Antibodies to plasminogen activator inhibitor-1 (PAI-1) and uses thereof

Abstract

To provide an antibody that specifically binds to plasminogen activator inhibitor type-1 (PAI-1), and methods comprising administering the antibody for recovering plasmin formation.SOLUTION: Provided herein is an isolated monoclonal antibody that specifically binds to PAI-1, the antibody comprising: (a) a heavy chain framework region and a heavy chain variable region, where the heavy chain variable region comprises heavy chain CDR1 region comprising a specific sequence, heavy chain CDR2 region comprising a specific sequence, and heavy chain CDR3 region comprising a specific sequence; and (b) a light chain framework region and a light chain variable region, where the light chain variable region comprises light chain CDR1 region comprising a specific sequence, light chain CDR2 region comprising a specific sequence, and light chain CDR3 region comprising a specific sequence.SELECTED DRAWING: None

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to U.S. Provisional Application No. 61 / 865,451, filed August 13, 2013, and European Patent Application No. 14305757.8, filed May 22, 2014, which are incorporated by reference in their entireties. [Background technology]

[0002] background Plasminogen activator inhibitor type 1 (PAI-1) is a key player in plasmin generation. The serine proteases tissue-type plasminogen activator (tPA) and urokinase PAI-1 is the major inhibitor of uPA type plasminogen activator (uPA). PAI-1 regulates fibrinolysis by inhibiting plasminogen activation in the vascular compartment. Fibrinolysis is a tightly coordinated process to degrade fibrin clots formed by activation of the coagulation cascade. Dysregulation of the coagulation / fibrinolysis balance can lead to bleeding or thrombotic disorders. PAI-1 also binds to receptor-bound plasminogen activators, resulting in abnormal hemostatic events such as hemostasis. PAI-1 is a key regulator of plasminogen activation in the pericellular compartment (in blood vessels and tissues), where it is primarily activated by urokinase bound to the urokinase receptor (uPAR). By inhibiting pericellular proteolysis, PAI-1 inhibits plasminogen activation in the extracellular matrix. PAI-1 regulates multiple cellular functions such as extracellular matrix (ECM) degradation, growth factor activation and release from the ECM, matrix metalloproteinase (MMP) activation, and cell apoptosis. Recently, protease-independent effects of PAI-1 have been demonstrated (e.g., vitronectin, heparin, and glycosaminoglycans). cofactors, uPAR-urokinase complex or cell receptor (LRP: low density lipoprotein receptor PAI-1 was identified through its interaction with integrins (PAI-associated proteins) or integrins, which affect cellular functions such as adhesion / deadhesion, migration, proliferation, and intracellular bioactivity. These cellular mechanisms and antifibrinolytic effects have led to the pathogenic role of PAI-1 in tumor growth and metastasis, fibrosis, acute myocardial infarction, and other diseases. It has been established in infarction and metabolic disorders such as atherosclerosis, obesity and diabetes.

[0003] The human SERPINE1 (PAI-1) gene is located on chromosome 7 and contains eight introns and nine It consists of 12,169 exons and has a size of 12,169 b (Non-Patent Document 1). A single-chain glycoprotein of approximately 50 kDa (379 amino acids) from the serine protease inhibitor superfamily. PAI-1 is a protein that is synthesized in an active conformation but spontaneously becomes latent in the absence of vitronectin (Vn). Vitronectin, the major cofactor for PAI-1, is a surface The mechanism of inhibition of the two major targets of PAI-1 (tPA and uPA) is suicide inhibition. The RCL region of PAI-1 contains the bait peptide bond (R346-M347, also called P1-P'1), which contains the cleavage site for this serine protease. After first forming a Michaelis complex with tPA or uPA, the catalytic triad then reacts with the bait peptide bond to form an acyl-enzyme complex, which induces a strong conformational change after cleavage of the P1-P'1 peptide bond. The conformational change indicates that the protease is covalently bonded to PAI-1 as an acyl-enzyme. This complex results in the insertion of the cleaved RCL into the β-strand. Under non-physiological conditions, hydrolysis of this acyl-enzyme complex can induce the release of cleaved PAI-1 and free active protease (Non-Patent Document 2).

[0004] PAI-1 circulates in the blood at highly variable levels (nM range) and in excess of t-PA or uPA concentrations. PAI-1 exhibits structural flexibility and can be found in one of three conformations. PAI-1 can be expressed in three different conformations: (1) a latent conformation, (2) an active conformation, or (3) a substrate conformation (see FIG. 1). PAI-1 is found primarily as a non-covalent complex with vitronectin (Kd approx. 1 nM), which reduces the latency transition by 1.5-3 fold. The affinity of latent, cleaved, or complexed PAI-1 for vitronectin was Matrix-bound vitronectin was also significantly reduced in the pericellular matrix along with PAI-1. PAI-1 is localized in the endothelial cells, monocytes, macrophages, and vascular smooth muscle cells. PAI-1 is synthesized by platelets, which can then be stored in large amounts in a latent form by platelets (in α-granules). , a fast and specific inhibitor of tPA and uPA in solution (second-order rate constant 10 6 ~10 7 M -1 s -1 (having However, they are inactive against proteases bound to fibrin or to either their cellular receptors. They are also inactive against other proteases such as thrombin, plasmin, and activated protein C. is also inhibited by PAI-1, but less efficiently.

[0005] Several 3D structures of human PAI-1 have been solved in latent conformations since the first one was described in 1992 (Non-Patent Document 3). These 3D structures include mutant forms of PAI-1 in substrates (Non-Patent Document 4), a stabilized active conformation (Non-Patent Document 5), PAI complexed to the vitronectin-somatomedin B domain (Non-Patent Document 6) or PAI complexed with an inhibitory pentapeptide from the RCL loop (Non-Patent Document 7). More recently, latent conformations have been described. The structure of mouse PAI-1 in its native conformation was solved by Dewilde et al. (Non-Patent Document 8) and revealed differences from human PAI-1 in the RCL position, the location of the gate region and α-helix A. Structure / function relationships in PAI-1 were investigated by using more than 600 mutant proteins to identify the locations of domains involved in the various activities of this multifunctional serpin (reviewed by Dewilde et al. (Non-Patent Document 9)).

[0006] PAI-1 binds to hepatocytes, adipocytes, mesangial cells, fibroblasts, myofibroblasts, and epithelial cells. Since it can be synthesized by almost all cell types, including epithelial cells, its expression is important in physiological (e.g., circadian variations in plasma PAI-1 levels) and pathological conditions (e.g., obesity, metabolic syndrome, insulin resistance ... PAI-1 is considered an acute phase protein. Transcriptional regulation of PAI-1 mRNA expression is regulated by several cytokines and growth factors (e.g. TGFβ, TNFα, EGF, FGF, insulin, angiotensin II and IV), hormones (e.g., aldosterone, glucocorticoids, PMA, high glucose) and stress factors (e.g., It is induced by various factors, such as hypoxia, reactive oxygen species, and lipopolysaccharides.

[0007] Furthermore, a polymorphism in the promoter (position -675) of the PAI-1 gene affects expression levels. The 4G allele increases PAI-1 levels, and the 4G / 4G variant (occurring in about 25% of the population) increases plasma PAI-1 levels by about 2 times compared to 5G / 5G (25% occurrence and 4G / 5G 50% occurrence). It induces a 5% increase in the 4G / 4G polymorphism. The 4G / 4G polymorphism has been associated with myocardial infarction (Non-Patent Document 10), certain types of pulmonary fibrosis (idiopathic interstitial pneumonia) (Non-Patent Document 11), and the 4G / 4G genotype donor group is an independent risk factor for kidney transplant loss due to interstitial fibrosis and tubular atrophy (Non-Patent Document 12).

[0008] Several pathogenic roles have been attributed to PAI-1 in thrombotic diseases such as arterial and venous thrombosis, acute myocardial infarction, and atherosclerosis. Its involvement in metabolic disorders such as phosphorus resistance syndrome and obesity is well recognized. is also known as a profibrotic factor in several organs and is involved in the formation of fibrotic tissues. It has been shown to be overexpressed in the liver, lungs, kidneys, heart, abdominal heal Skin: scarring or scleroderma) (reviewed by 13). PAI-1 knockout (KO) Mice are protected from fibrosis in various models, such as liver (bile duct ligation or xenobiotics), kidney (unilateral ureteral obstruction model (UUO)), and lung (bleomycin inhalation) (Non-Patent Document 14; Non-Patent Document 2). Patent Literature 15; Non-Patent Literature 16), whereas in the heart, its deletion protects against induced fibrosis (Non-Patent Literature 17), but predisposes to age-dependent cardiac selective fibrosis (Non-Patent Literature 18). Downregulation of PAI-1 expression by siRNA (Non-Patent Document 19) or inhibition by chemical compounds (Non-Patent Document 20) It has been reported that overexpression of PAI-1 (Reference 20; Non-Patent Document 21) reduces pulmonary fibrosis, whereas overexpression of wild-type PAI-1 (Non-Patent Document 22) or a PAI-1 mutant that retains only vitronectin binding but not tPA inhibitor function exacerbates pulmonary fibrosis (Non-Patent Document 23).

[0009] Bile duct ligation (BDL) liver fibrosis is attenuated by antibodies neutralizing PAI-1 (Patent Document 1), while downregulation by siRNA attenuates BDL and xenobiotic-induced liver fibrosis (Non-Patent Document 24). PAI-1 KO mice were protected from cholestatic-induced liver injury and fibrosis in BDL (Non-Patent Document 25; Non-Patent Document 26; Non-Patent Document 27) and from angiotensin II-induced liver fibrosis (Non-Patent Document 28).

[0010] PAI-1 KO mice have been shown to be effective in the UUO model (Non-Patent Document 29), diabetic nephropathy (Non-Patent Document 30), and angiotensin II-induced nephropathy (Non-Patent Document 31; see for review). , 32 and 33) protects against renal fibrosis. In contrast, PAI-1 overexpressing mice exhibit more severe fibrosis and increased macrophage recruitment after UUO (Non-Patent Document 34; Non-Patent Document 35). R) Urinary protein expression and glomerular function in experimental glomerulonephritis (thy1) in rats. It has been shown that PAI-1-blocking peptides protect mice from the development of fibrosis by reducing matrix accumulation (Non-Patent Document 36). It inhibits nectin accumulation in UUO mice (Non-Patent Document 37). [Prior art documents] [Patent documents]

[0011] [Patent Document 1] U.S. Patent No. 7,771,720 [Non-patent literature]

[0012] [Non-Patent Document 1] Klinger, KW et al. Proc. Natl. Acad. Sci. USA 84:8548, 1987 [Non-Patent Document 2] Blouse et al., Biochemistry, 48:1723, 2009 [Non-Patent Document 3] Mottonen et al., Nature 355:270, 1992 [Non-Patent Document 4] Aertgeerts et al., Proteins 23:118, 1995

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[0013] PAI-1, a target for many pathologies, has been the subject of much research over the last 20 years, with many studies aimed at inhibiting its activity or treating it. It has been the focus of intensive research attempting to modulate its expression. Chemical compounds (Suzuki et al., Expert Opin. Investig. Drugs 20:255, 2011), monoclonal antibodies (Gils and Declerk, Thromb Haemost; 91:425, 2004), peptides, mutants (Cale and Lawrence, Curr. Drug Targets 8:971, 2007), siRNA or antisense RNA have been used to inhibit its various functions or suppress its expression. However, despite intensive research, the therapeutic potential of PAI-1 has not been fully established. The problem of developing therapeutically effective modulators remains unsolved. Thus, there is a need in the art for novel agents that inhibit PAI-1 activity for use in treating PAI-1 mediated human pathologies. [Means for solving the problem]

[0014] Summary of disclosure In one aspect, a monoclonal antibody that specifically binds human plasminogen activator type 1 (PAI-1) is Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, [the heavy chain variable region comprises CDR1 (SEQ ID NO:34), CDR2 (SEQ ID NO:33), and CDR3 (SEQ ID NO:32) of SEQ ID NO:6], and a light chain variable region, [the light chain variable region comprises CDR1 (SEQ ID NO:34), CDR2 (SEQ ID NO:33), and CDR3 (SEQ ID NO:32) of SEQ ID NO:7]. In a further aspect, the present invention includes a CDR comprising CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 35). In one embodiment, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO:6, and the light chain comprises a light chain variable region comprising SEQ ID NO:7. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:6, and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:7. All percent identities approximate. Values ​​indicate minimum % identity; % identities higher than the stated values ​​are also encompassed by the disclosure.

[0015] In another aspect, the present invention comprises (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 34, a heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) a light chain framework region. an isolated monoclonal antibody that specifically binds to PAI-1, the monoclonal antibody comprising a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 36, and a light chain CDR3 region comprising SEQ ID NO: 35; Disclosed herein is a null antibody. In certain aspects, the antibody heavy chain comprises the heavy chain of SEQ ID NO:6. and the antibody light chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO:7. Includes the area.

[0016] In one aspect, disclosed herein is an isolated monoclonal antibody that specifically binds to human plasminogen activator inhibitor type 1 (PAI-1), wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:22), CDR2 (SEQ ID NO:21), and CDR3 (SEQ ID NO:31) of SEQ ID NO:2. 20)], and a light chain variable region, [the light chain variable region comprises CDR1 of SEQ ID NO: 3 (SEQ ID NO: In a further aspect, the present invention includes a CDR comprising CDR1 (SEQ ID NO:25), CDR2 (SEQ ID NO:24), and CDR3 (SEQ ID NO:23). wherein the heavy chain comprises a heavy chain variable region comprising SEQ ID NO:2 and the light chain comprises a light chain variable region comprising SEQ ID NO:3. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:2 and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:3.

[0017] In a further aspect, an isolated monoclonal antibody that specifically binds to PAI-1 comprises: (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO:22, a heavy chain CDR2 region comprising SEQ ID NO:21, and a heavy chain CDR3 region comprising SEQ ID NO:20; and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO:25, a light chain CDR2 region comprising SEQ ID NO:24, and a light chain CDR3 region comprising SEQ ID NO:23. Disclosed herein are monoclonal antibodies. In certain aspects, the antibody heavy chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO:26, and the antibody light chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO:3. In some embodiments, the light chain framework region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to

[0018] In one aspect, the method specifically binds to human plasminogen activator inhibitor type 1 (PAI-1). Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:28), CDR2 (SEQ ID NO:27), and CDR3 (SEQ ID NO:26) of SEQ ID NO:4, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO:31), CDR2 (SEQ ID NO:30), and CDR3 (SEQ ID NO:29) of SEQ ID NO:5. In a further aspect, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO:4, and the light chain comprises a light chain variable region comprising SEQ ID NO:5. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:4 and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:5.

[0019] In a further aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO:28, a heavy chain CDR2 region comprising SEQ ID NO:27, and a heavy chain CDR3 region comprising SEQ ID NO:26; and (b) a light chain An isolated monoclonal antibody that specifically binds to PAI-1, comprising a framework region, a light chain CDR1 region comprising SEQ ID NO:31, a light chain CDR2 region comprising SEQ ID NO:30, and a light chain CDR3 region comprising SEQ ID NO:29. Disclosed herein are monoclonal antibodies. In certain aspects, the antibody heavy chain comprises a heavy chain framework region of SEQ ID NO:4 and a nucleotide sequence similar to that of SEQ ID NO:5, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% of the heavy chain framework region of SEQ ID NO:5. The antibody light chain comprises a heavy chain framework region that is identical to that of SEQ ID NO:5. The light chain framework region comprises a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the light chain framework region.

[0020] In one aspect, the method specifically binds to human plasminogen activator inhibitor type 1 (PAI-1). Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO: 40), CDR2 (SEQ ID NO: 39), and CDR3 (SEQ ID NO: 38) of SEQ ID NO: 8, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO: 41), CDR2 (SEQ ID NO: 39), and CDR3 (SEQ ID NO: 38) of SEQ ID NO: 9, number 43), CDR2 (SEQ ID NO: 42), and CDR3 (SEQ ID NO: 41). In a further aspect, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO: 8 and the light chain comprises a light chain variable region comprising SEQ ID NO: 9. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO: 8 and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO: 9.

[0021] In another aspect, the present invention comprises (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO:40, a heavy chain CDR2 region comprising SEQ ID NO:39, and a heavy chain CDR3 region comprising SEQ ID NO:38; and (b) a light chain framework region. an isolated monoclonal antibody that specifically binds to PAI-1, the monoclonal antibody comprising a light chain CDR1 region comprising SEQ ID NO: 43, a light chain CDR2 region comprising SEQ ID NO: 42, and a light chain CDR3 region comprising SEQ ID NO: 41; Disclosed herein is a null antibody. In certain aspects, the antibody heavy chain comprises the heavy chain of SEQ ID NO: 8. and the antibody light chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO:9. Includes the area.

[0022] In one aspect, the method specifically binds to human plasminogen activator inhibitor type 1 (PAI-1). Disclosed herein is an isolated monoclonal antibody comprising a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:52), CDR2 (SEQ ID NO:51), and CDR3 (SEQ ID NO:50) of SEQ ID NO:10, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO:52), CDR2 (SEQ ID NO:51), and CDR3 (SEQ ID NO:50) of SEQ ID NO:11. SEQ ID NO:55), CDR2 (SEQ ID NO:54), and CDR3 (SEQ ID NO:53). In one embodiment, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO: 10, and the light chain comprises a light chain variable region comprising SEQ ID NO: 11. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO: 10, and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO: 11.

[0023] In a further aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO:52. and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO:55, a light chain CDR2 region comprising SEQ ID NO:54, and a light chain CDR3 region comprising SEQ ID NO:53. Disclosed herein are monoclonal antibodies. In certain aspects, the antibody heavy chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO: 10, and the antibody light chain comprises a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO: 11.

[0024] In one aspect, the method specifically binds to human plasminogen activator inhibitor type 1 (PAI-1). Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:58), CDR2 (SEQ ID NO:57), and CDR3 (SEQ ID NO:56) of SEQ ID NO:12, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO:61), CDR2 (SEQ ID NO:60), and CDR3 (SEQ ID NO:59) of SEQ ID NO:13. In a further aspect, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO:12, and the light chain comprises a light chain variable region comprising SEQ ID NO:13. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:12 and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:13.

[0025] In another aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO:58, and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO:61, a light chain CDR2 region comprising SEQ ID NO:60, and a light chain CDR3 region comprising SEQ ID NO:59. Disclosed herein are antibody fragments comprising a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO: 12, and a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO: 13.

[0026] In one aspect, the method specifically binds to human plasminogen activator inhibitor type 1 (PAI-1). Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:64), CDR2 (SEQ ID NO:63), and CDR3 (SEQ ID NO:62) of SEQ ID NO:14, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO:67), CDR2 (SEQ ID NO:66), and CDR3 (SEQ ID NO:65) of SEQ ID NO:15. In a further aspect, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO:14, and the light chain comprises a light chain variable region comprising SEQ ID NO:15. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:14 and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:15.

[0027] In a further aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO:64. and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 67, a light chain CDR2 region comprising SEQ ID NO: 66, and a light chain CDR3 region comprising SEQ ID NO: 65. Disclosed herein are monoclonal antibodies. In certain aspects, the antibody heavy chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO: 14, and the antibody light chain comprises a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO: 15.

[0028] In one aspect, the method specifically binds to human plasminogen activator inhibitor type 1 (PAI-1). Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:70), CDR2 (SEQ ID NO:69), and CDR3 (SEQ ID NO:68) of SEQ ID NO:16, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO:73), CDR2 (SEQ ID NO:72), and CDR3 (SEQ ID NO:71) of SEQ ID NO:17.

[0029] In a further aspect, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO: 16, and the light chain comprises a light chain variable region comprising SEQ ID NO: 17. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO: 16, and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO: 17.

[0030] In a further aspect, (a) a heavy chain framework region, the heavy chain CDR1 region comprising SEQ ID NO: 70 and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 73, a light chain CDR2 region comprising SEQ ID NO: 72, and a light chain CDR3 region comprising SEQ ID NO: 71. Disclosed herein are monoclonal antibodies. In certain aspects, the antibody heavy chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO: 16, and the antibody light chain comprises a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO: 17.

[0031] In one aspect, the method specifically binds to human plasminogen activator inhibitor type 1 (PAI-1). Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:46), CDR2 (SEQ ID NO:45), and CDR3 (SEQ ID NO:44) of SEQ ID NO:80, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO:49), CDR2 (SEQ ID NO:48), and CDR3 (SEQ ID NO:47) of SEQ ID NO:81.

[0032] In a further aspect, the heavy chain comprises a heavy chain variable region comprising SEQ ID NO: 80 and the light chain comprises and a light chain variable region comprising SEQ ID NO: 81. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:80 and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:81.

[0033] In one aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising the sequence SEQ ID NO:46, and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 49, a light chain CDR2 region comprising SEQ ID NO: 48, and a light chain CDR3 region comprising SEQ ID NO: 47. Antibodies are disclosed herein. In certain aspects, the antibody heavy chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO: 80, and the antibody light chain comprises a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO: 81.

[0034] In another aspect, a method for the treatment of plasminogen activator inhibitor type 1 (PAI-1) comprising administering to the subject a therapeutically effective amount of plasminogen activator inhibitor (PAI-1) specific binding protein (PAI-1) that specifically binds to plasminogen activator inhibitor type 1 (PAI-1) is provided. Disclosed herein is an isolated monoclonal antibody, wherein the antibody comprises a heavy chain variable region, wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:76), CDR2 (SEQ ID NO:75), and CDR3 (SEQ ID NO:74) of SEQ ID NO:18, and a light chain variable region, wherein the light chain variable region comprises CDR1 (SEQ ID NO:79), CDR2 (SEQ ID NO:78), and CDR3 (SEQ ID NO:77) of SEQ ID NO:19. In a further aspect, the heavy chain comprises a variable region comprising heavy chain SEQ ID NO:18 and the light chain comprises a light chain variable region comprising SEQ ID NO:19. In a further aspect, the heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:18 and the light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to SEQ ID NO:19.

[0035] (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 76, a heavy chain CDR2 region comprising SEQ ID NO: 75, and a heavy chain CDR3 region comprising SEQ ID NO: 74; and (b) a light chain framework region, comprising the sequence An isolated monoclonal antibody comprising a light chain CDR1 region comprising SEQ ID NO: 79, a light chain CDR2 region comprising SEQ ID NO: 78, and a light chain CDR3 region comprising SEQ ID NO: 77. In certain aspects, the antibody heavy chain comprises a heavy chain framework region of SEQ ID NO: 18 and 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical, and the antibody light chain comprises a heavy chain framework region of SEQ ID NO: 19. The light chain framework region comprises a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the light chain framework region.

[0036] In one aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising the sequence SEQ ID NO: 33, (b) a heavy chain CDR2 region comprising SEQ ID NO:146, and a heavy chain CDR3 region comprising SEQ ID NO:32; and (b) a light chain frame a light chain CDR1 region comprising SEQ ID NO: 37; a light chain CDR2 region comprising SEQ ID NO: 145; and An isolated monoclonal antibody that specifically binds to PAI-1, comprising a light chain CDR3 region comprising SEQ ID NO:35. Disclosed herein are clonal antibodies.

[0037] In one aspect, an isolated monoclonal antibody that specifically binds to PAI-1 comprises: (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 147, a heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 36, and a light chain CDR3 region comprising SEQ ID NO: 35. Antibodies are disclosed herein.

[0038] In one aspect, the present invention comprises (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 147, a heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 145, and a light chain CDR3 region comprising SEQ ID NO: 32. An isolated monoclonal antibody that specifically binds to PAI-1 and contains a light chain CDR3 region that includes sequence number 35. Disclosed herein are antibodies.

[0039] In one aspect, the present invention comprises (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 146, a heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 145, and a light chain CDR3 region comprising SEQ ID NO: 32. An isolated monoclonal antibody that specifically binds to PAI-1 and contains a light chain CDR3 region that includes sequence number 35. Disclosed herein are antibodies.

[0040] In one aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising the sequence SEQ ID NO: 34, (b) a heavy chain CDR2 region comprising SEQ ID NO:33, and a heavy chain CDR3 region comprising SEQ ID NO:32; and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO:37, a light chain CDR2 region comprising SEQ ID NO:145, and a light chain CDR3 region comprising SEQ ID NO:36. An isolated monoclonal antibody that specifically binds to PAI-1 and contains a light chain CDR3 region that includes sequence number 35. Disclosed herein are antibodies.

[0041] In a further aspect, a heavy chain variable region, [wherein the heavy chain variable region comprises CDR1 (SEQ ID NO:34), CDR2 (SEQ ID NO:33), and CDR3 (SEQ ID NO:32) of SEQ ID NO:6], and a light chain variable region, Disclosed herein is an isolated monoclonal antibody that binds to essentially the same epitope on PAI-1 as an isolated monoclonal antibody comprising the CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 35) of SEQ ID NO: 7, wherein the light chain variable region comprises CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 35) of SEQ ID NO: 7.

[0042] In a particular aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 76; and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 79, a light chain CDR2 region comprising SEQ ID NO: 78, and a light chain CDR3 region comprising SEQ ID NO: 77. Disclosed herein are monoclonal antibodies.

[0043] In one aspect, a humanized monoclonal antibody that specifically binds to human PAI-1 is provided herein. The antibody is disclosed in, wherein the antibody comprises: (a) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 82, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 91, or an antigen-binding fragment thereof; (b) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 83, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 92, or an antigen-binding fragment thereof; (c) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 84, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 93, or an antigen-binding fragment thereof; (d) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 85, or or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO:91, or an antigen-binding fragment thereof; (e) a heavy chain having a heavy chain variable region comprising SEQ ID NO:85, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO:93, or an antigen-binding fragment thereof; (f) a heavy chain having a heavy chain variable region comprising SEQ ID NO:86, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO:94, or an antigen-binding fragment thereof; (g) a heavy chain having a heavy chain variable region comprising SEQ ID NO:87, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO:95, or an antigen-binding fragment thereof; (h) a heavy chain having a heavy chain variable region comprising SEQ ID NO:88, or an antigen-binding fragment thereof. (i) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 89, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 97, or an antigen-binding fragment thereof; (j) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 90, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 98, or an antigen-binding fragment thereof; (k) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 86, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 93, or an antigen-binding fragment thereof; (l) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 86, or an antigen-binding fragment thereof. (m) a heavy chain having a heavy chain variable region comprising SEQ ID NO:89, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO:93, or an antigen-binding fragment thereof; or (n) a heavy chain having a heavy chain variable region comprising SEQ ID NO:89, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO:95, or an antigen-binding fragment thereof. In a further aspect, the humanized heavy chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the previously disclosed human heavy chain variable regions, and the humanized light chain variable region is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the previously disclosed human light chain variable regions.

[0044] In one aspect, (a) a heavy chain framework region, and a heavy chain variable region comprising SEQ ID NO:86; and (b) a light chain framework region, and a light chain variable region comprising SEQ ID NO: 93. In certain aspects, the isolated monoclonal antibody heavy chain comprises a heavy chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework region of SEQ ID NO: 86, and the isolated monoclonal antibody light chain comprises a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework region of SEQ ID NO: 93. In certain other aspects, the isolated monoclonal antibody heavy chain comprises a heavy chain framework region that is 95% identical to the heavy chain framework region of SEQ ID NO: 86, or The isolated monoclonal antibody light chain has 95% identity to the framework region of SEQ ID NO:93. It includes certain light chain framework regions.

[0045] In another aspect, a humanized monoclonal antibody that specifically binds to human PAI-1 is provided herein. and wherein the antibody comprises a heavy chain having a heavy chain variable region comprising SEQ ID NO: 154, or and a light chain having a light chain variable region comprising SEQ ID NO: 153, or an antigen-binding fragment thereof. In another aspect, the humanized monoclonal antibody specifically binds to human PAI-1. Disclosed herein is a monoclonal antibody, wherein the antibody comprises a polypeptide comprising SEQ ID NO: 155. A heavy chain having a chain variable region, or an antigen-binding fragment thereof, and a light chain comprising SEQ ID NO: 153. In a further aspect, the humanized heavy chain variable region comprises a light chain having a variable region identical to any of the previously disclosed human heavy chain variable regions, or an antigen-binding fragment thereof. , 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the humanized light chain variable region of It is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the human light chain variable regions previously disclosed.

[0046] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is provided herein. The antibody is disclosed in the literature, wherein the antibody binds to a polypeptide comprising SEQ ID NO: 158. In one embodiment, the isolated monoclonal antibody comprises a polypeptide comprising SEQ ID NO:158. In yet another embodiment, the antibody specifically binds to a fragment of PAI-1. The isolated monoclonal antibody binds to a polypeptide comprising SEQ ID NO: 156 and / or SEQ ID NO: 158. In another embodiment, the isolated monoclonal antibody specifically binds to PAI-1. The null antibody is directed to a polypeptide comprising SEQ ID NO:156, SEQ ID NO:158, and / or SEQ ID NO:157. In yet another embodiment, an isolated monoclonal antibody that specifically binds to PAI-1 is provided. The isolated monoclonal antibodies disclosed herein include specific binding affinity for residues 160, 262, 296-297, 300-307, and / or 310-316 of SEQ ID NO:1. In certain embodiments, the isolated monoclonal antibodies disclosed herein interact with at least residues 311, 312, and 313 (DQE) of SEQ ID NO:1. In certain embodiments, the PAI-1 bound by the antibody is human PAI-1. In other embodiments, the PAI-1 bound by the antibody is the active form of human PAI-1.

[0047] In another embodiment, an isolated antibody that specifically binds to PAI-1 as disclosed herein is provided. The monoclonal antibody binds to a polypeptide comprising SEQ ID NO: 161. In one embodiment, the isolated monoclonal antibody binds to a polypeptide comprising SEQ ID NO: 159 and / or SEQ ID NO: 161. In yet another embodiment, the isolated monoclonal antibody binds to a polypeptide comprising SEQ ID NO: 159, SEQ ID NO: 160, and / or SEQ ID NO: 161. In another embodiment, the isolated monoclonal antibody that specifically binds to PAI-1 is Residues 44-64 and / or residues 307-321 of cynomolgus monkey (cyno)-PAI-1 (SEQ ID NO: 162) In certain embodiments, the PAI-1 bound by the antibody has a specific binding affinity to the antibody. In another embodiment, the PAI-1 bound by the antibody is a crab-PAI-1. This is the latent form of quis-PAI-1.

[0048] In a further aspect, a compound that competitively inhibits the binding of any of the disclosed antibodies to PAI-1 is provided. Disclosed herein are isolated monoclonal antibodies that compete for and / or competitively inhibit binding with any of the isolated monoclonal antibodies disclosed herein. In certain embodiments, the isolated monoclonal antibodies compete or competitively inhibit binding to human PAI-1. The selected monoclonal antibodies compete or competitively inhibit binding to a polypeptide comprising SEQ ID NO: 156, SEQ ID NO: 157, and / or SEQ ID NO: 158. The isolated monoclonal antibodies compete or competitively inhibit binding to a polypeptide comprising SEQ ID NO:159, SEQ ID NO:160, and / or SEQ ID NO:161. In one embodiment, the isolated antibody binds to a polypeptide comprising SEQ ID NO: 156, 157, and / or 158. (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 34, and (b) a light chain framework region, a light chain CDR1 region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 145, and a light chain CDR3 region comprising SEQ ID NO: 35.

[0049] In another aspect, disclosed herein are nucleotide sequences encoding any of the isolated monoclonal antibodies disclosed herein.

[0050] In one aspect, disclosed herein is a method for treating a condition resulting from increased expression of or increased sensitivity to PAI-1, the method comprising administering to a patient or other subject a pharma- tically effective amount of a PAI-1 antibody orally, parenterally via an injectable solution, by inhalation, or topically. This includes administering.

[0051] In one aspect, a pharma- tically effective amount of a PAI-1 antibody is administered to a patient or other subject in need thereof. Disclosed herein is a method of restoring plasmin production, comprising administering orally, parenterally by injectable solution, by inhalation, or topically. Parenteral administration disclosed herein includes intravenous, infusion, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal, intravenous, intraarterial, subcutaneous, and intramuscular parenteral dosage forms. In some embodiments, administration to a patient or other subject includes multiple doses. In another aspect, the method of restoring plasmin production facilitates therapeutic treatment of a condition involving an increased level of fibrotic tissue. In some aspects, the condition is characterized by fibrosis. In some aspects, the condition is fibrosis, dermal fibrosis, systemic sclerosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, and chronic lung disease. In other aspects, plasmin generation facilitates the therapeutic treatment of liver fibrosis, renal fibrosis, including chronic kidney disease, thrombosis, venous and arterial thrombosis, deep vein thrombosis, peripheral limb ischemia, disseminated intravascular coagulation thrombosis, acute ischemic stroke with or without thrombolysis, or stent restenosis.

[0052] In another aspect, a pharmacopoietin for the manufacture of a medicament for treating a condition caused by increased expression of or increased sensitivity to PAI-1, comprising administering to a patient or other subject orally, parenterally by injectable solution, by inhalation, or topically, a pharmacopoietin for the manufacture of a medicament for treating a condition caused by increased expression of or increased sensitivity to PAI-1. Uses of the I-1 antibody are disclosed herein.

[0053] In one aspect, the medicament is for treating a condition that includes an increased level of fibrotic tissue. In some aspects, the condition is characterized by fibrosis. In some aspects, the condition is fibrosis, dermal fibrosis, systemic sclerosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, and chronic lung disease. In other aspects, the medicament is for treating a condition that includes liver fibrosis, renal fibrosis including chronic kidney disease, thrombosis, venous and arterial thrombosis, deep vein thrombosis, peripheral limb ischemia, disseminated intravascular coagulation thrombosis, acute ischemic stroke with or without thrombolysis, or stent restenosis.

[0054] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is provided herein. In some embodiments, the antibody inhibits pulmonary fibrosis. In certain embodiments, the antibody inhibits fibrosis in the lungs of a subject. In certain embodiments, the antibody inhibits fibrosis in the lungs of a subject with idiopathic pulmonary fibrosis (IPF). In some embodiments, the antibody inhibits fibrosis in the lungs of a subject with idiopathic pulmonary fibrosis (IPF). Thus, the isolated monoclonal antibodies disclosed herein induce increased fibrin degradation in the subject. In certain embodiments, the antibodies increase fibrin degradation in the plasma of the subject. In some other embodiments, the isolated monoclonal antibodies disclosed herein inhibit collagen accumulation in the lungs of the subject. In some embodiments, the subject has IPF. In some other embodiments, The isolated monoclonal antibody disclosed herein increases D-dimer levels in bronchoalveolar lavage fluid (BALF) of a subject. In some embodiments, the subject has IPF. In some other embodiments, the isolated monoclonal antibody disclosed herein increases D-dimer levels in bronchoalveolar lavage fluid (BALF) of a subject ... The monoclonal antibody specifically binds to PAI-1, wherein the antibody inhibits fibrosis in a subject. In one embodiment, the subject has IPF.

[0055] In another aspect, the method comprises administering to a patient orally, parenterally by injection solution, by inhalation, or topically, a method for treating a condition resulting from increased expression or increased sensitivity to PAI-1. The use of a pharma- tically effective amount of a PAI-1 antibody for the manufacture of a medicament for the treatment of wherein the condition is idiopathic pulmonary fibrosis.

[0056] In another aspect, the patient or other subject is orally administered a pharma- tically effective amount of a PAI-1 antibody. Disclosed herein are methods of restoring plasmin generation, including administering to a subject parenterally via injectable solution, by inhalation, or topically, plasmin generation facilitating therapeutic treatment of idiopathic pulmonary fibrosis.

[0057] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is provided herein. In one embodiment, the antibody restores fibrinolytic activity in a subject. In a particular embodiment, the antibody restores fibrinolytic activity in a subject having an acute ischemic attack. The acute ischemic attack may or may not be accompanied by thrombolysis. In some embodiments, the isolated monoclonal antibody restores clot lysis. In a particular embodiment, the antibody restores clot lysis in vitro. In yet another embodiment, the antibody has an IC of about 2 nM in vitro. 50 to restore clot lysis.

[0058] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is provided herein. In some embodiments, the subject has an acute ischemic stroke, wherein the antibody restores fibrin breakdown in the subject.

[0059] In another aspect, the present invention provides the use of a pharma- tically effective amount of a PAI-1 antibody for the manufacture of a medicament for treating a condition resulting from increased expression of or increased susceptibility to PAI-1, including administering the antibody orally, parenterally by injection solution, by inhalation, or topically to a patient. Disclosed herein is a method for treating acute ischemic stroke, wherein the condition is acute ischemic stroke with or without thrombolysis.

[0060] In another aspect, a pharma- tically effective amount of a PAI-1 antibody is administered to a patient or other subject in need thereof. Disclosed herein are methods of restoring plasmin generation, including administering to the body orally, parenterally by injection solution, by inhalation, or topically, where plasmin generation facilitates therapeutic treatment of acute ischemic attacks with or without thrombolysis.

[0061] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is provided herein. The present invention relates to a method for the treatment of osteoarthritis of a subject, comprising administering to said subject an antibody capable of inhibiting the formation of adhesions in said subject. In some embodiments, the adhesion formation is following surgery or trauma. In some embodiments, the adhesion formation in the subject is in the abdomen. In other embodiments, the adhesion formation occurs in the shoulder, pelvis, heart, spine, hands, and other body areas of the subject.

[0062] In another aspect, the present invention relates to the use of a pharma- tically effective amount of a PAI-1 antibody for the manufacture of a medicament for treating or preventing a condition resulting from increased expression of or increased susceptibility to PAI-1, comprising administering the antibody orally, parenterally by injection solution, by inhalation, or topically to a patient. The use is disclosed herein, where the condition is abdominal adhesion formation.

[0063] In another aspect, a pharma- tically effective amount of a PAI-1 antibody is administered to a patient or other subject in need thereof. Disclosed herein are methods of restoring plasmin generation, comprising administering to a subject orally, parenterally by injection solution, by inhalation, or topically, wherein plasmin generation promotes treatment or prevention of adhesion formation. In some embodiments, the adhesion formation in the subject is abdominal.

[0064] In another aspect, disclosed herein is an isolated monoclonal antibody that binds to PAI-1 / vitronectin complex. In another aspect, disclosed herein is an isolated monoclonal antibody that neutralizes PAI-1 activity by inducing PAI-1 substrate conformation. In one embodiment, the antibody restores or is capable of restoring plasmin generation. In another embodiment, the isolated monoclonal antibody induces or is capable of inducing fibronectin degradation. In yet another embodiment, the isolated monoclonal antibody induces or is capable of inducing matrix metalloproteinase (MMP) activation.

[0065] In another aspect, the isolated monoclonal antibody disclosed herein is an antibody fragment. In some embodiments, the antibody is a single chain Fv antibody. In other embodiments, the heavy and light chains are connected by a flexible linker to form a single chain antibody. In other embodiments, the antibody is a Fab, Fab', or (Fab')2 antibody.

[0066] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is provided herein. In one embodiment, disclosed herein is an isolated crystal comprising a Fab' fragment of monoclonal antibody A44, wherein the Fab' fragment consists of light chain sequence SEQ ID NO:7 and heavy chain sequence SEQ ID NO:6. In another embodiment, disclosed herein is an isolated crystal comprising a Fab' fragment comprising light chain sequence SEQ ID NO:93 and heavy chain sequence SEQ ID NO:86. Disclosed herein is an isolated crystal comprising the fragment. In one embodiment, the isolated crystal comprises asymmetric unit cell dimensions a=105 Å, b=152 Å, and c=298 Å. In one embodiment, the isolated crystal belongs to the P212121 space group. In another embodiment, In one embodiment, the isolated crystals comprise an x-ray diffraction resolution of 3.3 Å. In one embodiment, the isolated crystals retain the biological activity of the crystallized antibody. In some embodiments, the isolated crystals exhibit greater in vivo activity than the soluble counterpart of the crystallized antibody. It has a long half-life.

[0067] In one aspect, the present invention provides a method for the production of a crystallized antibody that specifically binds to PAI-1, and a method for producing the same. Disclosed herein are pharmaceutical compositions comprising at least one pharmaceutical excipient that is or encapsulates the at least one pharmaceutical excipient.

[0068] In another aspect, disclosed herein is a pharmaceutical composition comprising a pharma- ceutically acceptable carrier and a therapeutically effective amount of any of the antibodies disclosed herein.

[0069] In one aspect, the present invention is directed to a method for treating a pulmonary artery disease comprising administering to a patient a therapeutically effective amount of PAI-1 or a fragment thereof, and vitronectin. A method for producing antibodies against PAI-1 is provided herein, which comprises immunizing a mammal with the conjugate. Disclosed in the specification.

[0070] In another aspect, disclosed herein is a method for screening a PAI-1 antibody by ELISA for its ability to block the function of PAI-1 as a tPA activity inhibitor, the method comprising the steps of: (a) binding PAI-1 to an ELISA plate; (b) incubating the ELISA plate with a PAI-1 antibody; (c) incubating the ELISA plate with tPA; (d) labeling. (e) incubating the ELISA plate with the labeled anti-tPA antibody; and (f) measuring the OD emitted by the labeled anti-tPA antibody. 405 wherein a positive reading is obtained; The results indicate that the PAI-1 antibody binds to PAI-1 but blocks the formation of a covalent bond between PAI-1 and tPA, and the negative scores indicate that the PAI-1 antibody blocks the interaction of tPA with PAI-1. This indicates that you want to check.

[0071] In another aspect, a method for screening hybridomas is disclosed herein. In a particular embodiment, the method for screening comprises using an anti-mouse immobilized anti-PAI-1 antibody. In another embodiment, the screening method includes a forward screening method using free PAI-1 as a ligand or against immobilized vitronectin. In certain embodiments, the method includes or comprises a screening assay. In some embodiments, the method is applied to determine the affinity of an antibody for a PAI-1 / vitronectin complex. In some embodiments, the method comprises: immobilizing vitronectin on a surface; contacting the surface-immobilized vitronectin with PAI-1, thereby forming a complex; contacting the surface containing the complex with an antibody; separating the antibody bound to the complex from unbound antibody; detecting the antibody bound to the complex, and determining the level of antibody bound to the complex. The method includes analyzing the antibody to determine the affinity of the antibody for the complex. [Brief description of the drawings]

[0072] [Figure 1]FIG. 1 shows a schematic diagram of the mechanism between PAI-1 and serine proteases of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA). PAI-1 exhibits structural flexibility and can exist in a latent or active conformation when it binds to vitronectin (Vn). The RCL region of PAI-1 carries the bait peptide bond (also called P1-P1'), which is the cleavage site by the serine proteases. A Michaelis complex with tPA or uPA is formed first, and then the catalytic triad reacts with the bait peptide bond to form an acyl-enzyme complex, which induces a strong conformational change after cleavage of the P1-P'1 peptide bond. The acyl-enzyme is an unstable complex formed by a covalent bond between a serine residue (black triangle) from the catalytic triad from the serine protease (tPA) and an amino acid from the substrate (black circle) that undergoes further hydrolysis. The conformational change leads to the insertion of the cleaved RCL into the β-strand while the protease remains covalently bound to PAI-1 as an acyl-enzyme. Under non-physiological conditions, hydrolysis of this acyl-enzyme complex can lead to the release of cleaved PAI-1 and free active protease. [Diagram 2] Figure 2 shows a typical standard curve for antibody titration in a binding ELISA as described in Example 2. Antibodies 31C9, 33B8 and 33H1 were positive controls and IgG1 was as a negative control. [Diagram 3] Figure 3 shows the expression curves for the functional ELISA to select antibodies that block the interaction of PAI-1 with tPA, as described in Example 4. Antibody 33H1 was a positive control, IgG1 was a negative control, and A44 was identified as a positive antibody clone. [Figure 4] FIG. 4 shows the neutralization of human PAI-1, which blocks the activity of tPA, by A44 and commercial antibodies (33B8 and 33H1) in the chromogenic assay described in Example 4. [Diagram 5]FIG. 5 shows neutralization of human PAT-1 blocking activity of tPA by a selection of antibodies generated by different fusions (see Example 4). [Figure 6] FIG. 6 shows that human PAI-1 and its orthologs block human tPA activity in a chromogenic assay with similar potency. [Figure 7] FIG. 7 shows the neutralization of the blocking activity of human tPA by A44 and 33B8 (commercially available) antibodies in cynomolgus monkey (cyno) and mouse PAI-1 in the chromogenic assay described in Example 4. [Figure 8] Figure 8 shows SDS-Page analysis of the mechanism of action of antibodies 33H8 (which converts PAI-1 from active to latent conformation), 33H1 (which converts PAI-1 from active to substrate conformation) and A44 in blocking the interaction of PAI-1 with tPA. Lane 1: molecular weight standard; lane 2: PAI-1 only; lane 3: tPA only; lane 4: PAI-1 in the presence of tPA; lane 5: 33B8 + PAI-1 + tPA; lane 6: 33H1 + PAI-1 + tPA; lane 7: A44 + PAI-1 + tPA; lane 8: mAb is an isotype control antibody. [Figure 9] Figure 9 shows SDS-Page analysis of the mechanism of action of antibodies 33H8 (which converts PAI-1 from active to latent conformation), 33H1 (which converts PAI-1 from active to substrate conformation) and antibodies resulting from fusions C26, E16 and E21 to block the interaction of PAI-1 with tPA. Lane 1: molecular weight standard; lane 2: PAI-1 only; lane 3: tPA only; lane 4: PAI-1 in the presence of tPA; lane 5: 33B8 + PAI-1 + tPA; lane 6: 33H1 + PAI-1 + tPA; lane 7: C26 + PAI-1 + tPA; lane 8: E16 + PAI-1 + tPA; lane 9: E21 + PAI-1 + tPA; lane 10: mAb is an isotype control antibody. [Figure 10]Figure 10 shows SDS-Page analysis of the mechanism of action of antibodies 33H8 (which converts PAI-1 from active to latent conformation), 33H1 (which converts PAI-1 from active to substrate conformation) and antibodies resulting from fusions A39, B109 and C45 to block the interaction of PAI-1 with tPA. Lane 1: molecular weight standard; lane 2: PAI-1 only; lane 3: tPA only; lane 4: PAI-1 in the presence of tPA; lane 5: 33B8 + PAI-1 + tPA; lane 6: 33H1 + PAI-1 + tPA; lane 7: A39 + PAI-1 + tPA; lane 8: B109 + PAI-1 + tPA; lane 9: C45 + PAI-1 + tPA; lane 10: mAb is an isotype control antibody. [Figure 11] Figure 11 shows the alignment of the light chains of the following murine antibodies: A105 (SEQ ID NO:3), A39 (SEQ ID NO:5), A44 (SEQ ID NO:7), A71 (SEQ ID NO:9), A75 (SEQ ID NO:81), B109 (SEQ ID NO:11), B28 (SEQ ID NO:13), C45 (SEQ ID NO:15), E16 (SEQ ID NO:17), and E21 (SEQ ID NO:19). The CDRs are highlighted in bold. [Figure 12] Figure 12 shows an alignment of the heavy chains of the following murine antibodies: A105 (SEQ ID NO:2), A39 (SEQ ID NO:4), A44 (SEQ ID NO:6), A71 (SEQ ID NO:8), A75 (SEQ ID NO:80), B109 (SEQ ID NO:10), B28 (SEQ ID NO:12), C45 (SEQ ID NO:14), E16 (SEQ ID NO:16), and E21 (SEQ ID NO:18). The CDRs defined by IMGT are highlighted in bold. [Figure 13] FIG. 13 shows the alignment of mouse A44 light chain (SEQ ID NO:7) with vk1 (SEQ ID NO:101) and vlambda3 (SEQ ID NO:102). [Figure 14] FIG. 14 shows the alignment of mouse A44 heavy chain (SEQ ID NO:6) with vh2 (SEQ ID NO:103) and vh4 (SEQ ID NO:104). [Figure 15]Figure 15 shows the clone A44 humanized VL with all constructs aligned. All aligned sequences (SEQ ID NOs: 91-98) are further described below in Table 25. The black boxes represent the CDR domains. The highlighted residues differ in sequence from the residue immediately above them in the alignment. Residue numbering is as described by IMGT. [Figure 16] Figure 16 shows the clone A44 humanized VH with all constructs aligned. All aligned sequences (SEQ ID NOs: 82-90) are further described below in Table 25. Black boxes represent CDR domains. Highlighted residues differ in sequence from the residue immediately above in the alignment. Residue numbering is as described by IMGT. [Figure 17] FIG. 17 shows that the percent inhibition of PAI-1 activity was plotted as a function of mAb concentration and IC50 was determined Imax using Biostat speed software. [Figure 18] FIG. 18 shows the purification of homogeneous recombinant 6-His tagged Fab A44. [Figure 19] Figure 19 shows SPR analysis using Biacore 2000 using single kinetic analysis of human PAI-1 glycosylation binding to immobilized APG antibody. Sensorgrams from single-cycle kinetic are shown in grey. Fitted model is shown in black. [Figure 20] Figure 20 shows human plasma PAI-1 neutralization by APG, APGv2, and APGv4 antibodies, as determined by detection of UK-PAI-1 complex formation by ELISA. Percent inhibition of PAI-1 activity was plotted as a function of APG, APGv2, or APGv4 antibody concentration. [Figure 21] FIG. 21 shows restoration of human plasma clot lysis by A44V11 (1, 3 or 10 nM) in the presence of 1 nM tPA and 3 nM PAI-1 as detected by turbidimetric kinetic measurements with absorbance readings at 340 nm as a function of time (min). [Figure 22]FIG. 22 shows the absence of restoration of human plasma clot lysis by human IgG1 negative control (1, 3 or 10 nM) in the presence of tPA 1 nM and PAI-1 3 nM, as detected by absorbance at 340 nm as a function of time (min). [Diagram 23] FIG. 23 shows restoration of human plasma clot lysis by A44V11 or a human IgG1 isotype negative control at various concentrations. [Figure 24] FIG. 24 shows restoration of human plasma clot lysis by 3 nM APG, APGV2 or APGV4 in the presence of 1 nM tPA and 3 nM PAI-1, detected by absorbance at 340 nm as a function of time (min). [Diagram 25] FIG. 25 shows the restoration of human plasma clot lysis by APG variants 2 and 4 at various concentrations. [Figure 26] FIG. 26 shows immunoblot anti-PAI-1 on human LL29 myofibroblast supernatants 48 hours after treatment with A44V11 at 50 nM or IgG isotype control mAb and TGFβ 5 ng / ml. [Figure 27] FIG. 27 shows generic MMP activity in human primary lung fibroblasts after treatment of cells with PBS (control), plasminogen (Pg), A44v11 and plasminogen (A+Pg) or negative human IgG and plasminogen (Neg+Pg) for 48 hours. [Figure 28] Figure 28 shows human active PAI-1 levels in bronchoalveolar lavage fluid (BALF) (A) and lung lysates (B) from bleomycin-treated mice at 7 and 9 days after day 4 treatment with A44 or IgG1 or PBS at 10 mg / kg administered intraperitoneally (ip). Active PAI-1 determined by ELISA (#HPAIKT Molecular Innovation). Percentage of inhibition was calculated by dividing the difference between A44 bleomycin (bleo) and IgG bleo by the difference between IgG bleo and untreated (PBS) mice groups. [Figure 29]Figure 29 shows mouse D-dimer levels in BALF from bleomycin-treated mice 7 and 9 days after treatment with 10 mg / kg A44 or IgG1 or PBS administered ip on day 4, as determined by ELISA (Asserachrom D-Di, Diagnostica Stago). The fold increase in D-dimer induced by A44 compared to IgG is shown. [Diagram 30] FIG. 30 shows right lung masses from transgenic humanized mice 21 days after either saline or bleomycin treatment followed by ip administration of PBS (vehicle), IgG1 or A44 10 mg / kg every 3 days from day 4 to day 20. [Diagram 31] FIG. 31 shows hydroxyproline lung content in transgenic humanized mice after 21 days of either saline or bleomycin treatment followed by ip administration of PBS (vehicle), IgG1 or A44 10 mg / kg every 3 days from day 4 to day 20. [Diagram 32] Figure 32 shows active PAI-1 levels in plasma from monkeys treated with A44V11 (A) mAb (n=5) or IgG1 isotype control (B) (n=4) (5mg / kg intraperitoneally (ip)) 24 hours prior to LPS challenge (100ug / kg intravenous (iv)). Blood samples were taken at the indicated time points and active PAI-1 levels were determined in plasma using ELISA (# HPAIKT from Molecular Innovation). [Diagram 33] Figure 33 shows active PAI-1 levels in liver biopsies from monkeys treated with A44V11 (A) mAb (n=5) or IgG1 isotype control (B) (n=4) (5mg / kg ip) 24 hours prior to LPS challenge (100ug / kg iv). Liver biopsies were taken at the indicated time points in anesthetized monkeys and active PAI-1 levels were determined in lysates using ELISA (#HPAIKT from Molecular Innovation). [Diagram 34]Figure 34 shows D-dimer levels in plasma from monkeys treated with A44V11 (A) mAb (n=5) or IgG1 isotype control (B) (n=4) (5mg / kg ip) 24 hours prior to LPS challenge (100ug / kg iv). Blood samples were taken at the indicated time points and D-dimer levels were measured in plasma using ELISA. [Diagram 35] Figure 35 shows plasmin alpha 2 antiplasmin (PAP) complex levels in plasma from monkeys treated with A44V11 (A) mAb (n=5) or with IgG1 isotype control (B) (n=4) (5mg / kg ip) 24 hours prior to LPS challenge (100ug / kg iv). Blood samples were taken at the indicated time points and PAP levels were determined in plasma using ELISA (# Asserachrom PAP from Diagnostica Stago). [Diagram 36] Figure 36 shows the levels of active PAI-1 in intraperitoneal fluid (IPF) and uterine horn lysates. Active PAI-1 levels in intraperitoneal fluid (A) and uterine horn lysates (B). At 6 hours and 7 days, active PAI-1 levels were lower in both intraperitoneal fluid (IPF) and uterine horn (UH) lysates in animals treated with A44V11 antibody compared to animals treated with isotype control antibody, with no differences observed at 72 hours. (*p<0.001 as calculated by Student's t-test) [Figure 37] FIG. 37 shows another example of the purification of homogeneity recombinant 6-His tagged Fab A44. [Figure 38] FIG. 38 shows purification of homologous recombinant 6-His tagged Fab A44 complexed with human wt PAI-1 protein. [Figure 39] FIG. 39(a) shows the complex crystallization of the Fab A44 / PAI-1 complex, and FIG. 39(b) shows the best optimized crystal. [Diagram 40] FIG. 40 shows rod-shaped single crystals of the Fab A44 / PAI-1 complex. [Diagram 41]FIG. 41 shows that Fab A44 recognizes the active form of human PAI-1 and the latent form of cyno PAI-1. [Diagram 42] FIG. 42 shows the PAI-1 epitope recognized by Fab A44 on (A) active human PAI-1, and (B) latent cyno PAI-1. [Diagram 43] FIG. 43 shows the heavy chain paratope of the Fab A44 / PAI-1 complex. [Diagram 44] FIG. 44 shows the light chain paratope of the Fab A44 / PAI-1 complex. [Diagram 45] Figure 45 shows a sequence alignment of the putative A44 binding epitopes of cynomolgus monkey, human, rat, and mouse PAI-1. Sequences are taken from SEQ ID NO:1 (PAI-1 human), SEQ ID NO:162 (PAI-1 cynomolgus monkey), SEQ ID NO:163 (PAI-1 mouse), and SEQ ID NO:164 (PAI-1 rat). [Figure 46] FIG. 46 shows a comparison of the mouse PAI-1 structure with the human PAI-1 / A44V11 complex. [Figure 47] FIG. 47 shows the structure of the human PAI-1 / A44V11 complex and a model of vitronectin binding to PAI-1. [Figure 48] FIG. 48 shows peptic peptide coverage of cyno-PAI-1 (SEQ ID NO: 162); 95.3% sequence coverage is obtained from 150 overlapping peptic peptides. [Figure 49]FIG. 49 shows representative deuterium uptake plots for cyno-PAI-1 peptides in the unbound (circle lines), APGv2-bound (x-lines), and A44v11-bound (diamond lines) states. Residue ranges / positions are from SEQ ID NO: 162. (A) Most peptic peptides showed no difference between cyno-PAI-1 alone and bound to either mAb. (B) A peptide spanning residues 44-64 showed similar protection from exchange in both mAb-bound states. (C) A peptide incorporating 295-322 uptakes less deuterium in both mAb-bound states, but the magnitude of protection is higher for A44v11. [Figure 50] Figure 50 shows a hydrogen / deuterium exchange (HDX) comparison of cyno-PAI-1 alone and bound to A44v11. (A) Butterfly plot of average relative fractional exchange with unbound state at top and bound state at bottom. Lines correspond to data acquired at 10 seconds, 1 minute, 5 minutes, and 240 minutes. (B) Plot of difference data (in Daltons) from the plot in (A) above for cyno-PAI-1 alone or bound to A44v11. [Figure 51] Figure 51 shows HDX comparison of cyno-PAI-1 alone and bound to APGv2. In (A), butterfly plot of average relative fractional exchange with unbound state on top and bound state on bottom. Lines correspond to data acquired at 10 seconds, 1 minute, 5 minutes, and 240 minutes. In (B), plot of difference data (Daltons) from panel (A) above for cyno-PAI-1 alone or bound to APGv2. [Figure 52]Figure 52 shows HDX comparison of cyno-PAI-1 bound to A44v11 and cyno-PAI-1 bound to APGv2. In (A), butterfly plot of average relative fractional exchange with APGv2 bound state on top and A44v11 bound state on bottom. Lines correspond to data acquired at 10 seconds, 1 minute, 5 minutes, and 240 minutes. In (B), plot of difference data (Daltons) from panel (A) above for cyno-PAI-1 bound to APGv2 or A44v11. [Diagram 53] Figure 53 shows the cyno-PAI-1:A44v11 epitope as determined by HDX MS. Residues in cynoPAI-1 (SEQ ID NO: 162) that show protection from exchange upon A44v11 antibody binding are shown in bold. Residues in the cyno-PAI-1:A44v11 epitope as determined from crystallization studies are boxed. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0073] Detailed Description The present invention provides antibodies and fragments thereof that specifically bind to human PAI-1 and modulate the biological function of PAI-1. Such antibodies are useful in treating PAI-1-related diseases or disorders (e.g., fibrosis). The present invention also relates to pharmaceutical compositions and methods for treating PAI-1 antibodies. The present invention provides nucleic acids encoding such antibodies, recombinant expression vectors and host cells for producing such antibodies, or fragments thereof. Methods of using the antibodies disclosed herein for the treatment of cancer or for modulating PAI-1 activity are also provided. These are also encompassed by the present invention.

[0074] I. Definition In order that the present invention may be more readily understood, certain terms are first defined.

[0075] As used herein, the term "human PAI-1" includes the amino acid sequence set forth below: or a peptide consisting of: VHHPPSYVAHLASDFGVRVFQQVAQASKDRNVVFSPYGVASVLAMLQLTTGGETQQQIQAAMGFKIDDKGMAPALRHLYKELMGPWNKDEISTTDAIFVQRDLKLVQGFMPHFFRLFRSTVKQVDFSEVERARFIINDWVKTHTKGMISNLLGKGAVDQLTRLVLVNALYFNGQWKTPFPDSSTHRRLFHKSDGSTVSVPMMAQTNKFNYTEFTTPDGHYYDILELPYHGDTLSMFIAAPYEKEVPLSALTNILSAQLISHWKGNMTRLPRLLVLPKFSLETEVDLRKPLENLGMTDMFRQFQADFTSLSDQEPLHVAQALQKVKIEVNESGTVASSSTAVIVSARMAPEEIIMDRPFLFVVRHNPTGTVLFMGQVMEP (SEQ ID NO: 1), or Fragment of.

[0076] As used herein, the term "antibody" refers to an immunoglobulin molecule comprising four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (V H or VH) and Heavy chain constant region (C H The heavy chain constant region contains three domains, C H 1. C H 2 and C H 3 Each light chain comprises a light chain variable region (V L or VL) and a light chain constant region (C L The light chain constant region consists of one domain (C L 1) is included. H and V L The area is more conserved Each V can be further subdivided into regions of hypervariability called complementarity determining regions (CDRs) interspersed with regions called framework regions (FRs). H and V LThe amino-terminus is carboxyl It is composed of three CDRs and four FRs arranged towards the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0077] The term "antigen-binding fragment" of an antibody, as used herein, includes any naturally occurring, enzymatically derived, synthetic or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be prepared, for example, from intact antibody molecules by any suitable standard technique, such as proteolytic digestion, or recombinant DNA techniques, including the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Antigen-binding moieties can be derived using recombinant genetic engineering techniques. Non-limiting examples of antigen-binding moieties include: (i) Fab fragments; (ii) F(ab')2 fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of amino acid residues that mimic hypervariable regions of antibodies (e.g., isolated complementarity determining regions (CDRs)). Other engineered molecules, such as diabodies, triabodies, ), tetrabodies and minibodies are also referred to as "antigen-binding The term "fragment" is included in the "

[0078] As used herein, the term "CDR" or "complementarity determining region" refers to a region of a heavy or light chain polypeptide. "antigen binding sites" refers to discontinuous antigen binding sites found within both variable regions of a polypeptide. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and Chothia et al., J. Mol. Biol. 196:901-917 (1987) and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the definition refers to overlapping or non-overlapping amino acid residues when compared against each other. Includes a subset. The Kabat definition is based on sequence variability. All IG and TR V regions of all species The IMGT unique numbering for each sequence relies on the high conservation of the structure of the variable regions (Lefranc, Mp et al., Dev comp. Immunol. 27:55-77, 2003). The IMGT numbering, established after aligning more than 5,000 sequences, takes into account and combines the framework and CDR definitions. The Clothia definition is based on the location of structural loop regions. The contact definition (MacCallum et al.) is Based on the analysis of the complex crystal structure and antibody-antigen interactions. The amino acid residues which encompass the CDRs as defined by others are shown for comparison. In one embodiment disclosed herein, the term "CDR" refers to a CDR defined by the Kabat definition. In another embodiment disclosed herein, the CDR refers to a CDR defined by the IMGT definition. It is.

[0079] As used herein, the term "framework (FR) amino acid residues" refers to amino acids in the framework region of an Ig chain. As used herein, the term "framework region" or "FR region" refers to amino acid residues that are part of the variable region but are not part of the CDRs. Variable region frameworks thus include amino acids outside the CDRs, but are approximately 100-120 amino acids long.

[0080] The present invention also relates to "conservative modifications" in the CDR amino acid sequences of the antibodies disclosed herein. "Amino acid substitutions" i.e., amino acids that do not abolish the binding of the antibody to the antigen, i.e., PAI-1. Conservative substitutions include acid modifications. A conservative substitution is a substitution of a native amino acid residue with a non-native residue that has little or no effect on the polarity or charge of the amino acid residue at that position. For example, a conservative substitution results from the replacement of a non-polar residue in a polypeptide with any other non-polar residue. In addition, any native residue in a polypeptide can also be substituted with alanine, as previously described for "alanine scanning mutagenesis" (Cunningham et al., Science 244:1081-85 (1989)). Conservative Amino acid substitutions include the replacement of one class of amino acid with an amino acid of the same class, where a class is determined by common physicochemical amino acid side chain properties and, for example, standard Dayhoff frequencies. Determined by frequency exchange matrix or BLOSUM matrix Classes are defined by the high frequency of substitutions in homologous proteins found in nature, which are determined by the degree of substitution. Six general classes of amino acid side chains have been classified, including: Class I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gln, Glu); Class IV (His, Arg, Lys); Class V (Ile, Leu, Val, Met); and Class VI (Phe, Tyr, Trp). For example, another Substitution of Asp for a class III residue, such as Asn, Gln, or Glu, is considered a conservative substitution. Thus, the predicted nonessential amino acid residues in the PAI-1 antibody are consistent with other amino acids from the same class. The amino acid residue is replaced with a 5'-amino acid residue. Methods for identifying conservative amino acid substitutions that do not eliminate antigen binding are well known in the art (see, e.g., Brummell et al., Biochem. 32:1180, 1993; Kobayashi et al. Protein Eng. 12:879, 1999; and Burks et al. Proc. Natl. Acad. Sci. USA 94:412, 1997). General rules for conservative amino acid substitutions are shown in Table 1 below.

[0081] [Table 1]

[0082] Conservative amino acid substitutions also include non-naturally occurring amino acid residues that are typically incorporated by chemical peptide synthesis rather than by synthesis in living systems. These include peptidomimetics and other reverse or inverted forms of amino acid moieties.

[0083] Conservative modifications to the amino acid sequence (and corresponding modifications to the coding nucleotides) are expected to result in PAI-1 antibodies having functional and chemical characteristics similar to those of naturally occurring PAI-1 antibodies. In contrast, substantial modifications in the functional or chemical characteristics of a PAI-1 antibody are not expected to result in PAI-1 antibodies having functional and chemical characteristics similar to those of naturally occurring PAI-1 antibodies. (a) a change in the conformation of the molecule in a region of substitution, e.g., a sheet or helix conformation; This can be achieved by selecting substitutions that differ significantly in their effect on maintaining the structure of the molecular backbone, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Naturally occurring residues can be divided into groups based on common side chain properties: 1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; 2) Neutral hydrophilic: Cys, Ser, Thr; 3) Acidic: Asp, Glu; 4) Basic: Asn, Gln, His, Lys, Arg; 5) Residues that affect chain orientation: Gly, Pro; and 6) Aromatics: Trp, Tyr, Phe.

[0084] Non-conservative substitutions may involve the exchange of a member of one of these classes for a member of another class. Such substituted residues may be introduced into regions of the human PAI-1 antibody that are homologous with the non-human PAI-1 antibody, or into non-homologous regions of the molecule.

[0085] In certain aspects, the heavy or light chain variable region may be 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the variable region sequences disclosed herein.

[0086] As used herein, the term "specifically binds to" means to bind to a specific -6 M, 1x10 -7 M, 1x10 -8 M, 1x10 -9 M, 1x10 -10 M, 1x10 -11 M, 1x10 -12 Binds to antigen with a Kd lower than M or The term also refers to the ability of an antibody or its antigen-binding fragment to bind to an antigen with an affinity that is at least two-fold higher than its affinity for a nonspecific antigen. It encompasses and refers to the ability of the combined fragments to bind to one another.

[0087] The present disclosure also provides antibodies that competitively inhibit the binding of an antibody to an epitope disclosed herein, as determined by any method known in the art for determining competitive binding, such as the immunoassays described herein, In certain embodiments, the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50%.

[0088] As used herein, the term "antigen" refers to the binding site or epitope recognized by an antibody or antigen-binding fragment thereof.

[0089] The term "vector" as used herein is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid molecule to which it has been linked. One type of vector is a "plasmid," which refers to a circular double stranded DNA loop into which additional DNA segments can be ligated. Another type of vector is a viral vector, in which additional DNA segments are ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be used for introduction into a host cell. Upon infection, the vector may be integrated into the genome of the host cell, and thereby be replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "recombinant expression vectors" (also referred to as "recombinant expression vectors"). Generally, expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. The terms "plasmid" and "vector" can be used interchangeably. However, the invention is directed to such other forms of expression vectors, e.g. For example, viral vectors (e.g., replication defective retroviruses, adenoviruses and adenoviruses). The term "virus" is intended to include viruses and / or associated viruses, which serve equivalent functions.

[0090] A number of expression vector systems can be used for the purposes of the present invention. For example, one class of vectors utilizes DNA elements derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, baculovirus, retroviruses (RSV, MMTV, or MOMLV) or SV40 virus. Others utilize internal ribosomal It also contains the specification of polycistronic systems with binding sites. The integrated cells may be selected by introducing one or more markers which allow for selection of transfected host cells. The markers may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene may be directly linked to the DNA sequence to be expressed or may be expressed by other methods such as by introducing a marker gene into the host. They may be introduced into the same cell by co-transformation. Additional elements may also be required for optimal synthesis of mRNA. These elements may include signal sequences, splice signals, as well as transcription promoters, enhancers, and termination signals. In certain embodiments, the cloned variable region genes are inserted into an expression vector together with synthetic heavy and light chain constant region genes (e.g., human) as discussed above.

[0091] More generally, a vector or DNA sequence encoding the antibody or fragment thereof is prepared. Once the expression vector is prepared, it can be introduced into a suitable host cell; i.e., the host cell can be transformed. Introduction of the plasmid into the host cell can be accomplished by a variety of techniques well known to those of skill in the art. These include, but are not limited to, transfection (electrophoresis and electroporation), and the like. troporation), protoplast fusion, calcium phosphate precipitation, cell fusion, envelope These include cell fusion with enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG, Mammalian Expression Vectors, Chapter 24.2, pp. 470-472, Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). An embodiment disclosed herein is introduction of the plasmid into the host by electroporation. The transformed cells are grown under conditions appropriate to produce the light and heavy chains, and assayed for heavy or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, and immunoassay. These include radioimmunoassay (RIA), fluorescence activated cell sorter analysis (FACS), and immunohistochemistry. can be.

[0092] As used herein, the term "transformation" refers to the introduction of DNA into a recipient host cell, which changes the genotype and results in a change in the recipient cell. The term "company" is used in a broad sense to refer to a company or organization that provides services to companies.

[0093] A "host cell" is a cell that has been constructed using recombinant DNA techniques and contains at least one heterologous It refers to cells transformed with a vector encoding the gene. In the description of methods for isolation of polypeptides from recombinant hosts, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the antibody, unless expressly specified otherwise. In other words, recovery of polypeptides from "cells" can mean either from sedimented whole cells or from the cell culture containing both the medium and the suspended cells.

[0094] It should be understood that this term is intended to refer not only to the particular subject cell but also to the progeny of such a cell. Because certain modifications may occur in successive generations due to mutations or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are included within the scope of the term "host cell" as used herein.

[0095] As used herein, the terms "treat", "treating" and "treatment" refer to therapeutic or prophylactic measures described herein. A "treatment" method includes preventing, curing, delaying one or more symptoms of a disease or disorder, or the recurrence of a disease or disorder. In one embodiment, a subject is treated with a PAI-1-associated disease or disorder to reduce or ameliorate the severity of, or to prolong the survival of, the subject beyond that which would be expected in the absence of such treatment. or a disorder (e.g., a fibrotic disease) or susceptible to such a disease or disorder. The method involves administering to a subject an antibody or antigen-binding fragment disclosed herein.

[0096] As used herein, the term "PAI-1-associated disease or disorder" includes disease states in which altered levels or activity of PAI-1 is observed, with or without symptoms associated with the disease state. Exemplary PAI-1-associated diseases or conditions include various types of fibrosis.

[0097] As used herein, the term "effective amount" refers to an amount that is sufficient, when administered to a subject, to effect treatment, prognosis, or diagnosis of a PAI-1-related disease or disorder as described herein. A therapeutically effective amount refers to the amount of antibody or antigen-binding fragment thereof that binds to PAI-1. The dosage varies depending on the subject and disease state to be administered, the weight and age of the subject, the severity of the disease state, the method of administration, etc., and can be easily determined by one of ordinary skill in the art. The dosage for administration can range, for example, from about 1 ng to about 10,000 mg, from about 1 μg to about 5,000 mg, from about 1 mg to about 1,000 mg, or from about 10 mg to about 100 mg of the antibody or antigen-binding fragment thereof disclosed herein. The dosage regimen may be adjusted to provide the optimal therapeutic response. An effective amount is also one in which any toxic or detrimental effects (i.e., side effects) of the antibody or antigen-binding fragment thereof are minimized or outweighed by the beneficial effects.

[0098] As used herein, the term "subject" or "mammal" includes any human or non-human animal.

[0099] The term "epitope" as used herein refers to an antigenic determinant that interacts with a specific antigen-binding site in the variable region of an antibody molecule known as the paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind to different regions of an antigen and have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is one that is generated by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is one that is generated by adjacent amino acid residues in a polypeptide chain.

[0100] As used in this specification and the appended claims, the singular forms "a," "an," and " "The" includes plural references unless expressly indicated otherwise. is pointed out here.

[0101] II. Anti-PAI-1 antibody In one aspect, the present invention provides an antibody or an antigen-binding fragment thereof that specifically binds to human PAI-1. Exemplary VH, VL and CDR amino acid sequences and nucleotide sequences disclosed herein are provided. The nucleotide sequences are shown in Table 2. The CDR regions shown in Table 2 are defined by IMGT.

[0102] [Table 2]

[0103] [Table 3]

[0104] [Table 4]

[0105] [Table 5]

[0106] [Table 6]

[0107] In another embodiment, the invention provides an anti-PAI-1 antibody that competitively inhibits an antibody or antigen-binding fragment thereof that binds to the same epitope or that comprises the VH and VL region amino acid sequences shown in SEQ ID NOs: 6 and 7, respectively. Such an antibody can be, for example, a surface plasmon can be identified using conventional competitive binding assays, including resonance (SPR)-based competitive assays. .

[0108] III. Engineered Anti-PAI-1 Antibodies In certain embodiments, the anti-PAI-1 antibodies disclosed herein comprise one or more Modified forms of the anti-PAI-1 antibodies disclosed herein may include modifications known in the art. The nanoparticles may be manufactured using any known technique.

[0109] i) Reduced immunogenicity In certain embodiments, the anti-PAI-1 antibodies disclosed herein, or antigen-binding fragments thereof Segments may be modified to reduce immunogenicity using art-recognized techniques, for example, antibodies or fragments thereof may be chimerized, humanized, or deimmunized.

[0110] In one embodiment, the antibodies, or antigen-binding fragments thereof, disclosed herein may be chimeric. A chimeric antibody is an antibody in which different portions of the antibody are derived from different animal species, such as an antibody having a variable region derived from a mouse monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies or fragments thereof are known in the art. See, e.g., Morrison, Science 229:1202, 1985; Oi et al., BioTechniques 4:214, 1986; Gillies et al., J. Immunol. Methods 125:191, 1989; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816,397, which are incorporated herein by reference in their entireties. See, for example, the techniques developed for producing "chimeric antibodies" (Morrison et al., Proc. Natl. Acad. Sci. 81:851, 1984; Neuberger et al., Nature 312:604, 1984; The methods described in Takeda et al., Nature 314:452, 1985) can be used to synthesize the above molecules. The gene sequence encoding the binding specificity of the mouse anti-PAI-1 antibody molecule is required for the appropriate biological activity. It may be fused together with sequences from a human antibody molecule. As used herein, a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.

[0111] In another embodiment, the antibodies, or antigen-binding fragments thereof, disclosed herein are humanized. A humanized antibody has a binding specificity that comprises one or more complementarity determining regions (CDRs) from a non-human antibody and a framework region from a human antibody molecule. Often In addition, framework residues in the human framework regions are replaced with the corresponding residues from the CDR donor antibody to alter or improve antigen binding. Substitutions can be made by methods well known in the art, for example, by modeling the interactions of CDR and framework residues to identify framework residues important for antigen binding and by profiling specific positions. Antibodies are identified by sequence comparison to identify unusual framework residues in the antibody. See, e.g., Queen et al., US Patent No. 5,585,089; Riechmann et al., Nature 332:323, 1988, which are incorporated by reference in their entireties. For example, CDR-grafting (EP 239,400; International Publication No. WO 91 / 09967; U.S. Pat. No. 5,225,539 Nos. 5,530,101 and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular Immunology 28:489, 1991; Studnicka et al., Protein Engineering 7:805, 1994; Roguska. et al., PNAS 91:969, 1994), and chain shuffling (U.S. Pat. No. 5,565,332). The humanized polypeptides can be produced using a variety of techniques known in the art, including those disclosed herein.

[0112] In certain embodiments, humanization methods based on the influence of molecular mobility of antibodies during or upon immune recognition are used (see International Publication No. WO 2009 / 032661, which is incorporated herein by reference in its entirety). Protein mobility is related to the molecular movement of protein molecules. Protein mobility is the ability of an entire protein, a part of a protein, or a single amino acid to adopt a population of conformations that are significantly different from each other. Information about protein mobility can be obtained by performing protein X-ray crystallography experiments (see, e.g., Kundu et al., Biophys. J. 83:723, 2002), nuclear magnetic resonance experiments (see, e.g., Freedberg et al., J. Am. Chem. Soc. 120:7916, 1998), or molecular dynamics (MD) simulations. A MD simulation of a protein is performed on a computer, and by calculating the physical interactions of the atoms with each other it is possible to determine the motion of all protein atoms over a period of time. The output of the MD simulation is the trajectory of the examined protein over the period of the simulation. A trajectory is a set of protein conformations, also called snapshots, which are sampled periodically, for example every 1 picosecond (ps), over the period of the simulation. By analyzing the set of snapshots, the mobility of protein amino acid residues can be quantified. A mobile residue is thus one that fits into a set of different conformations in the context of the polypeptide in which it resides. MD methods are known in the art, see for example Brooks et al. "Proteins: A Theoretical Perspective of Dynamics, Structure and Thermodynamics" (Wiley, New York, 1988). Amber (Case et al. J. Comp. Chem. 26:1668, 2005; Brooks et al. J. Comp. Chem. 4:187, 1983; and MacKerell et al. (1998) "The Encyclopedia of Computational Chemistry" vol. 1:271-177, Schleyer et al., eds. Chichester: John Wiley & Sons) or I Some software, such as mpact (see Rizzo et al. J. Am. Chem. Soc.; 122:12898, 2000), allows for MD simulations.

[0113] It has been shown that most protein complexes share relatively large flat buried surfaces, and the mobility of the binding partners provides the basis for their flexibility, allowing them to conformationally fit with each other (Sundberg and Mariuzza, Structure 8, R137-R142, 2000). Thus, examples of "induced fit" have been shown to play a major role in protein-protein interactions. Furthermore, there is a growing body of data showing that proteins do in fact bind ligands of diverse shapes, sizes and compositions (Protein Science 11:184-187, 2002), and that conformational diversity appears to be a crucial component of the ability to recognize different partners (James et al., Science 299:1362, 2003). Flexible residues are involved in the binding of protein-protein partners (Grunberg et al., Structure 14, 683, 2006).

[0114] Mobile residues may be recognized by memory B cells and induce immunogenic responses. Antibodies can adopt a variety of conformations resulting in a set of interacting regions. Thus, antibodies can be humanized by modifying a number of residues from the framework so that the set of conformations and recognition regions exhibited by the modified antibody resembles as closely as possible those adopted by human antibodies. This can be achieved by: (1) constructing a homology model of the parent mAb; This can be achieved by: (1) constructing a non-human antibody molecule and running MD simulations; (2) analyzing the flexible residues and identifying the most flexible residues of the non-human antibody molecule, as well as identifying residues or motifs that may be the source of heterogeneity or degradation reactions; (3) identifying a human antibody that exhibits a set of recognition regions that are most similar to the parent antibody; (4) determining the flexible residues to be mutated, residues or motifs that are the source of heterogeneity and degradation and may also be mutated; and (5) modifying by checking for the presence of known T-cell or B-cell epitopes. Flexible residues can be found using MD calculations using an implicit solvent model as taught herein, which accounts for the interaction of the water solvent with protein atoms over the duration of the simulation.

[0115] Once a set of flexible residues has been identified within the variable light and heavy chains, a set of human heavy and light chain variable region frameworks that closely resemble the antibody of interest is identified. This can be done by, for example, using a BLAST search for the set of flexible residues against a database of antibody human germline sequences. Also, the kinetics of the parent mAb can be compared to the kinetics of a library of germline standard structures. Alternatively, the CDR residues and adjacent residues may be selected to provide a high affinity for the antigen. The flexible residues are then replaced.

[0116] In cases where some human residues show similar homology, the selection is also driven by the nature of the residues that may affect the solution behavior of the humanized antibody. For example, polar residues are often located in exposed mobile loops on hydrophobic residues. Residues that are sources of instability and heterogeneity are also mutated, even if they are found in the CDRs. This is due to the fact that sulfoxide formation can arise from oxygen radicals, such as exposed methionine and the Asp-Pro dipeptide bond. Proteolytic cleavage of acid-labile bonds (Drug Dev. Res. 61:137, 2004), exposing Deamidation sites found with asparagine residues followed by small amino acids such as Gly, Ser, Ala, His, Asn, or Cys (J. Chromatog. 837:35, 2006) and N-glycosylation sites For glycosylation sites (Asn-X-Ser / Thr), for example, Asn-X-Ser / Thr sites. Typically, exposed methionines are replaced with Leu, exposed asparagines are replaced with glutamine or aspartic acid, or the subsequent residue is altered. For glycosylation sites (Asn-X-Ser / Thr), Asn or Ser / Thr The residue is changed.

[0117] The resulting composite antibody sequence is checked for the presence of known B-cell or linear T-cell epitopes. For example, a search is performed using the publicly available Immune Epitope Data Base (IEDB) (PLOS Biol. (2005) 3(3)e91). If a known epitope is found within the composite sequence, another set of human sequences is imported and substituted. Thus, the surface area of ​​U.S. Pat. No. 5,639,641 is Unlike the grafting method, both B cell-mediated and T cell-mediated immunogenic responses are addressed by this method. This method also avoids the problem of activity loss that can be observed with CDR grafting (U.S. Pat. No. 5,530,101). Furthermore, stability and solubility issues are also considered during the engineering and selection process, resulting in antibodies optimized for low immunogenicity, high antigen affinity, and improved biophysical properties.

[0118] In some embodiments, de-immunization involves the deactivation of an antibody or its antigen binding. As used herein, the term "deimmunization" refers to the modification of an antibody, or antigen-binding fragment thereof, to alter T cell epitopes, thereby reducing the immunogenicity of the antibody or antigen-binding fragment thereof. For example, the VH and VL sequences from a starting antibody can be analyzed and the complementarity determining regions (CDRs) and other key regions within the sequences can be modified to include, but are not limited to, modifications to the VH and VL sequences (e.g., International Publication Nos. WO9852976A1 and WO0034317A2). A human T cell epitope "map" can be generated from each V region showing the location of the epitopes in relation to different residues. Individual T cell epitopes from the T cell epitope map are analyzed to identify alternative amino acid substitutions with low risk of altering the activity of the final antibody. A variety of alternative VH and VL sequences containing combinations of amino acid substitutions can be designed and these sequences can be used to generate a variety of PAI-1 specific antibodies for use in the diagnostic and treatment methods disclosed herein. The modified V regions and human V-regions are then engineered into a recombinant human antibody or fragment thereof and then tested for function. Typically, between 12 and 24 mutant antibodies are generated and tested. The complete heavy and light chain genes, including the C region, were cloned into expression vectors and The subsequent plasmids are introduced into cell lines for production of complete antibodies. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variants are identified.

[0119] ii) Effector Function and Fc Modifications The anti-PAI-1 antibodies disclosed herein may comprise an antibody constant region (e.g., an IgG constant region, a human IgG constant region, a human IgG1 or IgG4 constant region) that mediates one or more ejector functions. For example, binding of the C1 component of complement to an antibody constant region may activate the complement system. Complement activation is important in opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may also be involved in autoimmune hypersensitivity. Additionally, antibodies may mediate the expression of various antigens via the Fc region, with Fc receptor binding sites on the antibody Fc region binding to Fc receptors (FcR) on cells. It binds to a receptor on a cell. There are numerous Fc receptors specific for different classes of antibodies, including IgG (gamma receptors), IgE (epsilon receptors), IgA (alpha receptors) and IgM (mu receptors). Binding of antibodies to Fc receptors on the cell surface mediates the phagocytosis and degradation of antibody-coated particles, clearance of immune complexes, and lysis of antibody-coated target cells by killer cells (antibody-dependent cell-mediated cytotoxicity). These cells induce a number of important and diverse biological responses, including proliferation, metabolism, and cell death (called ADCC), release of inflammatory mediators, placental transfer, and regulation of immunoglobulin production. In certain embodiments, the antibodies or fragments thereof disclosed herein are Fc- In an alternative embodiment, the anti-PAI-1 antibodies disclosed herein bind to gamma receptors. lacks one or more effector functions (e.g., ADCC activity) or lacks Fc It may contain a constant region that is unable to bind to a receptor.

[0120] Certain embodiments disclosed herein include at least one amino acid sequence in one or more constant region domains. Decreased or enhanced effector function, ability to non-covalently dimerize, localize to a specific site in the body (e.g., at the site of a tumor or at a specific time point) when compared to a complete unmodified antibody that has at least one amino acid deleted or is otherwise of approximately the same immunogenicity. Anti-PAI-1 antibodies that have been modified to produce desirable biochemical characteristics, such as increased ability to bind to PAI-1, decreased plasma half-life, or increased serum half-life. Certain antibodies, or fragments thereof, for use in the diagnostic and treatment methods described herein are similar to immunoglobulin heavy chains but lack at least a portion of one or more heavy chain domains. For example, in certain antibodies, an entire domain of the constant region of the modified antibody is deleted, e.g., all or part of the CH2 domain. A part is deleted.

[0121] In certain other embodiments, the anti-PAI-1 antibody comprises constant regions derived from different antibody isotypes. In another embodiment, the anti-PAI-1 antibody comprises a chimeric hinge (i.e., a hinge portion derived from a hinge domain of a different antibody isotype, e.g., a hinge comprising an upper hinge domain from an IgG4 molecule and an IgG1 middle hinge domain). In one embodiment, the anti-PAI-1 antibody comprises an Fc region or other portion thereof from a human IgG4 molecule and a Ser228Pro mutation (Kabat numbering) in the core hinge region of that molecule.

[0122] In certain anti-PAI-1 antibodies, the Fc portion can be linked to effectors using techniques known in the art. Constant region modifications may be mutated to increase or decrease their function. For example, deletion or inactivation of constant region domains (by point mutation or other means) may decrease Fc receptor binding of circulating modified antibodies, thereby increasing tumor localization. In other cases, constant region modifications consistent with the present invention may moderate complement binding and thus decrease serum half-life and non-specific binding of conjugated cytotoxins. Still other modifications of the constant region may be used to modify disulfide bonds or oligosaccharide moieties, allowing enhanced localization due to increased antigen specificity or mobility. The resulting physiological profile, such as tumor localization, biodistribution and serum half-life, bioavailability and other biochemical effects of the modifications can be readily measured and quantified using well-known immunological techniques without undue experimentation.

[0123] In certain embodiments, the Fc domain used in the antibodies disclosed herein is an Fc variant. As used herein, the term "Fc variant" refers to an Fc domain that has at least one amino acid substitution compared to the wild-type Fc domain from which it is derived. For example, the Fc domain is derived from a human IgG1 antibody, and the human IgG1 Fc domain The Fc variants of this invention contain at least one amino acid substitution compared to the Fc domain.

[0124] The amino acid substitutions in the Fc variants may be located anywhere within the Fc domain (i.e., at any EU convention amino acid position). In one embodiment, the Fc variants comprise a substitution at an amino acid position located in the hinge domain or a portion thereof. In another embodiment, the Fc variants comprise a substitution at an amino acid position located in the CH2 domain or a portion thereof. In one embodiment, the Fc variant comprises an amino acid sequence located at a CH3 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution located in the CH4 domain or a portion thereof. Contains substitutions at amino acid positions.

[0125] The antibodies disclosed herein may have improved effector function or FcR binding (e.g., Any art-recognized Fc variant known to confer immunosuppressive properties (decreased or enhanced) may be used. The above Fc variants are described, for example, in International PCT Publication Nos. WO88 / 07089A1, WO96 / 14339A1, WO98 / 05787A1, WO98 / 23289A1, WO99 / 51642A1, WO99 / 58572A1, WO00 / 09560A2, WO00 / 32767A1, WO00 / 42072A2, WO02 / 44215A2, WO02 / 060919A2, WO03 / 074569A2, WO04 / 016750A2, WO04 / 029207A2, WO04 / 035752A2, WO04 / 063351A2, WO04 / 074455A2, WO04 / 099249A2, WO05 / 040217A2, WO05 / 070963A1, WO05 / 077981A2, WO05 / 092925A2, WO05 / 123780A2, WO06 / 019447A1, WO06 / 047350A2, and WO06 / 085967A2 or U.S. Patent Nos. 5,648,260; 5,739,277; 5,834,250; 5,869,046; 6,096,871; 6,121,022; 6,194,551; 6,242 Nos. 6,195; 6,277,375; 6,528,624; 6,538,124; 6,737,056; 6,821,505; 6,998,253; and 7,083,784, each of which is incorporated herein by reference. In one exemplary embodiment, the antibody disclosed herein comprises an amino acid substitution at EU position 268 (e.g., H268D In another exemplary embodiment, the antibodies disclosed herein may comprise an Fc variant comprising an amino acid substitution at EU position 239 (e.g., S239D or S239E) or an amino acid substitution at EU position 332 (e.g., I332D or I332Q).

[0126] In certain embodiments, the antibodies disclosed herein may comprise Fc variants that contain amino acid substitutions that alter the antigen-independent effector functions of the antibody, in particular the circulating half-life of the antibody. Such antibodies exhibit increased or decreased binding to FcRn when compared to antibodies lacking these substitutions, and therefore have increased or decreased half-lives in serum, respectively. Fc variants with improved affinity for FcRn are expected to have longer serum half-lives, and such molecules have utility in methods of treating mammals where a long half-life of the administered antibody is desirable, e.g., for treating chronic diseases or disorders. In contrast, Fc variants with reduced FcRn binding affinity are expected to have shorter half-lives, and such molecules are also useful for administration to mammals where, for example, a reduced circulation time may be advantageous, e.g., for in vivo diagnostic imaging methods, or in situations where the starting antibody has toxic side effects if present in the circulation for an extended period of time. Fc variants with reduced FcRn binding affinity are also less likely to cross the placenta, and therefore are also useful in treating diseases or disorders in pregnant women. Additionally, other applications in which reduced FcRn binding affinity may be desirable include applications in which brain, kidney or liver localization is desirable. In one exemplary embodiment, the modified antibodies disclosed herein exhibit reduced transport from the vasculature across the epithelium of the renal glomerulus. In another embodiment, the modified antibodies disclosed herein exhibit reduced transport across the blood-brain barrier (BBB) ​​from the brain into the vascular space. In one embodiment, the antibody with altered FcRn binding comprises an Fc domain with one or more amino acid substitutions in the "FcRn-binding loop" of the Fc domain. The FcRn-binding loop is composed of amino acid residues 280 to 299 (according to Kabat numbering). FcRn binding activity Exemplary amino acid substitutions that alter are disclosed in International PCT Publication No. WO05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, the antibodies disclosed herein, or fragments thereof, comprise an Fc domain having one or more of the following substitutions: V284E, H285E, N286D, K290E, and S304D (Kabat numbering).

[0127] In other embodiments, antibodies for use in the diagnostic and treatment methods described herein have a constant region that has been altered to reduce or eliminate glycosylation, e.g., an IgG1 or IgG4 heavy chain constant region. For example, the antibodies disclosed herein can also include Fc variants that contain amino acid substitutions that alter the glycosylation of the antibody. For example, the Fc variants can have reduced glycosylation (e.g., N- or O-linked glycosylation). In an exemplary embodiment, the Fc variants have an N-linked glycosylation that is normally found at amino acid position 297 (EU numbering). In another embodiment, the antibody comprises an amino acid substitution near or within a glycosylation motif, e.g., an N-linked glycosylation motif containing the amino acid sequence NXT or NXS. In a particular embodiment, the antibody comprises an Fc variant comprising an amino acid substitution at amino acid position 228 or 299 (EU numbering). In a more particular embodiment, the antibody comprises an IgG1 or IgG4 constant region comprising S228P and T299A mutations (EU numbering).

[0128] Exemplary amino acid substitutions that confer reduced or altered glycosylation are disclosed in International PCT Publication No. WO05 / 018572, which is incorporated herein by reference. In certain embodiments, the antibodies disclosed herein, or fragments thereof, are modified to remove glycosylation. Such antibodies or fragments thereof are referred to as "agly" antibodies, or The fragments thereof may be referred to as "agly" antibodies. Without being bound by theory, It is believed that the "Agri" antibody, or fragment thereof, may have an improved safety and stability profile in vivo. An exemplary Agri antibody, or fragment thereof, comprises an aglycosylated Fc region of an IgG4 antibody, which lacks Fc-effector functions, thereby reducing the risk of Fc-mediated toxicity to normal vital organs expressing PAI-1. In yet another embodiment, the antibody or fragment thereof disclosed herein comprises an altered glycan. For example, the antibody may have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., afucosylated. In another embodiment, the antibody may have an altered number of sialic acid residues on the N-glycan at Asn297 of the Fc region.

[0129] iii) Covalent bond The anti-PAI-1 antibodies disclosed herein are directed to a covalent bond that binds the specificity of the antibody to its cognate epitope. The antibody may be modified by covalent attachment of a molecule to the antibody so as not to interfere with the covalent binding of the antibody to the antibody. For example, and not by way of limitation, the antibodies or fragments thereof disclosed herein may be modified by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization with protecting / blocking groups, or the like. The polypeptides may be modified by proteolytic cleavage, linkage to cellular ligands or other proteins, etc. Any of a number of chemical modifications may be made by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, etc. Additionally, derivatives may be modified by one or more It may contain one or more non-classical amino acids.

[0130] The antibodies or fragments thereof disclosed herein may further be recombinantly fused to heterologous polypeptides at the N-terminus or C-terminus or chemically conjugated (including covalent and non-covalent) to polypeptides or other compositions. For example, anti-PAI-1 antibodies may be recombinantly fused or conjugated to molecules useful as labels in detection assays, and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, for example, International PCT Publication Nos. WO 92 / 08495; WO 91 / 14438; WO 89 / 12624; U.S. Patent No. 5,314,995; and EP 396,387.

[0131] The anti-PAI-1 antibodies may be modified to increase their in vivo half-life or by other methods known in the art. The antibodies may be fused to heterologous polypeptides for use in immunoassays to determine the binding of PAI-1 to a target polypeptide. For example, in one embodiment, PEG can be attached to the anti-PAI-1 antibodies disclosed herein to increase their half-life in vivo (Leong, SR, et al., Cytokine 16:106, 2001; Adv. in Drug Deliv. Rev. 54:531, 2002; or Weir et al., Biochem. Soc. Transactions 30:512, 2002).

[0132] Additionally, the anti-PAI-1 antibodies disclosed herein may be used in combination with other antibodies to facilitate their purification or detection. In certain embodiments, the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in the pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif., 91311), among others. Many of these are commercially available. Hexa-histidine provides for convenient purification of the fusion protein, as described, for example, in Gentz ​​et al., Proc. Natl. Acad. Sci. USA 86:821-824, 1989. Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 37:767, 1984), and the "Flag" tag.

[0133] The anti-PAI-1 antibodies disclosed herein can be used in unconjugated form or can be, for example, The anti-PAI-1 antibodies disclosed herein may be conjugated to at least one of a variety of molecules to improve the therapeutic properties of the antibody, to facilitate target detection, or for imaging or treatment of the patient. The anti-PAI-1 antibodies disclosed herein may be labeled or conjugated either before or after purification, if purification is performed. In particular, the anti-PAI-1 antibodies disclosed herein may be used as therapeutic agents, prodrugs, Conjugated to peptides, proteins, enzymes, viruses, lipids, biological response modifiers, drugs, or PEG It can be done.

[0134] The invention further encompasses anti-PAI-1 antibodies conjugated to diagnostic or therapeutic agents. Anti-PAI-1 antibodies may be used diagnostically, for example, to determine the effectiveness of a given treatment or prophylactic regimen, as part of a clinical testing procedure, for example, to monitor the onset or progression of an immune cell disorder (e.g., CLL). Detection may be accomplished by coupling the anti-PAI-1 antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, various positron emitting metals using positron emission tomography, and non-radioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be attached to antibodies for use as diagnostics in accordance with the present invention. Non-limiting examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase. non-limiting examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; non-limiting examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; A non-limiting example of a luminescent material is luminol; a non-limiting example of a bioluminescent material is Examples include luciferase, luciferin, and aequorin; and suitable radioactive materials. Non-limiting examples of radioactive substances include 125I, 131I, 111In, or 99Tc.

[0135] The anti-PAI-1 antibodies for use in the diagnostic and treatment methods disclosed herein are Cytotoxins (e.g., radioisotopes, cytotoxic drugs, or toxins), therapeutic agents, cytostatic agents, biotoxins, prodrugs, peptides, proteins, enzymes, viruses, lipids, biological response modifiers, pharmaceuticals, immunologically active ligands (e.g., lymphokines or derived molecules that inhibit tumor cells), other antibodies that bind to both mitochondrial cells and effector cells such as T cells, or are conjugated to PEG obtain.

[0136] In another embodiment, the anti-PAI-1 antibodies for use in the diagnostic and treatment methods disclosed herein can be conjugated to a molecule that reduces tumor cell growth. The disclosed compositions may include antibodies or fragments thereof coupled to drugs or prodrugs. Still other embodiments disclosed herein include the use of antibodies or fragments thereof coupled to specific biotoxins or their cytotoxic fragments, such as ricin, gelonin, Pseudomonas exotoxin, or diphtheria toxin. The choice of which conjugated or unconjugated antibody to use depends on the type and stage of the cancer, the use of adjunctive treatments (e.g., chemotherapy or external radiation), and the condition of the patient. Of course, one of skill in the art can readily make such a choice in view of the teachings herein.

[0137] Of course, in previous studies, isotope-labeled antitumor antibodies have been successfully used to destroy tumor cells in animal models and in some cases in humans. Exemplary radioisotopes include: 90Y, 125I, 131I, 123I, 111In, 105Rh, 153Sm, 67Cu, 67Ga, 166Ho, 177Lu, 186Re and 188Re. Radionuclides act by producing ionizing radiation that causes multiple strand breaks in nuclear DNA, resulting in cell death. Isotopes used to manufacture therapeutic conjugates typically produce high-energy alpha or beta particles with short trajectories. Such radionuclides kill cells to which they are in close proximity, such as tumor cells to which the conjugate is attached or has entered. They have little or no effect on cells to which they are not localized. Radionuclides are essentially non-immunogenic.

[0138] IV. Expression of Anti-PAI-1 Antibodies, or Antigen-Binding Fragments Thereof The genetic material isolated as described above was manipulated to obtain the anti-PAI-1 antibodies disclosed herein. The genes are then typically inserted into an expression vector for introduction into a host cell which can be used to produce desired quantities of the claimed antibody, or fragment thereof.

[0139] In other embodiments, the anti-PAI-1 antibodies or fragments thereof disclosed herein The polypeptides may be expressed using polycistronic constructs. In such expression systems, multiple gene products of interest, such as heavy and light chains of an antibody, may be produced from a single polycistronic construct. These systems advantageously use internal ribosome entry sites (IRES) to obtain relatively high levels of the polypeptides disclosed herein in eukaryotic host cells. Suitable IRES sequences are disclosed in U.S. Pat. No. 6,193,980, incorporated herein by reference. Those skilled in the art will appreciate that such expression systems may be used to efficiently produce the full range of polypeptides disclosed in the present application.

[0140] In one embodiment, the host cell line used for antibody expression is of mammalian origin; one of skill in the art will recognize the particular host cell line that is most suitable for the desired gene product to be expressed therein. The cell line can be determined. Exemplary host cell lines include, but are not limited to, DG44 and DUXB11 (Chinese hamster ovary lines, DHFR minus), HELA (human cervical carcinoma), CVI (monkey kidney line), COS (a derivative of CVI containing the SV40 T antigen), R1610 (Chinese hamster line), and ES cell lines. fibroblasts), BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), 293 (human kidney). In one embodiment, the cell lines provide altered glycosylation, such as afucosylation, of antibodies expressed therefrom (e.g., PER.C6.RTM. (Crucell) or a FUT8-knockout CHO cell line (Potelligent.RTM. Cells) (Biowa, Princeton, NJ)). In certain specific embodiments, NSO cells may be used. CHO cells may be used. Host cell lines are typically available from commercial services, the American Tissue Culture Collection, or from published literature.

[0141] In vitro production allows for scale-up to obtain large amounts of the desired polypeptide. Techniques for the cultivation of mammalian cells under tissue culture conditions are known in the art and include homogenous suspension cultures in airlift reactors or continuous stirred reactors, or immobilized or entrapped cell cultures, for example in hollow fibers, microcapsules, on agarose microbeads or ceramic cartridges. If necessary or desired, the solution of the polypeptide can be purified by conventional chromatographic methods, such as gel filtration, ion exchange chromatography, chromatography on DEAE-cellulose or (immuno-) affinity chromatography.

[0142] The genes encoding the anti-PAI-1 antibodies, or fragments thereof, disclosed herein also include The transformed cells may be non-mammalian cells, such as bacteria, yeast or plant cells. In this connection, it will be appreciated that unicellular non-mammalian microorganisms, such as various bacteria, may also be transformed; i.e., those capable of growing in culture or fermentation. Bacteria that are susceptible include members of the family Enterobacteriaceae, such as Escherichia coli or Salmonella; the family Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus, and Haemophilus influenzae. It will further be appreciated that when expressed in bacteria, the polypeptides can become part of inclusion bodies. The polypeptides must be isolated, purified, and then assembled into functional molecules.

[0143] In addition to prokaryotes, eukaryotic microbes can also be used. Saccharomyces cerevisiae, or common baker's yeast, is the most widely used yeast, although a number of other strains are commonly available. It is most commonly used among prokaryotic microorganisms. For expression in yeast, for example, plasmid YRp7 (Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al., Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)) is commonly used. This plasmid already contains the TRP1 gene, which creates a selection marker for yeast mutants that lack the ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1 (Jones, Genetics, 85:12 (1977)). The presence of the trpl lesion as a characteristic of the yeast host cell genome then provides an effective environment for detecting transformation by growth in the absence of tryptophan.

[0144] V. Pharmaceutical Formulations and Methods of Administration of Anti-PAI-1 Antibodies In another aspect, the present invention provides a pharmaceutical composition comprising an anti-PAI-1 antibody, or a fragment thereof. Provide something.

[0145] Methods of producing and administering the antibodies or fragments thereof disclosed herein to a subject are well known or readily determined by one of skill in the art. The route of administration of the antibodies or fragments thereof disclosed herein can be oral, parenteral, by inhalation or topical. The term parenteral as used herein includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal administration. Intravenous, intraarterial, subcutaneous and intramuscular forms of parenteral administration may be used in certain embodiments. Although all these forms of administration are expressly contemplated within the scope disclosed herein, the form for administration will be an injectable solution, particularly for intravenous or intraarterial injection or infusion. Typically, suitable pharmaceutical compositions for injection will include a buffer (e.g., acetate, acetic acid ... , phosphate or citrate buffers), surfactants (e.g., polysorbates), and optionally stabilizers (e.g., human albumin). However, other suitable buffers may be used consistent with the teachings herein. In the methods, the polypeptide can be delivered directly to the site of the harmful cell population, thereby increasing the exposure of the affected tissue to the therapeutic agent.

[0146] Preparations for parenteral administration include sterile aqueous or non-aqueous solutions, suspensions, and emulsions. Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcoholic / aqueous solutions, emulsions, or suspensions, including saline and buffered media. In the present invention, pharma- ceutically acceptable carriers include, but are not limited to, 0.01 to 0.1 M (e.g., 0.05 M) phosphate buffer or 0.8% saline. Other common parenteral Oral vehicles include sodium phosphate solutions, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils. Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers, such as those based on Ringer's dextrose, and the like. Preservatives and other additives may also be present, such as, for example, antibacterial agents, antioxidants, chelating agents, and inert gases and the like. More particularly, pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (water soluble in water) or dispersions and sterile powders for the extemporaneous preparation of injection solutions or dispersions. In such cases, the composition must be sterile and should be fluid to the extent that easy syringability exists. The carrier should be stable in water and, in one embodiment, preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier may be, for example, water, ethanol, polyol (e.g., glycol, cellulose, propylene glycol, and liquid polyethylene glycol), and The composition may be a solvent or dispersion medium containing a suitable mixture of the above. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In certain embodiments, isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride, are included in the composition. Prolonged absorption of the injectable compositions can be achieved by the use of agents which delay absorption, for example, aluminum monostearate. This can be achieved by including gum and gelatin in the composition.

[0147] In either case, a sterile injectable solution is prepared containing the active compound (e.g., by itself or in combination with other active agents). The dispersions can be prepared by incorporating the required amount of the combined antibody) in a suitable solvent with one or a combination of ingredients enumerated herein, as required, followed by sterile filtration. In general, dispersions are prepared by adding the active compound to a sterile vehicle containing a basic dispersion medium and the required other ingredients from those enumerated above. In the case of sterile powders for the preparation of sterile injectable solutions, the preparation method can be vacuum drying and freeze-drying, which results in a powder of the active ingredient and any additional desired ingredients from its previously sterile-filtered solution. The preparations for injection are processed, filled into containers such as ampoules, bags, bottles, syringes or vials, and sealed under aseptic conditions according to methods known in the art. In addition, the preparations can be packaged and sold in the form of a kit, as described in co-pending U.S. application Ser. No. 09 / 259,337 and U.S. application Ser. No. 09 / 259,338, each of which is incorporated herein by reference. Such an article of manufacture, in one embodiment, has a label or package insert indicating that the attached composition is useful for treating a subject suffering from or predisposed to an autoimmune or neoplastic disorder.

[0148] The effective dosage of the stabilized antibodies or fragments thereof disclosed herein for the treatment of the above conditions will vary depending on many different factors, including the means of administration, the target site, the physiological state of the patient, whether the patient is human or animal, other drugs administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, but non-human animals, including transgenic mammals, may also be treated. Treatment dosages may be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0149] For passive immunization with the antibodies disclosed herein, dosages can range from, for example, about 0.0001 to 100 mg / kg, and more usually 0.01 to 5 mg / kg (e.g., 0.02 mg / kg, 0.25 mg / kg, 0.5 mg / kg, 0.75 mg / kg, 1 mg / kg, 2 mg / kg, etc.) of host body weight. For example, dosages can be 1 mg / kg body weight or 10 mg / kg body weight or within the range of 1 to 10 mg / kg, or in certain embodiments at least 1 mg / kg. Intermediate doses within the above ranges are also contemplated to be within the ranges disclosed herein.

[0150] Subjects may be administered such doses daily, every other day, weekly, or according to any other schedule determined by empirical analysis. Exemplary treatments involve administration in multiple dosages over a long period of time, e.g., for at least six months. Further exemplary treatment regimens include: Exemplary dosing schedules include administration once every two weeks, once a month, or once every 3 to 6 months. Schedules include 1-10 mg / kg or 15 mg / kg on consecutive days, 30 mg / kg every other day, or 60 mg / kg weekly. In some methods, two or more antibodies with different binding specificities are administered. monoclonal antibodies may be administered simultaneously, in which case the dosage of each antibody administered may be within the ranges indicated.

[0151] The antibodies or fragments thereof disclosed herein may be administered on multiple occasions. The intervals between single doses may be, for example, daily, weekly, monthly or yearly. The intervals may also be irregular as indicated by measuring the blood levels of the polypeptide or target molecule in the patient. In some methods, the dosage is adjusted to achieve a particular plasma antibody or toxin concentration, for example, 1-1000ug / ml or 25-300ug / ml. Alternatively, the antibodies or fragments thereof may be administered as a sustained release formulation, in which case less frequent administration is required. The dosage and frequency will vary depending on the half-life of the antibody in the patient. In general, humanized antibodies exhibit the longest half-life, followed by chimeric and non-human antibodies. In one embodiment, the antibodies or fragments thereof disclosed herein may be administered in unconjugated form. In another embodiment, the antibodies disclosed herein may be administered in conjugated form. In yet another embodiment, the antibodies or fragments thereof disclosed herein can be administered in unconjugated form and then in conjugated form, or vice versa.

[0152] The dosage and frequency of administration vary depending on whether the treatment is prophylactic or therapeutic. In prophylactic applications, compositions containing the antibodies of the present invention or a cocktail thereof are administered to a patient not already in a disease state to enhance the patient's resistance. Such an amount is defined to be a "prophylactically effective dose." In this use, the exact amount again depends on the patient's state of health and general immunity, but generally ranges from 0.1 to 25 mg per dose, particularly 0.5 to 2.5 mg per dose. Relatively low dosages are administered at relatively infrequent intervals over an extended period of time. Some patients continue to receive treatment for the rest of their lives.

[0153] In therapeutic applications, relatively high dosages at relatively short intervals (e.g., about 1-400 mg / kg of antibody per dose, 5-25 mg for radioimmunoconjugates are more commonly used, and higher doses is used in the cytotoxin-drug conjugate molecule) may be required until disease progression is reduced or terminated, and in certain embodiments, until the patient shows partial or complete remission of disease symptoms. Thereafter, the patient may be administered a prophylactic regimen.

[0154] In one embodiment, a subject may be treated with a nucleic acid molecule (e.g., in a vector) encoding a polypeptide disclosed herein. Doses for nucleic acids encoding the polypeptides range from about 10 ng to 1 g, 100 ng to 100 mg, 1 ug to 10 mg, or 30 to 300 ug DNA per patient. Doses for infectious viral vectors range from 10 to 100 or more viruses per dose. It changes with illus particles.

[0155] Therapeutic agents may be administered by parenteral, topical, intravenous, oral, subcutaneous, intraarterial, intracranial, intraperitoneal, intranasal or intramuscular means for prophylactic or therapeutic treatment. Intramuscular injection or intravenous infusion may be used for administration of the antibodies disclosed herein. In some methods, the therapeutic antibody or fragment thereof is injected directly into the skull. In some methods, the antibody or fragment thereof is administered in a sustained release composition or Medipad. TM Devices like It is administered as a device.

[0156] The agents disclosed herein can optionally be administered in combination with other agents that are effective in treating (e.g., prophylactically or therapeutically) the disorder or condition in need of treatment. The additional agents are those recognized in the art and normally administered for the particular disorder.

[0157] An effective single treatment dosage (i.e., a therapeutically effective amount) of the 90Y-labeled antibodies disclosed herein is , ranging between about 5 and about 75 mCi, and in one embodiment, ranging between about 10 and about 40 mCi. Effective single treatment non-marrow ablative dosages of 131I-labeled antibodies range between about 5 and about 70 mCi, and in one embodiment, between about 5 and about 40 mCi. Effective single treatment ablative dosages of 131I-labeled antibodies (i.e., may require autologous bone marrow transplantation) range between about 30 and about 600 mCi, and in one embodiment, between about 50 and less than about 500 mCi. In conjunction with chimeric modified antibodies, due to their longer circulating half-life compared to mouse antibodies, effective single treatment non-marrow ablative dosages of iodine-131-labeled chimeric antibodies range between about 5 and about 40 mCi, and in one embodiment, is less than about 30 mCi. For example, imaging standards for 111In labeling are typically less than about 5 mCi.

[0158] Although most clinical experience has been gained with 131I and 90Y, other radiolabels are known in the art. and have been used for similar purposes. Still other radioisotopes are used for imaging. Additional radioisotopes that are compatible with the scope of the present invention include, but are not limited to, I, 123I, 125I, 32P, 57Co, 64Cu, 67Cu, 77Br, 81Rb, 81Kr, 87Sr, 113In, 127Cs, 129Cs, 132I, 197Hg, 203Pb, 206Bi, 177Lu, 186Re, 212Pb, 212Bi, 47Sc, 105Rh, 109Pd , 153Sm, 188Re, 199Au, 225Ac, 211A 213Bi. In this regard, alpha, gamma, and beta emitters are all compatible with the present invention. Moreover, in view of the present disclosure, it is believed that one of ordinary skill in the art will be able to readily determine, without undue experimentation, which radionuclides are compatible with a selected course of treatment. To this end, further radionuclides already used in clinical diagnosis include 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, In addition to 111In, antibodies have also been developed for potential use in targeted immunotherapy. These radionuclides include 188Re and 186Re, and to a lesser extent 199Au and 67Cu. U.S. Patent No. 5,460,785 provides further information regarding such radioisotopes. No. 6,399,411, and is incorporated herein by reference.

[0159] As previously discussed, the antibodies or fragments thereof disclosed herein may be administered in a pharmacologic effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be appreciated that the disclosed antibodies or fragments thereof are formulated to facilitate administration and promote stability of the active agent. In certain embodiments, the pharmaceutical compositions according to the present invention comprise a pharmacologic acceptable non-toxic sterile carrier, such as saline, non-toxic buffers, preservatives, and the like. For purposes of this application, a pharmacologic effective amount of the antibodies disclosed herein, conjugated or unconjugated to a therapeutic agent, will be held to mean an amount sufficient to achieve effective binding to the target and achieve a benefit, for example, to ameliorate symptoms of a disease or disorder, or to detect a substance or cell. In the case of tumor cells, the polypeptides, in certain embodiments, can interact with selected immunoreactive antigens on neoplasms or immunoreactive cells, resulting in increased killing of those cells. It will be appreciated that the pharmaceutical compositions disclosed herein may be administered in single or multiple doses to provide a pharmacologic effective amount of the polypeptide.

[0160] In accordance with the scope of the present disclosure, the antibodies disclosed herein may be administered to humans or other animals in amounts sufficient to produce a therapeutic or prophylactic effect according to the treatment methods described above. The polypeptides disclosed herein may be administered to such humans or other animals in conventional dosage forms prepared by mixing the antibodies disclosed herein with conventional pharma- ceutically acceptable carriers or diluents in accordance with known techniques. It will be recognized by those of skill in the art that the form and characteristics of the pharma- ceutically acceptable carrier or diluent will be determined by the amount of active ingredient to be mixed, the route of administration and other well-known variables. Those of skill in the art will be able to easily incorporate one or more of the polypeptides according to the present invention. It will further be appreciated that cocktails including polypeptide species may prove particularly effective.

[0161] VI. Methods of Treating PAI-1-Associated Diseases or Disorders The anti-PAI-1 antibodies or fragments thereof disclosed herein are useful for antagonizing PAI-1 activity. Thus, in another aspect, the present invention provides methods for treating a PAI-1-associated disease or disorder by administering to a subject in need thereof a pharmaceutical composition comprising one or more of the anti-PAI-1 antibodies or antigen-binding fragments thereof disclosed herein. do.

[0162] PAI-1 related diseases or disorders amenable to treatment include, but are not limited to, kidney, These include pathophysiological conditions such as liver or pulmonary fibrosis, or abdominal adhesion formation.

[0163] The development of intraperitoneal adhesions is a major cause of morbidity in humans. Complications of adhesions can be as serious as life-threatening intestinal obstruction, but chronic pelvic pain and infertility in women are also common sequelae of peritoneal adhesions. The majority of adhesions arise from surgery, but in some cases they are caused by inflammation, intrauterine It has also been shown to be caused by membraneosis, chemical peritonitis, radiation therapy, foreign body reactions, and continuous ambulatory peritoneal dialysis. Peritoneal injury induces a local inflammatory response that leads to fibrin deposition. A decrease in tissue plasminogen activator (tPA) and an increase in plasminogen activator inhibitors are associated with increased risk of developing peritoneal insufficiency. It is believed that a post-traumatic dysfunction in peritoneal fibrinolytic activity caused by an increase in the toxic agents PAI-1 and PAI-2 allows deposited fibrin to organize into permanent adhesions.

[0164] Seprafilm (R) Currently available and effective treatment options include open access (open laparotomy) and It has limitations on its use only in open surgery and cannot be used in laparotomy. The search for a treatment continues.

[0165] In certain exemplary embodiments, the antibodies disclosed herein may be administered to treat chronic kidney disease, which is the leading cause of end stage renal failure as well as renal fibrosis and associated acute kidney injury.

[0166] One of ordinary skill in the art would be able, by routine experimentation, to determine what an effective, non-toxic amount of an antibody (or additional therapeutic agent) would be for the purpose of treating a PAI-1-associated disease or disorder. For example, a therapeutically active amount of a polypeptide may be determined based on the disease state (e.g., stage I vs. stage IV), age, sex, medical complications (e.g., immunosuppressive conditions or diseases) and weight of the subject, as well as the severity of the disease. The dosage may vary depending on factors such as the ability of the antibody to elicit a desired response in a subject. Dosage regimens may be adjusted to provide an optimal therapeutic response. For example, several divided doses may be administered daily, or the dose may be proportionally reduced as indicated by the exigencies of the therapeutic situation. In general, however, an effective dosage is expected to be in the range of about 0.05 to 100 milligrams per kilogram of body weight per day, and in one embodiment, in the range of about 0.5 to 10 milligrams per kilogram of body weight per day.

[0167] The different aspects and embodiments thereof disclosed herein can be combined with each other. Furthermore, any of these aspects and embodiments described above can be combined with any of the specific aspects and embodiments described herein below.

[0168] Some specific aspects and embodiments that further serve to illustrate the invention are set forth below:

[0169] Description of Specific Aspects and Embodiments Item 1. (a) a heavy chain framework region and a heavy chain variable region, [the heavy chain variable region comprises a heavy chain CDR1 region comprising SEQ ID NO: 34, a heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32]; and (b) a light chain framework region and a light chain variable region, wherein the light chain variable region has the sequence represented by SEQ ID NO: 37. a light chain CDR1 region comprising SEQ ID NO: 145, a light chain CDR2 region comprising SEQ ID NO: 35, and a light chain CDR3 region comprising SEQ ID NO: 35. [including the area] 2. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

[0170] Item 2. (a) a heavy chain framework region and a heavy chain variable region comprising SEQ ID NO: 86; and (b) a light chain framework region and a light chain variable region comprising SEQ ID NO: 93. 2. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

[0171] Item 3. (a) a heavy chain variable region that is at least 95% identical to the heavy chain variable region of the antibody of Item 2, and / or (b) a light chain variable region that is at least 95% identical to the light chain variable region of the antibody of item 2. 2. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

[0172] Item 4. An isolated monoclonal antibody that binds to essentially the same epitope as the antibody of item 1.

[0173] Item 5. (a) A heavy chain framework region and a heavy chain variable region, [the heavy chain variable region is a sequence a heavy chain CDR1 region comprising sequence number 34, a heavy chain CDR2 region comprising sequence number 33, and a heavy chain CDR3 region comprising sequence number 32; and (b) a light chain framework region and a light chain variable region, wherein the light chain variable region has the sequence represented by SEQ ID NO: 37. a light chain CDR1 region comprising SEQ ID NO: 36, a light chain CDR2 region comprising SEQ ID NO: 36, and a light chain CDR3 region comprising SEQ ID NO: 35. 2. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

[0174] Item 6. The antibody described in Item 5, wherein the heavy chain variable region comprises SEQ ID NO: 6 and the light chain variable region comprises SEQ ID NO: 7.

[0175] Item 7. An isolated monoclonal antibody that binds to essentially the same epitope as the antibody of Item 5.

[0176] Item 8. A humanized monoclonal antibody that specifically binds to human PAI-1, the antibody teeth: (a) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 82, or an antigen-binding fragment thereof; and a light chain having a light chain variable region comprising SEQ ID NO: 91, or an antigen-binding fragment thereof; (b) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 83, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 92, or an antigen-binding fragment thereof; (c) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 84, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 93, or an antigen-binding fragment thereof; (d) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 85, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 91, or an antigen-binding fragment thereof; (e) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 85, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 93, or an antigen-binding fragment thereof; (f) a heavy chain variable region comprising SEQ ID NO: 86, or an antigen-binding fragment thereof; and a light chain having a light chain variable region comprising SEQ ID NO:94, or an antigen-binding fragment thereof; (g) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 87, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 95, or an antigen-binding fragment thereof; (h) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 88, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 96, or an antigen-binding fragment thereof; (i) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 89, or an antigen-binding fragment thereof and a light chain having a light chain variable region comprising SEQ ID NO: 97, or an antigen-binding fragment thereof; (j) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 90, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 98, or an antigen-binding fragment thereof; (l) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 86, or an antigen-binding fragment thereof and a light chain having a light chain variable region comprising SEQ ID NO: 95, or an antigen-binding fragment thereof; (m) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 89, or an antigen-binding fragment thereof. and a light chain having a light chain variable region comprising SEQ ID NO: 93, or an antigen-binding fragment thereof; or (n) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 89, or an antigen-binding fragment thereof and a light chain having a light chain variable region comprising SEQ ID NO: 95, or an antigen-binding fragment thereof. Includes.

[0177] Item 9. (a) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 22, a heavy chain CDR2 region comprising SEQ ID NO: 21, and a heavy chain CDR3 region comprising SEQ ID NO: 20; and a light chain comprising a light chain CDR1 region comprising SEQ ID NO: 25, a light chain CDR2 region comprising SEQ ID NO: 24, and a light chain CDR3 region comprising SEQ ID NO: 23. Variable region, (b) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 28, a heavy chain CDR2 region comprising SEQ ID NO: 27, and a heavy chain CDR3 region comprising SEQ ID NO: 26; and a light chain CDR1 region comprising SEQ ID NO: 31, a light chain variable region comprising a light chain CDR2 region comprising sequence number 30 and a light chain CDR3 region comprising sequence number 29; (c) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 40, a heavy chain CDR2 region comprising SEQ ID NO: 39, and a heavy chain CDR3 region comprising SEQ ID NO: 38; and a light chain CDR1 region comprising SEQ ID NO: 43, a light chain variable region comprising a light chain CDR2 region comprising sequence number 42 and a light chain CDR3 region comprising sequence number 41; (d) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 46, a heavy chain CDR2 region comprising SEQ ID NO: 45, and a heavy chain CDR3 region comprising SEQ ID NO: 44; and a light chain CDR1 region comprising SEQ ID NO: 49, a light chain variable region comprising a light chain CDR2 region comprising sequence number 48 and a light chain CDR3 region comprising sequence number 47; (e) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 52, a heavy chain CDR2 region comprising SEQ ID NO: 51, and a heavy chain CDR3 region comprising SEQ ID NO: 50; and a light chain CDR1 region comprising SEQ ID NO: 55, a light chain variable region comprising a light chain CDR2 region comprising sequence number 54 and a light chain CDR3 region comprising sequence number 53; (f) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 58, a heavy chain CDR2 region comprising SEQ ID NO: 57, and a heavy chain CDR3 region comprising SEQ ID NO: 56; and a light chain CDR1 region comprising SEQ ID NO: 61, a light chain variable region comprising a light chain CDR2 region comprising sequence number 60 and a light chain CDR3 region comprising sequence number 59; (g) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 64, a heavy chain CDR2 region comprising SEQ ID NO: 63, and a heavy chain CDR3 region comprising SEQ ID NO: 62; and a light chain CDR1 region comprising SEQ ID NO: 67, a light chain variable region comprising a light chain CDR2 region comprising sequence number 66 and a light chain CDR3 region comprising sequence number 65; (h) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 70, a heavy chain CDR2 region comprising SEQ ID NO: 69, and a heavy chain CDR3 region comprising SEQ ID NO: 68; and a light chain CDR1 region comprising SEQ ID NO: 73, a light chain variable region comprising a light chain CDR2 region comprising SEQ ID NO: 72 and a light chain CDR3 region comprising SEQ ID NO: 71; or (i) a heavy chain variable region comprising a heavy chain CDR1 region comprising SEQ ID NO: 76, a heavy chain CDR2 region comprising SEQ ID NO: 75, and a heavy chain CDR3 region comprising SEQ ID NO: 74; and a light chain CDR1 region comprising SEQ ID NO: 79; An isolated monoclonal antibody that specifically binds to PAI-1, comprising a light chain variable region comprising a light chain CDR2 region comprising SEQ ID NO:78, and a light chain CDR3 region comprising SEQ ID NO:77.

[0178] Item 10. A humanized monoclonal antibody according to item 8 or 9 having essentially the same endonucleases on PAI-1. An isolated monoclonal antibody that specifically binds to a pitope that binds to PAI-1.

[0179] Item 11. A method of restoring plasmin production comprising administering to a subject in need thereof a pharma- ceutical effective amount of a PAI-1 antibody orally, parenterally via an injectable solution, by inhalation, or topically.

[0180] Item 12. The method of item 11 for treating a condition involving increased levels of fibrotic tissue.

[0181] Item 13. The method of item 12, wherein the condition is fibrosis, skin fibrosis, systemic sclerosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, chronic lung disease, liver fibrosis, renal fibrosis, chronic kidney disease, thrombosis, venous and arterial thrombosis, deep vein thrombosis, peripheral limb ischemia, disseminated intravascular coagulation thrombosis, acute ischemic stroke with or without thrombolysis, or stent restenosis.

[0182] Item 14. The method according to any one of Items 11, 12, or 13, wherein the PAI-1 antibody comprises an antibody according to any one of Items 1 to 10.

[0183] Item 15. Use of a pharma- ceutical effective amount of a PAI-1 antibody for the manufacture of a medicament for treating a condition caused by increased levels of PAI-1 or increased sensitivity to PAI-1, comprising administering to a patient orally, parenterally by injectable solution, by inhalation, or topically. EXAMPLES

[0184] The present invention is further illustrated by the following examples, which should not be construed as further limiting. The sequence listing, figures, and all references, patents and published patent applications cited throughout this application are hereby expressly incorporated by reference.

[0185] Moreover, in accordance with the present invention, conventional molecular biology, microbiology, and recombinant DNA techniques may be employed. Such techniques are fully explained in the literature. See, e.g., Sambrook, Fritsch & Maniatis, Molecular Cloning: A Laboratory Manual, Second Edition (1989) Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York (referred to herein as "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (ed. D. N. Glover, 1985); Oligonucleotide Synthesis (ed. M. J. Gait, 1984); Nucleic Acid Hybridization [eds. B. D. Hames & S. J. Higgins, 1985]; Transcription And Translation [eds. B. D. Hames & S. J. Higgins, 1984]; Animal Cell Culture [eds. R. I. Freshney, 1986]; Immobilized Cells And Enzymes [IRL Press, (1986)]; B. Perbal, A Practical Guide To Molecular Cloning (1984); F. M. Ausubel et al. (eds.), Current Protocols in Molecular Biology, John Wiley & Sons, Inc. (1994).

[0186] Example 1: Hybridoma generation: Immunization of mice with PAI-1 protein and antibody generation It is cross-reactive with human (h) and cynomolgus monkey (cyno) active PAI-1 (glycosylated or non-glycosylated forms) and neutralizes the inhibitory activity of PAI-1 to restore downstream production of plasmin. thereby providing an effective therapeutic method for the treatment of renal, hepatic or pulmonary fibrosis. Antibodies have been developed that prevent abdominal adhesion formation and keloid scar formation. Neutralization of PAI-1 inhibitory function by monoclonal antibodies has been described to fall into three categories of mechanisms: (1) establishment (2) blocking PAI-1 against tPA or uPA by somatic damage; or (3) convert PAI-1 to a substrate conformation. Convert it to:

[0187] a) Antigen PAI-1 binds to vitronectin with subnanomolar affinity. PAI-1 is secreted from cells in an active conformation stabilized by binding to PAI-1. PAI-1 undergoes a spontaneous conformational change from an active to a latent conformation within minutes at room temperature and within a few hours at room temperature. This resistance is mediated by a conformational change that reduces the half-life of the active conformation of PAI-1 by several minutes. The half-life of active conformational PAI-1 in immunized animals is extended from 0.1 to several hours. To extend the lifespan of PAI-1 and to allow the mouse immune system to recognize the active PAI-1 conformation. To make this possible, a complex of vitronectin and PAI-1 was used for immunization.

[0188] Human glycosylated PAI-1 produced in insect cells was purchased from Innovative Research (Catalog). Vitronectin (catalog number IHVN) and tPA (catalog number IGLYHPAI-A) were purchased from Biosciences. To prepare the immunogen, PAI-1 was incubated with vitronectin in a 1:1 molar ratio for 1 hour at room temperature or with tPA in a 1:1 molar ratio for 15 minutes at 37°C. All immunogens were prepared using sterile saline as the diluent.

[0189] b) Immunization Standard hybridoma production protocols known in the art were performed to generate antibodies. Standard approaches previously described in the literature used PAI-1 alone or PAI-1 / tPA complexes. The inventors instead generated antibodies against the active conformation of PAI-1. The PAI-1 / vitronectin complex was used as a new approach to generating antibodies against PAI-1. To generate antibodies, a three-pronged approach was taken, as outlined below: (1) classical immunization of mice with PAI-1 / vitronectin complexes to obtain mouse splenocytes for fusion with a mouse myeloma cell line as a fusion partner to produce hybridomas; (2) classical immunization of mice with the PAI-1 / tPA complex to obtain mouse splenocytes for fusion with a mouse myeloma cell line as a fusion partner to produce hybridomas; and (3) Classical immunization of mice with PAI-1 alone to obtain mouse splenocytes for fusion with a mouse myeloma cell line as a fusion partner to produce hybridomas.

[0190] Three mice per antigen (PAI-1 alone, Vn / PAI-1 complex, tPA / PAI-1 complex) were used in the study. These mice were 9-20 week-old naïve female BALB / c mice (Charles River, strain Cortez-La). On day 0, nine mice were immunized intraperitoneally in phosphate-buffered saline (PBS) with PAI-1 alone, Vn / PAI-1, or tPA / PAI-1 complex. A total of 10 ug of antigen was administered per mouse. The mice were boosted with the same amount of antigen on day 14 and then mixed with Sigma Adjuvant System (Sigma Cat. No. 6322) at a 1:1 volume-to-volume ratio in a total volume of 200 μl per mouse. On day 21, blood samples were collected for PAI-1 specific antibody titer assessment. Mice immunized with PAI-1 / tPA complexes showed very low specific reactivity to PAI-1 and high anti-tPA titers and were not used for downstream fusions.

[0191] On day 51, the highest anti-PAI-1 specific antibody titers and the highest PAI-1 complexed protein (i.e. i.e., either Vn or tPA), but mouse and rat PAI-1 oncoproteins Mice with the highest titers against lusologus were selected for fusion. Mice selected for fusion were boosted in PBS with PAI-1 alone or PAI-1 / Vn complex for a total of 10ug of antigen per mouse mixed with Sigma Adjuvant System (Sigma Cat. No. 6322) at a 1:1 ratio in a total volume of 200μl per mouse, as above. On day 55, mice were sacrificed by CO2 chamber and Blood was collected by cardiac puncture and spleens were harvested for hybridoma production. Four other mice underwent the same procedure at later time points (2-4 months after the first mouse was used for fusion).

[0192] Serum titrations were performed in 3 mice for PAI-1 alone and PAI-1 / tPA, and in 2 mice for PAI-1 / Vn using the ELISA protocol described in Example 2 (binding ELISA).

[0193] [Table 7]

[0194] Mice immunized with PAI-1 / tPA complexes did not reach the high specific titer criterion and were not used for fusions. Based on the serum titers shown in Table 3, a total of five mice with high specific titers to PAI-1 were selected for fusion.

[0195] b) fusion The five mice with the highest specific titers to PAI-1 were selected for fusion. On the day of injection, mice were sacrificed in a CO2 chamber, blood was collected by cardiac puncture, and spleens were removed. The spleen cells were then removed from the fibroelastic membrane using forceps and placed in a Petri dish containing 10 ml of serum-free hybridoma fusion medium (IMDM; 500 ml of Iscove's modified Dulbecco's medium (HyClone SH30259.01)). Squeeze and wash twice with 10 ml of serum-free IMDM (including an initial spin).

[0196] Cells were counted using a Countess automated cell counter. Fusion partner cells (myeloma:FO (ATCC reference CRL-1646)) and splenocytes were then mixed in a 50 ml tube at a ratio of 1:2 to 1:10 (cell number). The cells were combined and spun down at 970 rpm for 10 minutes (slow spin) to form a soft pellet. Pre-warmed (37°C) 1 ml PEG (PEG 1500) in 75 mM Hepes 50% w / v, Roche catalogue number 783641 (10783641001) was added dropwise to the cell pellet over 1 minute and the cells were mixed after each drop of PEG was added. The pellet was incubated with PEG for an additional minute, followed by the addition of 10 ml of serum-free IMDM medium over 1 minute, with the first ml of 10 being added over 30 seconds. The cells were spun down at 970 rpm for 10 minutes (slow spin) to preserve viability. The fused cells were plated in 96-well plates in selection medium (200 ml Gibco Hybridoma (SFM #12045), 20 ml 10% HyClone SuperLow IgG Defined FBS (#SH30898.03), 2 ml penicillin / streptomycin, 4 ml (Hybridoma Fusion and Cloning Supplement (Roche Diagnostics 11 363 735 001 (50X)) and 4 ml HAT (hypoxanthine-aminopterin-thymine). The fusions were then plated at 200 ul in Sigma-Aldrich No. HO262 (50X) fusion medium. After approximately 10-14 days, or when the medium in the wells turned yellow, the fusions were ready for screening. Supernatants from the resulting hybridomas were then plated at 200 ul in Sigma-Aldrich No. HO262 (50X) fusion medium. The presence of antibodies was tested by ELISA (Example 2).

[0197] Example 2: Hybridoma analysis for specificity for PAI-1-vitronectin complex Binding ELISA for serum screening Each fusion from the spleens of the five selected mice yielded approximately 5000 clones that needed to be screened for binding to the PAI-1 / Vn complex as a first stage primary screen. Primary screening of hybridoma supernatants was performed using either PAI-1 or PAI-1-vitronectin complex. ELISA was performed in parallel to select for hybridomas that specifically bound to PAI-1 conjugated to vitronectin. Materials used for the ELISA were: Immulon 4 HBX ELISA plates (Dynax Cat. No. N0541216); human monomeric vitronectin 5ug / ml (Innovative Research Cat. No. IHVN); glycosylated human PAI-1 (active form) (Molecular Innovations Cat. No. GLYHPAI-A); several fusions of non-glycosylated mouse PAi-1 (Molecular Innovations Cat. No. MPAI-A); secondary antibody, HRP-goat anti-mouse IgG (H+L) (Jackson ImmunoResearch Labs No. 115-035-166); and ABTS substrate: Roche Diagnostics (No. 115-035-167). 204 521 001).

[0198] Control antibodies used were: a) 33B8, a mouse monoclonal inhibitory antibody against PAI-1 (IgG1; Innovative Research catalogue no. IMA-33B8); b) 33H1, a mouse monoclonal inhibitory antibody against PAI-1 (IgG1; Innovative Research Catalog Number IMA-33H1); c) 31C9, a mouse monoclonal non-inhibitory antibody against PAI-1 (IgG1; Innovative Research Catalog Number IMA-31C9); and d) 1B7.11, an IgG1 isotype control antibody (anti-TNP mAb - purchased from ATCC (catalog no. TIB-191) Produced in-house from imported hybridoma cell lines It was.

[0199] The ELISA method was as follows: 50 ul / well of Vn was coated overnight at 4°C with 5 ug / ml in PBS. The next day, plates were blocked with 200ul 1% bovine serum albumin in PBS (BSA / PBS) for 1 hour; plates were washed 4 times with 200ul / well PBS; active PAI-1 at 2ug / ml in 1% BSA / PBS was added to the plates at 50ul / well and incubated for 1 hour; plates were washed 4 times with 200ul / well PBS; antibody dilutions or hybridoma supernatants from the original 96-well plates in 1% BSA / PBS were added to the ELISA plates at 50ul / well; plates were incubated for 1 hour. Incubated at room temperature (RT); plates were washed 4 times with 200 ul / well PBS; 1% BSA / PBS HRP-anti-mouse IgG 50ul 1:2000 was added and incubated for 1 hour at room temperature; plates were washed 4 times with 200ul / well PBS; ABTS substrate (1 pill dissolved in 5ml) was added at 50ul / well The plates were then incubated at OD for 1 h on a BioTek Synergy HT instrument. 405A typical standard curve for antibody titration in a binding ELISA is shown in Figure 2. Antibodies 31C9, 33B8 and 33H1 served as positive controls, and IgG1 served as a negative control. Table 4 shows that of the approximately 5,000 clones generated, 675 clones were positive for both PAI-1 and PAI-1 / Vn. These clones were then subjected to PAI-1 affinity We screened them accordingly.

[0200] [Table 8]

[0201] Example 3: Biacore screening of hybridoma supernatants by affinity ranking Further selection of high affinity antibodies with low off-rates was performed by Biacore. Biacore hybridoma supernatant screening was performed using: (1) a reverse screening using anti-mouse immobilized anti-PAI-1 antibody, or (2) a forward screening assay using free PAI-1 as a ligand or against immobilized Vn. The instruments used were BIACORE 2000 or BIACORE 3000 (GE Healthcare) designed for real-time biomolecular interaction analysis (BIA). The sensor chips used were CM5 chips (GE Healthcare) with a carboxymethylated dextran matrix on the surface. Each sensor chip had four parallel flow cells (Fc). All flow cells were coupled with anti-mouse IgG Fc mAb by standard amine coupling according to the manufacturer's protocol for chip fabrication.

[0202] In the Biacore reverse screening assay, ELISA-positive hybridoma supernatants were selected and filtered through a 0.2 μm filter before being injected onto the Biacore chip surface. The hybridoma supernatant was injected onto one of the flow cells Fc2 to Fc4, and the IgG in the hybridoma supernatant was captured onto the chip surface by the anti-mouse IgG Fc mAb. Fc1 was left intact on the reference cell. Human PAI-1 protein in PBS was then injected onto Fc1 to Fc4. PBS buffer was also injected onto the chip surface as a blank. Fc1 and the blank Binding of antibodies from the supernatant to PAI-1 protein after subtraction of the signal from the buffer run The affinity (KD) / dissociation rate (kd) was analyzed and ranked using Scrubber 2 software. Damn it.

[0203] Purified human vitronectin protein in a Biacore sequential screening assay The proteins were immobilized on CM5 chip flow cells Fc1 to Fc4. Human or cynomolgus monkey PAI-1 was captured on all flow cells. The filtered selected hybridoma supernatants were then added to the flow cells except for Fc1. One flow cell was injected over the captured PAI-1, and Fc1 was reserved as a reference flow cell. PBS buffer was also injected over the chip surface as a blank. After subtracting the signals of Fc1 and the blank buffer run, the antibody in the hybridoma supernatant against the vitronectin-captured PAI-1 was Binding affinities of the target proteins were determined using Scrubber 2 software (version 2.0a, 2005; BioLogic Software, BioLogic Software Rty Ltd., 116 Blamey Court, Campbell, ACT 2612 Australia). were used to analyze and rank the results.

[0204] Table 5 shows a selection of positive and negative antibody clones from fusions A, B, C, D and E. Because a large number of antibodies were screened, not all data are shown. Excellent (kd<10-10) antibody activity against human and cynomolgus PAI-1 proteins. 4 Antibody clones that showed a binding / dissociation rate of 1 / sec Only the lawns were selected for functional chromogenic assays.

[0205] [Table 9]

[0206] Example 4: Functional ELISA for screening hybridoma supernatants to select antibodies that block the interaction of PAI-1 with tPA To allow for the selection of functional antibodies, a new ELISA was developed to distinguish between antibodies that only bind to PAI-1 versus those that block the function of PAI-1 as tPA inhibitors (functional ELISA). This was developed to make this possible.

[0207] Hybridoma supernatants were analyzed to identify different clones with the ability to block tPA-PAI-1 interaction. To identify hybridoma supernatants from the clones, we screened them with a novel functional ELISA. The design of a functional ELISA is as follows: (1) if the antibody binds to PAI-1 but the antibody binding does not block the formation of a covalent bond between PAI-1 and tPA, the anti-tPA antibody will bind to the plate-bound tPA through PAI-1 and produce a positive record; (2) if the antibody blocks PAI-1, thereby either changing the PAI-1 conformation or blocking the tPA interaction by steric hindrance, the anti-tPA antibody will not be able to bind to the plate and will produce a positive record. The record was negative (lower OD 405 In parallel, the hybridoma supernatant was cultured as described in Example 2. Hybridoma supernatants were tested for binding to PAI-1 in an ELISA using a ELISA kit. Since the amount of antibody in the hybridoma supernatants was unknown, a lower than control reading (i.e., lower than the isotype control reading) was considered as identification of the antibody of interest. Due to the variable antibody concentration in the supernatants, some In the case of , blocking was only partial.

[0208] Streptavidin coated plates (NUNC #436014) were incubated with 2ug / ml Biotin-PAI-1 (Human PAI-1 with N-terminal biotinylated active fraction; Molecular Innovations Catalog #NTBIOPAI-A) at 50ul / ml in 1% BSA / PBS for 2 hours at room temperature (RT). Plates were blocked with 200 ul 1% BSA / PBS for 1 hour at RT and washed 4 times with 200 ul / well PBS. Purified antibody dilutions and hybridoma supernatants were added to the wells at 50 ul / well. and incubated for 15 minutes. The plate was washed 4 times with 200 ul / well PBS. Chain tPA (Innovative Research Cat# HTPA-TC) at 1ug / ml was added to the plate at 50ul / well and incubated for 30 minutes at RT. Plates were washed 4 times with 200ul / well PBS. Anti-tPA HRP conjugated antibody (Life Span Technologies, Cat# LS-C39721) was added to the plate at 1:3000 dilution and incubated for 45 minutes. Plates were washed 4 times with 200ul / well PBS. ABTS substrate (dissolve 1 tablet in 5ml; Roche Diagnostics #11 204 521 001) was added to the plate at 50ul / well and allowed time for color development. Plates were read at OD 100 on a BioTek Synergy HT instrument. 405 An OD lower than the IgG isotype control indicates blocking of tPA binding to PAI-1.

[0209] In some cases, functional ELISA was performed prior to Biacore supernatant screening and serves as a selection step that is more important for hybridoma development. Representative curves using 33H1 as a positive control, IgG1 as a negative control, and A44 as an identified positive antibody clone are shown in Figure 3. vinegar.

[0210] [Table 10]

[0211] More than 200 supernatants were screened. Table 6 shows a selection of positive and negative hybridoma supernatants. Approximately 10 hybridomas per fusion were found to block the binding of PAI-1 to tPA in a functional ELISA. Based on the data from the hybridoma supernatants, hybridomas were selected for sequencing and mid-scale antibody production. D4 bound well to non-glycosylated PAI-1. Although no IgG antibody was identified, it was purified and sequenced based on its Biacore binding to glycosylated PAI-1. The purified antibodies were analyzed by Biacore for affinity kinetics and The potency compared to commercially available antibodies was further characterized in chromogenic and cellular assays.

[0212] Example 5: Sequencing by 5'-RACE (rapid amplification of cDNA ends) and mouse antibody purification Antibodies against a specific target produced from a series of fusions can have the same sequence. By sequencing the antibody genes at an early stage of antibody generation, any potentially duplicated antibodies can be eliminated, and the precise antibody gene sequence guides antibody selection and humanization, as well as chimeric antibody construction.

[0213] 5'-RACE is a procedure for the amplification of nucleic acid sequences from messenger RNA templates between a defined intermediate site and an unknown sequence at the 3' or 5' end of the mRNA. This methodology of amplification with single-sided specificity has been described as "one-sided" PCR or "anchored" PCR. The original variable mouse anti-human PAI-1 antibody sequence of the lead antibody The sequence was determined by 5'-RACE cDNA sequencing and confirmed by N-terminal protein sequencing.

[0214] To determine the variable heavy (VH) and light (VL) IgG sequences, total RNA from hybridoma cells was isolated using the RNeasy Mini Kit (QIAGEN, Cat. No. 74104) according to the manufacturer's instructions. Briefly, cells (5x106 cells) were lysed in 350ul of RLT buffer from the kit, followed by capture of total RNA on a spin column. RNA was eluted with TE buffer from the kit and stored on ice.

[0215] The first strand cDNA was purified using SMARTer TM RACE cDNA Amplification Kit (ClonTech, Catalog No. 634923) The VH and VL chain cDNAs were prepared using the SMARTer 5'-RACE protocol according to the manufacturer's instructions. TM The DNA was amplified separately using the 5'-primer provided in the kit and the 3' VH and VL gene specific primers described below. Ta: Heavy chain 3'-primer: 5'-TATGCAAGGCTTACAACCACA -3' (SEQ ID NO: 105) Light chain 3'-primer: 5'-CTCATTCCTGTTGAAGCTCTTGAG -3' (SEQ ID NO: 106)

[0216] The amplified VH and VL genes were separately cloned into a TOPO vector using the TOPO TA cloning kit (Invitrogen, Cat. No. K4520-01). These procedures were performed according to the manufacturer's instructions. To transform bacteria, the reaction mixture was transferred to competent E. coli cells. The cells were then added to 100 μl of LB agar containing 100 μg of ampicillin per ml and incubated on ice for 20 minutes. The tubes containing the E. coli cells and reaction mixture were heated to 42° C. for 40 seconds and 250 microliters of lit's SOC medium was added. After incubating the E. coli at 37° C. for 60 minutes with shaking at 300 rpm, the bacteria were spread onto LB agar plates containing 100 micrograms of ampicillin per ml and subsequently incubated overnight at 37° C.

[0217] Upon confirmation of the inserted VH and VL genes by PCR, five bacterial clones were selected and The plasmid DNA was then grown in LB medium containing 100 micrograms of ampicillin per ml for plasmid DNA preparation. Plasmid DNA was prepared using the QIAprep Spin Miniprep Kit (QIAGEN, Catalog). The VH and VL IgG genes of the hybridomas were sequenced by the Sanger method and the CDRs were aligned according to the Contact definition (MacCallum et al.). It was decided to use.

[0218] Monoclonal antibodies were produced in serum-free medium (Gibco Cat. No. 12045) according to the manufacturer's instructions in CELLine bioreactor flasks (Wilson Wolf Manufacturing Corp.; Cat. No. CL350 or Cat. No. CL1000) and purified by Protein A / G chromatography (GE Healthcare Life Sciences, Cat. Nos. 28-4083-47 and 28-4082-53). Purified antibodies were further characterized for affinity kinetics on Biacore and for color and cell proliferation. The antibodies were characterized for potency in the assay relative to commercially available antibodies.

[0219] Example 6: Functional chromogenic assay using purified antibodies The purified antibodies were tested in a chromogenic assay for their ability to block PAI-1, which inhibits tPA function, and therefore antibodies that block PAI-1 would result in restoration of tPA function. Chromogenic assays detect their natural products by hydrolyzing one or more peptide bonds. They utilize proteolytic enzymes acting on natural substrates (proteins and peptides). The method is usually highly specific in the sense that only peptide bonds adjacent to specific amino acids are cleaved. Chromogenic substrates are peptides that react with proteolytic enzymes resulting in the formation of a quantifiable color. Chromogenic substrates are synthetically produced and designed to have a selectivity similar to that of the enzyme's natural substrates. A chemical group is attached to the peptide portion of the chromogenic substrate that produces color when released after enzyme cleavage. The color change can be followed spectrophotometrically and is proportional to the proteolytic activity.

[0220] A chromogenic assay was used to confirm the ability of the antibodies to neutralize PAI-1 function as tPA inhibitors. tPA can release pNA from the chromogenic substrate S2288. S228 in solution is colorless, but after exposure to tPA and the subsequent release of pNA, the solution turns OD 405 This produces a yellow color that can be read at RT. Color formation can be observed over a period of 2-3 hours to determine the kinetics of the enzymatic reaction. PAI-1 can block the enzymatic activity of tPA in a concentration-dependent manner.

[0221] A two-stage chromogenic assay was performed. All reagents were at 10x concentration until they were added to the substrate solution. In the first stage, the potency of PAI-1 in inhibiting tPA was assessed using a chromogenic assay ( tPA activity was measured using PAI-1 titration (with fixed tPA concentration). PAI-1 titration curves were analyzed to determine tPA activity. The IC50 for PAI-1 blocking tPA was determined. The IC80 calculated from the curve was then selected for further antibody matching for the ability to neutralize PAI-1 blocking function and restore tPA enzymatic activity. Equal volumes (25ul) of tPA (14nM) (Innovative Research, Cat# IHTPA-TC) and glycosylated (active form) human PAI-1 (Molecular Innovations, Cat# GLYHPAI-A) or non-glycosylated (active form) mouse PAI-1 (Molecular Innovations Cat# IMPAI) were mixed and incubated with 3-fold serial dilutions of PAI-1 starting at 108nM and a fixed concentration of tPA. All protein dilutions were made in 1%BSA / PBS. The mixtures were then plated in 96 well plates. The wells of a 100-μl microtiter plate were incubated for 15 min at room temperature. Then, 200 ul of chromogenic substrate S2288 (1.25 mM) (Chromogenix, Cat. No. S-820852) diluted according to the manufacturer's instructions was added to the wells, and the OD at 405 nm was measured every 10 min for 2 h. 405 Change in absorbance Recordings were made to measure the residual tPA activity. For controls, background was measured in the absence of tPA (no enzymatic reaction), the positive control was no PAI-1 (100% tPA activity), and the negative control was a 10-fold excess of PAI-1 over tPA (complete block of tPA activity). See Figure 4 for representative curves for 33B8, A44, 33H1 and IgG1.

[0222] For the second step, the functional properties of the antibodies were determined by assessing their ability to inhibit active PAI-1 and restore tPA function using a PAI-1 neutralization assay. 12.5ul of active PAI-1 (56nM) was diluted with an equal volume of either PBS (Invitrogen, Cat. No. 14190-144) containing 1% BSA (Sigma, Cat. No. A3059) or serial 3-fold dilutions of the antibody starting at 2uM. Control and unknown antibodies were incubated at concentrations ranging from 0.1 to 300 nM (5-fold dilutions) with 3 nM PAI-1 and tPA added to the mix. All components were incubated at room temperature at 10x concentrations and further showed clear to yellow color upon cleavage by tPA. The mixture was diluted 10-fold with tPA substrate S2288, which changes color to red. Samples were read at OD 405 every 10 minutes for 2 hours at 37°C. The mixture was then transferred to a 96-well plate to achieve antibody-antigen complex formation. The wells were incubated at room temperature for 30 minutes in a microtiter plate. Then, 25 μl of tPA (IC value of tPA activity) was added. 80 (14 nM, corresponding to Finally, 200 ul 1.25 mM substrate S2288, diluted according to the manufacturer's instructions, was added to the mixture. Residual tPA activity was measured every 10 min for 2 h by recording the absorbance change at 405 nm. Zero percent PAI-1 activity is defined as the PAI-1 activity observed in the absence of antibody. Neutralization of PAI-1 activity by antibody was calculated from the residual PAI-1 activity measured in the presence of antibody. Controls were IgG1 as an isotype control (negative) and 33H1 mAb and 33B8 mAb as positive controls. See Figure 5 for representative curves for B28, E16, E21, A75 and IgG1.

[0223] Orthologues of human PAI-1 that inhibit human tPA were tested in a two-stage chromogenic assay system. Titrations of the orthologues were performed as described above for human PAI-1 (see FIG. 6 for representative curves of titration) and tPA activity was determined chromogenically (see FIG. 7 for representative curves for 33B8 and A44 against cynomolgus monkey and mouse PAI-1). The final concentration of human tPA used in the assay was 1.4 nM. 12.5 ul active PAI-1 (56 nM) was mixed with an equal volume of either PBS containing 1% BSA or serial 3-fold dilutions of antibody starting at 2 uM. The mixture was incubated in a well of a 96-well microtiter plate for 30 minutes at room temperature. Then, 25 ul of tPA (14 nM) was added to the well, and 15 The mixture was incubated at room temperature for 1 min. To terminate the reaction, 200 ul tPA substrate S2288 (Chromogenix) (1.25 mM) was added to the mixture. Ortholog PAI-1 was obtained from Molecular Innovations. The following PAI-1 proteins were produced: mouse PAI-1 (wild-type active fraction; catalog number MPAI); rat PAI-1 (wild-type active fraction; catalog number RPAI); and rabbit PAI-1 (stable mutant; catalog number RbPAI-I91L). Cynomolgus PAI-1 (active cynomolgus PAI-1) was produced in-house in E. coli. Biacore Insufficient off-rates for the rabbit and rat orthologs in the screen (data not shown) Because of this, we did not screen antibodies against these orthologs.

[0224] [Table 11]

[0225] [Table 12]

[0226] One or more antibodies from each fusion were identified in this assay using cynomolgus monkeys. ) and human PAI-1 inhibitory function, and about 14 antibodies showed moderate to strong A39 and B28 showed that these two antibodies blocked glycosylated hPAI-1 but had no activity against human or cynomolgus monkey non-glycosylated PAI-1. All of the antibodies had a unique profile in that none of them could effectively block mouse PAI-1 activity (within 10-fold of human PAI-1) except for C26.

[0227] Example 7: Mechanism of action for monoclonal antibodies Monoclonal antibodies can inhibit PAI-1 by three different mechanisms: a) by steric hindrance, b) by converting PAI-1 into a latent conformation upon binding, and c) by converting PAI-1 into a substrate for the tPA conformation instead of an inhibitor ("substrate conformation"). PAI-1 binds covalently to tPA upon interaction with the serine protease .

[0228] Chromogenic assay and SDS-PAGE techniques were used to identify the antibody mechanism of action. The reaction between monoclonal antibodies (or control antibodies), PAI-1 and tPA was carried out as described above for the functional chromogenic assay. Samples were mixed with Laemmli sample buffer and loaded onto an SDS-PAGE gel under non-reducing conditions and run for 30 minutes. The gel was then stained with Coomassie Blue. The protein, complexes, and cleaved forms of PAI-1 were visualized by staining with . Control monoclonal antibodies that convert PAI-1 to a latent conformation were used as comparators. 33B8 is known to convert PAI-1 to a latent conformation, and 33H1 converts PAI-1 to a substrate conformation. It is known that the conformational transition of the substrate can be converted into a conformational transition. This assay can reliably identify substrate conformations, but cannot distinguish between cryptic conformations or steric hindrance. Representative SDS gels are shown in Figures 8, 9 and 10.

[0229] [Table 13]

[0230] A44, C26, C45 and E21 convert the active conformation of PAI-1 into the substrate conformation. Although A39 and B109 have different mechanisms of action, the assay shows that these antibodies convert PAI-1 from an active conformation to a latent conformation. Either by changing the PAI-1 activity to a 3-amino acid, or by steric hindrance. It was not possible to distinguish between the

[0231] Example 8: Purified antibody binding kinetics In the kinetic measurements, the antibodies were assayed in reverse at 25°C. In the reverse assay, the PAI-1 antibodies The antibody was then captured by anti-mouse IgG Fc antibody prepared on a CM5 chip, followed by serial two-fold dilutions of PAI-1 protein (human or cynomolgus monkey) starting at 40 nM. The flow rate was selected. 2000 seconds was used for the dissociation time to accommodate the slow off-rate of the selected antibody. After each round of antibody-PAI-1 binding, the chip was regenerated with glycine-HCl, pH 1.7 buffer. Kinetic data analysis was performed using Biacore BIAevaluation software. Sensorgrams were analyzed by subtracting the reference flow cell values ​​and the blank buffer values. The sensorgrams were fitted by using a simulated kinetics 1:1 (Langmuir) model with local Rmax. Data for the antibodies tested is shown in Table 9 below.

[0232] [Table 14]

[0233] The binding kinetics of representative antibodies were further analyzed and compared using a complex of vitronectin and PAI-1 in a Biacore forward assay. Tranectin protein was coupled to CM5 chip by amine coupling at Fc1-Fc4 of the flow cell. Human PAI-1 was then captured on the vitronectin surface as a ligand in flow cells Fc2-Fc4. Fc1 was kept as a reference cell. Antibodies were added starting at 40 nM. The samples were diluted 2-fold and injected into Fc1-4. Kinetic data analysis was performed using Biacore BIAevaluation software. Sensorgrams were first double-referenced by subtracting the reference cell and blank buffer values, and then fitted with a 1:1 (Langmuir) model. , together with the overall Rmax.

[0234] [Table 15]

[0235] The data in Table 10 showed that A44 binds to free human PAI-1 as well as to PAI-1 complexed with vitronectin.

[0236] Example 9: Functional assays in primary human cytoplasm To further examine the ability of each antibody to restore downstream plasmin production by primary human cells, a plasmin generation assay was used. Only antibodies that showed good affinity in this assay were used and tested in this assay.

[0237] On day 1, human primary hepatic stellate cells (Sciencell CA, Catalog No. SC5300) were cultured in starvation medium (DMEM Gibco + glutamax-1 4.5g / L D-glucose, pyruvate (31966-021), 0.2% fetal bovine serum gold Cells were plated at 20,000 cells / well in 1000 mM PAA (A11-152) at 37°C under 5% CO2. On day 2, antibodies were added to recombinant PAI-1 (Molecular Innovation, Cat. No. 136116) to neutralize PAI-1 activity. The cells were preincubated with tPA (Molecular Innovations (catalog no. HTPA-TC), 5 nM in DMEM without red phenol) for 15 min at room temperature. At the same time, tPA (Molecular Innovations (catalog no. HTPA-TC), 5 nM in DMEM without red phenol) was incubated with the cells for 15 min at 37°C. After washing away unbound tPA, the PAI-1 / mAb mixture was added to the cells, and the residual tPA activity was then assayed by addition and subsequent addition of a glu-plasminogen / substrate mixture (Glu-Pg: Sigma catalog no. IGLYHPAI-A, recombinant glycosylated human PAI-1, final concentration 5 nM). No. 9001-91-6; 0.5 μM final concentration) and plasmin chromogenic substrate: (CBS00.65 Stago Cat. No. 00128, 0.5 mM final concentration).

[0238] Plasminogen activation to plasmin was measured using a spectrophotometer (IEMS) thermostated at 37°C. Detected by kinetic reading of A405 / 492 every 45 seconds using a 3D chromatograph (Thermofisher). Biolise software (Thermofischer) calculated the maximum rate of chromogenic substrate cleavage: Plasmin generation was expressed as Vmax: the maximum rate of A405 / 492 nm per minute (mDO / min) was calculated. PAI-1 inhibition was then calculated using tPA alone and PAI-1 (no mAb, as no inhibition) as reference (100% inhibition). Calculate and plot the IC using Biostat speed software. 50 and calculate Imax did.

[0239] [Table 16]

[0240] Example 10: Antibody binding epitopes investigated by Biacore competitive assay A selected group of anti-PAI-1 antibodies with excellent binding and blocking activity were examined for their potential binding epitopes in a Biacore competition assay. In addition to the newly identified antibodies, several antibodies with known binding sites on human PAI-1 were also examined in the assay. Commercially available anti-PAI-1 antibodies were set up to compete for binding to human PAI-1 protein. Each antibody was immobilized onto a flow cell of a CM5 chip using a standard amine coupling reaction. All antibodies tested, except for clone B28, showed binding site activity after amine coupling. The human PAI-1 protein was captured by the antibody immobilized on the chip, followed by The immobilized antibodies and antibodies with different binding sites on human PAI-1 were injected as samples. Only the analyte antibodies that are compatible with the antibody will show additional binding signals in the Biacore. The body was repeated twice and the results are shown in the table below.

[0241] [Table 17]

[0242] When A44 is immobilized and binds to PAI-1, C45 (the analyte antibody) binds to the PAI-1 to which A44 is bound. Thus, C45 cannot bind to the same binding site on PAI-1 that A44 does. A44, which competes for (denoted as "c / c" in Table 12) or binds to PAI-1, prevents C45 binding to PAI-1. This analysis is supported when the experiment is repeated in the reverse order. Specifically, when C45 is the immobilized antibody and binds to PAI-1, A44 as the analyte antibody cannot bind to the C45-bound PAI-1 (denoted as "c / c" in Table 12). In a similar analysis, A71 and A75 compete for the same site on PAI-1. Biacore analysis shows whether A44 and C45, as well as A71 and A75, compete with each other for binding to PAI-1. It has been confirmed that they interfere with each other.

[0243] In contrast, the commercially available antibodies, 33H1 and 33B8, do not compete with A44. When A44 is the immobilized antibody and bound to PAI-1, both 33H1 and 33B8 still bind to PAI-1 bound to A44. (shown as "b / b" in Table 12). This was confirmed in a reverse experiment. When PAI-1 is bound to immobilized 33H1 or immobilized 33H8, A44 still binds to PAI-1. Thus, the commercially available antibodies 33H1 and 33B8 compete with the binding of A44 to PAI-1. It does not interfere or interfere with anything.

[0244] Interestingly, some of the immobilized antibodies (i.e., B109) were not associated with the analyte antibodies (i.e., 33B8) to the captured PAI-1 protein; When the antibody pairs are swapped for analyte antibodies (e.g., by flipping the pairs on the chip), the antibody pairs no longer compete with each other for binding to PAI-1. For example, when B109 is the immobilized antibody bound to PAI-1, 33B8 was unable to bind to PAI-1. However, when 33B8 is the immobilized antibody bound to PAI-1, B109 was unable to bind to PAI-1. This result One possible explanation for this result is that when an immobilized antibody is bound to PAI-1, PAI-1 may change into a conformation unfavorable to the second or analyte antibody and prevent the binding of the analyte antibody (e.g., if B109 is the immobilized antibody and 33B8 is the analyte antibody, However, if the antibody pair is reversed, the immobilized antibody may bind in a manner that leaves the conformation of PAI-1 relatively unchanged, thus preventing the analyte antibody from binding to the bound PAI-1. (i.e., the analyte antibody B109 can bind to PAI-1 bound by immobilized antibody 33B8). Thus, the competition observed between 33B8 and B109 is not due to overlapping binding sites on PAI-1, but rather to a conformational change in PAI-1 when bound to B109. This appeared to be due to changes in

[0245] Another interesting observation was that B28 lost binding to human PAI-1 when immobilized via amine coupling, suggesting that the CDR regions of B28 contain amino acids with primary amino groups.

[0246] Example 11: Selection of Mouse Monoclonal Antibodies for Humanization Table 13 shows a summary of in vitro data characterizing the most active monoclonal antibodies from the five fusions performed. Based on these data, A44 was selected as the most effective in the chromogenic assay and plasmin generation, while having the highest affinity in Biacore. was selected for humanization.

[0247] [Table 18]

[0248] The heavy and light chain sequences shown in Table 1 are aligned in Figure 12, and the CDRs defined by IMGT are highlighted in bold. Based on the in vitro data shown in Table 13, A44 was selected for humanization.

[0249] Example 12: Engineering of anti-PAI-1 A44 Fab: humanization, stabilization and mutation of unnecessary sequence motifs Several approaches, discussed below, were taken to humanize, stabilize and optimize the sequence motifs of the A44 murine antibody against PAI-1.

[0250] 1) Humanization The humanization protocol used is described in PCT / US08 / 74381 (US20110027266) (reference is made in its entirety). The mouse A44 variable light (VL) and variable heavy (VH) sequences were used to build homology models of the anti-PAI-1 A44 light chain (LC) and heavy chain (HC) in Molecular Operating Environment (MOE; v. 2010.10; Chemical Computing Group). The following templates were used: light chain framework - 1D5I (94% identity in the framework regions), heavy chain framework - 3KSO (96% identity in the framework regions), L1 - 1D5I (94% identity), L2 - 1D5I (94% identity), L3 - 1AXS (72% identity), H1 - 1IC7 (82% H2 - 1MBU (68% identity), H2 - 1MBU (68% identity) and H3 - 2WDB (62% identity). The H3 loop was particularly difficult to model because Trp is the first residue. 2WDB is also a shorter loop, but has a Trp at the start of the loop and the same Phe-Asp-Tyr sequence at the end of the H3 loop. Glu-105 (LC) and The side chains of His-99 and His-100 were reconstructed and the model was subsequently modified using standard procedures implemented in MOE. The protein was energy minimized using the NMR spectroscopy (NMR spectroscopy) tool. Molecular dynamics (MD) simulations of a minimal 3D homology model of mouse A44 were then performed using restraints on the protein backbone at a temperature of 500 K for 1.1 nanoseconds (ns) in Generalized Born implicit solvent. Ten diverse conformations were extracted from this initial MD run every 100 picoseconds (ps) for the last 1 ns. These were then Multiple conformations were analyzed for 2.3 ns at 300 K without any constraints on the protein backbone. Then, for each of the 10 MD runs, the last 2,000 snapshots from the MD trajectory (one per 1 ps) were used to analyze each mouse A44 alpha. For amino acids, their root mean square deviations (rmsd) compared to a reference medoid position were calculated. By comparing the average rmsd of 10 separate MD runs for a given amino acid to the overall average rmsd of all A44 mouse amino acids, it was determined that the amino acid was sufficiently likely to interact with the T cell receptor and be responsible for the activity of the immune response, as seen during MD. To determine whether the amino acids are flexible in the mouse A44 antibody, 37 amino acids were identified as flexible, excluding the CDRs and the surrounding 5 angstroms.

[0251] The movements of the 62 most mobile mouse A44 amino acids over a 20 ns (10x2 ns) time period were then compared to the movements of the corresponding mobile amino acids in 49 human germline homology models, each of which was run in a 10x2 ns MD simulation. The 49 human germline models were compared to the 7 most common Systematically combined human germline light chains (vk1, vk2, vk3, vk4, vlambda1, vlambda2, vlambda3) and the seven most common human germline heavy chains (vh1a, vh1b, vh2, vh3, vh4, vh5, vh6) The vk1-vh2 human germline antibody showed 0.58 4D similarity of its flexible amino acids compared to the flexible amino acids of the mouse A44 antibody; therefore, the vk1-vh2 germline antibody The vlambda3-vh4 human germline showed the second highest 4D similarity, 0.57, and was also used as the basis for humanizing the A44 antibody. For pairwise amino acid associations between mouse A44 and vk1-vh2 amino acids, the two sequences were aligned by optimal 3D superposition of the alpha carbons of the two corresponding homology models. Pairwise amino acid associations between mouse A44 and vlambda3-vh4 was performed in a similar manner. Figure 13 shows the alignment of mouse A44 light chain with vk1 and vlambda3. FIG. 14 shows the alignment of mouse A44 heavy chain with vh2 and vh4.

[0252] 2) Stabilization a) Knowledge-based approach Light and heavy chains with low occurrence frequency relative to their respective canonical sequences The amino acids in the chain (excluding CDRs) are the most frequently occurring amino acids (ΔΔGth > 0.5 kcal / mol (E. Monsellier, H. Bedouelle. J. Mol. Biol. 362, 2006, p. 580-593)). This initial list of consensus mutations for the LC and HC was limited to amino acids found in the closest human germline (vk1-vh2). Proposed mutations near the periphery of the CDRs (5 angstrom "Vernier" zones (J. Mol. Biol. 224, 1992, p. 487-499)) The changes that were identified were removed from consideration. This resulted in two stabilizing mutations in the LC (see Table 15). ) and five stabilizing mutations in HC (see Table 16). Other criteria were considered to consider these mutations for potential stabilization of the anti-PAI-1 A44 antibody. These criteria were favorable changes in hydropathy at the surface or molecular dynamics-based putative stabilization of the variant. Further stabilizing mutations were also reported as successful in the literature (E. Monsellier & H. Bedouelle, J. Mol. Biol., 362, 2006, p. 580-593; BJ Steipe et al. J. Mol. Biol., 1994, 240, 188-192) and were considered (see Tables 17 and 18), but no further mutations were suggested.

[0253] [Table 19]

[0254] [Table 20]

[0255] [Table 21]

[0256] [Table 22]

[0257] b) 3D and MD-based approaches 3D and MD-based approaches have been reported previously (Seco J., Luque FJ, Barril X., J. Med. Chem. 2009 Apr 23:52(8):2363-71; Malin Jonsson et al., J. Phys. Chem. B 2003, 107:5511-5518). The hydrophobic regions of the antibody were analyzed by elution with a binary solvent (20% isopropanol in water). The Fab was unambiguously identified by analyzing molecular dynamics simulations of the Fab in a 20 ns production simulation (Figure 1). Further analysis was completed using a hydrophobic surface map within Schrodinger's maestro software (v. 8.5.207). The protein surface analyzed by these two methods was highly hydrophilic. Using both these techniques, there were no residues that contributed to any hydrophobic patches on the surface, thus suggesting no anti-aggregation mutations.

[0258] 3) Humanization through grafting Humanization using grafting techniques has been reported previously (PT Jones, PH Dear, J. Foote, MS Neuberger, G. Winter, Nature 1986, 321:522-525). Humanization was performed by identifying the two closest human germline sequences to the anti-PAI1 A44 variable domain light and heavy chains. This was done by performing BLAST searches against all systematically enumerated human germline sequences. (V and J domains for kappa and lambda chains; all possible combinations of V, D and J domains for the heavy chain). BLAST searches were performed using an internet application linked to the Sequence Information Retrieval and Analysis (SIRA) service provided by the National Center for Biotechnology Information (NCBI).

[0259] The closest human germline is 70% for the anti-PAI1 A44 variable domain light and heavy chains, respectively. and 67% sequence identity. Using the internal VBASE germline, the light chain was found to be close to the V I-O18 (approximately 64% identity) locus, and the heavy chain was found to be close to the 4-30 locus of the VH4 subfamily (approximately 69% identity). The CDR regions (based on Kabat) and Vernier residues were similar to the mA44 light chain. The humanization mutations (Ball, IGVK1-33-01_IGKJ4-01 (IGVK1) and IGVK1-33-01_IGKJ4-01 (IGVK1) are shown in italics. Vernier residues as defined in J. Mol. Biol., 1992, 224, 487 are underlined. The 2011 humanized mouse heavy chain was generated by performing a pairwise comparison of the two aligned sequences, excluding the CDR and Vernier zone residues defined above (also underlined in mouse). T46L and Q69T from the mouse light chain and M2V (Vernier zone residues) in the mouse heavy chain were mutated to the mostly conserved human germline sequence as part of the humanization by a grafting approach (LC5a, HC5a). In another variant, these three Vernier zone residues were mutated to the 2011 humanized mouse heavy chain (LC5a, HC5a) by replacing the 2011 humanized mouse heavy chain (LC5a, HC5a). The LC5b and HC5b were maintained as LC5b and HC5b, respectively.

[0260] mA44 - light chain (SEQ ID NO: 141) [ka]

[0261] IGKV1-33-01_IGKJ4-01 (SEQ ID NO: 107) [ka]

[0262] mA44 - heavy chain (SEQ ID NO: 140) [ka]

[0263] IGHV4-59-02_IGHD6-13-01_IGHJ4-02 (SEQ ID NO: 108) [ka]

[0264] The next closest human germline was identified with 59% and 58% sequence identity to the anti-PAI1 A44 variable domain light and heavy chains, respectively. Using the internal VBASE germline, the light chain was VκIII-L6 (about 56% identity) locus was found to be close, and the heavy chain was of the VH6 subfamily. of 6-01 The CDR regions (based on Kabat) and the Vernier region were found to be close to the locus. The CDRs and Vernier band residues (as defined in J. Mol. Biol., 1992, 224, 487) are shown in bold and underlined. The humanizing mutations were obtained by performing a pairwise comparison of the two aligned sequences, excluding the CDRs and Vernier band residues (also underlined in mouse) as defined above and shown in bold.

[0265] mA44 - light chain (SEQ ID NO: 141) [ka]

[0266] IGKV3-11-02_IGKJ4-01 (SEQ ID NO: 143) [ka]

[0267] mA44 - heavy chain (SEQ ID NO: 140) [ka]

[0268] IGHV6-1-02_IGHD6-13-01_IGHJ4-02 (SEQ ID NO: 144) [ka]

[0269] 4) Mutation of unwanted sequence motifs The following sequence motifs were considered: Asp-Pro (acid-labile bond), Asn-X-Ser / Thr (glycoside bond), X = any amino acid except Pro), Asp-Gly / Ser / Thr (succinimide / iso-asp formation in flexible regions), Asn-Gly / His / Ser / Ala / Cys (exposed deamidation sites), and Met (oxidation in exposed regions). The VL and VH domains of mouse anti-PAI1 A44 contain two potential Contains glycosylation sites: N in LC 52 RS (in CDR2) and N in HC 72 TS. One exposed A deamidation site is present in the CDR1 of HC (N 31 G). Three potential succinimide formation sites were identified in the original murine sequence: D in the LC 56 G (end of CDR2), and D 27 S (in CDR1) and D in HC 89 T. LC problem motif, N 52 RS and D 56 Both Gs are in CDR2. Because these mutations occur in the CDRs, they are the basis for the two proposed engineered sequences: This was addressed by mutations in the sequences (LC2 and LC4). 52 is conservatively mutated to Gln, and D 56 was mutated to Glu. There were four existing problematic residues in the HC. The first two occur in CDR1: a potential succinimide formation site, D 27 S, and deamidation site N 31 G. Two further A problematic motif also exists in the third framework region. In CDR1, D 27 was mutated to E, but N 31 Changed to Q. N 72 and D. 89 were changed to Q and E, respectively. These problematic motifs were addressed in the engineered sequences HC2a and HC4 described below. The HC2b mutant 31Only G deamidation site mutations Includes.

[0270] The resulting humanized sequences were identified in the IEDB database (found on the World Wide Web at immuneepitope.com, version June 2009; Vita R., Zarebeski L., Greenbaum JA, Emami H., Hoof I., Salimi N., Damle R., Sette A., Peters B. The immune epitope database 2.0 Nucleic Acids Res. 2010, Jan, 38 (Database issue):D854-62. Epub 2009, Nov 11) to ensure that none of the sequences contained any known human B or T cell epitopes (70% sequence identity was used as the cutoff for results obtained by BLAST search, and only results from the human species were considered). DeClerck et al. (International Publication No. WO 2002034776) identified an epitope for PAI-1. Each of the present publications discloses antibodies that bind to epitopes, none of which are relevant to the epitopes disclosed herein.

[0271] For mouse A44 LC, there is one human epitope from Kirschmann et al. (The Journal of Immunology, 1995, 155, 5655-5662), which consists of 14 amino acids as shown below. It has approximately 71% identity across the chain. The control sequence was a partial sequence that was not confirmed by mass spectrometry. No binding data was reported for this peptide. This epitope was seen in all proposed LV variants. No potentially problematic epitopes were identified when a similar search was performed for HC.

[0272] 5) Original sequence of anti-PAI1 variable domain The CDRs are highlighted in bold and the Vernier regions (as defined by Foote and Winter, J. Mol. Biol., 1992, 224:487-499) are underlined.

[0273] Light chain (SEQ ID NO: 142) [ka]

[0274] Germinality index = 70% for IGKV1-33-01_IGKJ4-01 [V I-O18]

[0275] Heavy Chain (SEQ ID NO: 140) [ka]

[0276] Embryonic index = IGHV4-59-02_IGHD6-137-01_IGHJ4-02 [VH4 4-30] 67%

[0277] 6) Manipulated sequences The 4D humanization and grafting approach was applied to the two closest human germline sequences. a) Engineered light chain sequences LC1a contains 7 mutations derived from 4D humanization using the closest germline sequence vk1. LC1b has 12 mutations derived from 4D humanization against the second closest human germline sequence vl3. LC2 contains 2 additional mutations in CDR2 compared to LC1a. These mutations are responsible for the potential glycosylation sites (N 52 RS) and potential succinimide forming sites ( D 56G). LC3 contains mutations from 4D humanization to the closest germline sequence, including two additional stabilizing mutations. LC4 combines humanized, stabilizing, and unnecessary motif mutations. CDRs and vernier bands are italicized, vernier residues are underlined, and humanized mutations are The differences are in bold, the problematic motif is double-strikethrough, and the stabilizing mutations are shown in the lower box. Figures 16 and 17 show a summary of the mutations.

[0278] LC1a (SEQ ID NO:91): [ka]

[0279] No further human epitopes for sequence LC1a were found in the IEDB database. LC1a embryonic index = 76% for IGKV1-33-01_IGKJ4-01 [VκI-O18].

[0280] LC1b (SEQ ID NO:92): [ka]

[0281] In addition to the epitopes described in section 4 above, K39PGQSPKTLI shares 70% sequence identity with KPGQPPRLLI (Kirschmann et al. J. Immun., 1995, 155, 5655-5662). This peptide has an IC50 >100,000 nM against all HLA-DR alleles for which it was tested. It has been reported that LC1b embryonic index = 67% for IGKV1-33-01_IGKJ4-01 [VκI-O18].

[0282] LC2 (SEQ ID NO:93): [ka]

[0283] No further human epitopes were found for sequence LC2 in the IEDB database. Ta. LC2 embryonic index=76% for IGKV1-33-01_IGKJ4-01 [VκI-O18].

[0284] LC3 (SEQ ID NO:94): [ka]

[0285] No further human epitopes were found for sequence LC3 in the IEDB database. LC3 embryonic index = 78% for IGKV1-33-01_IGKJ4-01 [VκI-O18].

[0286] LC4 (SEQ ID NO:95): [ka]

[0287] No further human epitopes were found for sequence LC4 in the IEDB database. LC4 embryonic index = 78% for IGKV1-33-01_IGKJ4-01 [VκI-O18].

[0288] LC5a (SEQ ID NO:96): [ka]

[0289] In addition to the epitopes described in section 4 above, A43PKLLIYRAN shares 80% sequence identity with APKLLIYAASSL (Kirschmann et al. J. Immun., 1995, 155, 5655-5662). No molecular weight was determined for this peptide and no binding data were reported. LC5a Embryonic index=IGKV1-33-01_IGKJ4-01 [VκI-O18], 85%.

[0290] LC5b (SEQ ID NO:97): [ka]

[0291] No further human epitopes were identified for sequence LC5b in the IEDB database. LC5b embryonic index=83% for IGKV1-33-01_IGKJ4-01 [VκI-O18].

[0292] LC5c (SEQ ID NO:98): [ka]

[0293] In addition to the epitopes listed in Section 4 above, 39 PGQAPRTLI has 80% sequence identity to KPGQPPRLLI (Kirschmann et al. J. Immun., 1995, 155, 5655-5662) This peptide has an IC50 of >100,000 nM against all HLA-DR alleles for which it was tested. It was reported to have LC5c. Germline index=79% for IGKV3-11-02_IGKJ4-01 [VκIII-L6]. A diagram of all light chain mutations is shown in FIG. 15.

[0294] b) Engineered Heavy Chain Sequences HC1a contains eight mutations derived from the 4D humanization method relative to the closest human germline sequence. HC1b contains six nucleotides derived from the 4D humanization method against the second closest germline sequence. HC2a contains four additional mutations compared to HC1a to address unwanted sequence motifs. HC2b contains a deamidation site (N 31 G). HC3 contains humanized mutations from HC1a, including five additional stabilizing mutations. HC4 contains humanized mutations from HC1a, stabilizing mutations from HC3, and the problematic motif from HC2a. The CDRs and vernier bands are in italics, vernier residues are underlined, Humanizing mutations are in bold, problematic motifs are double strikethrough, and stabilizing mutations are shown in the lower box.

[0295] HC1a (SEQ ID NO:82): [ka]

[0296] No human epitopes were identified for the sequence HC1a in the IEDB database. HC1a embryonic index = 68% for IGHV4-31-03_IGHD6-25-01_IGHJ4-02.

[0297] HC1b (SEQ ID NO:83): [ka]

[0298] No human epitopes were identified for sequence HC1b in the IEDB database. HC1b embryonic index = 73% for IGHV4-31-03_IGHD6-25-01_IGHJ4-02.

[0299] HC2a (SEQ ID NO:84): [ka]

[0300] No human epitopes were identified for sequence HC2a in the IEDB database. HC2a embryonic index = 67% for IGHV4-31-03_IGHD6-25-01_IGHJ4-02.

[0301] HC2b (SEQ ID NO:85): [ka]

[0302] No human epitopes were identified for sequence HC2b in the IEDB database. HC2b embryonic index = 67% for IGHV4-31-03_IGHD6-25-01_IGHJ4-02.

[0303] HC3 (SEQ ID NO:86): [ka]

[0304] No human epitopes were identified for sequence HC3 in the IEDB database. HC3 embryonic index=IGHV4-31-03_IGHD6-25-01_IGHJ4-02, 72%.

[0305] HC4 (SEQ ID NO:87): [ka]

[0306] No human epitopes were identified for sequence HC4 in the IEDB database. Embryonic index=IGHV4-31-03_IGHD6-25-01_IGHJ4-02, 70%.

[0307] HC5a (SEQ ID NO:88): [ka]

[0308] No human epitopes were identified for the sequence HC5a in the IEDB database. HC5a Embryonic Index=IGHV4-59-02_IGHD6-13-01_IGHJ4-02 [VH4 4-59] 84%.

[0309] HC5b (SEQ ID NO:89): [ka]

[0310] No human epitopes were identified for the sequence HC5b in the IEDB database. HC5b Embryonic Index=IGHV4-59-02_IGHD6-13-01_IGHJ4-02 [VH4 4-59] 84%.

[0311] HC5c (SEQ ID NO:90): [ka]

[0312] No human epitopes were identified for the sequence HC5c in the IEDB database. HC5c Embryonic Index=IGHV6-1-02_IGHD6-13-01_IGHJ4-02 [VH6 6-01] 78%.

[0313] A diagram of all heavy chain mutations is shown in FIG.

[0314] c) Combinations of heavy and light chain variant sequences For grafting, three versions of the light chain (LC5a, LC5b, LC5c) and three versions of the heavy chain (HC5a, HC5b, HC5c) were generated. LC5a contains 16 mutations derived from grafting to the closest human germline sequence and retains most of the mouse CDR and mouse Vernier zone residues. Two mouse Vernier residues, T46 and N69, are not found in either human germline. LC5b contained 14 mutations derived from grafting to the closest human germline sequence and retained the mouse CDR and all mouse Vernie zone residues. LC5c contained 14 mutations derived from grafting to the second closest human germline sequence and retained the mouse CDR and all mouse Vernie zone residues. The antibody contained 22 mutations derived from the mouse CDRs and retained all of the mouse Vernier zone residues.

[0315] HC5a contained 20 mutations derived from grafting to the closest human germline sequence and retained most of the mouse Vernier zone residues except for the mouse CDRs and M2V. , which occurs at this position in human germline sequences with a very low propensity. HC5b contains 20 mutations derived from grafting to the closest human germline sequence and has the same mutations as the mouse CDRs and All mouse Vernier zone residues are retained. HC5c is the second closest chromosome to the human germline sequence. It contains 23 mutations derived from rafting and retains the mouse CDRs and all the mouse Vernier zone residues.

[0316] A total of 10 combinations were prepared (summarized in Table 19): LC1axHC1a (mutations addressing 4D humanization based on closest germline sequence) LC1bxHC1b (mutations addressing 4D humanization based on the second closest germline sequence) LC2xHC2a (4D humanization and mutations to address unwanted sequences) LC2xHC2b (4D humanization and mutations to address unwanted sequences) LC1axHC2b (4D humanization and mutations addressing unwanted sequences) LC3xHC3 (mutations addressing 4D humanization and stabilization) LC4xHC4 (4D humanization, unwanted sequences and stabilizing mutations) LC5axHC5a (humanized by grafting, retaining the CDRs and three conservative Vernier Mutations that address the incorporation of modifications) LC5bxHC5b (mutations allowing for the retention of humanized CDRs and Vernier regions by grafting) LC5cxHC5c (mutations allowing for the retention of humanized CDRs and Vernier regions by grafting)

[0317] [Table 23]

[0318] [Table 24]

[0319] [Table 25]

[0320] [Table 26]

[0321] [Table 27]

[0322] In summary, 10 mutants were generated during the humanization process, which were expressed and characterized in several in vitro assays described below.

[0323] 7) Characterization of humanization in mutants Based on the computational modeling shown in the examples above, ten variants were generated (variants 1-8 by 4D humanization and variants 9-10 by CDR grafting; variant 3). and 10 were generated relative to the next closest germline. The variable regions were prepared for HEK293 expression. The proteins were cloned into the corresponding DNA pXL plasmids (New England Biolabs; NheI / Eco47III for HC, NheI / BsiWI for LC). The humanized sequence was codon optimized for HEK expression and the gene was synthesized by GeneArt (a subsidiary of Life Technologies). The resulting plasmid was Transfected with FreeStyle TM Transient expression was performed in the 293 expression system (Invitrogen, Cat. No. K9000-01). Mutants 3 and 10 were poorly expressed and were not pursued further. All other mutants were expressed and purified using a Protein A column. Analysis gels showed partial glycosylation (approximately 5-10%) of the light chain in variants 6 and 9 and of the heavy chain in variants 5 and 7 (data not shown). The remaining eight variants were assayed for plasmin activity in human astrocytes using a chromogenic assay using hPAI and human glycosylated PAI. The results are shown in Table 23.

[0324] [Table 28]

[0325] Mutants 6 and 9 showed the best potency in the plasmin generation assay, but had partial (5-10%) glycosylation in the light chain. Based on these results, new mutants 11-14 were produced using a combination of heavy chains from mutants 6 and 9 and light chains from mutants 5 and 7. Table 24 shows a summary of all mutants made.

[0326] [Table 29]

[0327] [Table 30]

[0328] [Table 31]

[0329] [Table 32]

[0330] [Table 33]

[0331] [Table 34]

[0332] All variants except for variants 3 and 10, which were poorly expressed, were tested in Biacore against human and cynomolgus PAI-1 and vitronectin-PAI-1 complexes. The data are shown in Table 26. show.

[0333] [Table 35]

[0334] [Table 36]

[0335] The Biacore data showed no significant differences between the humanized variants. All except variant 8 The humanized variants of A44 showed affinity for both cynomolgus monkey PAI-1 and human PAI-1, and PAI-1 complexed to vitronectin within an acceptable range. Compared to the parent A44, humanization did not appear to alter antibody affinity. The affinity and potency of the humanized variants were not significantly different in chromogenic and Biacore assays, but were significantly higher than that of the humanized variants in the cell assay. The ability of the mutants to restore sumin production was significantly lower than the parental mouse antibody for some mutants (see Table 27 below for a comparison of the chromogenic and cellular assays). Humanized variants 11–14 were examined for their ability to block PAI-1 in cellular assays. Tested.

[0336] [Table 37]

[0337] Mutants 11-14 showed good potency in the plasmin generation assay and were further characterized in additional in vitro assays.

[0338] 8) Characterization of humanized mutants in human liver Further screening of humanized mutants 11–14 was performed using endogenously produced human PAI-1 PAI-1 activity was measured in human plasma and human fibrotic liver samples using a 96-well platelet counting assay. The activity of this serpin was assessed by measuring its ability to form a stable complex with urokinase immobilized on a gel plate. After washing away unbound PAI-1, uPA-PAI-1 complexes were detected by the use of a polyclonal anti-PAI-1 antibody. Bound polyclonal anti-PAI-1 antibody, which is proportional to active PAI-1 in the sample, was then detected using horseradish peroxidase conjugated to a secondary antibody (Molecular Innovation Cat. No. HPAIKT). Various concentrations of the A44 humanized variants were incubated with either human or cynomolgus recombinant PAI-1 (0.31 nM final concentration). After incubation at room temperature for 15 minutes, the uPA-PAI-1 complexes were then tested for functionally active PAI-1 using the ELISA described above. Samples were compared to a human PAI-1 standard. Highly active PAI-1 Human plasma from high BMI patients with uPA levels was diluted 4-fold and incubated with increasing amounts of the A44 humanized variant. Residual active PAI-1 levels were determined using uPA-PAI-1 complexes detected by ELISA. Cynomolgus monkey recombinant PAI-1 neutralization was also tested by plasmin generation to confirm cross-reactivity.

[0339] [Table 38]

[0340] Human fibrotic liver samples (provided by Biopredic International, Rennes, France, from surgical resection of hepatic colon metastases) were homogenized as follows: weighed frozen liver samples were homogenized. The pull was filled with ceramic beads (Cat No. 03961-1-003, Bertin Technology, France). Homogenization was performed in a dry tube using a Precellys homogenizer (Bertin Technology, France; 2x30 sec at 6800 rpm at 4°C) and then lysed using 1 ml / g lysis buffer (1.5 M Tris buffer 0.1 M Tris + 0.15 M NaCl in NaCl TBS pH 7.4). Centrifugation was performed for 10 min at 5000 g at 4°C. Afterwards, liver lysates in the supernatant were harvested and stored frozen at -80°C. Total protein concentration using a standard BCA assay and active and total PAI-1 levels (determined by UK-PAI combined ELISA provided by Mol Innov Cat. No. HPAIKT and Cat. No. MPAIKT-TOT) were performed according to the manufacturer's instructions by plotting standard human PAI-1 concentration against A450nm using Biostat Calibration software. Increasing concentrations of A44 humanized variants incubated with liver lysates diluted to 2.5nM active PAI-1 were analyzed by the ELISA kit as previously described. The data were analyzed by assessing the inhibition of PAI-1 activity (PAI-1 activity without mAb was 0% inhibited, and IgG1 did not cause significant and dose-dependent inhibition of PAI-1) for each mAb concentration. The percent inhibition of PAI-1 activity was plotted as a function of mAb concentration and IC50 was determined and Imax was calculated using Biostat speed software. The data are shown in Figure 17 and Table 29. As shown in.

[0341] [Table 39]

[0342] Based on the above data, A44-hv11 was selected for further characterization in additional structural studies as well as additional in vitro and in vivo studies.

[0343] Example 13: Humanization of APG antibody by grafting Humanization using grafting techniques has been reported previously (PT Jones, et al., Nature 1986, 321:522-525). Humanization of the anti-PAI1 murine antibody APG was reported in German patent application DE200015 We started with the mouse light chain (SEQ ID NO: 148) and mouse heavy chain (SEQ ID NO: 149) from 3251; The mouse antibody was prepared by Debrock et al., Biochimica et Biophysica Acta, 1337(2):257-266 (1997). has also been described. Identifying the germline and canonical class of the HC and LC chains of the mouse antibody yielded muIGHV1-39 and muIGKV14-111, respectively. Next, a list of human germlines close to the anti-PAI1 APG variable domain light and heavy chains was identified and ranked by percent identity. Both steps were performed using BLAST searches against all systematically listed human germlines. A BLAST search was performed by performing a 3D search (all possible combinations of V and J domains for kappa and lambda chains; V, D and J domains for the heavy chain). BLAST searches were performed using the IMGT / DomainGapAlign tool provided at http: / / www.imgt.org. (See Ehrenmann, et al. Cold Spring Harbor Protocols 2011.6 (2011)). The closest human germline was identified with 67.4% and 63.3% sequence identity to the anti-PAI1 APG variable domain light and heavy chains, respectively. Using the IMGT database, the light chain was found to be close to HuIGKV1-33 and the heavy chain was close to HuIGHV1-46. The closest human germline to the anti-PAI1 APG variable domain heavy chain with matching reference classes was found to be HuIGHV7-4-1 with 62.2% sequence identity.

[0344] CDR regions (based on Kabat and IMGT combination for APG) and Vernier residues are shown in italics for the parent mouse APG (mAPG) light chain (SEQ ID NO: 148), IGKV1-33-01_IGKJ4-01 (IGKV1a) (SEQ ID NO: 107) and IGKV1-33-01_IGKJ2-02 (IGKV1b) (SEQ ID NO: 150) (see Table 30 below). The Vernier residues as defined in Foote, et al. J. Mol. Biol. 224(2):487-99 (1992) are underlined. The CDR and Vernier zone residues as defined above (also in the mAPG sequence) are underlined. Humanization was performed by performing a pairwise comparison of the two aligned sequences, except for those marked with an underline (Table 30). The mutations (in bold) were obtained. No further manipulations were performed on the mouse APG antibody. These The humanized antibodies were named APGv2 and APGv4.

[0345] [Table 40]

[0346] [Table 41]

[0347] The manipulated array The 4D humanization and grafting approach was applied to the human germline sequence matches described above. For the engineered light chain sequence, APGv2 was a mouse light chain spliced ​​into the human IGKV1-33 germline. For the engineered heavy chain sequences, APGv2 and APGv4 were derived from human IGHV7-4-1 and IGHV1-46 germline CDRs (APGv2 germline index = 94% with IGKV1-33-01_IGKJ2-01). 91% for APG_VH2 Embryonic Index=IGHV7-4-1-02_IGHD6-25-01_IGHJ4-02; 91% for APG_VH4 Embryonic Index=IGHV1-46-01_IGHD6-25-01_IGHJ4-02) containing mouse heavy chain CDRs grafted into the sequence. See Table 30 above.

[0348] Combinations of heavy and light chain variant sequences For grafting, one version of the light chain (APGv2_VL2; SEQ ID NO: 153) and two versions of the heavy chain (APGv2_VH2; SEQ ID NO: 154 and APGv4_VH4; SEQ ID NO: 155) were generated. APG_VL2 contains 15 mutations derived from grafting to the closest germline sequence. APG_VH2 retains the mouse CDR and Vernier zone residues. APG_VH2 is the most suitable VH2-dependent nucleotide sequence with a matching canonical class. It contains 21 mutations derived from grafting to the closest germline sequence, and contains the mouse CDRs and APG_VH4 contains 20 mutations derived from grafting to the closest human germline sequence, retaining the murine CDR and Vernier zone residues. The delimitation of the CDRs for this grafting protocol was based on a variety of different sequences available in the literature. Based loosely on the definition given by

[0349] APG_VL2xAPG_VH2 (mutations addressing humanization by grafting that preserves the CDRs and Vernier regions) APG_VL2xAPG_VH4 (humanized by grafting that retains the CDR and Vernier regions) Mutations to be addressed) Two mAPG variants were generated during this humanization campaign, designated APGv2 and APGv4, and were expressed and characterized in several in vitro assays, as described below.

[0350] Example 14: Affinity kinetics for APG antibodies by surface plasmon resonance Affinity for human glycosylated PAI-1 (GLYHPAI-A, Molecular Innovation) was assayed for mouse APG and two humanized variants (APGv2 and APGv4) on a Biacore 2000 instrument (GE Healthcare The fluorophores were probed by surface plasmon resonance (SPR) using a fluoroscopy scanner (Uppsala, Sweden).

[0351] First, the surface of a sensor chip CM5 (GE Healthcare, Uppsala, Sweden) was Human anti-Fc (anti-human IgG (Fc) antibody and anti-mouse IgG antibody kit, GE Healthcare) was prepared using standard amine coupling for capture. All monoclonal antibodies (mAbs) were diluted to 5 nM using HBS-EP running buffer. Each purified mAb was incubated with different mAbs for 3 min. Human PAI-1 was captured on the surface of a flow cell consisting of 100 μM ... A short dissociation time was used in the middle and a long dissociation time at the end (contact time: 120 s, short dissociation time: (Long dissociation: 90 sec; Long dissociation: 1800 sec, Flow rate: 50 μl / min). The chip was regenerated with glycine-HCl, pH 1.7 buffer after each antibody-PAI-1 binding. Kinetic data analysis was performed using Biacore BIAevaluation software. Sensorgrams were double referenced by subtracting the reference flow cell values ​​and blank buffer values. Sensorgrams were fitted using a simulated kinetic 1:1 (Langmuir) model with local Rmax. (See FIG. 19). Data for the three APG antibodies are shown in Table 31.

[0352] [Table 42]

[0353] Example 15: Characterization of APG antibodies in human plasma Mouse APG and the humanized variants APGv2 and APGv4 were used in the functional assays disclosed herein. The serpins were screened for their ability to block PAI-1 according to the method described above (see, e.g., Examples 6 and 9 above). Briefly, PAI-1 activity was assessed by the ability of this serpin to form a stable complex with urokinase immobilized on a 96-well plate. After washing away unbound PAI-1, the uPA-PAI-1 complex was isolated by using a polyclonal anti-PAI-1 antibody. The bound polyclonal anti-PAI-1 antibody (relative to the active PAI-1 in the sample) was then detected. For example, use horseradish peroxidase conjugated to a secondary antibody according to the manufacturer's instructions. (Molecular Innovation, Cat. No. HPAIKT).

[0354] Various concentrations of APG humanized variants (APGv2, APGv4) or parental mouse APG antibody were incubated with undiluted human plasma with high activity PAI-1 levels at room temperature for 15 minutes. Residual active PAI-1 levels were determined by ELISA using uPA-PAI-1 complex detection (see, e.g., Example 6) according to the manufacturer's instructions.

[0355] Inhibition of PAI-1 activity was calculated for each mAb concentration. Percent inhibition of PAI-1 activity was plotted as a function of the concentration of APG humanized variants (APGv2, APGv4) or parental mouse APG antibody. IC was calculated after three independent experiments (in duplicate) using Biostat speed software. 50 and I max was determined (see Figure 20). The data is shown in Table 32 below.

[0356] [Table 43]

[0357] Example 16: Clot lysis assay in human plasma: A44V11, mAPG, and APG mutant activity The fibrinolytic system is often altered in patients with stroke. Clot lysis assays can be used to determine fibrinolytic activity by measuring the extent of fibrin degradation. See generally Lindgren, A. et al. Stroke 27:1066-1071 (1996). Clot lysis assays are described in detail elsewhere. See, e.g., Beebe, et al. Thromb. Res. 47:123-8 (1987); Tilley et al., J. Vis. Exp.67:e3822.

[0358] The functional activity of A44V11 and other PAI-1 neutralizing antibodies was assessed using a human plasma clot lysis assay. Briefly, the assay applied here determined that tPA inhibits clot lysis. Clot formation is induced using a mixture of tissue factor / Ca2+ in the presence of known concentrations of PAI-1. Fibrin polymerization is monitored by turbidimetry, detected by absorbance measurements at 340 nm. The ability of the antibodies to restore clot lysis was determined by incubating normal human platelet poor plasma with increasing concentrations of the antibodies.

[0359] Briefly, clot lysis experiments were performed in microtiter plates. Citrated human plasma (Biopredic International, Rennes, France) was incubated with anti-PAI-1 antibodies or isotype control IgG diluted in assay buffer (NaCl, Tris-HCl pH=7.4). After 15 min incubation at room temperature, human glycosylated PAI-1 (GLYHPAI-A) was incubated with 100 μg / mL of IgG. t-PA (sctPA, Molecular Innovation) was then added to a final concentration of 1 nM and incubated for an additional 10 min. Clot formation was then monitored with tissue factor (Innovin) diluted in calcium assay buffer (CaCl2) to a final concentration of 7.5 mM. (R) The antibody was induced by an activation mixture containing 100 mM NaCl, 100 mM MHC class I (Siemens Healthcare Diagnostics, Marburg, Germany).

[0360] Dynamic readings of absorbance at 340 nm were taken every 30 s for 5 h using an iEMS microplate reader (ThermoFischer) or a Spectrostar Nano (BMG Labtech). To quantify the effect on clot lysis, the area under the curve (AUC), which reflects the balance between clot formation and clot lysis, was calculated. was calculated using GraphPad Prism software. Restoration of clot lysis after antibody treatment was determined according to the following calculation:

number

[0361] t-PA at a concentration of 1 nM caused complete lysis of normal plasma within 2 hours. PAI-1 at a concentration of 3 nM inhibited t-PA-induced clot lysis. Addition of either t-PA or PAI-1 alone had no effect on clot formation. Addition of neither t-PA nor PAI-1 had no effect on clot formation.

[0362] The A44V11 anti-PAI-1 antibody restored human platelet-poor plasma clot lysis (see FIG. 21). At 100 nM, isotype IgG1 was not restored (see FIG. 22). A44V11 had an I of 103%. max with an IC of 2 nM 50(See FIG. 23).

[0363] A humanized variant of the APG anti-PAI-1 antibody also restored human platelet-poor plasma clot lysis (Figure 24 APGv2 has an IC of 2.1 nM. 50 and 114% I max APGv4 had an IC of 2.8 nM. 50 and 116% I max at 100 nM (see FIG. 25). The clot lysis data are shown in the table below. To summarize: 33.

[0364] [Table 44]

[0365] Example 17: Evaluation of A44V11 Neutralization of PAI-1 in Primary Human Lung Cells The effect of antibody A44V11 on neutralizing PAI-1 was examined in a lung cell-based system. TGFβ was considered to be the most potent and widespread profibrotic cytokine. TGFβ was shown to induce PAI-1 expression and t-PA and plasmin activity, as well as collagen degradation in cultured mouse embryo fibroblasts (NIH3T3 cells). See Liu, RM. Antioxid Redox Signal. 10(2): 303-319 (2008). Primary lung cells from ATCC (Manassas, Virginia) were cultured in vitro and in vitro. Fibroblast cell lines LL29 (CCL-134) and LL97A (CCL-191) were plated overnight at 200,000 cells per well in 12-well plates. Cells were incubated with either the A44V11 antibody or an isotype control. The cells were incubated with IgG and TGFβ (R&D Systems, Minneapolis, Minn., Cat. No. 100-B-001) at a concentration of 5 ng / ml for 48 hours. After 48 hours, cell supernatants were harvested and probed with rabbit pAb anti-PAI-1 (abcam, ab66705) for detection of PAI-1 forms by Western blot. The analysis was carried out using

[0366] Cells treated with A44V11 antibody after TGFβ stimulation showed a PAI-1 band as a doublet, which corresponds to a truncated form of PAI-1 (see FIG. 26, lane 5). Cells that were transfected with A44V11 did not show this doublet formation (Figure 26, lane 6). Treatment of human lung cells induces an endogenous PAI-1 substrate conformation, which allows PAI-1 to be cleaved by proteases.

[0367] Example 18: A44V11 increases MMP activation Plasmin activates MMPs, enzymes capable of degrading most ECM proteins, including collagen, the major proteinaceous component of fibrous tissue. In this regard, plasmin is often cited as a general activator of MMPs (Loskutoff, et al. J. Clin. Invest. 106(12):1441-43 (2000). PAI-1 blocks plasmin generation. and subsequently inhibits MMP activation and matrix fragmentation by inhibiting fibroblast apoptosis. The ability of A44v11 to stimulate MMP activation was examined in a lung cell-based system. Primary lung fibroblasts LL29 (CCL-134) and LL97A (CCL-191) from ATCC (Manassas, Virginia) were used. , plated overnight at a concentration of 250,000 cells per well in 12-well plates. The cells were incubated with A44V11 or isotype control (IgG) and Lys-plasminogen activator for 48 h. The mice were incubated with 0.1 μM of 100 μM ... After 48 hours, cell supernatants were harvested and analyzed for various MMPs (e.g., MMP-1, 2, 3, 7, 8 , 9, 12, 13, and 14) was detected using the Sensoryte 520 Generic MMP Assay Kit (AnaSpec, Fremont, Calif., catalog no. 71158) according to the manufacturer's instructions.

[0368] As shown in FIG. 27, A44V11 inhibited the activity of plasmin-dependent MMPs in human lung fibroblasts. This chart shows a representative of two separate experiments. Cells treated with A44V11 and plasminogen showed substantially increased activation compared to cells treated with the negative IgG1 antibody. This study demonstrated that A44V11 stimulates MMP activity in a plasmin-mediated event. Demonstrate that it will.

[0369] Example 19: Analysis of the efficacy of A44V11 in a pulmonary fibrosis mouse model (bleomycin challenge) Bleomycin-induced experimental pulmonary fibrosis is a well-studied model of fibrosis that is well-documented. This model of pulmonary fibrosis resembles that seen in humans and has been used to evaluate the effects of potential therapeutic agents as well as basic research (see, e.g., Molina-Molina et al. Thorax 61:604-610 (2006)).

[0370] Pharmacodynamic studies in bleomycin-treated mice (fibrosis model) Transgenic mice expressing human PAI-1 (humanized PAI-1 transgenic mice) The mouse PAI-1 (SERPINE1) gene CDS (exons and introns) (NCBI Ref. No. NM_008871) was cloned with the corresponding human wild-type PAI-1 gene CDS (NCBI Ref. Nos. NM_000602.3; NC_000007.13) (see Klinger, KW et al. Proc. Natl. Acad. Sci. USA 84:8548 (1987)). The transgenic mice were generated by placing the endogenous mouse PAI-1 gene under the control of regulatory sequences in C57BL / 6x129 mice (The Jackson Laboratory, Bar Harbor, Maine). Molecular cloning and generation of transgenic mice were performed according to conventional techniques and in accordance with the manufacturer's and breeder's instructions. Expression of human PAI-1 and non-expression of mouse PAI-1 were confirmed in the transgenic mice. Both mRNA and protein levels were analyzed by standard qPCR and ELISA, respectively. The female zygotic humanized PAI-1 transgenic mice were 8-9 weeks old and weighed 22-25g. were used for these procedures. Rodent chow and water were provided ad libitum.

[0371] Mice were treated with bleomycin dissolved in 0.9% NaCl. (R) (Sanofi, France) 50 μl was administered by intratracheal injection with a microspayer at a dose of 2 mg / kg. For these procedures, mice were anesthetized with isoflurane (TEM, Lormont, France) by inhalation and then intubated with an 18G cannula. Anesthesia was maintained by connecting the mouse to a ventilator supplied with a bleomycin / isoflurane mixture. After anesthesia, a microsprayer was cannulated directly into the lungs for bleomycin infusion. The mouse was then Mice were extubated and allowed to recover from anesthesia. On day 4, they were randomized into three groups and then treated once with either A44v11 or negative control mouse IgG1 administered intraperitoneally at 10 mg / kg (1 mg / ml) in PBS.

[0372] At the indicated time points (day 7 or 9) after bleomycin challenge, mice were anesthetized with a xylazine / ketamine mixture and euthanized by thoracotomy in a citrate-coated tube. Blood collection was performed by intracardiac withdrawal of 100 µL ... (R) The lungs were then fixed using a 30-mm CT scanner (Leica Biosystems, Buffalo Grove, IL). A cannula was then placed in the trachea for a bronchoalveolar lavage (BAL) procedure (1.5 ml of 0.9% NaCl was instilled and withdrawn with three injections of 0.5 ml). The four lobes of the right lung were then harvested, cut into two pieces, and lysed for protein analysis. All experiments were performed in accordance with European ethical lows and approved by the internal ethical comity (CEPAL, sanofi).

[0373] A44V11 levels were measured using coated biotinylated human PAI-1 plates by ELISA (Molecular Innovation Anti-mouse IgG was detected using a sulfo-tagged secondary anti-mouse IgG (MesoScale Discovery, Gaithersburg, Maryland) for 7 days. treated with A44V11, the results were 200 nM in plasma, 11 nM in BALF, and 12 nM in lung lysates. .

[0374] As shown in FIG. 28, administration of a single intraperitoneal dose (10 mg / kg) of A44V11 on day 4 inhibited bleomycin-induced leukemia. Inhibition of BAL fluid and lung lysates in animals sacrificed 7 days after mycobacterial challenge For day 9 animals, A44V11 (10 mg / kg) achieved almost complete inhibition of human active PAI-1 in lung lysates, but not in BALF. However, only partial inhibition was achieved.

[0375] D-dimer, a fibrin degradation product, can be measured to evaluate the degree of fibrin degradation. To measure fibrin degradation, the level of D-dimer in BALF was detected by ELISA (Asserachrom D-Di, Diagnostica Stago, Asnieres, France) according to the manufacturer's instructions. The D-dimer level in BALF of A44V11 treatment group was increased by about 2.8 times on day 7 and 1.6 times on day 9 when compared with IgG1 negative control group, suggesting that A44V11 treatment increases fibrin degradation (see Figure 29).

[0376] Additional studies were conducted to further evaluate A44V11 activity in reducing fibrosis in bleomycin-challenged mouse lungs. For these studies, the study period length was 21 days from bleomycin challenge and antibody treatment (either A44V11 at 10 mg / kg or IgG1 control antibody) was repeated every 3 days starting on day 4 until day 20. Mice underwent a similar protocol to the pharmacodynamic study described above, except that on day 21 after bleomycin challenge, animals were sacrificed as described above.

[0377] Increased lung weight is known to be an indicator of increased fibrosis. Right lung weight, as a measure of fibrosis, was determined for mice in all experimental groups. As shown in Figure 30, bleomycin instillation partially suppressed the fibrosis observed with repeated dosing of A44V11 antibody at 10 mg / kg. Repeated dosing with the IgG1 negative control antibody did not inhibit the increase in right lung weight resulting from bleomycin challenge. The reduction in bleomycin-induced right lung weight gain in A44V11-treated mice was statistically significant (p<0.001) when compared to similar bleomycin-challenged mice treated with the IgG1 negative control antibody. Statistics Analysis was performed by one-way ANOVA followed by Newman-Keuls test. The results showed that A44V11 was Simulated PAI-1, but not control IgG1 antibody, inhibits bleomycin-induced fibrosis in mouse lungs It shows that it will not harm.

[0378] Collagen accumulation in the lung is another known indicator of fibrosis. To assay collagen accumulation, lung tissue from mice sacrificed on day 21 was prepared and separated by HPLC, followed by measurement of hydroxyproline. This technique has been described elsewhere, e.g., Hattori, et al. J Clin. Invest. 106(11):1341-1350 (2000). Briefly, lung tissue was prepared by hydrolysis under acidic conditions (6M HCl) for 22 hours at 105°C, followed by evaporation. Primary amines were blocked in the lung tissue with OPA (phthalaldehyde) and proline. Proline / hydroxyproline were specifically labeled using NBD (4-chloro-7-nitrobenzofurazan) (Santa Cruz Biotech., Santa Cruz, CA). The hydrolyzates were then purified using Synergistic TM The elution was performed on a 4 μm Hydro-RP 80Å, LC Column 150x3mm column (Phenomenex, Torrance, CA, Cat. No. 00F-4375-Y0) using HPLC (Shimazu Corp., Kyoto, Japan) with an acetonitrile gradient. The peaks were separated below. A standard curve of known amounts of hydroxyproline was used as a reference to quantitate the peaks. A representative of the quantitated data is shown in FIG.

[0379] Pulmonary collagen accumulation detected by hydroxyproline content was significantly increased in bleomycin-induced In mice treated with A44V11, the increase in pulmonary collagen accumulation was statistically increased in mice with bleomycin challenge. The increase in pulmonary collagen accumulation was statistically increased in mice with bleomycin challenge. The increase in pulmonary collagen accumulation was statistically increased in mice with bleomycin challenge. The increase in pulmonary collagen accumulation was statistically increased in mice with bleomycin challenge. The increase in pulmonary collagen accumulation was statistically significant when compared to similar mice with bleomycin challenge treated with IgG1 negative control antibody. The A44V11 treated mice showed approximately 44% less increase in collagen accumulation than the IgG1 control treated mice.

[0380] Example 20: Evaluation of A44V11 activity in an LPS challenge model in monkeys An acute lipopolysaccharide (LPS) challenge model in monkeys was applied to determine the PAI-1 neutralization efficacy of A44V11 in vivo. The LPS challenge model is described in Hattori, et al. J Clin Invest. 106(11):1341-1350 (2000). The activity of A44V11 mAb against PAI-1 in monkey plasma and liver samples was evaluated. Specifically, the experiment was performed using A44V11 (5 mg / kg, IP) The study was designed to evaluate the effect of a high dose of LPS (100 μg / kg-IV) on plasma and tissue levels of PAI-1 in anesthetized monkeys pretreated (24 h) with either IgG1 (negative control, 5 mg / kg, i.p.). The study was performed in accordance with European ethical laws. and recognized by the internal ethical comity (CEPAL, sanofi).

[0381] Cynomolgus Macaca fascicularis (male and female) weighing 4-9 kg were fasted overnight prior to chronic anesthesia (at least 8 h), which consisted of intramuscular (IM) injection of Zoletil 50 (Virbac, Taguig City, Philippines) at 0.12-0.16 mL / kg, followed by air / oxygen and isoflurane. The experiment consisted of the inhalation of a gas mixture of orchid (1–3%). The monkeys' body temperature was maintained at 37°C using a heating pad. After catheter insertion, LPS (serotype 0127-B8) was administered as a 1-min bolus in the cephalic accessory vein at a dose of 100 μg / kg (0.4 mL / kg). Blood and liver samples were taken at various time points. Platelet-poor plasma was isolated by centrifugation from the liver biopsies and terminal necropsies.

[0382] Active PAI-1, D-dimer and plasmin-α2 antiplasmin levels were determined using commercially available ELISA assays (Mol. Innovation, catalogue no. HPAIKT; Asserachrom D-Dimer; Plasmin-A2 antiplasmin, Diagnostica Stago) according to the manufacturer's instructions.

[0383] In plasma, active PAI-1 levels were reduced from about 30 ng / ml to less than 10 ng / ml in all monkeys administered A44v11 (see FIG. 32(A)). There was no increase in active PAI-1 levels following LPS challenge (100 ug / kg) (see FIG. 32(A)). In contrast, monkeys treated with the negative IgG1 control showed a strong increase in active PAI-1 levels following LPS challenge, with a maximum occurring at about 4 hours (from about 50 to about 250 ng / ml) (see FIG. 32(B)). Thus, treatment with the negative IgG1 control did not result in an increase in active PAI-1 levels following LPS challenge. It does not reduce the subsequently strongly increased levels of active PAI-1 in plasma (see FIG. 32(B)).

[0384] A similar phenomenon was observed in liver biopsy lysates. Monkeys treated with A44V11 mAb showed no increase in active PAI-1 levels after LPS treatment (Figure 33(A)). In contrast, LPS administration induced a strong increase (up to 3 ng / mg) in active PAI-1 in liver biopsy lysates from negative IgG1 control-treated monkeys. (See Figure 33(B)).

[0385] Concomitant with PAI-1 neutralization, D-dimer levels in A44V11-treated monkeys (see Figure 34(A)) were generally found to be higher than in negative IgG control-treated monkeys (see Figure 34(B)), thus suggesting that A44V11 treatment in monkeys also induces increased fibrinolysis in plasma.

[0386] Finally, plasma samples from A44V11-treated monkeys showed similar PAP levels to those in the negative IgG control-treated monkeys. In comparison, it showed increased levels of plasmin-α2 antiplasmin (PAP) complexes (see Figures 35(A) and (B)). The increase in PAP complexes and D-dimers in the presence of A44V11 indicates increased plasmin generation.

[0387] Example 21: Evaluation of A44V11 activity in an abdominal adhesion mouse model The effect of treatment with anti-PAI-1 antibody A44V11 on adhesion formation was examined in surgically injured mouse uterine horn models. The mouse uterine horn approximation and electrical cauterization procedures were performed to assess the uterine horn apposition to the serosal membrane. The procedure disrupts the surface, causes thermal damage to the uterine tissue, and approximates the damaged tissue surface during the healing process, which ultimately results in postoperative adhesions in 100% of untreated animals. The model and surgical procedure have been previously described in Haney AF et al., (1993). Fertility and Sterility, 60(3): 550-558.

[0388] For these adhesion studies, approximately 9-week-old rats were generated expressing the PAI-1 transgene. Humanized transgenic female mice weighing approximately 20 g were used. Forty-two adult transgenic female mice were divided into two groups and were cultured as described in detail in Haney AF et al. (1993). The animals underwent a surgical procedure designed to create adhesions between the uterine horns (UH) so that they could be cultured. Briefly, each animal was anesthetized with isoflurane for surgery according to IACUC guidelines and then A standard midline laparotomy was performed approximately 1.0 cm caudal to the xyphoid process. The UH was identified and approximated medially with one 7-0 Prolene suture (Ethicon Inc., Somerville, NJ) in each horn carefully placed through the muscle wall and the horns were then attached to the uterofallopian tubes. The ovarian vascular supply was then tied together just below the uterotubal junction. Care was taken not to injure the uterine horn. To induce electrocautery lesions, a bipolar electrocautery unit (Valley Lab Surgistat, Solid state Electrosurgery Unit, model number B-20) was used on the inner surface of each uterine horn over an area of ​​approximately 2x6mm. The cautery unit was set as follows: volts 100, 130Hz, 50-60amps. A 3mm wide cautery tip was placed over the inside of the horn with a pure coagulation current setting of 3. A current was applied, power was started, and contact was made for 1 second at two burn spots per corner. The muscle incision was closed in a continuous suture pattern using 5-0 Vicryl, BV-1 tapered needle (Ethicon Inc.). The skin was sutured horizontally with 5-0 Prolene, BV-1 tapered needle (Ethicon Inc.). It was closed with a mattress suture pattern.

[0389] After creating the UH injury, animals in group 1 were treated with a 0.16 mL volume of isotype control antibody (30 mg / kg), which was applied to the cautery burn. Animals in group 2 were treated in the same manner with a 0.16 mL volume of A44V11 For each group, animals were euthanized at 6 hours (n=5), 72 hours (n=4), or day 7 (n=12). (See Table 34 below.) Euthanasia was scheduled at 72 hours and day 7. The animals were then injected intraperitoneally (IP) with a second dose of antibody (30 mg / kg) 48 hours after surgery. did.

[0390] [Table 45]

[0391] Effectiveness evaluation and analysis: At the indicated time points, animals were euthanized and adhesion formation was assessed. Briefly, horn length was measured from the uterine bifurcation to a proximal suture placed just below the oviduct. The two external sutures surrounding the uterine horns were removed and the length of adhesions between the uterine horns was measured microscopically, documented and noted as present or absent (yes / no). Any tissue involved in adhesion formation is also recorded but may not be included in the length of the attached area. The distribution of the mean percent attached length between the uterine horns was checked for normality using the Shapiro-Wilk test. The groups were compared with each other using Tukey Kramer analysis if normally distributed and Wilcoxon rank sum analysis if not normally distributed. In all cases, a p value ≦0.05 was considered statistically significant. Treated animals showed a significantly lower percentage of length of adhesion formation between adjacent uterine horns (see Table 35).

[0392] [Table 46]

[0393] Detection of active PAI-1 and tPA levels After euthanasia, animals were provided with blood (plasma), intraperitoneal fluid (IPF), and uterine horn samples for evaluation. Sample collection was performed using conventional techniques. Plasma, IPF, and uterine horn samples The levels of active PAI-1 and tPA were assessed using ELISA. ELISA kit, catalog number HPAIKT, Molecular Innovations, Novi, MI). Data were processed using Excel, JMP, and Prism Graph Pad software. In all cases A p value of ≦0.05 was considered statistically significant. Decreased levels of activity at 6 hours and 7 days PAI-1 was secreted into the intraperitoneal (IP) fluid and (See FIG. 36.) Relative to isotype control, decreased levels of active PAI-1 at 6 hours in IPF were seen in A44-treated animals at 6 hours in IP fluid. The results were statistically significant at time points (p<0.001 by Student's t-test).

[0394] Example 22: Crystal structure of humanized antibody A44V11 Expression and purification of Fab A44V11 Recombinant Fab (rFab) was obtained from transiently transfected HEK293 cells using two plasmids encoding either the light chain or the C-terminal His-tagged heavy chain. After elution from the resin, the rFab was dialyzed extensively against PBS and stored at 4°C.

[0395] Source of Macaca fascicularis PAI-1 (also called cynomolgous or cyno PAI-1): Recombinant mature cynomolgus PAI-1(24-402) was expressed as inclusion bodies in E. coli and the recombinant protein was purified using conventional methods.

[0396] Source of Human PAI-1: Recombinant mature human PAI-1 (24-402) was purchased from Molecular Innovations Inc. (catalog number CPAI) and synthesized as described by Berkenpas et al. (1995, EMBO J., 14, 2969-2977). The active conformation was stabilized by introducing mutations (N150H, K154T, Q319L, M354I) into the ribosome.

[0397] Complex preparation and purification: Recombinant Fab and antigen were mixed in a 1.5:1 molar ratio, incubated for 30 min at room temperature, and the complex was further purified by preparative size exclusion on a Superdex 200 PG column (GE Healthcare) equilibrated with 25 mM MES pH 6.5, 150 mM NaCl.

[0398] Crystallization of Fab A44V11+Cyno PAI-1 complex The complex was concentrated to 10 mg / ml in 25 mM MES pH 6.5, 150 mM NaCl. It was crystallized in 16-24% ethanol, 100 mM Tris pH 8.5. Ethylene glycol (30%) was used as a cryoprotectant. The crystals diffracted to 3.3 Å in space group P321 (a=b=193 Å, c=144 Å) at the ID29 beamline at the ESRF. The data were processed with a combination of XDS and Scala (GlobalPhasing Ltd., Cambridge, UK).

[0399] Structure determination of the complex Fab A44V11 / Cyno-PAI-1: Models of Fab variable domains were generated using Prime in Maestro (Schrodinger, New York, NY). The constant domain was derived from the published structure 3FO2. Different models were used: the latent conformation was obtained from 1LJ5 and the active conformation from 1OC0. The Matthews coefficients (V M、 Calculations of the crystal volume per unit of protein molecular weight (V M 2. 2 predicts a complex size of 90 kilodaltons (KD). Molecular replacement was performed using Phaser (CCP4 suite) (McCoy, et al. J. Appl. Cryst. 40: 658-674 (2007)), which identifies the two monomers of latent PAI-1. Two variable domains, Fab and Fab, were identified. Further density was evident for the constant domain. This had to be manually placed. M This solution, which corresponds to (71% solvent), was also carefully examined for packing consistency. The structure was run using Buster (GlobalPhasing) The non-crystallographic symmetry was used to refine the sequence to an Rfree of 29.2% (Rfactor of 25.8%). was not stabilized by crystal packing and was poorly resolved in the electron density map.

[0400] Crystallization of Fab A44V11 + human PAI-1 complex Protein crystallization is an obstacle to biomolecular structure determination by x-ray crystallography. The success of protein crystallization is directly proportional to the quality of the protein molecules used in the crystallization experiment, where the most important quality criteria are the purity and homogeneity (both molecular and conformational) of the protein in solution.

[0401] First, to determine the PAI-1 / Fab mAb complex structure, the native mAb A44 was used to produce its Fab fragment by papain digestion. This large-scale Fab production resulted in a heterogeneous Fab fragment that was conjugated and purified in complex with human wild-type (wt) PAI-1 protein. The resulting protein complex was concentrated to a concentration of 7 mg / ml and incubated at two different temperatures, 4°C and 19°C. The Fabs were screened for crystallization under individual crystallization conditions at 800 °C. No crystallization hits were detected. To improve protein complex homogeneity, recombinant 6-His tagged Fab A44 was produced, purified, and complexed with human wild-type PAI-1 protein. (See Figure 36).

[0402] The complex crystallization screening was performed under the conditions of 20% PEG10K + 0.1M sodium acetate pH 4.6. Crystallization optimization by conventional crystallization methods, microseed matrix seeding, and in situ trypsinolysis crystallization did not significantly improve the quality of the crystals. The crystals obtained were needle-like and diffracted x-rays to a resolution (10 Å) insufficient for structure determination.

[0403] The failure of the complex crystals to crystallize could potentially be explained by the conformational heterogeneity of the complex. Wild-type PAI-1 molecules are known to adopt three different conformations (active, latent and substrate), which may hinder crystallization. To improve the quality of the crystals, a 6-His tagged A44 Fab in complex with latent PAI-1 was produced. (See FIG. 37).

[0404] The corresponding complexes were prepared and the 6-His tagged Fab A44 / wt PAI-1 protein complex was Crystallization was screened anew under the previously used conditions. The only crystallization hit from over 1000 conditions tested was identified for the complex under the condition of 20% PEG3350 + 0.2M nH4 acetate + 4% MPD + 50 mM Mes pH 6 (see Figure 39(a)). After extensive optimization, 3D crystals were obtained. X-ray diffraction studies using a synchrotron high-intensity X-ray beam showed no evidence of diffraction (see FIG. 39(b), which shows a representative optimized crystal).

[0405] The A44 Fab fragment was engineered to reduce the mobility of the protein portion. They decided to produce it using a new tag, but without the artificial tag 6-His that they had used previously. To further increase the chances of successful crystallization, we isolated an active mutant form of PAI-1 (N150H , K154T, Q319L, and M354I) were purchased from Molecular Innovations (catalog no. CPAI, Novi, MI). The Fab A44 protein was then inserted into the IgG1-binding domain and used to prepare a conjugate with the Fab A44 protein lacking the artificial tag. The coalescence was concentrated to 12 mg / ml in 25 mM MES pH 6.5, 150 mM NaCl. Acceptable rod-shaped single crystals were obtained in 10 % PEG3350, 100 mM ammonium sulfate and cryoprotected by adding 30% ethylene glycol (see Figure 40). These crystals diffract to 3.7 Å and show extensive cryoprotection. After protection optimization, an x-ray diffraction data set suitable for structure determination was obtained (3.3 Å). The beamline Proxima 1 at the synchrotron SOLEIL (Saint-Aubin, France) was used to analyze the crystals down to 3.3 Å. The space group is P212121 (a=105, b=152 c=298). The samples were processed using the XDS ref (XDS ref, Xdsme ref).

[0406] Structure determination of the complex Fab A44V11 / human-PAI-1: Pointless (CCP4) showed only 40% reliability in space group recognition. As a result, initial molecular replacement was performed using Amore (CCP4) to test all possible space group variations of the P222 point group. The final molecular replacement using Phaser (CCP4) was Four dimers of active PAI-1 / variable domains of Fab were identified in the symmetric unit. The lines were added manually to the electron density map. The structure was analyzed using Buster (GlobalPhasing) for non-crystallography. Using local symmetry, the structure was refined to an Rfree of 28% (Rfactor of 24.1%).

[0407] Epitope and paratope structure analysis The epitope and paratope regions formed in the cynomolgus and human complexes were identified and these complexes were compared. The crystal structures were compared with the complexes with human and cynomolgus PAI-1. The alignment of the A44V11 conformation was determined to 3.3 Å. The superposition of both structures (see Figure 40) is , that the paratope of A44V11 resembles both the latent and active forms of PAI-1. Fab A44 recognized the active form of human PAI-1 and the latent form of cynomolgus monkey PAI-1. Figure 42 shows the PAI-1 epitope recognized by Fab A44 in both active human PAI-1 (Figure 42(A)) and latent cynomolgus PAI-1 (Figure 42(B)). The paratope recognizing the latent conformation is part of the paratope that recognizes the active conformation.

[0408] Analysis of the interaction surface area revealed that PAI-1 interacts primarily with the heavy chain of A44V11. The surface area of ​​interaction between active human PAI-1 and the heavy chain (average of four complexes) is 674 Å. 2 is The surface area of ​​interaction between active human PAI-1 and the light chain (average of four complexes) is 372 Å. 2 is The surface area between the latent cyno PAI-1 and the heavy chain (average of the two complexes) is 703 Å. 2 It is. The surface area of ​​interaction between latent cynomolgus PAI-1 and the light chain (average of the two complexes) is 360 Å. 2 See Figures 43 and 44 for depictions of the heavy and light chain paratopes, respectively.

[0409] The residues in the A44V11 portion of the paratope are shown in Table 36 below. Residues in italics indicate correlation with active PAI-1. The underlined residues are involved in the interaction with the cryptic form but not with the cryptic form, whereas the underlined residues interact only with the cryptic form. All other residues are involved in both interfaces.

[0410] [Table 47]

[0411] Despite the different conformations of human and cynomolgus PAI-1 molecules, the same residues is involved in the interaction with Fab A44 (bold residues shown below in the sequence of human PAI-1) (SEQ ID NO:1): [ka]

[0412] An abbreviation for the A44V11 binding epitope on human PAI-1 is as follows: EXXQ (SEQ ID NO:156); LXR (SEQ ID NO:157); TDXXRQFQADFTXXSDQEPL (SEQ ID NO:158)

[0413] In summary, the cynomolgus monkey and human epitopes of PAI-1 that recognize FabA44 are consistent with both co-immunoglobulins and human epitopes. Fab A44 recognizes both human and cynomolgus monkey PAI-1, but does not appear to recognize mouse or rat PAI-1.

[0414] Example 23: Determination of A44V11 specificity and cross-reactivity To determine the specificity and reactivity of A44V11, the sequence of the A44V11 epitope (see above) was used to perform a sequence search using the ScanProsite (SIB Swiss Institute of Bioinformatics) database. A motif search was used to search for similar epitopes in other proteins. For further details, see Artimo, P. et al. Nucleic Acids Res. 40(W1):W597-603 (2012). All epitope sequence matches found in the search were associated with PAI-1. , suggesting that the A44V11 antibody is specific for PAI-1.

[0415] The A44V11 epitope was also compared to other known x-ray structures (3D search) using computational profiling and molecular modeling according to Med-SuMo, which showed e.g. hydrogen bonds, electrical charges, and It detects and compares biochemical functions on protein surfaces, including charge, hydrophobic and aromatic groups. Med-SuMo molecular modeling is described in Jambon, et al. Bioinformatics 21(20):3929-30 (2005). A 3D search of the A44V11 epitope identified a similar motif in human alpha-1-antitrypsin (AAT1). However, upon further investigation, the AAT1 motif It was found that the A44V11 epitope has significant differences between the A44V11 epitope and the other human proteins such that A44V11 does not bind to the A44V11 epitope. Therefore, the sequence pattern and 3D pattern analysis of the A44V11 epitope are similar to those of other human proteins. This suggests that there will be minimal cross-reactivity with proteins.

[0416] The human and cynomolgus PAI-1 epitopes for A44V11 were compared to the epitopes derived from mouse and rat PAI-1. Sequences were extracted from SEQ ID NO:1 (PAI-1 human), SEQ ID NO:162 (PAI-1 cynomolgus), SEQ ID NO:163 (PAI-1 mouse), and SEQ ID NO:164 (PAI-1 rat). Rat and mouse PAI-1 have 75% and 79% sequence identity with human PAI-1, respectively. Alignment of the different PAI-1 sequences still showed significant differences between rat / mouse and human / cynomolgus in their respective epitopes, indicating that A44V11 does not bind to either rat PAI-1 or mouse PAI-1. For example, mouse PAI-1 amino acids Ser300, Thr302, and Gln314 differ from their human / cynomolgus PAI-1 counterparts. The difference in residues in the β-terminal region of ... , representing the changes in the proposed epitope. Structural comparison of PAI-1 (Figure 46) shows that it is not possible to obtain both human and mouse activity from the A44V11 antibody. This further demonstrates that this is possible.

[0417] To further validate the epitopes identified for A44V11, human and cynomolgus A44V11 epitopes were compared with the binding region of vibronectin. The structure of human PAI1 in complex with the somatomedin B domain of vibronectin has been published (1OC0). The structures of these two complexes were compared (see Figure 47). The structural comparison suggests that the binding of A44V11 does not affect the interaction of PAI-1 with vibronectin. .

[0418] The A44V11 epitope was compared to the epitopes of other published anti-PAI1 antibodies. No overlap of the A44V11 epitope was observed with the other published anti-PAI-1 antibodies MA-55F4C2 and MA-33H1. They bind to residues in the region 128-156 (Debrock et al. Thromb Haemost, 79:597-601 (1998)).

[0419] Finally, the specificity and lack of cross-reactivity of the A44V11 antibody was confirmed by Biacore. Based on the sequence and 3D structure of the A44V11 epitope, molecular modeling studies strongly indicate that A44V11 is specific for human and cynomolgus PAI-1.

[0420] Example 24: Epitope Mapping by Hydrogen / Deuterium Exchange Mass Spectrometry (HDX MS) Hydrogen / deuterium exchange (HDX) monitored by mass spectrometry (MS) was applied to the PAI-1 binding antibodies disclosed herein to further characterize the epitope of each antibody. HDX MS is a particularly useful technique for comparing multiple states of the same protein. Detailed methodology and application of HDX MS to protein therapeutics can be found in Wei, et al., Drug Discovery Today, 19(1): 95-102. (2014). Briefly, this exchange process can be followed if the aqueous, all-H2O solvent is replaced with an isotope of hydrogen that has unique spectroscopic properties. Most state-of-the-art HDX Deuterated or "heavy" water (D2O) is used in the experiments. In particular, hydrogens attached to backbone nitrogens (also called backbone amide hydrogens) are useful for probing protein conformation. See, e.g., Marcsisin, et al. Anal Bioanal Chem. 397(3): 967-972 (2010). Exposed, dynamic regions of the protein exchange faster and protected, inflexible regions exchange slower. All relevant conditions (pH, temperature, ionic strength, etc.) are held constant. Thus, only structural differences (solvent accessibility, hydrogen bonding) affect this exchange. The interaction of the antibody with PAI-1 blocks the labeling of certain parts of the antigen, thus resulting in a different readout based on the site of binding (epitope).

[0421] Testing Method: Cynomolgus monkey-PAI-1 (10 μM), Cynomolgus monkey-PAI-1 bound to A44v11 (each 10 μM) and Cynomolgus-PAI-1 bound to APGv2 (10 μM each) was prepared in PBS, pH 7.2. Protein solutions were allowed to reach binding equilibrium by incubation at room temperature for 1 h. K d Based on the values, each of the antibody:antigen complexes was >99% bound under the labeling conditions described below.

[0422] Deuterium exchange, quenching, and sample injection were operated by an automated robotics system (LEAP Tech., Carrboro, NC). Aliquots of protein solution were diluted 10-fold with labeling buffer (PBS in 99.9% D2O, pD 7.2) and incubated at 20°C for 10 s, 1 min, 5 min, or 4 h. At the end of the deuterium exchange period, the protein was quenched by adding 50 μL of labeling solution to an equal volume of pre-chilled (0°C) 100 mM sodium phosphate, 4 M guanidine hydrochloride, 0.5 M TCEP, pH 2.5. This quenched the labeling reaction. A non-deuterated control was prepared in the same manner by diluting 10-fold with PBS in H2O.

[0423] Each quenched sample (50 μL, 50 pmol of each protein) was immediately injected into a Waters nanoAcquity using HDX Technology (Waters Corp., Milford, MA). The protein was digested online using a 2.1 mm x 30 mm Enzymate BEH pepsin column (Waters Corp.) maintained at 20 °C. All chromatographic elements were maintained at 0.0 ± 0.1 °C in the cooling chamber of the ultra-performance liquid chromatography (UPLC) system. The resulting peptides were captured and desalted at 100 μL / min for 3 min, then separated on a 1.0 x 100.0 mm ACQUITY UPLC HSS T3 column (Waters Corp.) using a 2-40% acetonitrile:water gradient at 40 μL / min for 12 min. Deuterium levels were not corrected for back-exchange and are reported relative. All comparative experiments were performed under identical conditions, negating the need for back-exchange correction. All experiments were performed in triplicate. Peptide carryover between injections was eliminated by injecting 50 μL of 1.5 M guanidine hydrochloride, 0.8% formic acid, and 4% acetonitrile over the column after each run.

[0424] Mass spectra were obtained using a standard electrospray source (Waters Corp.) operated in HDMSe mode. The data were acquired using a Waters Synapt G2-Si instrument fabricated by I.M.S. The instrument settings were as follows: capillary at 3.5 kV, sampling cone at 30 V, and source offset at 30 V. The source temperature was 80°C, the desolvation temperature was 175°C, the cone gas was 50L / h, and the desolvation temperature was 175°C. The solvation gas was 600 L / h and the nebulizer gas was 6.5 bar. Mass spectra were acquired over an m / z range of 50-1700. Mass accuracy was maintained throughout each run by co-injection of 100 fmol / uL human [Glu1]-Fibrinopeptide B with a lock mass probe.

[0425] MSE identification of non-deuterated peptic peptides was performed using ProteinLynx Global Server software (Waters Corp.). Deuterium uptake of each peptide was determined using DynamX 2.0 software (Waters Corp.). Relative deuterium levels were calculated by subtracting the centroid of the isotope distribution of the non-deuterated peptide from the corresponding centroid of the deuterium-labeled peptide. Deuterium uptake plots were generated automatically by the software.

[0426] Monitoring deuterium uptake for PAI-1 status After online pepsin digestion, 150 overlapping cynomolgus-PAI-1 peptic peptides were synthesized. Deuterium incorporation was determined using three different The state of the proteins bound to each other was monitored (10 s to 4 h) for all 150 peptides: (1) cynomolgus-PAI-1 alone; (2) A44v11 bound to cynomolgus-PAI-1; and (3) APGv2 bound to cynomolgus-PAI-1.

[0427] Most of the cyno-PAI-1 peptides showed nearly identical deuterium uptake between the three conditions, indicating no interaction between cyno-PAI-1 and any of the mAbs in these regions. See Figure 49(A), which shows one representative example with this result. In contrast, the peptide incorporating residues 44–64 shows , significant protection from exchange (reduced deuterium oxide) when bound to either A44v11 or APGv2 In addition, a peptide incorporating residues 295-322 also showed significant protection from exchange when bound to either A44v11 or APGv2 (Figure 49(C)). For this region, the magnitude of conservation was greater in cyno-PAI-1 than in APGv2, suggesting that APGv2 is more likely to be involved in the A44v11 gene. The results were larger when A44v11 was bound to the crab (see Figure 49(C)). When bound to Quisal-PAI-1, it provided greater overall protection from exchange than APGv2. This shows that you can get it.

[0428] Comparative study: For comparative studies, deuterium uptake was monitored for all 150 peptides generated from each of the three cynomolgus-PAI-1 states (see generally, Wei, et al. al., Drug Discovery Today, 19(1): 95-102 (2014). Data plots from each were compared with each other and were then butterflied to facilitate data interpretation. Lie plots were generated (see, e.g., Figures 50(A), 51(A), and 52(A)). For each butterfly plot, the x-axis is the calculated peptide midpoint position i for each of the 150 peptides compared; the y-axis is the average relative fractional exchange (ratio).

[0429] Difference plots were also generated for each comparison between cynomolgus-PAI-1 conditions (see, e.g., Figures 50(B), 51(B), and 52(B)). In these plots, the difference in the mean mean mean difference from one condition to the next was 1.5%. Deuterium uptakes were subtracted from the others and plotted in butterfly plots as well. The sum of the differences for each peptide is represented by the vertical bars. The dashed horizontal lines represent the individual measurements ( Either the sum of the differences (±0.5 Da) or the sum of the differences (±1.1 Da) exceeds the measurement error, and the two conditions These represent values ​​that may be considered as actual differences. Further details regarding this technique are disclosed in Houde D. et al., J. Pharm. Sci. 100(6):2071-86 (2011).

[0430] First, Cyno-PAI-1 alone was compared to the A44v11:Cyno-PAI-1 bound state (Figure 50). A butterfly plot for this comparison is shown in Figure 50(A). A difference plot for this comparison is shown in Figure 50(B). Cyno-PAI-1 bound to A44v11 and free form Cyno-PAI-1 The observed differences between cynomolgus-PAI-1 and cynomolgus-PAI-1 are located primarily in two regions of cynomolgus-PAI-1: one region near the N-terminus (residues 44-64) and the other region near the C-terminus (residues 307-321) ( See Figure 50(B).

[0431] Next, cyno-PAI-1 alone was compared to the APGv2:cyno-PAI-1 bound state (Figure 51). A butterfly plot for this comparison is shown in Figure 51(A). The APGv2-bound cyno-PAI-1 is shown in Figure 51(B). The observed differences between cyno-PAI-1 bound to APGv2 and free form cyno-PAI-1 are primarily located in two regions of cyno-PAI-1. One region is near the N-terminus and the other is missing the C-terminus, which is consistent with A44v11:cyno-PAI-1. The results are similar to those of the previous study. The A44v11 and APGv2 complexes with cynomolgus monkey-PAI-1 in the bound state The two antibodies share peptides that show reduced deuterium uptake when compared, which may indicate that the epitopes of the two antibodies are similar.

[0432] Finally, the two antibody-binding cyno-PAI-1 antibodies were compared to each other (Figure 52). A butterfly plot for this comparison is shown in Figure 52(A). A difference plot for this comparison is shown in Figure 52(B). The observed differences between A44v11:cyno-PAI-1 and APGv2:cyno-PAI-1 The difference is located in the C-terminal region of cyno-PAI-1 (see Figure 52(B)).

[0433] Example 25: Epitope comparison of antibodies A44v11 and APGv2 HDX MS was used to further define the epitopes of the A44v11 and APGv2 antibodies. By using overlapping peptides generated using the HDX MS technique, the antibody epitope can be refined slightly better than peptide level elucidation (see, for example, FIG. 48). The HDX MS data for peptides that showed significant protection from exchange upon A44V11 binding was further analyzed to determine the epitope for the cyno-PAI-1:A44v11 interaction. The HDX data for the cyno-PAI-1 A44V11 epitope was found to be consistent with the epitope determined using a crystallographic approach. The cyno-PAI-1 A44V11 epitope identified using HDX MS is shown in FIG. 53 (bold type) and in abbreviated form below: TTGGETRQQIQ (SEQ ID NO:159); RHL (SEQ ID NO:160); TDMXXXFQADFTSLSNQEPLH-V (SEQ ID NO: 161)

[0434] Cynomolgus-PAI-1 peptide bound APGv2 and showed significant protection from exchange The HDX MS data for A44v11 was analyzed to further determine the epitope for the cynomolgus monkey-PAI-1:APGv2 interaction. HDX MS epitope mapping data for A44v11 and APGv2 indicates that the epitopes are in the same region, as generally seen in FIG. In the region of residues 307-321, the same peptide showed antibody binding to both A44v11 and APGv2. However, the magnitude of protection was significantly greater for cynomolgus-PAI-1 than for APGv2. and higher when bound to A44v11 (see FIG. 49(C)). This finding is more evident in FIG. 52(B), which shows a difference peak in the region of residues 307-321 of cynomolgus-PAI-1. This indicates the specific binding between cynomolgus-PAI-1 and each of the A44V11 and APGv2 antibodies. Thus, although the epitopes for both A44V11 and APGv2 map to similar regions of PAI-1, the epitopes for each antibody are likely not the same.

Claims

1. a heavy chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO:88 or 89, and that comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:34, a CDR2 region comprising the amino acid sequence of SEQ ID NO:33, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:32; and a light chain variable region that is at least 90% identical to the amino acid sequence of SEQ ID NO: 91, 93, 94, 95, 96 or 97, and that comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO: 37, a CDR2 region comprising the amino acid sequence of SEQ ID NO: 36 or SEQ ID NO: 145, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:

35. An isolated monoclonal antibody which specifically binds to PAI-1, comprising:

2. a heavy chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO:88 or 89, and that comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:34, a CDR2 region comprising the amino acid sequence of SEQ ID NO:33, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:32; and a light chain variable region that is at least 95% identical to the amino acid sequence of SEQ ID NO:91, 93, 94, 95, 96 or 97, and that comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:37, a CDR2 region comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:145, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:

35.

2. An isolated monoclonal antibody that specifically binds to PAI-1 according to claim 1, comprising:

3. a heavy chain variable region that is at least 96% identical to the amino acid sequence of SEQ ID NO:88 or 89, and that comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:34, a CDR2 region comprising the amino acid sequence of SEQ ID NO:33, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:32; and a light chain variable region that is at least 96% identical to the amino acid sequence of SEQ ID NO:96 or 97, and that comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:37, a CDR2 region comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:145, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:35; 3. An isolated monoclonal antibody that specifically binds to PAI-1 according to claim 1 or 2, comprising:

4. A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 88 or 89; and a light chain variable region that is at least 96% identical to the amino acid sequence of SEQ ID NO:96 or 97, and that comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:37, a CDR2 region comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:145, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:35; 4. An isolated monoclonal antibody that specifically binds to PAI-1 according to any one of claims 1 to 3, comprising:

5. a heavy chain variable region comprising the amino acid sequence of SEQ ID NO:89; and a light chain variable region that is at least 96% identical to the amino acid sequence of SEQ ID NO:97 and comprises a CDR1 region comprising the amino acid sequence of SEQ ID NO:37, a CDR2 region comprising the amino acid sequence of SEQ ID NO:36 or SEQ ID NO:145, and a CDR3 region comprising the amino acid sequence of SEQ ID NO:

35.

5. The isolated monoclonal antibody of claim 4, comprising:

6. Use of a pharma- ceutical effective amount of an isolated monoclonal antibody that specifically binds to PAI-1 according to any one of claims 1 to 5 for the manufacture of a medicament for restoring plasmin generation in a subject in need thereof.

7. 7. The use according to claim 6, wherein the medicament is administered orally, parenterally by injectable solution, by inhalation or topically.

8. 7. The use of claim 6, wherein the medicament treats a condition involving increased levels of fibrotic tissue.

9. 9. The use according to claim 8, wherein the condition is fibrosis, systemic sclerosis, interstitial lung disease, chronic lung disease, chronic kidney disease, peripheral limb ischemia, acute ischemic stroke with or without thrombolysis, or stent restenosis.

10. 10. The use of claim 9, wherein the medicament treats a condition including skin fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, liver fibrosis, or renal fibrosis.

11. 10. The use of claim 9, wherein the medicament treats a condition including venous and arterial thrombosis, deep vein thrombosis, or disseminated intravascular coagulation thrombosis.

12. A container comprising the isolated monoclonal antibody of any one of claims 1 to 5.

13. 13. The container of claim 12, wherein the container is a prefilled syringe, a vial, or an auto-injector.

14. 14. A kit comprising the container of claim 12 or 13 and a label or instructions for administration and / or use of the isolated monoclonal antibody.

15. Use of a pharma- ceutical effective amount of an isolated monoclonal antibody that specifically binds to PAI-1 according to any one of claims 1 to 5 for the manufacture of a medicament for restoring plasmin generation and treating increased levels of fibrotic tissue, fibrosis, systemic sclerosis, interstitial lung disease, chronic lung disease, chronic kidney disease, peripheral limb ischemia, acute ischemic attacks with or without thrombolysis, or stent restenosis, skin fibrosis, pulmonary fibrosis, idiopathic pulmonary fibrosis, liver fibrosis, renal fibrosis, venous and arterial thrombosis, deep vein thrombosis, or disseminated intravascular coagulation thrombosis.

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