of an antigen binding protein, antibody, fusion protein or conjugate according to any one of claims 1 to 34 in the manufacture of a medicament for treating, inhibiting, preventing or minimising the spread or progression of cancer, including inhibiting or preventing metastasis of cancer.

48. 35. A pharmaceutical composition comprising the antigen-binding protein, antibody, fusion protein, or conjugate of any one of claims 1 to 34 and a pharmaceutically acceptable excipient.

49. 49. The pharmaceutical composition of claim 48 for use in inhibiting plasmin activity in a subject in need thereof.

50. 49. The pharmaceutical composition of claim 48 for use in inhibiting fibrinolysis in a subject in need thereof.

51. 49. A pharmaceutical composition according to claim 48 or 49, wherein the composition is intended for use in inhibiting plasmin activity in subjects suffering from hemophilia, menorrhagia, von Willebrand syndrome or thrombolysis-induced bleeding, or for restoring hemostasis in subjects who have suffered trauma or are bleeding due to surgery or childbirth.

52. 49. A pharmaceutical composition according to claim 48 for use in the treatment or prevention of streptococcal infections.

53. 49. The pharmaceutical composition of claim 48 for use in treating, inhibiting, preventing or minimizing the spread or progression of cancer, including inhibiting or preventing metastasis of cancer.

Images