ANTIBODIES TO PLASMINOGEN ACTIVATOR INHIBITO -1 (PAI-1) AND USES

THEREOF

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

Plasminogen Activator inhibitor type- 3 (PAI-1) is the main inhibitor of tissue -type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA), the key serine proteases responsible for plasmin generation. 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 leads to abnormal haemostasis events like bleeding or thrombotic diseases. PAI- 1 is also a key regulator of plasminogen activation in the pericellul r compartment

(intravascular and tissular) where receptor bound plasminogen is activated mainly by urokinase bound to the urokinase receptor (uPAR). By inhibiting pericellular proteolysis, PAI-1 regulates numerous cellular functions like extracellular matrix (ECM) degradation, growth factors activation and release from ECM, matrix metalloproteinases (MMP) activation and cellular apoptosis. Recently, protease-independent effects of PAI-1 have been identified through its interaction wi th cofactors (like vitronectin, heparin, glycosaminoglycan), uPAR -urokinase complexes or cellular receptors (LRP: low-density Lipoprotein Receptor-related Protein) or integrins affecting cell functions like adhesion/de-adhesion, migration, proliferation and intracellular bioactivity. By these cellular mechanisms and anti-fibrinolytic effects, a pathogenic role of PAT- 1 has been established in tumor growth and metastasis, fibrosis, acute myocardial infarction and metabolic disorders like atherosclerosis, obesity and diabetes.

The Human SERPINE ! (PAT-1) gene is localized to chromosome 7, consists of eight introns and nine exons, and has a size of 12, 169 b (Klinger, .W. ei as. Proc. Nail Acad. Sci. USA 84: 8548, 1987). PAI-1 is a single chai glycoprotein of approximately 50 kDa (379 amino acids) from the SERPEN (serine protease inhibitor) superfamily that is synthesized in the active conformation but spontaneously becomes latent in the absence of vitronectin (Vn). Vitronectin, the mam cefaclor of PAI-1, stabilizes the active conformation with the Reactive Center Loop (RCL) which is approximately 20 amino acids that are exposed on the surface. The mechanism of inhibition of PAI-1 "s two main targets (tPA and uPA) is a suicide inhibition. The RCL region of PAI-1 bears the bait peptide bond (R346-M347, also called Pl -P" l), which bears the cleavage site for this serine protease. A Michaelis complex with tPA or uPA forms first, then the catalytic triad reacts with the bait peptide bond to form an acyl-enzyme complex that, after cleavage of the Pl -P" l peptide bond, induces strong conformation changes. The conformational changes cause insertion of the cleaved RCL into a β-strand with the protease staying covalently bound as an acyl enzyme with PAI-1. Under non-physiological circumstances, hydrolysis of this acyl-enzyme complex may induce release of the cleaved PAI-1 and free active protease (Blouse ei al, Biochemistry, 48:1723, 2009).

PAl-1 circulates in blood at highly variable levels (nM range) and in excess over t-PA or uPA concentrations. PAI-1 exhibits structural flexibility and can be found in one of three conformations: (1) a latent conformation, (2) an active conformation, or (3) a substrate conformation (see Figure 1). PAI-3 is mainly found as a noncovalent complex with vitronectin (Kd ~l nM) that decreases latency transition by 1.5 to 3 fold. Latent, cleaved or complexed PAI-1 affinity for vitronectin is significantly reduced. Matrix bound vitronectin also localizes with PAI-1 in the pericellular space. Endothelial cells, monocytes, macrophages and vascular smooth muscle cells synthesize this PAI- 1 which then can be stored in large amounts under latent form by platelets (in the a granule). PAI-1 is a fast and specific inhibitor (with the second order rate constant of 10" to 10" MTV1) of tPA and uPA in solution, but inactive against protease bound either to fibrin or their cellular receptors. Other proteases like thrombin, plasmin, activated Protein C can be also inhibited by PAI-1 but less efficiently.

Several 3D structures of human PAI-1 have been solved since the first one described in 1992 (Mottonen ei at., Nature 355:270, 1992) in the latent conformation. These 3D structures include mutant forms of PAI-1 in the substrate (Aertgeerts et al, Proteins 23:118, 1995), stabilized active conformation (Sharp et ah, Structure 7:111, 1999), PAI complexed to Vitronectin-somatomedin B domain (Zhou et ah, Nat. Struct. Biol. 10:541, 2003) or with inhibiting pentapeptide from the RCL loop (Xue et al., Structure 6:627, 1998). More recently, mouse PAl-1 structure in latent conformation was elucidated by Dewilde et al. (J Struct. Biol. 171:95, 2010) and revealed differences with human PAI-1 in the RCL position, gate region and position of α-helix A. Stracture/furtction relationships in PAI-1 have been studied by using more than 600 mutant proteins (reviewed by De Taeye et al, Thromb. Haemost. 92:898, 2004) to localize domains involved in t e various activities of this multi function al s erpi n .

Since PAI-1 can be synthesized by almost every cell type including hepatocyte, adipocyte, mesangial cell, fibroblast, myofibroblast, and epithelial cell, its expression greatly varies under physiological (e.g., circadian variation of plasma PAI-1 level) and pathological conditions (e.g., obesity, metabolic syndrome, insulin resistance, infection, inflammatory diseases, cancer). PAI-1 is considered to be an acute phase protein. Transcriptional regulation of PAI- 1 mRNA expression is induced by several cytokines and growth factors (e.g. , TGFp, TNFrx, EGF, FGF, Insulin, angiotensin II & IV), hormones (e.g., aldosterone, glucocorticoids, PMA, high glucose) and stress factors (e.g., hypoxia, reactive oxygen species, lipopolysaccharides).

Moreover, a polymorphism in the promoter (position■■ 675) of the PAI-1 gene affects expression level. The 4G allele increases PAI-1 level and the 4G/4G variant (occurring in around 25% of the population) induces an increase of approximately 25% of plasma PAI-1 level in comparison to 5G/5G (25 % occurrence and 4G/5G 50% occurrence). The 4G/4G polymorphism has been linked to myocardial infarction (Dawson el al , Arierioscler Thromb. 11: 183, 1991), a specific type of pulmonary fibrosis (idiopathic interstitial pneumonia) (Kim et al, Mo! Med. 9:52, 2003) and the 4G/4G genotype donor group is an independent risk factor for kidney graft loss due to Interstitial Fibrosis &Tubular Atrophy (Rerolle et al, Nephrol Dial Transplant 23:3325, 2008).

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 like insulin resistance syndrome and obesity is well recognized. PAI-1 is also known as a profibrotic factor for several organs and has bee shown to be over-expressed in fibrotic tissues (i.e., liver, lung, kidney, heart, abdominal adhesions, skin: scar or scleroderma) (reviewed by Ghosh and Vaughan, J. Cell Physiol 227:493, 2012). PAT- ! knock-out (KO) mice are protected from fibrosis in different models, such as liver (bile duct ligation or xenobiotic), kidney (unilateral ureteral obstruction model (UUO)), lung (bleomycin inhalation) (Bauman et al, J. Clin. Invest. 120: 1950, 2010; Hattori et al, Am. J. Pathol. 164:1091, 2004; Chuang-Tsai el al, Am. J. Pathol 163:445, 2003) whereas in heart this deletion is protected from induced fibrosis (Takeshita et al, AM. J. Pathol. 164:449, 2004) but prone to age-dependent cardiac selective fibrosis (Moriwaki et al, Cric. Res. 95:637, 2004). Down-regulation of PAI-1 expression by siRNA (Senoo et al, Thorax 65:334, 2010) or inhibition by chemical compounds (Izuhara et al, Arierioscler. Thromb. Vase. Biol. 28:672, 2008; Huang et al, Am. J. Respir. Cell Mol Biol. 46:87, 2012) have been reported to decrease lung fibrosis whereas PAI-1 overexpressioD of wild type (Eitzman et al, J. Clin. Invest. 97:232, 1996) or a PAI-1 mutant retaining only vitronectin binding but not tPA inhibitor function exacerbates lung fibrosis (Courey et al, Blood 118:2313, 20 ! 1).

Bile duct ligation (BDL) liver fibrosis is attenuated by antibody neutralizing PAI-1 (U.S. Patent No. 7,771 ,720) whereas down-regulation by siRNA attenuates BDL and xenobiotic induced liver fibrosis (Hu et al, J. Hepatol. 5 ! : 102, 2009). PAI-1 KO mice were protected from cholestatic-induced liver damage and fibrosis in BDL (Bergheim et al, J. Pharmacol. Exp. Ther. 316:592, 2006; Wang et al, FEBSLett. 581:3098, 2007; Wang el al, Hepaiology 42: 1099, 2005) and from angiotensin II induced liver fibrosis (Beier et al, Arch. Bioch. Biophys. 510: 19, 2011).

PAI-1 KO mice are protected from renal fibrosis in the UUO model (Oda et al, Kidney Int. 60, 587, 2001), in diabetic nephropathy (Nicholas et al, Kidney Int. 67: 1297, 2005) and in angiotensin II induced nephropathy (Knier et al, J. Hypertens. 29: 1602, 2011; for reviews see Ma et al. Frontiers Biosci. 14:2028, 2009 and Eddy A. A. Thromb. Haemost. 101 :656, 2009). In contrast,

PAI-i over expressing mice display more severe fibrosis and increased macrophage recruitment following UUO (Matsuo et al, Kidney Int. 67: 2221 , 2005; Bergheir et al, J. Pharmacol Exp. Then 316:592, 2006). Non-inhibitory PAI-i mutant (PAI-1 R) has been shown to protect mice from the development of fibrosis in experimental glomerulonephritis (thyl) in rat by decreasing urinary protein expression and glomerular matrix accumulation (Huang et al, Kidney Int. 70: 515, 2006). Peptides blocking ΡΑΪ-l inhibit collagen 3, 4 and fibronectin accumulation in UUO mice (Gonzalez et al, Exp. Biol Med. 234: 1511, 2009).

PAI-1, as a target for numerous pathologies, has been the focus of intensive research to inhibit its activity or to regulate its expression for the last 20 years. Chemical compounds (Suzuki et al, Expert Opin. investig. Drugs 20:255, 201 1 ), monoclonal antibodies (Gils and Declerk, Thromb

Haemost; 91 :425, 2004), peptides, mutants (Cale and Lawrence, Curr. Drug Targets 8:971 , 2007), siRNA or anti-sense RNA have been designed to inhibit its various function or to regulate its expression. Howe ver, despite the intensive research, the problem of developing a therapeutically effective modulator of PAI-1 still reinains to be solved. Accordingly, there is a need in the art for novel agents that inhibit the PAI-i activity for use in the treatment of PAI-1 -mediated human pathologies.

SUMMARY OF THE DISCLOSURE

in one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator Inhibitor type- 1 (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDR 1 (SEQ ID NO: 34), CDR2 (SEQ TD NO: 33), and CDR3 (SEQ ID NO: 32) of SEQ ID NO: 6, and a light chain variable region, said light chain variable region comprising CDR 1 (SEQ ID NO: 37), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 35) of SEQ ID NO: 7. In an additional aspect 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, 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 % identity approximations indicate the minimum % identity; higher % identity than the recited values are also encompassed by the disclosure.

In another aspect, disclosed herein is an isolated monoclonal antibody thai binds specifically to PAI-i, comprising: (a) heavy chain framework regions, a heavy chain CDR 1 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) light chain framework regions, a light chain CDR 3 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 T 0: 35, In certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework regions of SEQ ID NO: 6, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 7.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator Inhibitor type- 1 (PAi-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDR1 (SEQ ID NO: 22), CDR2 (SEQ ID NO: 21), and CDR3 (SEQ ID NO: 20) of SEQ ID NO: 2, and a light chain variable region, said light chain variable region comprising CDR1 (SEQ ID NO: 25), CDR2 (SEQ ID NO: 24), and

CDR3 (SEQ ID NO: 23) of SEQ ID NO: 3. In an additional aspect 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, 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.

In an additional aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a) heavy chain framework regions, 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) light chain framework regions, 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. In certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, or 90% identical to the heavy chain framework regions of SEQ ID NO: 26, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, or 90% identical to the framework regions of SEQ ID NO: 3.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator inhibitor type- ! (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDR 1 (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, said light chain variable region comprising CDR1 (SEQ ID NO: 31), CDR2 (SEQ ID NO: 30), and

CDR3 (SEQ ID NO: 29) of SEQ ID NO: 5. In an additional 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, 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.

in an additional aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a.) heavy chain framework regions, a heavy chain CDRl 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) light chain framework regions, a light chain CDRl 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, In certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, or 90% identical to the heavy chain framework regions of SEQ ID NO: 4, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 5.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator Inhibitor lype-1 (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDRl (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, said light chain variable region comprising CDRl (SEQ ID NO: 43), CDR2 (SEQ ID NO: 42), and CDR3 (SEQ 3D NO: 41) of SEQ ID NO: 9. In an additional 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, 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.

In another aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI- 1 , comprising: (a) heavy chain framework regions, a heavy chain CDRl 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) light chain framework regions, a light chain CDRl 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 , In certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% Identical to the heavy chain framework regions of SEQ ID NO: 8, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 9,

In one aspect, disclosed herein is An isolated monoclonal antibody that binds specifically to human Plasminogen Activator Inhibitor type- 1 (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDRl (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, said light chain variable region comprising CDRl (SEQ ID NO: 55), CDR2 (SEQ ID NO: 54), and

CDR3 (SEQ ID NO: 53) of SEQ ID NO: 11. In an additional aspect the heavy chain comprises a heavy chain variable region comprising SEQ ID NO: ! 0, and the light chain comprises a light chain variable region comprising SEQ ID NO: I I . in a further aspect 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.

In an additional aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1 , comprising: (a) heavy chain framework regions, 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 (b) light chain framework regions, 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. in certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, or 90% identical to the heavy chain framework regions of SEQ ID NO: 10, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 11.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator Inhibitor iype-1 (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising 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, said light chain variable region comprising CDR1 (SEQ ID NO: 61), CDR2 (SEQ ID NO: 60), and CDR3 (SEQ ID NO: 59) of SEQ ID NO: 13. in an additional 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, 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.

In another aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a) heavy chain framework regions, a heavy chain CDR1 region comprising SEQ ID NO: 58, a heavy chain CDR2 region comprising SEQ ID NO: 57, and heavy chain CDR3 region comprising SEQ ID NO: 56; and (b) light chain framework regions, 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. In certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework regions of SEQ ID NO: 12, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 13.

in one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator inhibitor type-1 (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDRl (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, said light chain variable region comprising CDRl (SEQ ID NO: 67), CDR2 (SEQ ID NO: 66), and CDR3 (SEQ ID NO: 65) of SEQ ID NO: 15. In an additional 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, 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.

In an additional aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAT- i , comprising: (a) heavy chain framework regions, a heavy chain CDRl 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 (b) light chain framework regions, a light chain CDRl 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. In certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, or 90% identical to the heavy chain framework regions of SEQ ID NO: 14, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, or 90% identical to the framework regions of SEQ ID NO: 15.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator inhibitor type- 1 (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDR 1 (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, said light chain variable region comprising CDRl (SEQ ID NO: 73), CDR2 (SEQ ID NO: 72), and CDR3 (SEQ ID NO: 71) of SEQ ID NO: 17.

In an additional 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, 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.

In an additional aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ TD NO: 70, a heavy chain CDR2 region comprising SEQ ID NO: 69, and a heavy chain CDR3 region comprising SEQ ID NO: 68; and (b) light chain framework regions, a light chain CDRl 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 , in certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91 %, or 90% identical to the heavy chain framework regions of SEQ ID NO: 16, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93 %, 92%, 91 %, or 90% identical to the framework regions of SEQ ID NO: 17.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to human Plasminogen Activator Inhibitor type-i (PAI- 1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CDRl (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, said light chain variable region comprising CDRl (SEQ ID NO: 49), CDR.2 (SEQ ID NO: 48), and CDR3 (SEQ ID NO: 47) of SEQ ID NO: 81.

in an additional aspect the heavy chain comprises a heavy chain variable region comprising SEQ ID NO: 80, and the light chain comprises a light chain variable region comprising SEQ ID NO: 81. In a further aspect, 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: 83 .

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a) heavy chain framework regions, a heavy chain CD l 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 (b) light chain framework regions, a light chain CDRl 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, In certain aspects, the antibody heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% Identical to the heavy chain framework regions of SEQ ID NO: 80, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 81.

In another aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to Plasminogen Activator inhibitor type-1 (PAI-1), wherein the antibody comprises a heavy chain variable region, said heavy chain variable region comprising CD l (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, said light chain variable region comprising CDR 3 (SEQ ID NO: 79), CDR2 (SEQ ID NO: 78), and CDR3

(SEQ ID NO: 77) of SEQ ID 19. Tn an additional aspect the heavy chain comprises a heavy chain variable region comprising SEQ ID NO: 18, and the light chain comprises a light chain variable region comprising SEQ ID NO: 19. In a further aspect, 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.

An isoiated monoclonal antibody that binds specifically to PAI- 1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ ID NO: 76, heavy chain CDR2 region comprising SEQ ID NO: 75, and a heavy chain CDR3 region comprising SEQ ID NO: 74; and (b) light chain framework regions, a light chain CDRl 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 heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the heavy chain framework regions of SEQ ID NO: i 8, and the antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 19,

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ ID NO: 33, heavy chain CDR2 region comprising SEQ ID NO: 146, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) light chain framework regions, a light chain CDRl 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.

in one aspect, disclosed herein is an isolated monoclonal antibody mat binds specifically to PAI-1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ ID NO: 147, heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) light chain framework regions, a light chain CDR 1 region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 36, and a light chain CD 3 region comprising SEQ ID NO: 35.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI- 1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ ID NO: 347, heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) light chain framework regions, a light chain CDRl 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.

in one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ ID NO: 146, heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) light chain framework regions, a light chain CDRl 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.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ ID NO: 34, heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) light chain framework regions, a light chain CDRl region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 1 5, and a light chain CDR3 region comprising SEQ ID NO: 35.

In an additional aspect, disclosed herein is an isolated monoclonal antibody that binds to essentially the same epitope on PAI-1 as an isolated monoclonal antibody, comprising a heavy chain variabie region, wherein the heavy chain variable region comprises CDRl (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 CDRl (SEQ ID NO: 37), CDR2 (SEQ ID NO: 36), and CDR3 (SEQ ID NO: 35) of SEQ ID NO: 7.

In a certain aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI- 1, comprising: (a) heavy chain framework regions, a heavy chain CDRl region comprising SEQ ID NO: 76, heavy chain CDR2 region comprising SEQ ID NO: 75, and a heavy chain CDR3 region comprising SEQ ID NO: 74; and (b) light chain framework regions, a light chain CDR 1 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 one aspect, disclosed herein is a humanized monoclonal antibody that binds specifically to human PAI- 1, wherein the antibody comprises: (a) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 82, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 91 , or an antigen-binding fragment thereof; (b) a heavy chain having a heavy chain variabie 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 variabie 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 variabie region comprising SEQ ID NO: 85, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 91, or an antigen-binding fragment thereof: (e) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 85, or an antigen-bindi ng 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 aniigen-binding fragment thereof; (g) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 87, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 95, or an antigen-binding fragment thereof; (h) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 88, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 96, or an antigen-binding fragment thereof; (i) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 89, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 97, or an antigen-binding fragment thereof; (j) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 90, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 98, or an antigen- binding fragment thereof; (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 aniigen-binding fragment thereof; (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 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.

In one aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI- 1 comprising: (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 monocionai heavy chain comprises heavy chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to

the heavy chain framework regions of SEQ ID NO: 86, and the isolated monoclonal antibody light chain comprises light chain framework regions that are 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to the framework regions of SEQ ID NO: 93. In certain other aspects, the isolated monoclonal antibody heavy chain comprises heavy chain framework regions that are 95% identical to the heavy chain framework regions of SEQ ID NO: 86, and the isolated monoclonal antibody light chain comprises light chain framework regions that are 95% identical to the framework regions of SEQ ID NO: 93.

In another aspect, disclosed herein is a humanized monoclonal antibody that binds specifically to human PAI-1, wherein the antibody comprises a heavy chain having a heavy chain variable region comprising SEQ ID NO: 154, or an antigen-binding fragment thereof; and a light chain having a light chain variable region comprising SEQ ID NO: 153, or an antigen-binding fragment thereof. In another aspect, disclosed herein is a humanized monoclonal antibody that binds specifically to human PAI-1, wherein the antibody comprises a heavy chain having a heavy chain variable region comprising SEQ ID NO: 155, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 153, 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%, 1%, or 90% identical to any of the previously disclosed human light chain variable regions.

In another aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to ΡΑΪ- 1 , wherein the antibody binds a polypeptide comprising SEQ ID NO: 158. In another embodiment, the isolate monoclonal antibody binds a fragment of a polypeptide comprising SEQ ID NO: 158. In yet another embodiment, the isolated monoclonal antibody that binds specifically to PAl-1 binds a polypeptide comprising SEQ ID NO: 156 and/or SEQ ID NO: 158. in another embodiment, the isolated monoclonal antibody that binds specifically to PAI- 1 binds a polypeptide comprising SEQ ID NO: 156, SEQ ID NO: 158, and/or SEQ ID NO: 157. In still another embodiment, the isolated monoclonal antibody that binds specifically to PAT- ! comprises 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 D-Q-E) 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.

In other embodiments, the isolated monoclonal antibody that binds specifically to PAI-1 disclosed herein binds a polypeptide comprising SEQ ID NO: 161 . In still other embodiments, the isoiated monoclonal antibody binds a polypeptide comprising SEQ ID NO: 159 and/or SEQ ID NO: 161 , In. still other embodiments, the isoiated monoclonal antibody binds a polypeptide comprising SEQ ID NO: 159, SEQ ID NO: 160, and/or SEQ ID NO: 161. In still another embodiment, the isoiated rnonoclonai antibody that binds specifically to PAI-1 comprises specific binding affinity for residues 44-64 and/or residues 307-321 of cyno-PAI-1 (SEQ ID NO: 162). In certain embodiments, the PAI-1 bound by the antibody is cyno-PAI-1. In other embodiments, the PAI-1 bound by the antibody is the latent form of cyno-PAI-1.

In a further aspect, disclosed herein is an isolated monoclonal antibody that competitively inhibits binding of any of the disclosed antibodi es to PA1- 1. In an embodiment, disclosed herein is an isoiated monoclonal antibody that competes for binding and/or competitively inhibits binding with any of the isolated monoclonal antibodies disclosed herein. In certain embodiments, the isolated monoclonal antibody competes or competitively inhibits binding to human PAI- i . In certai embodiments, the isolated monoclonal antibody competes or competitively inhibits binding to a polypeptide comprising SEQ ID NO: 156, SEQ ID NO: 1 7, and/or SEQ ID NO: 158. In another embodiment, the isolated monoclonal antibody competes or competitively inhibits binding to a polypeptide comprising SEQ ID NO: 159, SEQ ID NO: 160, and/or SEQ ID NO: 161. In an embodiment, the isolated antibody competes for binding to a polypeptide comprising SEQ ID NO: 156, 157, and/or 158 with an isolated monoclonal antibody comprising (a) heavy chain framework regions, a heavy chain CDR1 region comprising SEQ ID NO: 34, heavy chain CDR2 region comprising SEQ ID NO: 33, and a heavy chain CDR3 region comprising SEQ ID NO: 32; and (b) light chain framework regions, a light chain CDR1 region comprising SEQ ID NO: 37, a light chain CDR2 region comprising SEQ ID NO: 345, and a light chain C.DR3 region comprising SEQ ID NO: 35.

In another aspect, discloed herein are nucleotides encoding any of the isolated monoclonal antibodies disclosed herein.

In one aspect, disclosed herein is a method of treating a condition caused by increased expression of PAI- i or increased sensitivity to PAI- 1 comprising administering to a patient or other subject orally, pareoterally by a solution for injection, by inhalation, or topically a pharmaceutically effective amount of a PAI- 1 antibody.

In one aspect, disclosed herein is a method restoring plasmin generation comprising administering to a patient or other subject in need thereof orally, parenterally by a solution for injection, by inhalation, or topically a pharmaceutically effective amount of a PAI- 1 antibody. Parenteral administration disclosed herein includes intravenous, drip, intraarterial, intraperitoneal, intramuscular, subcutaneous, rectal or vaginal, intravenous, intraarterial, subcutaneous, and intramuscular forms of parenteral administration. In some embodiments, the administration to a

patient or other subject comprises multiple administrations. In another aspect, the method of restoring plasmin generation facilitates therapeutic treatment of a condition comprising increased levels of fibrotic tissue, in some aspects, the condition is characterized by iibrosis. In some aspects, the condition is iibrosis, skin fibrosis, systemic sclerosis, lung fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, and chronic lung disease. In other aspects, the plasmin generation facilitates therapeutic treatment of liver fibrosis, kidney fibrosis, including chronic kidney disease, thrombosis, venous and arterial thrombosis, deep vein thrombosis, peripheral limb ischemia, disseminated intravascular coagulation thrombosis, acute ischemic stroke with and without thrombolysis, or stent restenosis.

In another aspect, disclosed herein is the use of a pharmaceutically effective amount of a PAI-! antibody for the manufacture of a medicament for treating a condition caused by increased expression of PAI-1 or increased sensitivity to PAI-1 comprising administering to a patient or other subject orally, parenterally by a solution for injection, by inhalation, or topically.

in one aspect, the medicament is for treating a condition co prising increased levels of fibrotic tissue. In some aspects, the condition is characterized by fibrosis, in some aspects, the condition is fibrosis, skin fibrosis, systemic sclerosis, lung fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, and clironic lung disease. In other aspects, the medicament is for treating a condition comprising liver fibrosis, kidney fibrosis, including chronic kidney disease, thrombosis, venous and arterial thrombosis, deep vein thrombosis, peripheral limb ischemia, disseminated intravascular coagulation thrombosis, acute ischemic stroke wiih and without thrombolysis, or stent restenosis.

in another aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI- 1 , wherein the antibody inhibits lung fibrosis. In certain embodiments, the antibody inhibits fibrosis in the lung of a subject. In certain embodiments, the antibody inhibits fibrosis in the lung of a subject with idiopathic pulmonary fibrosis (IPF). in some embodiments, the isolated monoclonal antibody disclosed herein induces an increase in fibrin degradation in a subject. In certain embodiments, the antibody increases fibrin degradation in the plasma of the subject. In some other embodiments, the isolated monoclonal antibody disclosed herein inhibits collagen accumulation in the lung of a subject. In some embodiments, the subject lias IPF. in some other embodiments, the isolated monoclonal antibody disclosed herein increases D-dimer levels in the bronchoalveolar lavage fluid (BALF) of a subject. In some embodiments, the subject has IPF. In some other embodiments, the isolated monoclonal antibody disclosed herein binds specifically to PAI-1, wherein the antibody inhibits the increase in lung weight due to fibrosis in a subject. In one embodiment, the subject has IPF.

in another aspect, disclosed herein is the use of a pharmaceutic ally effective amount of a PAI-1 antibody for the manufacture of a medicament for treating a condition caused by increased expression of PAI-i or increased sensitivity to PAI-1 comprising administering to a patient orally, parenteral!)" by a solution for injection, by inhalation, or topically, wherein the condition is idiopathic pulmonary fibrosis.

In another aspect, disclosed herein is a method restoring piasmin generation comprising administering to a to a patient or other subject thereof orally, parenterally by a solution for injection, by inhalation, or topically a pharmaceutically effective amount of a PAI-1 antibody, wherein the piasmin generation facilitates therapeutic treatment of idiopathic pulmonary fibrosis,

In another aspect, disclosed herein is an isolated monoclonal antibody thai binds specifically to PAI-1, wherein the antibody restores fibrinolytic activity in a subject, in certain embodiments, the antibody restores fibrinolytic activity in a subject with acute ischemic stroke. The acute ischemic stroke can be either with or w ithout thrombolysi . In some embodiments, the isolated monoclonal antibody restores clot lysis. In certain embodiments, the antibody restores in vitro clot lysis, in still other embodiments, the antibody restores in vitro clot lysis with an TC 0 of about 2 nM.

In other aspects, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1, wherein the antibody restores fibrin breakdown in a subject. In some embodiments, the subject has acute ischemic stroke.

In another aspect, disclosed herein is 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 PAI-1 or increased sensitivity to PAI- I comprising administering to a patient orally, parenterally by a solution for injection, by inhalation, or topically, wherein the condition is acute ischemic stroke with and without thrombolysis.

In another aspect, disclosed herein is a method restoring piasmin generation comprising administering to a patient or other subject in need thereof orally, parenterally by a solution for injection, by inhalation, or topically a pharmaceutically effective amount of a ΡΑΪ- 1 antibody, wherein the piasmin generation facilitates therapeutic treatment of acute ischemic stroke with and without thrombo lysi s .

In another aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-i, wherein the antibody inhibits formation of adhesions in a subject, in some embodiments, the adhesion formation is following surgery or injury to the subject. In some emboidments, the adhesion formation in the subject is abdominal. In other embodiments, the adhesion formation occurs in the shoulder, pelvis, heart, spine, hand, and other body regions of the subject.

In another aspect, disclosed herein is the use of a pharmaceutically effective amount of a PAI-I antibody for the manufacture of a medicament for treating or preventing a condition caused by

increased expression of PAI- 1 or increased sensiti vity to PAI- 1 comprising administering to a patient orally, parenterally by a solution for injection, by inhalation, or topically, wherein the condition is abdominal adhesion formation.

In another aspect, disclosed herein is a method of restoring plasmin generation comprising administering io a io a patient or other subject in need thereof orally, parenterally by a solution for injection, by inhalation, or topically a pharmaceutically effective amount of a PAI- 1 antibody, wherein the plasmin generation facilitates therapeutic treatment or prevention of adhesion formation. In some emboidments, the adhesion formation in the subject is abdominal.

In another aspect, disclosed herein is an isolated monoclonal antibody that binds to a PAI-1 /"vitronectin complex. In another aspect, disclosed herein is an isolated monoclonal antibody that neutralizes PAI- 1 activity by inducing PAI-1 substrate conformation. In an 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

metalloproteinases (MMP) activation.

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 chain and light chain are connected by a flexible linker to form a single-chain antibody. In other embodiments, the antibody is a Fab, Fab", or (Fab1)? antibody.

In another aspect, disclosed herein is an isolated monoclonal antibody that binds specifically to PAI-1 , wherein the antibody is a crystallized antibody, in an embodiment, disclosed herein is an isolated crystal comprising the Fab" fragment of monoclonal antibody A44, wherein the Fab" fragment consists of light chain sequ ence SEQ ID NO:7 and heavy chain sequence SEQ ID NO:6. In another embodiment, disclosed herein is an isolated crystal comprising a Fab" fragment comprising light chain sequence SEQ ID NO:93 and heavy chain sequence SEQ ID NO: 86. In an embodiment, the isolated crystal comprises assimeiric unit cell dimensions a=105 A, b= 52 A and c~298 A. In an embodiment, the isolated crystal belongs to P212121 space group. In another embodiment, the isolated crystal comprises a 3.3 A resolution of x-ray diffraction. In an embodiment, the isolated crystal retains the biological activity of the crystallized antibody. In some embodiments, the isolated crystal lias a greater hal f li fe in vivo than the soluble counterpart of the crystallized antibody.

In one aspect, disclosed herein is a pharmaceutical composition comprising: (a) the crystallized antibody that binds specifically to PAI-1 and (b) at least one pharmaceutical excipient which embeds or encapsulates the crystal.

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,

in one aspect, disciosed herein is a method of generating an antibody against PAI-1 comprising immunizing a mammal with a complex composed of PAI- 1 , or a fragment thereof, and vitronectin.

In another aspect, disciosed herein is a method of screening a PAI- 1 antibody in an ELiSA for its ability to block PAl-l "s function as a tPA activity inhibitor, comprising the steps of: (a) binding PAI-1 to an ELISA plate; (b) incubating the ELISA plate with the PAI-i antibody; (c) incubating the ELISA plate with tPA; (d) incubating the ELISA plate with labeled anti-tPA antibody; and (e) measuring the OD405 emitted by the labeled anti-tPA antibody; wherein a positive readout indicates that the PAT- ! antibody binds to PAI-i but does not block formation of the covalent bond between PAI- 1 and tPA, and a negative readout indicates that the PAI- 1 antibody blocks tPA interaction with PAI- 1 .

In another aspect, disclosed herein is a method of screening hybridomas. In certain embodiments, the method of screening comprises a reverse screening method using anti-mouse immobilized anti-PAI-1 antibodies. In other embodiments, the method of screening comprises or a forward screening assay using free PAI-1 as a ligand or against immobilized vitronectin. In certain embodiments, the method is applied to determine the affinity of an antibody for a PAI-l/vitronectm complex. In some embodiments, the method comprises: immobilizing vitronectin to a surface;

contacting PAI-1 with the vitronectin immobilized to the surface, thereby forming a complex;

contacting the surface comprising the complex with the antibody; separating the antibody bound to the complex from unbound antibody; detecting the antibody bound to the complex, and analyzing the levels of antibody bound to the complex to determine the affinity of the antibody for the complex.

BRIEF DESCRIPTION OF THE DRAWINGS

Fsgisre 1 depicts a schematic representation of the mechanisms between PAI- 1 and the serine proteases tissue-type plasminogen activator (tPA) and urokinase-t pe plasminogen activator (uPA). PAI-I exhibits structural flexibility and can occur in a latent conformation or an active conformation when it is bound to vitronectin (Vn). The RCL region of PAI-1 bears the bait peptide bond (also called Pl-Pl ") that is the cleavage site by the serine protease. A Michaelis complex with tPA or uPA forms first, then the catalytic triad reacts with the bait peptide bond to form an acyl-enzyme complex that, after cleavage of the Pl-P" l peptide bond, induces strong conformation changes. Acyl enzyme is a labile complex formed by covalent bond between the serine residue (black triangle) from catalytic triad from serine protease (iPA) and amino-acid from the substrate (black circle) that undergoes

further hydrolysis. The conformational changes causes insertion of the cleaved RCL into a β-strand with the protease staying covalently bound as an acyl enzyme with PAi- i . Under non physiological circumstance, hydrolysis of this acyl-enzyme complex may induce release of the cleaved PAI-1 and free active protease.

Figure 2 depicts a typical standard curve for antibody titration in the binding ELISA as described in Example 2. The antibodies 31C9, 33B8 and 33H1 were positive controls and IgGl was as a negative control.

Figure 3 depicts a representation curve for a fixnctional ELISA to select antibodies that block the interaction of PAI-1 with tPA as described in Example 4, The antibody 33H1 is a positive control, IgGl is a negative control and A44 was identified as a positive antibody clone.

Figssre 4 depicts neutralization of human PAT-1 blocking activity of tPA by A44 and commercially available antibodies (33B8 and 33H1) in the chromogenic assay described in Example 4.

Figure 5 depicts neutralization of human PAI-1 blocking activity of tPA by a selection of antibodies produced from different fusions (see Example 4).

Figure 6 depicts human PAI-1 and its orthologs block human tPA activity in chromogenic assay with the similar potency.

Figure 7 depicts neutralization of cynomolgus (cyno) and mouse PAI-1 blocking activity of human tPA by A44 and 33B8 (commercially available) antibodies in the chromogenic assay described in Example 4.

Figure 8 depicts SDS-Page analysis of the mechanism of action for antibodies 33H8 (converts PAI-1 from active to latent confirmation), 33H1 (converts PAI-1 from active to substrate conformation) and A44 to block the interaction of PAI-1 with tPA. Lane 1 : molecular weight standards; Lane 2: PAI-1 only; Lane 3: tPA only; Lane 4: PAI-1 in the presence o tPA; Lane 5: 33B8 +PAI-1 +tPA; Lane 6: 33H1+PAI-1-KPA; Lane 7: A44+PAI-l +tPA; Lane 8: mAb is an isotype control antibody.

Figure 9 depicts SDS-Page analysis of the mechanism of action for antibodies 33H8 (converts PAI-1 from active to latent confirmation), 33H1 (converts PAI-1 from active to substrate conformation) and antibodies developed from fusions C26, E16 and E21 to block the interaction of PAI-1 with tPA. Lane 1: molecular weight standards; Lane 2: PAI-1 only; Lane 3: tPA only; Lane 4: PAI-1 in the presence oftPA; Lane 5: 33B8 +PAI-1 +tPA; Lane 6: 33H1+PAI-1-HPA; Lane 7: C26+PAI- l÷t A; Lane 8: E16+PAI-l+tPA; Lane 9: E21+PAI-l+tPA; Lane 10: mAb is an isotype control antibody.

Figure 10 depicts SDS-Page analysis of the mechanism of action for antibodies 33H8 (converts PAT- 1 from active to latent confirmation), 33H1 (converts PAT- 1 from active to substrate conformation) and antibodies developed from fusions A39, B109 and C45 to block the interaction of PAI-1 with tP , Lane 1 : molecular weight standards; Lane 2: PAi-1 only; Lane 3 : tPA only; Lane 4: PAI-1 in the presence of tPA; Lane 5: 33B8 +PAI- 1 +1PA; Lane 6: 33HH-PAI-1-HPA; Lane 7: A39+PAI-1-HPA; Lane 8: B109+PAI-1-HPA; Lane 9: C45+PAI-l+tPA; Lane 10: mAb is an isoiype control antibody.

Figure II depicts the alignment of the light chain 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). CDRs are highlighted in bold.

Figssre 12 depicts the alignment of the heavy chain of the following murine antibodies: A 105

(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), E ! 6 (SEQ ID NO: ! 6), and E21 (SEQ ID NO: 18). CDRs, as defined by IMGT, are highlighted in bold.

Figure 13 depicts the alignment of murine A44 light chain (SEQ ID NO: 7) with vkl (SEQ ID NO: 101) and vlambda.3 (SEQ ID NO: i 02).

Figure 14 depicts the alignment of murine A44 heavy chain (SEQ ID NO: 6) with vh2 (SEQ ID NO: 103) and vh4 (SEQ ID NO: 104).

Figure 15 depicts clone A44 humamzation 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 vary in sequence from the residue directly above in the alignment. Residue numbering is as described by IMGT.

Figure 16 depicts clone A44 humanization 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 vary in sequence from the residue directly above in the alignment. Residue numbering is as described by IMGT.

Figure 17 depicts percent inhibition of PAI-1 activity was plotted as a function of mAb concentration and IC50 was determined Imax using Biostat speed software.

Figure 18 depicts purification of homogeneity recombinant 6-His tagged Fab A44.

Figure 19 depicts SPR analysis with Biacore 2000 using single kinetic analysis of human PAI-1 glycosylated binding to immobilized APG antibody. A sensogram from single-cycle kinetic is shown in grey. Fit model is shown in black.

Figure 20 depicts human plasma PAI-1 neutralization by APG, APGv2, and APGv4 antibodies as determined by UK-PAl-1 compiex formation detection by ELISA. Percent inhibition of PAI-1 activity was plotted as a function of concentration of" APG, APGv2, or APGv4 antibodies.

Figure 21 depicts restoration of human plasma clot lysis by A44V 1 1 (1, 3 or 10 nM) in the presence of tPA 1 nM and PAI-I 3 nM as detected by turbidimetry kinetic measurement by absorbance reading a! 340 nm as a function of time (min).

Figure 22 depicts absence of restoration of human plasma clot lysis by human IgGl negative controle (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 depicts restoration of human plasma clot lysis by A44V11 or human IgGl isotype negative control at various concentrations.

Figssre 24 depicts restoration of human plasma clot lysis by APG, APGV2 or APGV4 at 3 nM in the presence of t A 1 nM and PAT- 1 3 nM as detected by absorbance at 340 am as a function of time (min).

Figure 25 depicts restoration of human plasma clot lysis by APG variant 2 and 4 at various concentrations.

Figure 26 depicts immunoblot anti-PAI-1 on human LL29 myofibroblast supernatants at 48H after treatment by A44V1 ! or TgG isotype control mAb at 50 nM and ΤΟΡβ 5 ng/rnl.

Figure 27 depicts generic MMP activity in human primary lung fibroblasts after cell treatment for 48hr with PBS (control), plasminogen (Pg), A44vi 1 and plasminogen (A+Pg) or negative human IgG and plasminogen (Neg+Pg).

Figure 28 depicts human active PAI- 1 level in broncho-alveolar lavage fluid (BALF) (A) and in lung lysate (B) from bleomycin treated mice at day 7 and day 9 following treatment at day 4 with A44 or IgGl at 10 mg/kg or PBS by i.p. administration. Active PAI-1 determined by ELISA (# HPAIKT Molecular innovation). Percentage of inhibition were calculated by dividing the difference between A44 bleo and IgG bleo by the difference between IgG bleo and untreated (PBS) mice group.

Figure 29 depicts mouse D-Dimer level in BALF from bleomycin treated mice at day 7 and day9 following treatment at day4 with A44 or IgGl at 10 mg/kg or PBS by i.p. administration as determined by ELISA (Asserachrom D-Di, Diagnostica Stago). Fold increase in D-dimer induced by A44 in comparison to IgG are indicated.

Figure 30 depicts right lung weight from transgenic humanized mice 21 days after either saline or bleomycin treatment followed by PBS (vehicle), IgGl or A44 i Omg/kg i.p, administration from day4 to day20 every 3 days.

Figure 31 depicts hydroxy proline lung content in transgenic humanized mice 21 days after either saline or bleomycin treatment followed by PBS (vehicle), IgGl or A44 IOmg/kg i.p.

administration from day4 to day20 every 3 days.

Figure 32 depicts active PAI-1 level in plasma from monkeys treated by A44 11 (A) mAb (n=5) or with IgGl isotype control (B) (n=4) (5 mg kg ip) 24 hours before LPS challenge (100 ug/kg iv). Blood samples were harvested at the indicated time point and active PAT- i levels were determined in plasma using the ELISA (# HPAIKT from Molecular Innovation).

Figure 33 depicts active PAI-1 level in liver biopsy from monkeys treated by A44V11 (A) mAb (n=5) or with IgGl isotype control (B) (n=4) (5 rng/kg ip) 24 hours before LPS challenge (100 ug/kg iv). Liver biopsies were harvested in anesthetized monkeys at the indicated time point and active PAI-1 levels were determined in lysates using the ELISA (# HPAIKT from Molecular innovation).

Figure 34 depicts D-dimer level in plasma from monkeys treated by A44V11 (A) mAb (n~5) or with IgGl isotype control (B) (n=4) (5 mg/kg ip) 24 hours before LPS challenge (100 ug/kg iv). Blood samples were harvested at the indicated time point and D-dimer levels were determined in plasma using the ELISA.

Figure 35 depicts Plasmin-o2 Antiplasmin (PAP) complexes level in plasma from monkeys treated by A44V1 1 (A) mAb (n-5) or with IgGl isotype control (B) (n= ) (5 mg/kg ip) 24 hours before LPS challenge ( 1 00 ug/kg iv). Blood samples were harvested at the indicated time point and PAP levels were determined in plasma using the ELISA (ft Asserachrom PAP from Diagnostics: Stago).

Figure 36 depicts level of active PAI-1 in intraperitoneal fluid (IFF) and uterine horn lysates. Active PALI levels in the intraperitoneal fluid (A) and uterine horn lysates (B). At the 6 hour and Day 7 time points active PAi-1 levels were lower in both intraperitoneal fluid (IFF) and Uterine Horn (UH) Lysates in the animals treated with A44V1 1 antibody in comparison to the isotype control antibody treated animlas, no difference was observed at 72 hour time point. (* p<0.001 calculated by the Student T-test)

Figure 37 depicts another example of purification of homogeneity recombinant 6-His tagged Fab A44.

Figure 38 depicts purification of homogeneity recombinant 6-His tagged Fab A44 complexed with t e human wt PAL i protein.

Figure 39(a) depicts the complex cry stallization of the Fab A44/PAI-1 complex, and Figure 39 (b) depicts the best optimized crystals.

Figure 40 depicts the rod-like single crystals of the Fab A44/PAI-1 complex.

Figure 41 depicts Fab A44 recognition the active form of human PAI-1 and the latent form of cyno PAI-1.

Figure 42 depicts the PAI-1 epitope recognized by Fab A44 in (A) active human PAl-i, and (B) latent cyno PAI-1.

Figure 43 depicts the heavy chain paratope of the Fab A44/PAI-1 complex.

Figure 44 depicts the light chain paratope of the Fab A44/PAT-1 complex.

Figure 45 depicts a sequence alignment of the proposed A44 binding epitopes of cyno, human, rat, and mouse PAI-1. Sequences are excerpted from SEQ ID NO: 1 (PAT- 1 human), SEQ ID NO: 162 (PAI-1 cyno), SEQ ID NO: 163 (PAI-1 mouse), and SEQ ID NO: 164 (PAI-1 rat).

Figure 46 depicts the comparison of the mouse PAI-1 structure with the structure of the human PAI- 1/A44V 11 complex.

Figure 47 shows the structure of human PAI-1/A44V11 complex and the model of vibronectin binding to PAI-1.

Figure 48 depicts peptic peptide coverage of cyno-PAI-1 (SEQ ID NO: 162); 95.3% sequence coverage is obtained from 150 overlapping peptic peptides.

Figssre 49 depicts representative deuterium uptake plots for cyno-PAI-1 peptides in the unbound (circle lines), APGv2 -bound (x-iines) and A44vl 1 -bound (diamond lines) states. Residue ranges/positions are from SEQ ID NO: 162. (A) most of the peptic peptides showed no difference between cyno-PAI-1 alone or bound to either rnAb. (B), peptides covering residues 44-64 showed similar protection from exchange in both mAb-bound states. (C), peptides incorporating residues 295-322 incorporate less deuterium in both rnAb-bound states, however the magnitude of protection is greater for A44v 11.

Figure 50 depicts hydrogen/deuterium exchange (HDX) comparison of cyno-PAI-1 alone and bound to A44vl 1. (A), butterfly plot of the average relative fractional exchange with the unbound state above and the bound state below. The lines correspond to data acquired for the 10 sec, 1 min, 5 min, and 240 min time points. In (B), plot of the difference data (in daltons) from the above plot in (A) for cyno-PAI-1 alone or bound to A44vl 1.

Figure 51 depicts HDX comparison of cyno-PAI-1 alone and bound to APGv2. i n (A), butterfly plot of the average relative fractional exchange with tlie unbound state above and tlie bound state below. The lines correspond to data acquired for the 10 sec, 1 min, 5 min and 240 min time points, in (B), plot of the difference data from panel (A) above for cyno-PAI-1 alone or bound to APGv2.

Figure 52 depicts HDX comparison of cyno-PAI-1 bound to A44v ! 1 and bound to APGv2. in (A), butterfly plot of the ave rage relative fractional exchange with the APGv2 bound state above and the A44vl 1 bound state below. The lines correspond to data acquired for the 10 sec, 1 min, 5 min and 240 min time points. In (B), plot of the difference data from panel (A) above for cyno-PAI-1 bound to APGv2 or A44v3 1.

Figure 53 depicts the cyno-PAI-1 :A44vl 1 epitope determined by HDX MS. The residues of cynoPAi-l (SEQ ID NO: 162) which show protection from exchange in the A44vl 1 antibody -bound state are shown in bold. The residues of cyno-PAI-1 :A44vl 1 epitope determined from the crystallization studies is shown in boxes.

DETAILED DESCRIPTION

The present invention provides antibodies and fragments thereof that speciiicaliy bind to human PAI-1 and modulate the biological functions ofPAI-1. Such antibodies are particularly useful for treating PAI-1 -associated disease or disorders (e.g., fibrosis). The invention also provides pharmaceutical compositions, as well as nucleic acids encoding PAI-1 antibodies, recombinant expression vec tors and host cells for making such antibodies, or fragments thereof. Methods of using antibodies as disclosed herein to detect PAI-1 or to modulate PAI-1 activity, either in vitro or in vivo, are also encompassed by the invention.

i. Definitions

In order that the present invention may be more readily understood, certain terms are first defined.

As used herein, the term "human PAI-1" refers to a peptide comprising or consisting of the amino acid sequence listed below:

VHHPPSYVAIILASDFGVRVFQQVAQASKDR VVFSPYGVASVXAMLQLTTG GETQQQIQAAMGFKIDDKGMAPALRIILYKELMGPWNKDEISTTDAIFVQRD L LVQGFMPHFFRLFRSTVKQVDFSEVERAR!TiND VKTHTKGMIS LLGK GAVIXJLTRLVLVNALYFNGQWKTPFPDSSTHRRLFHKSDGSTVSVPMMAQT NKFNYTEFTTPDGHYYD1LELPYHGDTLSMFIAAPYEKEVPLSALTNILSAQLI SHWKGNMTRLPRLLVLPKFSLETEVDLR PLENLGMTDMFRQFQADFTSLSD QEPLHVAQALQKVKIE ESGTVASSSTAVIVSARMAPEEIIMDRPFLFVVRH NPTGTVLFMGQVMEP (SEQ ID NO. 1), or a fragment thereof.

As used herein, the term "antibody" refers to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains inter-connected by disulfide bonds, as well as rnultimers thereof (e.g., IgM). Each heavy chain comprises a heavy chain variable region (abbreviated VH or VH) and a heavy chain constant region (CH or CH). The heavy chain constant region comprises three domains, C½l, CR2 and (¾3. Each light chain comprises a light chain variable region (abbreviated Vi or VL) and a light chain constant region (Q. or CL). The light chain constant region comprises one domain (Q 1). The VH and VL regions can be further subdivided into regions of hypervariability, termed complementarity determining regions (CDRs), interspersed with regions that are more conserved, termed framework regions (FR). Each VH and VL is composed of three CDRs

and four FRs, arranged from amino-terminus to carboxy-terininus in the following order: FRl, CDRl, FR2, CDR2, FRS, CDRS, FR4.

As used herein, the term "antigen-binding fragment" of an antibody includes any naturally occurring, enzyrnaticaliy obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds an antigen to form a complex. Antigen-binding fragments of an antibody may be derived, e.g., from full antibody molecules using any suitable standard techniques such as proteolytic digestion or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable and optionally constant domains. Non- limiting examples of antigen-binding portions include: (j) Fab fragments; (ii) F(ab")i fragments; (iii) Fd fragments; (iv) Fv fragments; (v) single-chain Fv (scFv) molecules; (vi) dAb fragments; and (vii) minimal recognition units consisting of the amino acid residues that mimic the hypervariable region of an antibody (e.g., an isolated complementarity determining region (CDR)). Other engineered molecules, such as diabodies, triabodies, tetrabodies and minibodies, are also encompassed within the expression "antigen-binding fragment"

As used herein, the term "CDR" or "complementarity determining region" means the noncontiguous antigen combining sites found within the variable region of both heavy and light chain polypeptides. These particular regions have been described by Kabat et al., J. Biol. Chem. 252, 6609-6616 (1977) and Kabat et al., Sequences of protein of immunological interest. (1991), and by Chothia et al, ,/. Mol Biol. 196:901-917 ( 1987) and by MacCall m et ai, J. Mol. Biol. 262:732-745 (1996) where the definitions include overlapping or subsets of amino acid residues when compared against each other. The Kabat definition is based on sequence variability. The IMGT unique numbering for all TG and TR V-regions of all species relies on the high conservation of the structure of the variable region (Lefranc, Mp et al., Dev comp. Immunol. 27:55-77, 2003), IMGT numbering, set up after aligning more than 5,000 sequences takes into account and combines the definition of the framework and CDRs. The Clotbia de finition is based on the location of the structural loop regions. The Contact definition (MacCallum et al.) is based on an analysis of the complex crystal structures and antibody-antigen interactions. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth for comparison, in one embodiment disclosed herein , the term "CDR" is a CDR as defined by the Kabat definition, in another embodiment disclosed herein , the CDR is a CDR as defined by IMGT.

As used herein the term "framework (FR) amino acid residues" refers to those amino acids in the framework region of an Ig chain. The term "framework region" or "FR region" as used herein, includes the amino acid residues that are part of the variable region, but are not part of the CDRs (e.g., using the Contact definition of CDRs). Therefore, a variable region framework is between about 100-120 amino acids in length but includes only those amino acids outside of the CDRs.

The present invention also encompasses "conservative amino acid substitutions" in the CDR amino acid sequences of the antibodies disclosed herein, i.e., amino acid sequence modifications which do not abrogate the binding of the antibody to the antigen, i.e., PAI-1. A conservative substitution is a substitution of a native amino acid residue with a nonnative residue such that there is 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. Furthermore, any native residue in the polypeptide may also be substituted with alanine, as has been previously described for "alanine scanning mutagenesis" (Cunningham et al„ Science 244:1081-85 (1989)), Conservative amino acid substitutions include the substitution of an amino acid in one class by an amino acid of the same class, where a class is defined by common physicochemical amino acid side chain properties and high substitution frequencies in homologous proteins found in nature, as determined, for example, by a standard Dayhoff frequency exchange matrix or BLOSUM matrix. Six general classes of amino acid side chains "nave been categorized and include: Ciass I (Cys); Class II (Ser, Thr, Pro, Ala, Gly); Class III (Asn, Asp, Gin, Glu); Class IV (His, Arg, Lys); Class V (He, Leu, Val, Met); and Class VT (Phe, Tyr, Tip). For example, substitution of an Asp for another ciass III residue such as Asn, Gin, or Glu, is a conservative substitution. Thus, a predicted nonessential amino acid residue in a PAI-1 antibody is replaced with another ammo acid residue from the same class. Methods of identifying amino acid conservative substitutions which do not eliminate antigen binding are well-known in the art {see, e.g., Brummell et ai., Biochem. 32: 1180, 1993; Kobayashi et al. Protein Eng. 12:879, 1999; and Burks et at. Proc. Nat!. Acad. Set. USA 94:412, 3997). General rules for conservative amino acid substitutions are set forth in Table 1 below.

Table 1: Conservative Amino Acid Snbsiitsstions

Original Exemplary Select

Residues Substitutions Substitutions

Ala Val, Leu, lie Val

Arg Lys, Gin, Asn Lys

Asn Gin, His, Lys, Arg Gin

Asp Glu Glu

Cys Ser Ser

Gin Asn Asn

Glu Asp Asp

Gly Pro, Ala Ala

His ASD, Gin, Lys, Arg Arg lie Leu, Vai, Met, Aia, Leu

Phe, Norieucine

Leu Norieucine, lie, lie

Val, Met, Ala, Phe

Lys Arg, Gin, Asn Arg

Met Leu, Phe, lie Leu

Phe Leu, Val, He, Ala, Leu

Tyr

Pro Ala Ala

Ser Thr Thr

Thr Ser Ser

Tip Tyr, Phe Tyr

Tyr Tip, Phe, Thr, Ser Phe

Val He, Met, Leu, Phe, Leu

Ala, Norieucine

Conservative amino acid substitutions also encompass 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 reversed or inverted forms of amino acid moieties.

Conservative modifications to the amino acid sequence (and the corresponding modifications to the encoding nucleotides) are expected to produce PAl-i antibodies having functional and chemical characteristics similar to those of naturally occurring PAl-i antibodies, in contrast, substantial modifications in the functional or chemical characteristics of PAI- 1 antibodies may be accomplished by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the molecular backbone in the area of the substitution, for example, as a sheet or helical conformation, (b) the charge or hy drophobic ity of the molecule at the target site, or (c) t e bulk of the side chain.

Naturally occurring residues may be divided into groups based on common side chain properties:

1) hydrophobic: norieucine, Met, Aia, Val, Leu, lie;

2) neutral hydrophilic: Cys, Ser, Thr;

3) acidic: Asp, Glu;

4) basic: Asn, Gin, His, Lys, Arg;

5) residues that influence chain orientation: Gly, Pro; and

6) aromatic: Tip, Tyr, Phe.

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

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

As used herein, the term "specifically binds to" refers to the ability of an antibody or an antigen-binding fragment thereof to bind to an antigen with an Kd that is lower than 1 x 10~6 M, 1 x 1 0"7 M, 1 x 1()-S M, 1 x 10"9 M, 1 x 10" l0 M, 1 x 1ίΤπ M, 1 x 10" !2 M, or less. The term also encompasses refers to the ability of an antibody or an antigen-binding fragment thereof to bind to an antigen with an affinity that is at least two-fold greater than its affinity for a nonspecific antigen.

The disclosure also provides antibodies that competitively inhibit binding of an antibody to an epitope disclosed herein as determined by any method known in the art for determining competitive binding, for example, 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%.

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

As used herein, the term "vector" is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid 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 may be ligated. Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) can be integrated into the genome of a host cell upon introduction into the host ceil, and thereby are replicated along with the host genome. Moreo ver, 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" (or simply, "expression vectors"), in general, expression vectors of utility in recombinant DNA techniques are often in the form of piasmids. The terms, "plasmid" and "vector" may be used interchangeably.

However, the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno- associated viruses), which serve equivalent functions.

Numerous expression vector systems may be employed for the purposes of this invention. For example, one class of vector utilizes DNA elements which are derived from animal viruses such as bovine papilloma virus, polyoma virus, adenovirus, vaccinia virus, bacuiovirus, retroviruses (RSV, MTV or MOMLV) or SV40 virus. Others involve the use of polycistronic systems with internal ribosome binding sites. Additionally, cells which have integrated the DNA into their chromosomes may be selected by introducing one or more markers which allow selection of transfected host cells. The marker may provide for prototrophy to an auxotrophic host, biocide resistance (e.g., antibiotics) or resistance to heavy metals such as copper. The selectable marker gene can either be directly linked to the DNA sequences to be expressed, or introduced into the same cell by ^transformation.

Additional eiements may also be needed for optimal synthesis of mRNA, These elements may include signal sequences, splice signals, as well as transcriptional promoters, enhancers, and termination signals. In particular embodiments the cloned variable region genes are inserted into an expression vector along with the heavy and light chain constant region genes (e.g. human) synthetic as discussed above.

More generally, once a vector or DNA sequence encoding an antibody, or fragment thereof, has been prepared, the expression vector may be introduced into an appropriate host cell. That is, the host cells may be transformed, introduction of the plasmid into the host cell can be accomplished by various techniques well known to those of skill in the art. These include, but are not limited to, transfection (including electrophoresis and electroporation), protoplast fusion, calcium phosphate precipitation, cell fusion with enveloped DNA, microinjection, and infection with intact virus. See, idgway, A. A. G. "Mammalian Expression Vectors" Chapter 24.2, pp. 470-472 Vectors, Rodriguez and Denhardt, Eds. (Butterworths, Boston, Mass. 1988). An embodiment disclosed herein is plasmid introduction into the host via electroporation. The transformed cells are grown under conditions appropriate to the production of the light chains and heavy chains, and assayed for heavy or light chain protein synthesis. Exemplary assay techniques include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence- activated cell sorter analysis (FACS), irtmranohistochemistry and the like.

As used herein, the term "transformation" shall be used in a broad sense to refer to the introduction of DNA into a recipient host cell that changes the genotype and consequently results in a change in the recipient cell.

"Host cells" refers to cells that have been transformed with vectors constructed using recombinant DNA techniques and encoding at least one heterologous gene, in descriptions of processes for isolation of polypeptides from recombinant hosts, the terms "cell" and "cell culture" are used interchangeably to denote the source of antibody unless it is clearly specified otherwise, in other words, recovery of polypeptide from the "cells" may mean either irons spun down whole cells, or from the cell culture containing both the medium and the suspended cells.

it should be understood thai this term is intended to refer not only to the particular subject cell but to the progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent ceil, but are still included within the scope of the term "host cell" as used herein.

As used herein, the term "treat," "treating," and "treatment" refer io therapeutic or preventative measures described herein. The methods of "treatment" employ administration to a subject, an antibody or antigen binding fragment disclosed herein , for example, a subject having a PAI- 3 -associated disease or disorder (e.g., a fibrotic disease) or predisposed to having such a disease or disorder, in order to prevent, cure, delay, reduce the severity of, or ameliorate one or more symptoms of the disease or disorder or recurring disease or disorder, or in order to prolong the survival of a subject bey ond that expected in the absence of such treatment.

As used herein, the term "PAI-I -associated disease or disorder" includes disease states with or without symptoms associated with a disease state, where altered levels or activity of PAI- 1 are found. Exemplary PAI- 1 -associated diseases or disorders include various types of fibrosis.

As used herein, the term "effective amount" refers to that amount of an antibody or an antigen binding fragment thereof that binds PAI-1, which is sufficient to effect treatment, prognosis or diagnosis of a PAI-1 -associated disease or disorder, as described herein, when administered to a subject. A therapeutically effective amount will vary depending upon the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like, which can readily be determined by one of ordinaiy skill in the art. The dosages for administration can range from, for example, about 1 ng to about 10,000 mg, about 1 ug to about 5,000 mg, about 1 mg to about 1 ,000 mg, about 10 mg to about 300 mg, of an antibody or antigen binding fragment thereof, disciosed herein . Dosage regiments may be adjusted to provide the optimum therapeutic response. An effective amount is also one in whic any toxic or detrimental effects (i.e., side effects) of an antibody or antigen binding fragment thereof are minimized or outweighed by t e beneficial effects.

As used herein, the term "subject" or "mammal" includes any human or non-human animal. 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 a paratope. A single antigen may have more than one epitope. Thus, different antibodies may bind io different areas on an antigen and may have different biological effects. Epitopes may be either conformational or linear. A conformational epitope is produced by spatially juxtaposed amino acids from different

segments of the linear polypeptide chain, A linear epitope is one produced by adjacent amino acid residues in a polypeptide chain,

it is noted here thai, as used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural reference unless the context clearly dictates otherwise.
What is claimed is:

Claim 1 , An isolated monoclonal antibody that binds specifically io PAI-l, comprising:

(a) a heavy chain framework region and a heavy chain variable region, the heavy chain variable region comprising 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, the light chain variable region comprising 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.

Claim 2. An isolated monoclonal antibody that binds specifically to PAI-l comprising:

(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.

Claim 3. An isolated monoclonal antibody that binds specifically to PAI-l comprising:

(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.

Claim 4, An isolated monoclonal antibody thai binds to essentially the same epitope as the

antibody of claim 1.

Claim 5. An isolated inonoclonal antibody that binds specifically to PAI- 1, comprising:

(a) a heavy chain framework region and a heavy chain variable region, the heavy chain variable region comprising 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, the light chain variable region 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.

Claim 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,

Claim 7, An isolated monoclonal antibody that binds to essentially the sarne epitope as the

antibody of claim 5.

Claim 8. A humanized monoclonal antibody that binds specifically to human PAi-1, wherein the antibody comprises:

(a) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 82, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 91, or an antigen-binding fragment thereof;

(b) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 83, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 92, or an antigen-binding fragment thereof:

(c) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 84, or an antigen-binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 93, or an antigen-binding fragment thereof;

(d) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 85, or 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, and a light chain having a light chain variable region comprising SEQ ID NO: 96, or an antigen -binding fragment thereof;

(i) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 89, or an antigen -binding fragment thereof, and a light chain having a light chain variable region comprising SEQ ID NO: 97, or an antigen-binding fragment thereof;

(j) a heavy chain having a heavy chain variable region comprising SEQ ID NO: 90, or an antigen-binding fragment thereof, and a light chai n havi ng a light chain variable region comprising SEQ ID NO: 98, or an antigen-binding fragment thereof;

(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 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,

m 9. An isolated monoclonal antibody that binds specifically to PAI-1, comprising

(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 CD 1 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: 3 1 , a light chain CDR2 region comprising SEQ ID NO: 30, and a l ight 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 CD 1 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: 5 ! , 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 CD 1 region comprising SEQ ID NO: 58, a heavy chain CDR2 region comprising SEQ ID NO: 57, and heavy chain CDR3 region comprising SEQ ID NO: 56; and a light chain variable region comprising a light chain CD 1 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,

(g) a heavy chain variable region comprising a heavy chain CDR 3 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 CDR i 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 CDRl 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 CDRl 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 CDRl region comprising SEQ ID NO: 76, 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 CDRl 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.

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

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

Claim 12. The method of claim 11, wherein the method treats a condition comprising increased levels of fibrotic tissue.

Claim 13. The method of claim 12, wherein the condition is fibrosis, skin fibrosis, systemic

sclerosis, lung fibrosis, idiopathic pulmonary fibrosis, interstitial lung disease, chronic lung disease, liver fibrosis, kidney fibrosis, chronic kidney disease, thrombosis, venous and arterial thrombosis, deep vein thrombosis, peripheral limb ischemia, disseminated intravascular coagulation thrombosis, acute ischemic stroke with and without thrombolysis, or stent restenosis.

Claim 14. The method of claim 11, 12, or 13 wherein the PAI-1 antibody comprises the antibody of any of the preceding claims.

Claim 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 to a patient, orally, parenterally by a solution for injection, by inhalation, or topically.