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

Abstract

To provide antibodies that specifically bind to plasminogen activator inhibitor type 1 (PAI-1).SOLUTION: Provided are isolated monoclonal antibodies that specifically bind to PAI-1, comprising a heavy chain framework region and a heavy chain variable region, wherein the heavy chain variable region comprises a heavy chain CDR1 region comprising a specific sequence, a heavy chain CDR2 region comprising a specific sequence, and a heavy chain CDR3 region comprising a specific sequence, and a light chain framework region and a light chain variable region, wherein the light chain variable region comprises a light chain CDR1 region comprising a specific sequence, a light chain CDR2 region comprising a specific sequence, and a 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 herein 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. PAI-1 regulates fibrinolysis by inhibiting plasminogen activation in the vascular compartment. Fibrinolysis is a tightly coordinated process for degrading 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 hemostatic disease. 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 promotes the degradation of plasminogen from the extracellular matrix. PAI-1 regulates numerous 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, glycosaminoglycans). cofactors, uPAR-urokinase complex or cellular 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 biological activity. These cellular mechanisms and antifibrinolytic effects have led to the pathogenic role of PAI-1 in tumor growth and metastasis, fibrosis, acute myocardial injury, and other conditions. 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 PAI-1 consists of 12,169 exons and has a size of 12,169 b (Non-patent Document 1). A single glycoprotein of approximately 50 kDa (379 amino acids) from the serine protease inhibitor superfamily. It is a protein that is synthesized in an active conformation but spontaneously becomes latent in the absence of vitronectin (Vn). Vitronectin, the main 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 binds covalently to PAI-1 as an acyl-enzyme. The cleaved RCL remains bound to the PAI-1 complex, resulting in 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 Figure 1). PAI-1 is found primarily as a noncovalent complex with vitronectin (Kd ∼1 nM), which reduces the latency transition by 1.5-3 fold. The affinity of latent, cleaved, or complexed PAI-1 for vitronectin is Matrix-bound vitronectin, along with PAI-1, is also significantly reduced in pericellular tissue. Endothelial cells, monocytes, macrophages, and vascular smooth muscle cells express PAI-1. 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, it is inactive against proteases bound to fibrin or any of their cellular receptors. 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), revealing differences from human PAI-1 in the RCL position, the location of the gate region, and α-helix A. The 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 highly correlated with physiological (e.g., circadian variations in plasma PAI-1 levels) and pathological conditions (e.g., obesity, metabolic syndrome, insulin resistance, etc.). PAI-1 is considered an acute phase protein. Transcriptional regulation of PAI-1 mRNA expression is mediated by several cytokines and growth factors (e.g., erythrocyte sedimentation, erythrocyte stenosis ... 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 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 approximately 25% of the population) increases plasma PAI-1 levels by approximately 2% compared to the 5G / 5G variant (25% occurrence and 4G / 5G variant 50% occurrence). The 4G / 4G polymorphism is 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 contributes to the formation of fibrotic tissues. It has been shown to be overexpressed in the liver, lungs, kidneys, heart, abdominal hemoglobin, and thyroid gland. 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 15). While this deletion protects against induced fibrosis in the heart (Non-Patent Document 17), it predisposes to age-dependent cardiac selective fibrosis (Non-Patent Document 18). Downregulation of PAI-1 expression by siRNA (Non-Patent Document 19) or inhibition by chemical compounds (Non-Patent Document 20) 20; Non-patent literature 21) have been reported to reduce pulmonary fibrosis, whereas overexpression of wild-type PAI-1 (Non-patent literature 22) or a PAI-1 mutant that retains only vitronectin binding but not tPA inhibitor function exacerbates pulmonary fibrosis (Non-patent literature 23).

[0009] Bile duct ligation (BDL)-induced 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 knockout 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 a review). , see Non-Patent Documents 32 and 33) protects against renal fibrosis. PAI-1 overexpressing mice show 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 of numerous pathologies, has been the subject of much research over the last 20 years, with many studies aimed at inhibiting its activity or targeting its function. 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 to human plasminogen activator type 1 (PAI-1) is 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: 34), CDR2 (SEQ ID NO: 33), and CDR3 (SEQ ID NO: 32) of SEQ ID NO: 6, and a light chain variable region, wherein 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. CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 35). wherein 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 % identities approximate Values ​​indicate minimum % identity; % identities higher than the stated values ​​are also encompassed by the present 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 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 monoclonal 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:3) 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: 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. and a light chain framework region that is 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to

[0018] In one aspect, 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: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, the monoclonal antibody 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 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% of the heavy chain framework region of SEQ ID NO: 4. 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, 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: 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. 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 are monoclonal antibodies. 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, 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: 11), CDR2 (SEQ ID NO: 52), 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 this 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, 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: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, an isolated monoclonal antibody that specifically binds to PAI-1, the monoclonal antibody comprising (a) a heavy chain CDR2 region comprising SEQ ID NO: 57, and a heavy chain CDR3 region comprising SEQ ID NO: 56; 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. 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: 12, 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: 13.

[0026] In one aspect, 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: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, 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: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, a 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, 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: 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 SEQ ID NO: 46, the sequence 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. Disclosed herein are 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: 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 plasminogen activator inhibitor type 1 (PAI-1)-specifically binds to Disclosed herein is an isolated monoclonal antibody comprising 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, sequence 1. 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 of the human serotype.

[0036] In one aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 33, the sequence (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, (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, the monoclonal antibody comprising a light chain CDR3 region containing sequence number 35. Disclosed herein are antibodies.

[0039] In one aspect, (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, the monoclonal antibody comprising a light chain CDR3 region containing sequence number 35. Disclosed herein are antibodies.

[0040] In one aspect, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 34, sequence (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, the monoclonal antibody comprising a light chain CDR3 region containing 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: a light chain variable region comprising CDR1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 35) of SEQ ID NO: 7.

[0042] In certain aspects, (a) a heavy chain framework region, a heavy chain CDR1 region comprising SEQ ID NO: 76; an isolated monoclonal antibody that specifically binds to PAI-1, comprising (a) 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, 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 described herein. (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 US Pat. No. 6,299,349, 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, a humanized monoclonal antibody that specifically binds to human PAI-1 is provided. 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, and the humanized light chain variable region is 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 described herein. and 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 clonal 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 monoclonal antibody comprises a 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 antibody disclosed herein interacts 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 other embodiments, the isolated monoclonal antibody specifically binds to PAI-1 disclosed herein. The monoclonal antibody binds to a polypeptide comprising SEQ ID NO: 161. In some embodiments, the isolated monoclonal antibody binds to a polypeptide comprising SEQ ID NO: 159 and / or SEQ ID NO: 161. In yet other embodiments, 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 comprises a specific binding affinity for the PAI-1. In another embodiment, the PAI-1 bound by the antibody is a crab-PAI-1. It 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 binding with and / or competitively inhibit binding to any of the isolated monoclonal antibodies disclosed herein. In certain embodiments, the isolated monoclonal antibody competes or competitively inhibits binding to human PAI-1. The selected monoclonal antibodies compete with 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 antibody competes with or competitively inhibits 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 CDR1 region comprising a heavy chain framework region, SEQ ID NO: 34; 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: 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 are methods for treating conditions resulting from increased expression of or increased sensitivity to PAI-1, the methods comprising administering to a patient or other subject a pharmaceutically 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 patient or other subject in need thereof is administered a pharmaceutically effective amount of a PAI-1 antibody. Disclosed herein are methods for restoring plasmin production, comprising administering orally, parenterally via an 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 administration forms. In some embodiments, administration to a patient or other subject comprises multiple doses. In another aspect, the method of restoring plasmin production facilitates the therapeutic treatment of a condition involving increased levels 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 pharmaceutically effective amount of PAI-1 is administered orally, parenterally by injectable solution, by inhalation, or topically to a patient or other subject 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 involving increased levels 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 involving liver fibrosis, renal fibrosis including chronic kidney disease, thrombosis, venous thrombosis 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 described 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 a 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 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, a method for treating a condition resulting from increased expression or increased sensitivity to PAI-1, comprising administering to a patient orally, parenterally by injection solution, by inhalation, or topically. The use of a pharmaceutically effective amount of a PAI-1 antibody for the manufacture of a medicament for treating wherein the condition is idiopathic pulmonary fibrosis.

[0056] In another aspect, the patient or other subject is orally administered a pharmaceutically effective amount of a PAI-1 antibody. Disclosed herein are methods for restoring plasmin generation, including administering to a subject a therapeutically effective amount of idiopathic pulmonary fibrosis (IDF), parenterally via an injectable solution, by inhalation, or topically, wherein plasmin generation promotes the therapeutic treatment of idiopathic pulmonary fibrosis.

[0057] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is described herein. The present invention relates to a method for treating thromboembolism, comprising administering to a subject a therapeutically effective amount of an antibody to a subject, the antibody being effective in treating a subject having an acute ischemic attack, the method comprising administering to a subject a therapeutically effective amount of an antibody to a subject having an acute ischemic attack, the method comprising administering to a subject having an acute ischemic attack, the antibody being effective in treating ... 50 to restore clot lysis.

[0058] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is described herein. In some embodiments, the subject has had 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 pharmaceutically effective amount of a PAI-1 antibody for the manufacture of a medicament for treating a condition caused by increased expression of or increased sensitivity to PAI-1, including administration to a patient orally, parenterally by injection solution, by inhalation, or topically. Disclosed herein is an acute ischemic stroke with or without thrombolysis.

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

[0061] In another aspect, an isolated monoclonal antibody that specifically binds to PAI-1 is described herein. The present invention relates to a method for treating a rheumatoid arthritis, comprising administering to a subject a therapeutically effective amount of an antibody against rheumatoid arthritis, the method ... In some embodiments, the adhesion formation occurs after 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 use of a pharmaceutically effective amount of a PAI-1 antibody for the manufacture of a medicament for treating or preventing a condition caused by increased expression of or increased sensitivity to PAI-1, comprising administering the antibody orally, parenterally by injection solution, by inhalation, or topically to a patient. The use is disclosed herein, wherein the condition is abdominal adhesion formation.

[0063] In another aspect, a patient or other subject in need thereof is administered a pharmaceutically effective amount of a PAI-1 antibody. Disclosed herein are methods for restoring plasmin generation, comprising administering orally, parenterally by injection solution, by inhalation, or topically to a subject, wherein plasmin generation promotes the 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 complexes. 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 described herein. In one embodiment, disclosed herein is an isolated crystal comprising a Fab' fragment of monoclonal antibody A44, wherein the Fab' fragment consists of the light chain sequence SEQ ID NO:7 and the heavy chain sequence SEQ ID NO:6. In another embodiment, disclosed herein is an isolated crystal comprising a Fab' fragment comprising the light chain sequence SEQ ID NO:93 and the 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, (a) a crystallized antibody that specifically binds to PAI-1, and (b) an embedded crystal. Disclosed herein are pharmaceutical compositions comprising at least one pharmaceutical excipient that is or encapsulates a pharmaceutical agent.

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

[0069] In one aspect, a composition comprising 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 an inhibitor of tPA activity, the method comprising the steps of: (a) binding PAI-1 to an ELISA plate; (b) injecting 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. indicates that the PAI-1 antibody binds to PAI-1 but blocks the formation of a covalent bond between PAI-1 and tPA, and a negative score indicates that the PAI-1 antibody blocks the interaction of tPA with PAI-1. Indicates that you want to check.

[0071] In another aspect, disclosed herein is a method for screening hybridomas. In certain embodiments, the screening method comprises screening hybridomas 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 comprises a forward 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 explanation of the drawings]

[0072] [Figure 1]Figure 1 shows a schematic diagram of the mechanism between PAI-1 and the serine proteases 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 bound to vitronectin (Vn). The RCL region of PAI-1 harbors the bait peptide bond (also called P1-P1'), which is the cleavage site for the serine protease. A Michaelis complex with tPA or uPA first forms, 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' peptide bond. The acyl-enzyme is an unstable complex formed by a covalent bond between a serine residue (closed triangle) from the catalytic triad of the serine protease (tPA) and an amino acid from the substrate (closed circle), which 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. [Figure 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. [Figure 3] Figure 3 shows the expression curves for a 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 blocking activity of tPA by A44 and commercial antibodies (33B8 and 33H1) in the chromogenic assay described in Example 4. [Figure 5]FIG. 5 shows the 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 against cynomolgus monkey (cyno) and mouse PAI-1 in the chromogenic assay described in Example 4. [Figure 8] Figure 8 shows an SDS-Page analysis of the mechanism by which antibodies 33H8 (which converts PAI-1 from an active to a latent conformation), 33H1 (which converts PAI-1 from an active to a substrate conformation), and A44 block the interaction of PAI-1 with tPA. Lane 1: molecular weight standard; lane 2: PAI-1 alone; lane 3: tPA alone; lane 4: PAI-1 in the presence of tPA; lane 5: 33H8 + PAI-1 + tPA; lane 6: 33H1 + PAI-1 + tPA; lane 7: A44 + PAI-1 + tPA; lane 8: mAb isotype control antibody. [Figure 9] Figure 9 shows an SDS-Page analysis of the mechanism by which antibodies 33H8 (which converts PAI-1 from an active to a latent conformation), 33H1 (which converts PAI-1 from an active to a substrate conformation), and antibodies derived from fusions C26, E16, and E21 block the interaction of PAI-1 with tPA. Lane 1: molecular weight standard; lane 2: PAI-1 alone; lane 3: tPA alone; lane 4: PAI-1 in the presence of tPA; lane 5: 33H1 + 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 an SDS-Page analysis of the mechanism by which antibodies 33H8 (which converts PAI-1 from an active to a latent conformation), 33H1 (which converts PAI-1 from an active to a substrate conformation), and antibodies derived from fusions A39, B109, and C45 block the interaction of PAI-1 with tPA. Lane 1: molecular weight standard; lane 2: PAI-1 alone; lane 3: tPA alone; lane 4: PAI-1 in the presence of tPA; lane 5: 33H1 + 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 the 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. Black boxes represent CDR domains. 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 them 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 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 a Biacore 2000 using single kinetic analysis of human PAI-1 glycosylation binding to immobilized APG antibody. The sensorgram from single-cycle kinetic is shown in gray. The 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 detecting UK-PAI-1 complex formation by ELISA. The percent inhibition of PAI-1 activity was plotted as a function of APG, APGv2, or APGv4 antibody concentration. [Figure 21] FIG. 21 shows the 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). [Figure 23] FIG. 23 shows the restoration of human plasma clot lysis by A44V11 or a human IgG1 isotype negative control at various concentrations. [Figure 24] FIG. 24 shows the 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 as detected by absorbance at 340 nm as a function of time (min). [Figure 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 in 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] Figure 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 at 10 mg / kg intraperitoneally (ip) or PBS. 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. [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 administered i.p. or PBS 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. [Figure 30] Figure 30 shows right lung masses from transgenic humanized mice 21 days after either saline or bleomycin treatment following ip administration of PBS (vehicle), IgG1, or A44 10 mg / kg every 3 days from day 4 to day 20. [Figure 31] FIG. 31 shows hydroxyproline lung content in transgenic humanized mice 21 days after either saline or bleomycin treatment following ip administration of PBS (vehicle), IgG1, or A44 10 mg / kg every 3 days from day 4 to day 20. [Figure 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) (5 mg / kg intraperitoneally (ip)) 24 hours prior to LPS challenge (100 μg / kg intravenously (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). [Figure 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) (5 mg / kg ip) 24 hours prior to LPS challenge (100 μg / kg iv). Liver biopsies were taken in anesthetized monkeys at the indicated time points, and active PAI-1 levels were determined in lysates using ELISA (#HPAIKT from Molecular Innovation). [Figure 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) (5 mg / kg ip) 24 hours prior to LPS challenge (100 μg / kg iv). Blood samples were collected at the indicated time points, and D-dimer levels were measured in plasma using ELISA. [Figure 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) (5 mg / kg ip) 24 hours prior to LPS challenge (100 μg / 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). [Figure 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 an isotype control antibody, with no differences observed at 72 hours. (*p<0.001 as calculated by Student's t-test) [Figure 37] Figure 37 shows another example of the purification of homogeneity recombinant 6-His tagged Fab A44. [Figure 38] FIG. 38 shows the purification of homologous recombinant 6-His tagged Fab A44 complexed with human wt PAI-1 protein. [Figure 39] Figure 39(a) shows the complex crystallization of the Fab A44 / PAI-1 complex, and Figure 39(b) shows the best optimized crystal. [Figure 40] FIG. 40 shows rod-shaped single crystals of the Fab A44 / PAI-1 complex. [Figure 41]FIG. 41 shows that Fab A44 recognizes the active form of human PAI-1 and the latent form of cyno PAI-1. [Figure 42] FIG. 42 shows the PAI-1 epitope recognized by Fab A44 on (A) active human PAI-1 and (B) latent cyno PAI-1. [Figure 43] FIG. 43 shows the heavy chain paratope of the Fab A44 / PAI-1 complex. [Figure 44] FIG. 44 shows the light chain paratope of the Fab A44 / PAI-1 complex. [Figure 45] Figure 45 shows a sequence alignment of the putative A44-binding epitopes of cynomolgus monkey, human, rat, and mouse PAI-1. Sequences are extracted 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] Figure 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]Figure 49 shows representative deuterium uptake plots for cynomolgus monkey (cyno)-PAI-1 peptides in the unbound (circle), APGv2-bound (x-line), and A44v11-bound (diamond) states. Residue ranges / positions are from SEQ ID NO: 162. (A) Most peptic peptides showed no difference between cyno-PAI-1 alone and cyno-PAI-1 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 residues 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 comparison of hydrogen / deuterium exchange (HDX) for cyno-PAI-1 alone and bound to A44v11. (A) Butterfly plot of average relative fractional exchange with the unbound state at the top and the bound state at the bottom. Lines correspond to data acquired at 10 seconds, 1 minute, 5 minutes, and 240 minutes. (B) Plot of the difference data (in Daltons) from the plot in (A) above for cyno-PAI-1 alone or bound to A44v11. [Figure 51] Figure 51 shows an HDX comparison of cyno-PAI-1 alone and cyno-PAI-1 bound to APGv2. In (A), butterfly plot of average relative fractional exchange with the unbound state at the top and the bound state at the bottom. Lines correspond to data acquired at 10 seconds, 1 minute, 5 minutes, and 240 minutes. In (B), plot of the difference data (Daltons) from panel (A) above for cyno-PAI-1 alone or cyno-PAI-1 bound to APGv2. [Figure 52]Figure 52 shows an 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 the APGv2-bound state on top and the A44v11-bound state on bottom. Lines correspond to data acquired at 10 seconds, 1 minute, 5 minutes, and 240 minutes. In (B), plot of the difference data (Daltons) from panel (A) above for cyno-PAI-1 bound to APGv2 or A44v11. [Figure 53] Figure 53 shows the cyno-PAI-1:A44v11 epitope determined by HDX MS. Residues of cynoPAI-1 (SEQ ID NO: 162) that show protection from exchange upon A44v11 antibody binding are shown in bold. Residues of the cyno-PAI-1:A44v11 epitope determined from crystallization studies are boxed. DETAILED DESCRIPTION OF THE INVENTION

[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-associated diseases or disorders (e.g., fibrosis). The present invention also relates to pharmaceutical compositions and methods for treating PAI-1 antibodies. Nucleic acids encoding such antibodies, recombinant expression vectors, and host cells for producing such antibodies, or fragments thereof, are provided. Methods of using the antibodies disclosed herein to inhibit or modulate 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, as well as multimers thereof (e.g., IgM), that comprise four polypeptide chains, two heavy (H) chains and two light (L) chains, interconnected by disulfide bonds. Each heavy chain contains a heavy chain variable region (V H or abbreviated as 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 contains a light chain variable region (V L or VL) and a light chain constant region (C L The light chain constant region is abbreviated as C or CL. L 1) V 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 to carboxyl It is composed of three CDRs and four FRs arranged from the C-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0077] As used herein, the term "antigen-binding fragment" of an antibody 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 obtained, for example, from intact antibody molecules by any appropriate standard technique, such as proteolytic digestion, or recombinant DNA technology, 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 Fragment”.

[0078] As used herein, the term "CDR" or "complementarity determining region" refers to a region of a heavy or light chain polypeptide. "Discontinuous antigen-binding sites" refers to discontinuous antigen-binding sites found within both variable regions of a given antibody. These specific 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. 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 complex crystal structures and analysis of antibody-antigen interactions. The amino acid residues which encompass the CDRs as defined by each are shown for comparison. In one embodiment disclosed herein, the term "CDR" refers to a CDR as defined by the Kabat definition. In another embodiment disclosed herein, a CDR refers to a CDR as defined by the IMGT definition. 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. The variable region framework thus includes amino acids outside the CDRs, but is approximately 100-120 amino acids long.

[0080] The present invention also provides "conservative amino acid substitutions" in the CDR amino acid sequences of the antibodies disclosed herein. "amino acid substitutions," i.e., amino acids that do not abolish 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. Additionally, 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 substitution of one class of amino acid with an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and, for example, standard Dayhoff frequencies. Determined by frequency exchange matrix or BLOSUM matrix 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, e.g., Asn, Gln, or Glu, is a conservative substitution. Therefore, predicted nonessential amino acid residues in PAI-1 antibodies are likely to be more specific than other amino acids from the same class. The amino acid residue is replaced with a non-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 biological 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 encoding nucleotides) are expected to result in PAI-1 antibodies with functional and chemical characteristics similar to those of naturally occurring PAI-1 antibodies. In contrast, substantial modifications in the functional or chemical characteristics of PAI-1 antibodies are not expected to result in substantial modifications. (a) in regions of substitution, e.g., sheet or helix conformation; This can be achieved by selecting substitutions that differ significantly in their effect on (a) the structure of the molecular backbone, (b) the charge or hydrophobicity of the molecule at the target site, or (c) maintaining 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 specifically bind to 1x10 -6 Medium, 1x10 -7 Medium, 1x10 -8 Medium, 1x10 -9 Medium, 1x10 -10 Medium, 1x10 -11 Medium, 1x10 -12 Binds to antigen with a Kd lower than M or lower 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.

[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, e.g., 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) are suitable for introduction into host cells. Upon replication, vectors 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 known as "recombinant expression vectors"). These vectors are often referred to simply as "expression vectors." In general, 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 also relates to the use of such other forms of expression vectors, e.g., plasmids. For example, viral vectors (e.g., replication defective retroviruses, adenoviruses and adenoviruses) The term "antigen" is intended to include viruses (including viruses associated with the virus) which serve equivalent functions.

[0090] Numerous 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 papillomavirus, polyomavirus, adenovirus, vaccinia virus, baculovirus, retrovirus (RSV, MMTV, or MOMLV), or SV40 virus. Others utilize internal ribosomal It contains the specification of polycistronic systems with binding sites. Integrated cells can be selected by introducing one or more markers which allow for selection of transfected host cells. Markers can provide for prototrophy to an auxotrophic host, resistance to biocides (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can be directly linked to the DNA sequence to be expressed, or it can be linked to a gene encoding a selectable marker gene. The variable region genes may be introduced into the same cell by cotransformation. 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 introduced into a suitable host cell, i.e., the host cell can be transformed. Introduction of the plasmid into the host cell can be achieved by a variety of techniques well known to those skilled 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 using enveloped DNA, microinjection, and infection with intact virus. See Ridgway, AAG, "Mammalian Expression Vectors," Chapter 24.2, pp. 470-472, in Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). An embodiment disclosed herein is the introduction of the plasmid into the host by electroporation. Transformed cells are grown under conditions appropriate for the production of light and heavy chains, and assayed for heavy or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), 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 alters the genotype and results in a change in the recipient cell. The term "contextual entity" is used in a broad sense to refer to a specific entity.

[0093] A "host cell" is a cell that has been constructed using recombinant DNA techniques and contains at least one heterologous " refers to cells transformed with a vector encoding the gene. In describing methods for isolating a polypeptide from a recombinant host, the terms "cells" and "cell culture" are used interchangeably to indicate the source of the antibody, unless clearly specified otherwise. In other words, recovery of polypeptide from "cells" can mean either from sedimented whole cells or from the cell culture containing both the medium and 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 mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still 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 is a method of preventing, curing, delaying one or more symptoms of a disease or disorder, or the recurrence of a disease or disorder. and / or administering to a subject, e.g., a patient suffering from a PAI-1-associated disease or condition, to reduce the severity of or ameliorate the disease, or to prolong the survival of the subject beyond that expected in the absence of such treatment. have a disease or disorder (e.g., a fibrotic disease) or are susceptible to such a disease or disorder The present invention 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 are 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, when administered to a subject, is sufficient 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 will vary depending on the subject and disease state, the weight and age of the subject, the severity of the disease state, the method of administration, etc., and these can be easily determined by one skilled in the art. The dosage for administration can range from about 1 ng to about 10,000 mg, about 1 μg to about 5,000 mg, about 1 mg to about 1,000 mg, or about 10 mg to about 100 mg of the antibodies or antigen-binding fragments 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] As used herein, the term "epitope" 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 formed by spatially juxtaposed amino acids from different segments of a linear polypeptide chain. A linear epitope is formed 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 antigen-binding fragment thereof that specifically binds to human PAI-1. The exemplary VH, VL and CDR amino acid sequences and nucleic acid 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 present invention provides anti-PAI-1 antibodies that competitively inhibit an antibody or antigen-binding fragment thereof that binds to the same epitope or that comprises the VH and VL region amino acid sequences set forth in SEQ ID NOs: 6 and 7, respectively. Such antibodies can be used, for example, to bind to surface plasmon can be identified using conventional competitive binding assays, including 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 The modified forms of the anti-PAI-1 antibodies disclosed herein may contain modifications known in the art. The polymeric material 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. Chimeric antibodies are antibodies in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine 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 used to generate 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. Humanized antibodies have a binding specificity comprising one or more complementarity-determining regions (CDRs) from a non-human antibody and framework regions from a human antibody molecule. Often, In these framework regions, framework residues in the human framework regions are substituted with the corresponding residue 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 interaction of CDR and framework residues to identify framework residues important for antigen binding and by determining specific positions. Antibodies are identified by sequence comparison to identify unusual framework residues in the antibody. See, e.g., Queen et al., U.S. 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 humanized using a variety of techniques known in the art, including those described 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 portion 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 mechanics (MD) simulations. MD simulations of proteins are performed on a computer, and by calculating the physical interactions of the atoms with each other, it is possible to determine the movements of all protein atoms over a period of time. The output of an MD simulation is the trajectory of the protein studied over the period of the simulation. The trajectory is a collection 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 collection of snapshots, the flexibility of protein amino acid residues can be quantified. A flexible residue is therefore one that adopts 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 binding partners provides the basis for their flexibility, allowing them to conformationally adapt to one another (Sundberg and Mariuzza, Structure 8, R137-R142, 2000). Thus, examples of "induced fit" have been shown to play a key role in protein-protein interactions. Furthermore, a growing body of data has demonstrated that proteins actually 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 their 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] Flexible residues may be recognized by memory B cells and induce immunogenic responses. Antibodies can adopt a variety of conformations that result in a set of interaction 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 most similar to that of the parent antibody; (4) determining the flexible residues to be mutated, residues or motifs that may be the source of heterogeneity and degradation and 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 course 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 can be identified, for example, by 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. The CDR residues and adjacent residues may be selected to maintain high affinity for the antigen. Flexible residues are then replaced.

[0116] If several 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 found in exposed, mobile loops above 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 sulfoxide formation can be generated 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 (J. Chromatog. 837:35, 2006) and N-glycosylation sites found with asparagine residues followed by small amino acids such as Gly, Ser, Ala, His, Asn, or Cys , e.g., Asn-X-Ser / Thr sites. Typically, exposed methionines are substituted with Leu, exposed asparagines are replaced with glutamine or aspartic acid, or the following 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 used. 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 loss of activity sometimes 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 deactivating the 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, which may be used to reduce 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 analyzed. A human T-cell epitope "map" can be generated from each V region showing the location of the epitope in relation to the residues that make up the V region. Individual T-cell epitopes from the T-cell epitope map are analyzed to identify alternative amino acid substitutions that have a 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 the human V-region or fragments 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 intact antibodies. The antibodies are then compared in appropriate biochemical and biological assays, and the optimal variant is identified.

[0119] ii) Effector Function and Fc Modification 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 eductor functions. For example, binding of the C1 component of complement to an antibody constant region can activate the complement system. Complement activation is important in opsonization and lysis of cellular pathogens. Complement activation also stimulates inflammatory responses and may be involved in autoimmune hypersensitivity. Furthermore, antibodies can bind to various antigens via the Fc region by allowing the Fc receptor binding site on the antibody Fc region to bind to an Fc receptor (FcR) on a cell. They bind to receptors on cells. 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 can result in 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 elicit a number of important and diverse biological responses, including the regulation of immune cell damage (called ADCC, or ADCC), the release of inflammatory mediators, placental transfer, and the 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. lack one or more effector functions (e.g., ADCC activity) or lack Fc It may contain a constant region that is unable to bind to a receptor.

[0120] Certain embodiments disclosed herein may 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 specific sites in the body (e.g., at the site of a tumor or at a specific time point), when compared to an otherwise intact, unmodified antibody of approximately the same immunogenicity, having at least one amino acid deleted. 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. For example, the antibodies described herein Certain antibodies, or fragments thereof, for use in the diagnostic and treatment methods described herein resemble 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 engineered 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 from different antibody isotypes. In another embodiment, the anti-PAI-1 antibody comprises a chimeric hinge (i.e., a hinge portion derived from hinge domains of different antibody isotypes, 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 derived 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 can be used to increase or decrease their function. For example, deletion or inactivation of constant region domains (by point mutation or other means) can decrease Fc receptor binding of circulating modified antibodies, thereby increasing tumor localization. In other cases, constant region modifications consistent with the present invention may attenuate complement binding and thus decrease serum half-life and nonspecific binding of conjugated cytotoxins. Still other modifications of the constant region can be used to modify disulfide bonds or oligosaccharide moieties, allowing for enhanced localization due to increased antigen specificity or mobility. The resulting physiological profile of the modifications, such as tumor localization, biodistribution, and serum half-life, bioavailability, and other biochemical effects, 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 here is derived from a human IgG1 antibody, and the human IgG1 Fc domain The Fc variants of the antibody 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 variants comprise an amino acid sequence at an amino acid position located in the CH3 domain or portion thereof. In another embodiment, the Fc variant comprises a substitution located in the CH4 domain or 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 can be prepared using the methods described in, for example, International PCT Publications 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 ,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 containing amino acid substitutions that alter the antigen-independent effector functions of the antibody, particularly 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 serum half-lives, 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., to treat chronic diseases or disorders. In contrast, Fc variants with decreased FcRn binding affinity are expected to have shorter half-lives, and such molecules are useful for administration to mammals where a shortened circulation time would be advantageous, e.g., for in vivo diagnostic imaging, 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 decreased FcRn binding affinity are also less likely to cross the placenta and therefore are useful in treating diseases or disorders in pregnant women. Additionally, other applications in which decreased 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 decreased transport from the vasculature across the epithelium of the renal glomerulus. In another embodiment, the modified antibodies disclosed herein exhibit decreased transport across the blood-brain barrier (BBB) ​​from the brain into the vascular space. In one embodiment, an 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 the following are disclosed in International PCT Publication No. WO 05 / 047327, which is incorporated herein by reference. In certain exemplary embodiments, the antibodies disclosed herein, or fragments thereof, comprise an Fc domain with 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, e.g., an IgG1 or IgG4 heavy chain constant region, that has been altered to reduce or eliminate glycosylation. For example, the antibodies disclosed herein can also include Fc variants containing 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 residue 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. WO 05 / 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 "agli" antibodies (e.g., "agli" antibodies). It is believed that "Agri" antibodies, or fragments thereof, may have an improved safety and stability profile in vivo. Exemplary Agri antibodies, or fragments thereof, contain an aglycosylated Fc region of an IgG4 antibody, which lacks Fc-effector functions, thereby reducing Fc-mediated toxicity to normal vital organs that express PAI-1. In yet another embodiment, the antibodies or fragments thereof disclosed herein comprise an altered glycan. For example, the antibody can have a reduced number of fucose residues on the N-glycan at Asn297 of the Fc region, i.e., is afucosylated. In another embodiment, the antibody can 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 characterized in that covalent binding of the antibody to its cognate epitope is The antibody may be modified by covalent attachment of a molecule to the antibody so as not to interfere with covalent binding. 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 made by one or more of the It may contain more than one non-classical amino acid.

[0130] The antibodies or fragments thereof disclosed herein may also be recombinantly fused at the N- or C-terminus to heterologous polypeptides or chemically conjugated (including covalent and non-covalent conjugations) 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, e.g., 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] Anti-PAI-1 antibodies may be used to increase their in vivo half-life or to enhance their overall survival using methods known in the art. The antibodies may be fused to heterologous polypeptides for use in immunoassays involving PAI-1. For example, in one embodiment, PEG can be attached to the anti-PAI-1 antibodies disclosed herein to increase their in vivo half-life (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. For example, hexa-histidine provides for convenient purification of the fusion protein, as described by 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 used in a conjugated form, e.g., 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, peptides, or the like. Conjugated to peptides, proteins, enzymes, viruses, lipids, biological response modifiers, drugs, or PEG It can be done.

[0134] The present invention further encompasses anti-PAI-1 antibodies conjugated to a diagnostic or therapeutic agent. Anti-PAI-1 antibodies can be used diagnostically, for example, to determine the effectiveness of a given treatment or prophylactic regimen, as part of a clinical testing procedure, or to monitor the development or progression of an immune cell disorder (e.g., CLL). Detection can be achieved 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. Pat. No. 4,741,900 for metal ions that can be conjugated 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 substance is luminol; a non-limiting example of a bioluminescent substance is luminol. Examples include luciferase, luciferin, and aequorin; and suitable radioactive materials. Non-limiting examples of radioactive materials 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 the growth of tumor cells. other antibodies that bind to both cytoplasmic 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 conjugated 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 light of the teachings herein.

[0137] Not surprisingly, 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 prepare therapeutic conjugates typically produce high-energy alpha or beta particles with short trajectories. Such radionuclides kill cells in their vicinity, such as tumor cells to which the conjugate has bound or entered. They have little or no effect on cells outside their localization. Radionuclides are inherently 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 gene is 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 Polycistronic constructs can be used to express polypeptides. In such expression systems, multiple gene products of interest, such as antibody heavy and light chains, can 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 the like. Cell lines include BALBC / 3T3 (mouse fibroblasts), HAK (hamster kidney line), SP2 / O (mouse myeloma), BFA-1c1BPT (bovine endothelial cells), RAJI (human lymphocytes), and 293 (human kidney). In one embodiment, the cell line provides altered glycosylation, e.g., 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 quantities of the desired polypeptide. Techniques for culturing 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 desirable, 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 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 growth in culture or fermentation. Bacteria susceptible to infection include members of the Enterobacteriaceae family, such as Escherichia coli or Salmonella species; Bacillaceae, such as Bacillus subtilis; Pneumococcus; Streptococcus; and Haemophilus influenzae. It should be further understood that when expressed in bacteria, polypeptides can become part of inclusion bodies. Polypeptides must be isolated, purified, and then assembled into functional molecules.

[0143] In addition to prokaryotes, eukaryotic microorganisms 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, the 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 provides a selection marker for yeast mutants lacking the ability to grow in tryptophan, e.g., 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 for 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. Routes of administration of the antibodies, or fragments thereof, disclosed herein can be oral, parenteral, by inhalation, or topical. As used herein, the term parenteral includes intravenous, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal, or vaginal administration. Intravenous, intraarterial, subcutaneous, and intramuscular forms of parenteral administration can be used in certain embodiments. While 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 contain 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 additives may be used consistent with the teachings herein. In the method, 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, pharmaceutically 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 solutions include 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 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) 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. cellulose, propylene glycol, and liquid polyethylene glycol), and The solvent or dispersion medium may be a solvent or dispersion medium containing an appropriate 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 injectable compositions can be achieved by the use of agents delaying 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). Dispersions can be prepared by incorporating the active compound (combined antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by sterile filtration. Generally, dispersions are prepared by incorporating the active compound into 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, preparation methods may be vacuum drying and freeze-drying, which yields a powder of the active ingredient and any additional desired ingredient from a previously sterile-filtered solution thereof. The injectable formulations 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. Furthermore, the formulations can be packaged and sold in the form of kits, such as those described in co-pending U.S. Application Nos. 09 / 259,337 and 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 dose of the stabilized antibodies or fragments thereof disclosed herein for treating 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 being administered, and whether the treatment is prophylactic or therapeutic. Typically, the patient is a human, although non-human animals, including transgenic mammals, can also be treated. Treatment dosages can be titrated using routine methods known to those of skill in the art to optimize safety and efficacy.

[0149] For passive immunization using 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 the host body weight. For example, dosages can be 1 mg / kg 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 as being within the scope disclosed herein.

[0150] Subjects may receive 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., at least six months. Further exemplary treatment regimens include: Exemplary dosing schedules include administration once every two weeks, once a month, or once every three to six 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 can be administered multiple times. The interval between single doses can be, for example, daily, weekly, monthly, or yearly. The intervals can also be irregular, as indicated by measuring the blood level of the polypeptide or target molecule in the patient. In some methods, the dosage is adjusted to achieve a specific plasma antibody or toxin concentration, for example, 1-1000 μg / ml or 25-300 μg / ml. Alternatively, the antibodies or fragments thereof can be administered as sustained-release formulations, in which case less frequent administration is required. The dosage and frequency vary depending on the half-life of the antibody in the patient. Generally, humanized antibodies exhibit the longest half-life, followed by chimeric antibodies and non-human antibodies. In one embodiment, the antibodies or fragments thereof disclosed herein can be administered in unconjugated form. In another embodiment, the antibodies disclosed herein can 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 precise 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 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 can 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 virus particles.

[0155] Therapeutic agents can be administered parenterally, topically, intravenously, orally, subcutaneously, intraarterially, intracranially, intraperitoneally, intranasally, or intramuscularly for prophylactic or therapeutic treatment. Intramuscular injection or intravenous infusion can be used to administer 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 art-recognized 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 from about 5 to about 70 mCi, and in one embodiment, from about 5 to about 40 mCi. Effective single-treatment ablative dosages of 131I-labeled antibodies (i.e., which may require autologous bone marrow transplantation) range from about 30 to about 600 mCi, and in one embodiment, from about 50 to less than about 500 mCi. In conjunction with chimeric engineered antibodies, due to their longer circulating half-lives compared to murine antibodies, effective single-treatment non-marrow ablative dosages of iodine-131-labeled chimeric antibodies range from about 5 to about 40 mCi, and in one embodiment, is less than about 30 mCi. For example, imaging criteria for the 111In label are typically less than about 5 mCi.

[0158] Although much 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. For example, additional radioisotopes that are compatible with the scope of the present invention include, but are not limited to: I, I, P, Co, Cu, Br, Rb, Kr, Sr, In, Cs, Cs, I, Hg, Pb, Bi, Lu, Re, Pb, Bi, Sc, Rh, Pd , 153Sm, 188Re, 199Au, 225Ac, 211A 213Bi. In this regard, alpha, gamma, and beta emitters are all compatible with the present invention. Furthermore, in view of the present disclosure, it is believed that one skilled 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, additional radionuclides already used in clinical diagnosis include 125I, 123I, 99Tc, 43K, 52Fe, 67Ga, 68Ga, In addition to 111In, antibodies are also being investigated for their 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. provides relevant data and is incorporated herein by reference.

[0159] As previously discussed, the antibodies or fragments thereof disclosed herein can be administered in a pharmaceutically effective amount for the in vivo treatment of mammalian disorders. In this regard, it will be understood that the disclosed antibodies or fragments thereof will be formulated to facilitate administration and promote stability of the active agent. In certain embodiments, pharmaceutical compositions in accordance with the present invention comprise a pharmaceutically acceptable, non-toxic, sterile carrier, such as saline, non-toxic buffers, preservatives, and the like. For purposes of this application, a pharmaceutically 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, e.g., ameliorate symptoms of a disease or disorder, or detect a substance or cell. In the case of tumor cells, the polypeptide, in certain embodiments, can interact with selected immunoreactive antigens on neoplastic or immunoreactive cells, resulting in increased killing of those cells. It will be understood that the pharmaceutical compositions disclosed herein can be administered in single or multiple doses to provide a pharmaceutically effective amount of the polypeptide.

[0160] In accordance with the scope of the present disclosure, the antibodies disclosed herein can 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 can be administered to such humans or other animals in conventional dosage forms prepared by combining the antibodies disclosed herein with conventional pharmaceutically acceptable carriers or diluents in accordance with known techniques. Those skilled in the art will recognize that the form and characteristics of the pharmaceutically acceptable carrier or diluent will be determined by the amount of active ingredient to be combined, the route of administration, and other well-known variables. Those skilled in the art will appreciate that the use of one or more polypeptides in accordance with the present invention will be readily apparent to those skilled in the art. It will further be appreciated that cocktails containing 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. Accordingly, 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 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 lung fibrosis, or abdominal adhesion formation.

[0163] The development of intraperitoneal adhesions is a major cause of human illness. Complications of adhesions can be as serious as life-threatening bowel 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 can also be 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 increased levels of plasminogen activator inhibitors It is believed that a post-traumatic dysfunction in peritoneal fibrinolytic activity caused by an increase in the inhibitors 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 (laparotomy) It has limitations on 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 a major 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 subject's disease state (e.g., stage I vs. stage IV), age, sex, medical complications (e.g., immunosuppressive conditions or diseases), and weight, as well as the patient's overall risk of developing a disease. The dosage regimen may vary depending on factors such as the ability of the antibody to elicit a desired response in the subject. Dosage regimens may be adjusted to provide the 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, 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, wherein 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 of SEQ ID NO: 37; a light chain CDR1 region comprising SEQ ID NO: 145, a light chain CDR2 region comprising SEQ ID NO: 145, and a light chain CDR3 region comprising SEQ ID NO: 35 [including the area] 1. 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 variable region comprising a light chain framework region and SEQ ID NO: 93 1. 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; 1. 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) Heavy chain framework region and heavy chain variable region, [the heavy chain variable region is a sequence 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 of 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] 1. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

[0174] Item 6. The antibody of 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, teeth: (a) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 82, or an antigen-binding fragment thereof 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 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 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 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 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 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 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 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 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 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 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 SEQ ID NO: 30 and a light chain CDR3 region comprising SEQ ID NO: 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 SEQ ID NO: 42 and a light chain CDR3 region comprising SEQ ID NO: 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 SEQ ID NO: 48 and a light chain CDR3 region comprising SEQ ID NO: 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 SEQ ID NO: 54 and a light chain CDR3 region comprising SEQ ID NO: 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 SEQ ID NO: 60 and a light chain CDR3 region comprising SEQ ID NO: 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 SEQ ID NO: 66 and a light chain CDR3 region comprising SEQ ID NO: 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 Item 9, which has essentially the same enzyme on PAI-1. An isolated monoclonal antibody that specifically binds to a pitope that binds to PAI-1.

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

[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 the antibody according to any one of Items 1 to 10.

[0183] Item 15. Use of a pharmaceutically 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 the antibody orally, parenterally by injectable solution, by inhalation, or topically to a patient. [Example]

[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 well 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 (hereinafter "Sambrook et al., 1989"); DNA Cloning: A Practical Approach, Volumes I and II (D.N. Glover, ed., 1985); Oligonucleotide Synthesis (M.J. Gait, ed., 1984); Nucleic Acid Hybridization [B.D. Hames & S.J. Higgins, eds., 1985]; Transcription and Translation [B.D. Hames & S.J. Higgins, eds., 1984]; Animal Cell Culture [R.I. Freshney, ed., 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 prevent downstream production of plasmin. thereby providing an effective therapeutic agent 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 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. PAI-1 undergoes a spontaneous conformational change from an active to a latent conformation within minutes at room temperature and within hours at room temperature. This resistance is mediated by a conformational change that shortens the half-life of PAI-1 in the active conformation by several minutes. The half-life of active conformational PAI-1 in immunized animals is extended from 100 to several hours. to prolong 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 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 produce antibodies. Standard approaches previously described in the literature used PAI-1 alone or a PAI-1 / tPA complex. The present inventors instead generated antibodies against the active conformation of PAI-1. A PAI-1 / vitronectin complex was used as a novel approach to generating antibodies against PAI-1. To generate antibodies, a three-pronged strategy 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 PAI-1 / tPA complexes 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 naive female BALB / c mice (Charles River, strain Cortez). 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 μg 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 PAI-1 complexed proteins (i.e., that is, the lowest titers against either Vn or tPA) but mouse and rat PAI-1 Mice with the highest titers against lushologues were selected for fusion. Mice selected for fusion were boosted with PAI-1 alone or PAI-1 / Vn complex in PBS as described above for a total of 10 μg of antigen per mouse mixed with Sigma Adjuvant System (Sigma Cat. No. 6322) at a 1:1 ratio in a total volume of 200 μl per mouse. On day 55, mice were sacrificed by CO chamber. 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 three mice for PAI-1 alone and PAI-1 / tPA, and in two 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 initiation, mice were sacrificed in a CO2 chamber, blood was collected by cardiac puncture, and spleens were removed. The splenocytes were 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 count). The cells were combined and spun down at 970 rpm for 10 minutes (low speed spin) to form a soft pellet. Preheated (37°C) 1 ml PEG (PEG 1500) in 75 mM Hepes 50% w / v, Roche catalog 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 1 minute, followed by the addition of 10 ml of serum-free IMDM medium over 1 minute, with the first 1 ml added over 30 seconds. The cells were spun down at 970 rpm for 10 minutes (low speed spin) to preserve viability. The fused cells were plated in a 96-well plate in selective medium (200 ml Gibco Hybridoma (SFM no. 12045), 20 ml 10% HyClone SuperLow IgG Defined FBS (no. 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-thymidine). The fusions were then plated at 200 ul in Sigma-Aldrich (Sigma-Aldrich #HO262 (50X)). After approximately 10-14 days, or when the medium in the wells turned yellow, the fusions were ready for screening. Supernatant from the resulting hybridomas was then added to the wells to detect PAI-1 and the PAI-1 / Vn complex binding. The presence of antibodies was tested by ELISA (Example 2).

[0197] Example 2: Hybridoma specificity for PAI-1-vitronectin complex Binding ELISA for serum screening Each fusion from the spleens of the five selected mice yielded approximately 5,000 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 the PAI-1-vitronectin complex. ELISA was performed in parallel to select 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 5 μg / 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); HRP-goat anti-mouse IgG (H+L) secondary antibody (Jackson ImmunoResearch Labs No. 115-035-166); and ABTS substrate: Roche Diagnostics (No. 11 204 521 001).

[0198] Control antibodies used were: a) 33B8, a mouse monoclonal inhibitory antibody against PAI-1 (IgG1; Innovative Research catalog number 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 μl / well of 5 μg / ml Vn in PBS was coated overnight at 4°C. The next day, the plates were blocked with 200ul 1% bovine serum albumin in PBS (BSA / PBS) for 1 hour; the plates were washed 4 times with 200ul / well PBS; 2ug / ml active PAI-1 in 1% BSA / PBS was added to the plates at 50ul / well and incubated for 1 hour; the plates were washed 4 times with 200ul / well PBS; antibody dilutions in 1% BSA / PBS or hybridoma supernatants from the original 96-well plates were added to the ELISA plates at 50ul / well; the plates were incubated for 1 hour. Incubated at room temperature (RT); plates were washed 4 times with 200 ul / well PBS; 1% BSA in PBS 50ul of HRP-anti-mouse IgG 1:2000 was added and incubated for 1 hour at room temperature; the plate was 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 0.5°C for 1 hour in 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 analyzed for PAI-1 affinity. We screened for this.

[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 assay 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 instrument used was a BIACORE 2000 or BIACORE 3000 (GE Healthcare) designed for real-time biomolecular interaction analysis (BIA). The sensor chip used was a CM5 chip (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. Hybridoma supernatant was injected onto one of flow cells Fc2 to Fc4, and IgG in the hybridoma supernatant was captured on the chip surface by anti-mouse IgG Fc mAb. Fc1 remained intact on the reference cells. 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 Affinity (KD) / dissociation rate (kd) was analyzed and ranked using Scrubber 2 software. That's terrible.

[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 transferred 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 from the Fc1 and blank buffer runs, the antibody activity of the hybridoma supernatants against vitronectin-captured PAI-1 was measured. Binding affinities were determined using Scrubber 2 software (version 2.0a, 2005; BioLogic Software, BioLogic Software 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. Due to the large number of antibodies screened, not all data are shown. Excellent (kd<10-) antibody activity against human and cynomolgus monkey PAI-1 proteins was observed. 4 Antibody clones that showed a binding / dissociation rate of 1 / sec Only 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 enable the selection of functional antibodies, a new ELISA was developed to distinguish between antibodies that only bind to PAI-1 versus antibodies that block the function of PAI-1 as tPA inhibitors (functional ELISA). It was developed to make this possible.

[0207] Hybridoma supernatants were analyzed for 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 altering 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 produce a positive record. Records are negative (lower OD 405 In parallel, hybridoma supernatants were cultured in the same manner as described in Example 2. The antibodies were tested for binding to PAI-1 in an ELISA using a hybridoma supernatant. Because the amount of antibody in the hybridoma supernatant was unknown, a lower than control reading (i.e., lower than the isotype control reading) was considered to be an identification of the antibody of interest. Due to the varying antibody concentrations in the supernatant, some In this case, blocking was only partial.

[0208] Streptavidin-coated plates (NUNC #436014) were incubated with 2 μg / ml biotin-PAI-1 (human PAI-1 with N-terminal biotin-labeled active fraction; Molecular Innovations Catalog #NTBIOPAI-A) at 50 μl / 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 four times with 200 μl / well of PBS. Chain tPA (Innovative Research Catalog No. HTPA-TC) at 1 ug / ml was added to the plate at 50 ul / well and incubated for 30 minutes at RT. Plates were washed 4 times with 200 ul / well PBS. Anti-tPA HRP conjugated antibody (Life Span Technologies, Catalog No. LS-C39721) was added to the plate at a 1:3000 dilution and incubated for 45 minutes. Plates were washed 4 times with 200 ul / well PBS. ABTS substrate (dissolve one tablet in 5 ml; Roche Diagnostics No. 11 204 521 001) was added to the plate at 50 ul / well and allowed time for color development. Plates were analyzed by OD spectrometry 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 is 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 the identified positive antibody clone are shown in Figure 3. vinegar.

[0210] [Table 10]

[0211] Over 200 supernatants were screened. Table 6 shows a selection of positive and negative hybridoma supernatants. Approximately 10 hybridomas per fusion blocked 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 not synthesized, 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 purified from a series of fusions may have the same sequence. By sequencing the antibody gene at the early stage of antibody generation, any potentially redundant antibodies are 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 single-sided specificity amplification methodology 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 an RNeasy Mini Kit (QIAGEN, catalog no. 74104) according to the manufacturer's instructions. Briefly, cells (5 x 106 cells) were lysed in 350 ul of the kit's RLT buffer, followed by capture of total RNA on a spin column. RNA was eluted with the kit's TE buffer and stored on ice.

[0215] The first strand cDNA was purified using SMARTer TM RACE cDNA Amplification Kit (ClonTech, catalog number 634923) The VH and VL chain cDNAs were prepared using 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 the 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 added to the tube and incubated on ice for 20 minutes. The tube containing the E. coli cells and reaction mixture was heated to 42°C for 40 seconds, and 250 microliters of lit 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 PCR confirmation of the inserted VH and VL genes, five bacterial clones were selected and The plasmid DNA was prepared by growing the plasmid in LB medium containing 100 micrograms of ampicillin per ml. Plasmid DNA was prepared using a QIAprep Spin Miniprep Kit (QIAGEN, Cat. No. 10011001). The VH and VL IgG genes of the hybridomas were sequenced by the Sanger method, and the CDRs were identified using 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 count analysis. The assay was characterized for potency compared 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 utilize the hydrolysis of one or more peptide bonds to produce their natural These methods utilize proteolytic enzymes that act on natural substrates (proteins and peptides). The methods are 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 to produce a quantifiable color. Chromogenic substrates are synthetically produced and designed to have 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 enzymatic cleavage. The color change can be followed spectrophotometrically and is proportional to 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. While S228 in solution is colorless, after exposure to tPA and subsequent release of pNA, the solution turns OD 405 This produces a yellow color that can be read in situ. Color formation can be observed over a 2-3 hour period 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 (using a 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 testing for its ability to neutralize PAI-1 blocking function and restore tPA enzymatic activity. Equal volumes (25 ul) of tPA (14 nM) (Innovative Research, Cat. No. IHTPA-TC) and glycosylated (active form) human PAI-1 (Molecular Innovations, Cat. No. GLYHPAI-A) or non-glycosylated (active form) murine PAI-1 (Molecular Innovations Cat. No. IMPAI) were mixed and incubated with 3-fold serial dilutions of PAI-1 starting at 108 nM 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 microtiter plate were incubated for 15 minutes at room temperature. Then, 200 μl 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 minutes for 2 hours. 405 Change in absorbance The residual tPA activity was measured by recording. 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. 12.5 ul of active PAI-1 (56 nM) 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 2 uM. Control and unknown antibodies were incubated with 3 nM PAI-1 and tPA in the mixture at concentrations ranging from 0.1 to 300 nM (5-fold dilutions). All components were incubated at 10x concentrations at room temperature and further incubated with 3 nM PAI-1 and tPA in the mixture at 10x concentrations, resulting in a clear to yellow color upon cleavage by tPA. The mixture was diluted 10-fold with tPA substrate S2288, which changes color to a red color. Samples were read at OD 405 every 10 minutes for 2 hours at 37°C. This mixture was then transferred to a 96-well plate to achieve antibody-antigen complex formation. The wells of the microtiter plate were incubated at room temperature for 30 minutes. Then, 25 μl of tPA (IC 80 (14 nM corresponding to 14 nM) was added to the wells and incubated for 15 minutes at room temperature. Finally, 200 μl of 1.25 mM substrate S2288, diluted according to the manufacturer's instructions, was added to the mixture. The residual tPA activity was measured every 10 minutes for 2 hours 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] Orthologs of human PAI-1 that inhibit human tPA were tested in a two-stage chromogenic assay system. Titrations of the orthologs were performed as described above for human PAI-1 (see Figure 6 for a representative curve of the titration), and tPA activity was determined chromogenically (see Figure 7 for a representative curve 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 of 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 μl of tPA (14 nM) was added to the well, and 15 μl of tPA was added to the well. The mixture was incubated at room temperature for 1 min. To terminate the reaction, 200 μl of 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 orthologues 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 as cynomolgus monkeys (cynomolgus monkeys). ) and human PAI-1 inhibitory function, and approximately 14 antibodies showed moderate to strong activity. A39 and B28 showed little blocking activity against glycosylated hPAI-1, but had no activity against non-glycosylated human or cynomolgus monkey PAI-1. None of the antibodies except C26 were able to effectively block mouse PAI-1 activity (within 10-fold of human PAI-1).

[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 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 mechanism of antibody action. The reaction between the monoclonal antibody (or control antibody), PAI-1, and tPA was performed as described above for the functional chromogenic assay. The 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, complex, and cleaved forms of PAI-1 were visualized by staining with . Control monoclonal antibodies were used as comparators. 33B8 is known to convert PAI-1 to a latent conformation, and 33H1 converts PAI-1 to a substrate conformation. This assay can reliably identify substrate conformations, but cannot distinguish between potential conformations or steric hindrances. Representative SDS gels are shown in Figures 8, 9, and 10.

[0229] [Table 13]

[0230] A44, C26, C45, and E21 differentiate the active conformation of PAI-1 from the substrate conformation. Although A39 and B109 have different mechanisms of action, the assay demonstrates that these antibodies convert PAI-1 from an active conformation to a latent conformation. It blocks PAI-1 activity by either changing it to a steroid or by steric hindrance. It was not possible to distinguish between the

[0231] Example 8: Purified antibody binding kinetics In kinetic measurements, the antibodies were assayed in reverse at 25°C. In the reverse assay, the PAI-1 antibodies were The antibody was 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 value and the blank buffer value. The sensorgrams were fitted by using a simulated kinetics 1:1 (Langmuir) model with local Rmax. Data for the antibodies tested are 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. The CM5 chiral stimulator was coupled to the flow cell Fc1-Fc4 by amine coupling. Human PAI-1 was then captured on the vitronectin surface as a ligand in flow cells Fc2-Fc4. Fc1 was reserved as a reference cell. Antibodies were added starting at 40 nM. The solution was 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. , used with 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 investigate 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, Cat. 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-well plated PAA (A11-152) at 37°C under 5% CO2. On day 2, antibodies were added to recombinant PAI-1 (Molecular Innovations, catalog 1) to neutralize PAI-1 activity. Cells were preincubated with tPA (Molecular Innovations (catalog no. HTPA-TC), 5 nM in red phenol-free DMEM) for 15 minutes at room temperature. After washing away unbound tPA, the PAI-1 / mAb mixture was added to the cells, and residual tPA activity was then assayed with a glu-plasminogen / substrate mixture (Glu-Pg; Sigma catalog no. IGLYHPAI-A, recombinant glycosylated human PAI-1, final concentration 5 nM) for 15 minutes at room temperature. Simultaneously, tPA (Molecular Innovations (catalog no. HTPA-TC), 5 nM in red phenol-free DMEM) for 15 minutes at 37°C. After washing away unbound tPA, the PAI-1 / mAb mixture was added to the cells, and residual tPA activity was then assayed with a glu-plasminogen / substrate mixture (Glu-Pg; Sigma catalog no. HTPA-TC). 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. The plasmin generation was detected by kinetic readings of A405 / 492 every 45 seconds using a chromatographic substrate cleavage system (Thermofischer). 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 measured using tPA alone and PAI-1 (without mAb) as references (100% inhibition). Calculate and plot 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 identified 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 standard amine coupling chemistry. All tested antibodies, except clone B28, showed binding site activity after amine coupling. Human PAI-1 protein was captured by an antibody immobilized on a chip, followed by Each antibody was injected as a sample. The immobilized antibody and antibodies with different binding sites on human PAI-1 were injected as a sample. 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 (analyte antibody) binds to the PAI-1 to which A44 is bound. Therefore, C45 cannot bind to the same binding site on PAI-1 that A44 does. A44, which competes for or binds to PAI-1 (denoted "c / c" in Table 12), prevents C45 from 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 revealed that 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 immobilized 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. Therefore, the commercially available antibodies 33H1 and 33B8 compete with A44 binding to PAI-1. It does not interfere or interfere.

[0244] Interestingly, some immobilized antibodies (i.e., B109) were not soluble in the analyte antibodies (i.e., blocked the binding of 33B8) to the captured PAI-1 protein; When the antibody pair is swapped with the analyte antibody (e.g., by flipping the pair on the chip), the antibody pair no longer competes with each other for binding to PAI-1. For example, when B109 is the immobilized antibody that binds to PAI-1, 33B8 was unable to bind to PAI-1. However, when 33B8 is the immobilized antibody that binds to PAI-1, B109 was unable to bind to PAI-1. This result One possible explanation for this effect is that when immobilized antibody is bound to PAI-1, PAI-1 may change into a conformation unfavorable to the second or analyte antibody and prevent 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 the 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 the

[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 antibody 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 variable light (VL) and variable heavy (VH) sequences of mouse A44 were used to construct homology models of the anti-PAI-1 A44 light chain (LC) and heavy chain (HC) in the 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 chain of His-99 was reconstructed, and the subsequent model was then reconstructed using standard procedures implemented in MOE. The energy was minimized using the NMR method. 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 Diverse 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 (one per 1 ps) from the MD trajectory were used to analyze the dynamics of each mouse A44 axis. For amino acids, their root mean square deviations (rmsd) compared to the reference medoid position were calculated. By comparing the average rmsd of 10 separate MD runs for a given amino acid with 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. 37 amino acids were identified as flexible in the murine A44 antibody, excluding the CDRs and the surrounding 5 angstroms.

[0251] The movements of the 62 most mobile mouse A44 amino acids over a 20 ns (10 x 2 ns) time period were then compared with the movements of the corresponding mobile amino acids in 49 human germline homology models, each of which was run in 10 x 2 ns MD simulations. The 49 human germline models were compared with the seven 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 murine A44 antibody; therefore, the vk1-vh2 germline antibody This body was used to humanize the A44 antibody, focusing on flexible amino acids. 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. Due to the pairwise amino acid association 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 were aligned based on the sequence alignment. 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 frequencies relative to their respective canonical sequences The amino acids of 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, pp. 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 Å "Vernier" zone (J. Mol. Biol. 224, 1992, pp. 487-499)) 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 examine these mutations for potential stabilization of the anti-PAI-1 A44 antibody. These criteria were favorable changes in surface hydropathy or molecular dynamics-based predicted stabilization of the mutant. Further stabilizing mutations have also been reported as successful in the literature (E. Monsellier & H. Bedouelle, J. Mol. Biol., 362, 2006, pp. 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 purified by a binary solvent (20% isopropanol in water). These were unambiguously identified by analyzing molecular dynamics simulations of the Fab in a 20 ns production simulation. 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, no residues contributed to any hydrophobic patches on the surface, and therefore no anti-aggregation mutations were suggested.

[0258] 3) Humanization by grafting Humanization using grafting techniques has been previously reported (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 initiated by performing a BLAST search 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. Using internal VBASE germlines, the light chain was found to be close to the V1-018 locus (approximately 64% identity), and the heavy chain was found to be close to the VH4 subfamily locus 4-30 (approximately 69% identity). The CDR regions (based on Kabat) and Vernier residues were similar to the mA44 light chain. The humanization mutations (Ball) are shown in italics for the IGVK1-33-01_IGKJ4-01 (IGVK1) and IGVK1-33-01_IGKJ4-01 (IGVK1). Vernier residues as defined in J. Mol. Biol., 1992, 224, 487 are underlined. The mouse light chain (LC5a, HC5a) was obtained by performing a pairwise comparison of the two aligned sequences, excluding the CDR and Vernier zone residues (also underlined in mouse) as defined above. 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 replaced with the residues found in the original mouse sequence. The LC5b and HC5b were maintained as they were.

[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, this light chain was VκIII-L6 (approximately 56% identity) locus, and the heavy chain is of the VH6 subfamily. of 6-01 The CDR regions (based on Kabat) and Vernier regions were found to be close to the locus. The CDRs and Vernier regions (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 residues in the CDRs and Vernier regions (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 (glycosylation), 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 CDR1 of HC (N 31 G). Three potential succinimide formation sites have been 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. Four existing problematic residues were 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 was 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 variant 31G deamidation site mutation only 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, Sequences were blasted for similarity against 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 searches, and only results from the human species were considered). DeClerck et al. (International Publication No. WO 2002034776) reported that the PAI-1 epitope The present invention discloses antibodies that bind to epitopes, none of which address 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 is a 14 amino acid sequence as shown below: It has approximately 71% identity across the chain. The control sequence was a partial sequence that had not been confirmed by mass spectrometry. No binding data were reported for this peptide. This epitope was found in all proposed LV variants. No potentially problematic epitopes were identified when a similar search was performed on 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 sequence LC1a contains seven 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 two 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 relative 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 Differences are in bold, problematic motifs are double-strikethrough, and stabilizing mutations are shown in the bottom box. Figures 16 and 17 show a summary of the mutations.

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

[0279] No further human epitopes for the 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 the LC1b embryonic index is 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 the 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). Molecular weight was not determined for this peptide, and no binding data were reported. LC5a Embryonic index = 85% for IGKV1-33-01_IGKJ4-01 [VκI-O18].

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

[0291] No additional human epitopes were identified for the 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 described in Section 4 above, 39 PGQAPRTLI shares 80% 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 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 Figure 15.

[0294] b) Engineered Heavy Chain Sequence 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 next 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 the 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 the 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 the 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 = 72% for IGHV4-31-03_IGHD6-25-01_IGHJ4-02.

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

[0306] No human epitopes were identified for the 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 = 84% for IGHV4-59-02_IGHD6-13-01_IGHJ4-02 [VH4 4-59].

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

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

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

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

[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 present 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. It contained 22 mutations derived from the mouse CDRs and retained all 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 CDR 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 murine CDRs and All mouse Vernier zone residues are retained. HC5c is the second closest nucleotide to the human germline sequence. It contains 23 mutations derived from rafting and retains the mouse CDR and all mouse Vernier zone residues.

[0316] A total of 10 combinations were prepared (summarized in Table 19): LC1axHC1a (mutation addressing 4D humanization based on closest germline sequence) LC1bxHC1b (mutations based on the second-closest germline sequence to accommodate 4D humanization) 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 (4D humanization and stabilization mutations) LC4xHC4 (4D humanized, unwanted sequences and stabilizing mutations) LC5axHC5a (humanized by grafting, retaining 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 variants were generated during the humanization process, which were expressed and characterized in several in vitro assays described below.

[0323] 7) Characterization of humanized variants Based on the computational modeling shown in the examples above, 10 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. Proteins were cloned into the corresponding DNA pXL plasmids (New England Biolabs; NheI / Eco47III for HC and 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 used in the same Transfected at time, and 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. The 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 biosynthesis 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 the poorly expressed variants 3 and 10 were tested in Biacore against human and cynomolgus monkey PAI-1 and vitronectin-PAI-1 complexes. The data are shown in Table 26. show.

[0333] [Table 35]

[0334] [Table 36]

[0335] 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 different from those of the parent A44 in the cellular assay. The ability of the mutants to restore sumin production was significantly lower than the parental murine antibody for some mutants (see Table 27 below for a comparison of the chromogenic and cellular assays). Humanized variants 11–14 were compared 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 variants 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 system. This serpin was assessed by measuring its ability to form a stable complex with urokinase immobilized on a plate. After washing away unbound PAI-1, uPA-PAI-1 complexes were detected using 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 variant 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 complex was 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 PAI-1 levels was diluted four-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. Neutralization of cynomolgus monkey recombinant PAI-1 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 pulley 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; 2 x 30 seconds at 6800 rpm at 4°C), followed by lysis using 1 ml / g of lysis buffer (1.5 M Tris buffer in NaCl TBS, 0.1 M Tris + 0.15 M NaCl pH 7.4). Centrifugation was performed at 5000 g for 10 minutes at 4°C. Liver lysates in the supernatant were subsequently collected 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 the A44 humanized variant incubated with liver lysates diluted to 2.5nM active PAI-1 were analyzed 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. Shown below.

[0341] [Table 39]

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

[0343] Example 13: Humanization of APG antibody by grafting Humanization using grafting techniques has been previously reported (PT Jones, et al., Nature 1986, 321:522-525). The humanization of the anti-PAI1 murine antibody APG is described in German Patent Application No. DE200015 We started with the mouse light chain (SEQ ID NO: 148) and mouse heavy chain (SEQ ID NO: 149) from No. 3251; The mouse antibody was prepared by Debrock et al., Biochimica et Biophysica Acta, 1337(2):257-266 (1997). Also described in

[1999] . Identifying the germline and reference class of the HC and LC chains of the murine antibody yielded muIGHV1-39 and muIGKV14-111, respectively. Next, a list of human germline sequences close to the anti-PAI1 APG variable domain light and heavy chains was identified and ranked by percent identity. Both steps were performed by BLAST searching against all systematically listed human germline sequences. This was done by performing a search (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 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 combinations for APG) and Vernier residues are shown in italics for the parental murine 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 defined in Foote et al. J. Mol. Biol. 224(2):487-99 (1992) are underlined. The CDR and Vernier band residues defined above (also underlined in the mAPG sequence) Humanization was performed by performing a pairwise comparison of the two aligned sequences (except for those underlined, Table 30). Mutations (in bold) were obtained. No further manipulations were performed on the mouse APG antibody. 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 engineered from a mouse light chain spliced ​​into the human IGKV1-33 germline. For the engineered heavy chain sequences, APGv2 and APGv4 contain the 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 contained 15 mutations derived from grafting to the closest germline sequence. APG_VH2 retains the mouse CDR and Vernier band residues. APG_VH2 is the most suitable VH2 with a matching canonical class. It contains 21 mutations derived from grafting to the closest germline sequence, and APG_VH4 contains 20 mutations derived from grafting to the closest human germline sequence, retaining the mouse CDR and Vernier zone residues. The delimitation of the CDRs for this grafting protocol was based on a variety of different methods available in the literature. Based loosely on the definition given by

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

[0350] Example 14: Affinity kinetics for APG antibodies by surface plasmon resonance The affinity to human glycosylated PAI-1 (GLYHPAI-A, Molecular Innovation) was assayed for mouse APG and two humanized variants (APGv2 and APGv4) using a Biacore 2000 instrument (GE Healthcare The samples were examined by surface plasmon resonance (SPR) using a 3000 keV spectroscopy (SPR) system (Uppsala, Sweden).

[0351] First, the surface of the sensor chip CM5 (GE Healthcare, Uppsala, Sweden) was coated with the mouse and 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 for 3 min at 4°C. Human PAI-1 was captured on the surface of a flow cell consisting of 1000 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: 120 s). (Long dissociation: 90 seconds; Long dissociation: 1800 seconds, 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 Figure 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). 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. (e.g., see below) using horseradish peroxidase conjugated to a secondary antibody according to the manufacturer's instructions. (Molecular Innovation, Cat. No. HPAIKT).

[0354] Varying concentrations of APG humanized variants (APGv2, APGv4) or parental mouse APG antibody were incubated with undiluted human plasma containing high activity PAI-1 levels at room temperature for 15 minutes. Residual activity 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 murine 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 was designed to determine whether tPA inhibits clot lysis. Clot formation is induced using a tissue factor / Ca2+ mixture in the presence of known concentrations of PAI-1. Fibrin polymerization is detected by turbidimetry, measuring absorbance 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 antibody or isotype control IgG diluted in assay buffer (NaCl, Tris-HCl pH = 7.4). After 15 min of incubation at room temperature, human glycosylated PAI-1 (GLYHPAI-A) was added. t-PA (sctPA, Molecular Innovation) was then added to a final concentration of 1 nM. Clot formation was monitored using tissue factor (Innovin) diluted in calcium assay buffer (CaCl) to a final concentration of 7.5 mM. (R) The cells were induced with an activation mixture containing 1000kJ of ATP (Siemens Healthcare Diagnostics, Marburg, Germany).

[0360] Dynamic readings of absorbance at 340 nm were taken every 30 seconds for 5 hours 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. Recovery of clot lysis after antibody treatment was determined according to the following calculation:

number

[0361] A 1 nM concentration of t-PA resulted in complete lysis of normal plasma within 2 hours. A 3 nM concentration of PAI-1 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 Figure 21). At 100 nM, isotype IgG1 was not restored (see Figure 22). A44V11 exhibited 103% I max with an IC of 2 nM 50(See Figure 23).

[0363] The humanized variant of the APG anti-PAI-1 antibody also restored clot lysis in human platelet-poor plasma (Figure 24 APGv2 has an IC of 2.1 nM. 50 and 114% of I max APGv4 had an IC of 2.8 nM. 50 and 116% of I max The clot lysis data are shown in the table below. Summarize in 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 is considered to be the most potent and widespread profibrogenic cytokine. TGFβ has been shown to induce PAI-1 expression and t-PA and plasmin activity, as well as collagen degradation in cultured mouse embryonic 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. 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 analyzed by Western blot using rabbit pAb anti-PAI-1 (Abcam, ab66705) for detection of PAI-1 forms. was used for the analysis.

[0366] Cells treated with A44V11 antibody after TGFβ stimulation showed a PAI-1 band as a doublet, which corresponds to the cleaved form of PAI-1 (see Figure 26, lane 5). Cells grown in A44V11 do not show this doublet formation (Fig. 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 that can degrade 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 degradation 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 a 12-well plate. The cells were incubated with A44V11 or an isotype control (IgG) and Lys-plasminogen activator for 48 hours. Incubated with HCl (Molecular Innovation, Cat. No. HGPG-712) at a concentration of 0.1 μM. After 48 hours, cell supernatants were collected and analyzed for various MMPs (e.g., MMP-1, 2, 3, 7, 8). , 9, 12, 13, and 14) was detected using the Sensolyte 520 Generic MMP Assay Kit (AnaSpec, Fremont, CA, catalog no. 71158) according to the manufacturer's instructions.

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

[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 efficacy of potential therapeutic agents as well as for 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)). 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. Female zygotic humanized PAI-1 transgenic mice, 8-9 weeks old and weighing 22-25g, were used. was used for these procedures. Rodent chow and water were available ad libitum.

[0371] Mice were treated with bleomycin 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 intraperitoneally with either A44v11 or negative control mouse IgG1 at 10 mg / kg (1 mg / ml) in PBS.

[0372] At the designated time points (day 7 or 9) after bleomycin challenge, mice were anesthetized with a xylazine / ketamine mixture and euthanized by thoracotomy in citrate-coated tubes. Blood collection was performed by intracardiac withdrawal. The left bronchus was clamped, and the left lung was removed and placed in a fixator (FineFix) under controlled pressure for histological analysis. (R) The lungs were fixed using a 3-mm syringe (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 0.5 ml injections). 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 laws 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). treated with A44V11, the results were 200 nM in plasma, 11 nM in BALF, and 12 nM in lung lysates. .

[0374] As shown in Figure 28, administration of a single intraperitoneal dose (10 mg / kg) of A44V11 on day 4 significantly reduced bleomycin levels. 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. only partial inhibition was achieved.

[0375] D-dimer, a fibrin degradation product, can be measured to assess 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 the A44V11-treated group increased by approximately 2.8-fold on day 7 and 1.6-fold on day 9 compared to the IgG1 negative control group, suggesting that A44V11 treatment increases fibrin degradation (see Figure 29).

[0376] Additional studies were conducted to further evaluate the activity of A44V11 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 an IgG1 control antibody) began on day 4 and was repeated every 3 days 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-induced mice treated with the IgG1 negative control antibody. 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 does no 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 previously 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) at 105°C for 22 hours, followed by evaporation. Primary amines were blocked in the lung tissue with OPA (phthalaldehyde), and proline was added. Hydroxyproline / hydroxyproline was specifically labeled using NBD (4-chloro-7-nitrobenzofurazan) (Santa Cruz Biotech., Santa Cruz, CA). The hydrolyzed product was then purified using Synergistic TM The eluate was analyzed by HPLC (Shimazu Corp., Kyoto, Japan) using a 4 μm Hydro-RP 80Å, LC Column 150x3 mm (Phenomenex, Torrance, CA, Cat. No. 00F-4375-Y0) with an acetonitrile gradient. The peaks were separated below. A standard curve of known amounts of hydroxyproline was used as a reference to quantify the peaks. Representative quantified data is shown in Figure 31.

[0379] Pulmonary collagen accumulation, detected by hydroxyproline content, was significantly associated with bleomycin chaperones. The increase in lung collagen accumulation was statistically significant in mice treated with A44V11 antibody at 10 mg / kg (p<0.08) (see Figure 31). Repeated dosing with the IgG1 negative control antibody did not inhibit the increase in lung collagen accumulation resulting from bleomycin challenge. The reduction in bleomycin-induced collagen accumulation in A44V11-treated mice was statistically significant (p<0.05) when compared with similar bleomycin-induced mice treated with the IgG1 negative control antibody. A44V11-treated mice showed approximately 44% less increase in collagen accumulation than 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 LPS or IgG1 (negative control, 5 mg / kg, intraperitoneally). The experiment was performed in accordance with European ethical laws. and recognized by the Internal Ethical Community (CEPAL, sanofi).

[0381] Cynomolgus Macaca fascicularis (male and female) weighing 4-9 kg were fasted overnight before long-term anesthesia (at least 8 hours), 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 inhalation of a gas mixture of ethanol (1-3%) was used. The monkeys' body temperature was maintained at 50°C using a heating pad. After catheter insertion, LPS (Serotype 0127-B8) was administered as a 1-minute bolus into the cephalic accessory vein at a dose of 100 μg / kg (0.4 mL / kg). At various time points, blood and liver samples were taken. Platelet-poor plasma was isolated by centrifugation. Liver biopsies and terminal necropsies were stored at -80°C.

[0382] Active PAI-1, D-dimer, and plasmin-α2 antiplasmin levels were determined using commercially available ELISA assays (Mol. Innovation, catalog number HPAIKT; Asserachrom D-Dimer; Plasmin-A2 antiplasmin, Diagnostica Stago) according to the manufacturer's instructions.

[0383] In plasma, active PAI-1 levels decreased from approximately 30 ng / ml to less than 10 ng / ml in all monkeys administered A44v11 (see Figure 32(A)). There was no increase in active PAI-1 levels after LPS administration (100 μg / kg) (see Figure 32(A)). In contrast, monkeys treated with the negative IgG1 control showed a strong increase in active PAI-1 levels after LPS administration, with the maximum occurring at approximately 4 hours (approximately 50 to approximately 250 ng / ml) (see Figure 32(B)). Thus, treatment with the negative IgG1 control did not significantly increase the active PAI-1 levels after LPS administration. It does not reduce the subsequently strongly increased levels of active PAI-1 in plasma (see Figure 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 monkeys treated with the negative IgG1 control. (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 those 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 PAP levels comparable to those in negative IgG control-treated monkeys. In comparison, increased levels of plasmin-α2 antiplasmin (PAP) complexes were observed (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 a mouse model of abdominal adhesions 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 electrocautery procedure was performed to assess the serosal membrane. This disrupts the surface, causing thermal injury to the uterine tissue and approximating 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 mice 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 maintained in a controlled manner as described in detail in Haney AF et al. (1993). Briefly, each animal was anesthetized with isoflurane for surgery according to IACUC guidelines and then placed in a suitable vein. 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 uterotubal canal. The ovarian vascular supply was tied together just below the uterotubal junction. Care was taken to avoid damaging the uterine horn. To induce electrocautery lesions, a bipolar electrocautery unit (Valley Lab Surgistat, Solid State Electrosurgery Unit, Model No. B-20) was used on the inner surface of each uterine horn over an area of ​​approximately 2 x 6 mm. The cautery unit was set as follows: 100 volts, 130 Hz, 50-60 amps. A 3 mm wide cautery tip was placed on the inner surface of each uterine horn, with a pure coagulation current setting of 3. The current was applied, power was turned on, 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 needles (Ethicon Inc.). The skin was closed horizontally using 5-0 Prolene, BV-1 tapered needles (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 given a second dose of antibody (30 mg / kg) intraperitoneally (IP) 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 an approximation 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 was also recorded, but may not be included in the length of the adhesion area. The distribution of the mean percent length of adhesion between the uterine horns was checked for normality using the Shapiro-Wilk test. These groups were compared with each other using Tukey-Kramer analysis if they were normally distributed, and Wilcoxon rank sum analysis if they were not. In all cases, a p-value of ≤0.05 was considered statistically significant. Treated animals showed a significantly lower percentage of adhesion formation length between adjacent uterine horns (see Table 35).

[0392] [Table 46]

[0393] Detection of active PAI-1 and tPA levels After euthanasia, animals were given blood (plasma), intraperitoneal fluid (IPF), and uterine horn samples for evaluation. Sample collection was performed using conventional techniques. Plasma, IPF, and uterine horn samples were collected. 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 in animals treated with A44V11 versus isotype control. (See Figure 36.) Relative to the isotype control, decreased levels of active PAI-1 at 6 hours in IPF were observed 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 extensively dialyzed against PBS and stored at 4°C.

[0395] Source of cynomolgus monkey (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 ribosomal protein.

[0397] Conjugate preparation and purification: The recombinant Fab and antigen were mixed at a molar ratio of 1.5:1, 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. Crystals diffracted to 3.3 Å in space group P321 (a = b = 193 Å, c = 144 Å) at the ID29 beamline at the ESRF. 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. Matthews coefficients (V M、 Calculations of the crystal volume per unit of protein molecular weight suggest the presence of up to four complexes in the asymmetric unit (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 domains. This had to be manually placed. M This solution, corresponding to (71% solvent), was also carefully examined for packing consistency. The structure was analyzed using Buster (GlobalPhasing). The non-crystallographic symmetry was used to refine the constant domain to an R-free of 29.2% (R-factor 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] To initially determine the PAI-1 / Fab mAb complex structure, we used the native mAb A44 to produce its Fab fragment by papain digestion. This large-scale Fab production yielded heterogeneous Fab fragments that were 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 first 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 complex crystals to crystallize could potentially be explained by the conformational heterogeneity of the complex. The wild-type PAI-1 molecule is known to adopt three different conformations (active, latent, and substrate), which may hinder crystallization. To improve the quality of the crystals, we prepared a 6-His-tagged A44 Fab in complex with latent PAI-1 (see Figure 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 Figure 39(b) showing a representative optimized crystal).

[0405] The A44 Fab fragment was engineered to reduce the mobility of the protein portion. We decided to produce it using a different tag, but without using an artificial tag like the 6-His tag we had used previously. To further increase the chances of successful crystallization, we investigated the active form of PAI-1 mutant (N150H , K154T, Q319L, M354I) were purchased from Molecular Innovations (catalog number CPAI, Novi, MI). The resulting Fab A44 protein was then introduced into the Fab and used to prepare a complex 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 exhibited 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 measure the nuclei down to 3.3 Å. The space group is P212121 (a=105, b=152 c=298). The samples were processed using the following: (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), testing all possible space group variations of the P222 point group. The final molecular replacement using Phaser (CCP4) was Four dimers of the active PAI-1 / variable domain 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 in complexes with human and cynomolgus PAI-1. The alignment of the two structures (see Figure 40) was determined to 3.3 Å for A44V11. , 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 monkey 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 cynomolgus PAI-1 and the heavy chain (average of the two complexes) is 703 Å 2 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, while 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 identical to both co- cDNA clones. 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 scan 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 revealed e.g. hydrogen bonds, electron transport Med-SuMo molecular modeling detects and compares biochemical functions on protein surfaces, including charge, hydrophobic, and aromatic groups. 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 there are significant differences between the A44V11 epitope and the A44V11 epitope such that A44V11 does not bind to it. 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 with 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 share 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, demonstrating 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 nucleotide sequence of the PAI-1 protein is such that mouse PAI-1 cannot be recognized by A44V11. , 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 it is a Noh play.

[0417] To further validate the epitopes identified for A44V11, the human and cynomolgus monkey A44V11 epitopes were compared with the binding region of vibronectin. The structure of human PAI-1 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 A44V11 binding does not affect the interaction of PAI-1 with vibronectin. .

[0418] The A44V11 epitope was compared with that of other published anti-PAI-1 antibodies. No overlap of the A44V11 epitope was observed with 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 are described 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 modern HDX Deuterated or "heavy" water (DO) 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, while protected, inflexible regions exchange more slowly, while all relevant conditions (pH, temperature, ionic strength, etc.) are held constant. Therefore, only structural differences (solvent accessibility, hydrogen bonding) affect this exchange. The interaction of the antibody with PAI-1 blocks the labeling of specific parts of the antigen, thus resulting in different readouts based on the site of binding (epitope).

[0421] Experimental Method: Cynomolgus monkey-PAI-1 (10 μM), Cynomolgus monkey-PAI-1 bound to A44v11 (10 μM each) and Cynomolgus-PAI-1 bound to APGv2 (10 μM each) was prepared in PBS at pH 7.2. The protein solution was allowed to reach binding equilibrium by incubation at room temperature for 1 hour. K values ​​of <50 pM were obtained. 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, pH 7.2) and incubated at 20°C for 10 seconds, 1 minute, 5 minutes, or 4 hours. At the end of the deuterium exchange period, 50 μL of labeling solution was added 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 components 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 manufactured by NIRS. The instrument settings were as follows: capillary voltage 3.5 kV, sampling cone voltage 30 V, and source offset 30 V. The source temperature was 80°C, the desolvation temperature was 175°C, the cone gas was 50 L / 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 the m / z range of 50 to 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 for 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 automatically generated by the software.

[0426] Monitoring deuterium uptake for PAI-1 status After online pepsin digestion, 150 overlapping cynomolgus-PAI-1 digest peptides were analyzed. Deuterium incorporation was determined using three different The protein states were monitored (10 seconds to 4 hours) 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] The majority of the cynomolgus-PAI-1 peptides showed nearly identical deuterium uptake between the three states, indicating no interaction between cynomolgus-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 heavy water) when bound to either A44v11 or APGv2 Furthermore, 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 protection was greater in cynomolgus PAI-1 than in APGv2. This suggests that A44v11 is a soluble form of the α-amyloid β ... When bound to quizal-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 butterfed 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 between the cynomolgus-PAI-1 conditions was 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 a vertical bar. The horizontal dashed lines represent the individual measurements ( Either the difference (±0.5 Da) or the sum of the differences (±1.1 Da) exceeds the measurement error, and the two states 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 primarily located 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 combined state (Figure 51). A butterfly plot for this comparison is shown in Figure 51(A). The A44v11:Cyno-PAI-1 fragment 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 near the N-terminus and the other lacking the C-terminus, which is the A44v11:Cyno-PAI-1 fragment. 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 a peptide that shows 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 were The difference is located in the C-terminal region of cynomolgus-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 HDX MS, the antibody epitope can be refined slightly better than peptide-level elucidation (see, e.g., Figure 48). 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 A44V11 epitope of cyno-PAI-1 was found to be consistent with the epitope determined using a crystallographic approach. The A44V11 epitope of cyno-PAI-1 identified using HDX MS is shown in Figure 53 (bold) 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 to APGv2 and showed significant protection from exchange The HDX MS data for A44v11 and APGv2 were 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 exhibited antibody binding for both A44v11 and APGv2. However, the magnitude of protection was significantly greater with cynomolgus-PAI-1 than with APGv2. This finding is more evident in Figure 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) a heavy chain framework region and a heavy chain variable region, wherein 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 comprises 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. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

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

3. (a) a heavy chain variable region that is at least 95% identical to the heavy chain variable region of the antibody of claim 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 claim 2. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

4. An isolated monoclonal antibody that binds to essentially the same epitope as the antibody of claim 1.

5. (a) a heavy chain framework region and a heavy chain variable region, wherein 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 comprises 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. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

6. The antibody of claim 5, wherein the heavy chain variable region comprises SEQ ID NO: 6 and the light chain variable region comprises SEQ ID NO:

7.

7. An isolated monoclonal antibody that binds to essentially the same epitope as the antibody of claim 5.

8. (a) a heavy chain, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO: 82, and a light chain, or an antigen-binding fragment thereof, having a light chain variable region comprising SEQ ID NO: 91; (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, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO: 85, and a light chain, or an antigen-binding fragment thereof, having a light chain variable region comprising SEQ ID NO: 91; (e) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 85, or an antigen-binding flag thereof; a light chain having a light chain variable region comprising SEQ ID NO: 93, or an antigen-binding fragment thereof; (f) a heavy chain, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO: 86, and a light chain, or an antigen-binding fragment thereof, having a light chain variable region comprising SEQ ID NO: 94; (g) a heavy chain, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO: 87, and a light chain, or an antigen-binding fragment thereof, having a light chain variable region comprising SEQ ID NO: 95; (h) a heavy chain, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO: 88, and a light chain, or an antigen-binding fragment thereof, having a light chain variable region comprising SEQ ID NO: 96; (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, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO: 90, and a light chain, or an antigen-binding fragment thereof, having a light chain variable region comprising SEQ ID NO: 98; (1) 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, or an antigen-binding fragment thereof, having a heavy chain variable region comprising SEQ ID NO: 89, and a light chain, or an antigen-binding fragment thereof, having a light chain variable region comprising SEQ ID NO: 93; 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. A humanized monoclonal antibody that specifically binds to human PAI-1, comprising:

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 variable region 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; (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 variable region comprising 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; (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 variable region 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; (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 variable region comprising 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. (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 variable region comprising 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. (i) a heavy chain variable region comprising a heavy chain CD1 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 heavy chain variable region comprising SEQ ID NO:

61. a light chain variable region comprising a light chain CD1 region comprising SEQ ID NO: 60, a light chain CDR2 region comprising SEQ ID NO: 60, and a light chain CDR3 region comprising SEQ ID NO: 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 variable region comprising 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. (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 variable region comprising 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; 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 variable region 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. An isolated monoclonal antibody that specifically binds to PAI-1, comprising:

10. An isolated monoclonal antibody that specifically binds to PAI-1, which binds to essentially the same epitope on PAI-1 as the humanized monoclonal antibody of claim 8 or claim 9.

11. A method of restoring plasmin generation comprising administering to a subject in need thereof a pharmaceutically effective amount of a PAI-1 antibody orally, parenterally by injectable solution, by inhalation, or topically.

12. 12. The method of claim 11 for treating a condition involving increased levels of fibrotic tissue.

13. 13. The method of claim 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.

14. The method of any one of claims 11, 12 or 13, wherein the PAI-1 antibody comprises an antibody according to any one of claims 1 to 10.

15. 1. Use of a pharmaceutically 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 the same orally, parenterally by injectable solution, by inhalation, or topically to a patient.

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