Matrix metalloproteinase-9 inhibitor and treatment of stroke

Abstract

Methods and pharmaceutical compositions for treating stroke, intracranial hemorrhage and ischemic injury. The methods include administering a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) antibody or antigen-binding fragment thereof that specifically binds MMP-9 and reduces MMP-9 activity, which treats stroke caused by intracranial hemorrhage and / or ischemic injury in a subject.

Description

PATENT COORP ERATION TREATY PATENT APPLICATIONFORMATRIX METALLOPROTEINASE-9 INHIBITOR AND TREATMENT OF STROKECROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims the benefit of and priority to U.S. Provisional Application No. 63 / 574,715, filed on April 4, 2024, the disclosure of which is incorporated by reference herein in its entirety.BACKGROUND OF THE INVENTION

[0002] Acute stroke is caused by intracranial hemorrhage (ICH) or by sudden ischemia induced by thrombotic vascular obstruction. ICH is a devastating medical event that affects ~ 1 million patients per year.1ICH includes brain bleeding such as intracerebral hemorrhage, subarachnoid hemorrhage and other brain hemorrhage. There is a significant 50-60% one year mortality7and nearly 80% of patients have permanent disability.2The frequency of ICH increases significantly with age, particularly in those > 85.3The frequency of ICH is not lessening and there is no effective medical therapy.3-4

[0003] With ICH, the blood volume affects the brain through multiple mechanisms. The ICH compresses the brain tissue and increases intracranial pressure. In addition, the blood from the hemorrhage appears to have cytotoxic effects on brain cells, promoting excitotoxicity, inflammation, breakdown of the blood brain barrier (BBB), edema, oxidative stress, etc.4

[0004] In addition to stroke caused by ICH, acute ischemic stroke is a leading cause of death and disability in the U.S. and worldwide. In acute ischemic stroke, arterial blood flow is restricted to the brain, which initiates a cascade of molecular events that include inflammation, excitotoxicity, oxidative stress, and the complement system activation.5Currently, the only clinically effective treatment for ischemic brain injury isto restore blood flow to the ischemic brain tissue, so that still viable brain cells, such as neurons, astrocytes, endothelial cells, etc. can recover. Effective approaches for restoring blood flow to the ischemic brain include physically extracting the thrombus by endovascular thrombectomy, or dissolving the thrombotic obstruction, such as by recombinant tissue plasminogen activator (r-tPA) or the r-tPA mutant, tenecteplase.

[0005] The goal of neuroprotection is to decrease the cellular damage and death that occur to neurons during ischemic stroke or ICH.6In contrast, brain cytoprotective therapies are directed to cells in both compartments of the neurovascular unit, such as neurons, astrocytes, endothelial cells and others.7Typically these therapies target molecular pathways that are deleterious during ischemia and reperfusion such as oxidative stress, glutaminergic neurotoxicity, and inflammation.8In ischemic stroke, neuroprotective therapies are often given together with reperfusion treatments, as many require perfusion to the ischemic tissue to be effective. How ever, reperfusion is frequently associated with increased secondary ICH, which is deleterious. In contrast to ischemic stroke, reperfusion does not have therapeutic value in ICH and may aggravate brain hemorrhage.

[0006] Within hours after ICH, levels of matrix metalloproteinases (MMPs) begin to rise, including MMP-2, 3, 7, 9 and 12. These MMPs are part of the acute inflammatory process and are believed to degrade the extracellular matrix causing disruption of the BBB4

[0007] MMP-9 is a calcium and zinc-dependent protease that can digest components of the extracellular matrix (collagen, laminin, fibronectin) and key components of the neurovascular matrix (e.g., tight junction components, growth factors, cell surface receptors and cell adhesion molecules).9'11MMP-9 levels rise acutely in the first 24 h after ICH and may peak by 48 hours, only to rise again after 7 days.4The acute dysregulation of MMP-9 that occurs in the setting of ICH may affect the integrity of the blood brain barrier (BBB), tissue injury and cell death.12, 13MMP-9 promotes neuronal death by disrupting cell-matrix interactions, attachment and integrin signaling (i.e., anoikis).14Despite these findings it is unclear whether MMP-9 contributes to the pathophysiology of ICH. MMP-9' ' mice have been reported to have reduced perihematomal edema in the autologous blood model of experimental ICH.15However, inother experimental models of ICH, MMP-9 / -mice suffer greater neurological deficits and mortality.16Similarly, MMP-9 inhibitors were cytotoxic in collagenase-induced experimental ICH.16. Broad spectrum inhibitors of various MMPs have been shown to be beneficial in experimental ICH, but these inhibitors are of low specificity and as noted by experts, it is impossible to determine through which MMP they may be acting.4

[0008] Acute dysregulation of MMP-9 also occurs in the setting of brain ischemia and affects the integrity of the blood brain barrier (BBB), tissue injury and cell death.12, 13In addition to MMP-9, several of the more than 20 known MMPs (MMP-2, MMP-3, MMP-7, MMP-9, MMP-10, MMP-12, MMP-13), have been reported to be active in ischemic brain injury.17-21Since multiple MMPs may participate in ischemic stroke, it was believed that broad spectrum inhibitors may be optimal therapeutic agents. At least 13 broad spectrum MMP inhibitors have entered clinical development, but none have been successful.22These agents inhibit several MMPs as w ell as the metalloproteinases of the ADAM (‘‘a disintegrin and metalloprotease’') and ADAM-TS families.20As noted for ICH, the significant adverse effects of these agents and their failure in clinical trials has been attributed to their lack of selectivity and their toxicity23in disease conditions.20, 24, 25For example, in some diseases, broad spectrum MMP inhibitors may inhibit deleterious MMPs as well as MMPs with beneficial effects. Recent studies have shown that selective inhibition of MMP-9 by antibodies can reduce ischemic brain injury, bleeding associated with reperfusion or recanalization therapy by recombinant tissue plasminogen activator (r- tPA), but the role of MMP-9 in ICH, or stroke with or without mechanical reperfusion alone is unknown.SUMMARY OF THE INVENTIONThe present disclosure provides methods and compositions for treating stroke, including acute stroke due to ischemic injury, hemorrhage and related conditions. In certain embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof directed against a matrix metalloproteinase 9 (MMP-9), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a heavy chain CDR1, CDR2, and CDR3 and a light chain variable region having a light chainCDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 comprises SEQ ID NO: 1. SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and wherein the light chain CDR1, CDR2, and CDR3 comprises SEQ ID NO: 5. SEQ ID NO: 6. and SEQ ID NO: 7, respectively as shown in Fig. 2AIn certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises SEQ ID NO: 4 and the light chain variable region comprises SEQ ID NO: 8.In certain embodiments, the invention provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof as described herein.In certain embodiments, the invention provides methods for treating stroke comprising: administering to a subject in need thereof a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) inhibitor, wherein the MMP-9 inhibitor is an antibody or antigen-binding fragment thereof that binds MMP-9, as described herein.In certain embodiments, the invention provides that the subject is a human.In certain embodiments, the invention provides that the MMP-9 inhibitor is administered following ischemic stroke in the subj ect. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered at least thirty minutes after ischemic stroke in the subject.In certain embodiments, the invention provides that the MMP-9 inhibitor is administered following intracranial hemorrhage (ICH) in the subject. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered at least thirty minutes after ICH in the subject.In certain embodiments, the invention provides that the stroke is caused by trauma, surgery or other mechanical injury.In certain embodiments, the invention provides that the treatment decreases cerebral infarction, ischemic cell death, brain lesion expansion, brain swelling, brain hemorrhage,brain injury7, fibrin deposition, inflammation or a combination thereof, in the subject. In certain embodiments, the invention provides that the treatment reduces disability, neurobehavioral impairment and / or dependency on caretakers. In certain embodiments, the invention provides that the treatment prevents tissue deterioration and improves recovery7from initial disability7, neurobehavioral impairment and / or dependency on caretakers.In certain embodiments, the invention provides that the administration is intravenous, intramuscular, subcutaneous or intranasal.In certain embodiments, the invention provides methods of treatment for reducing injury from hemorrhagic stroke, including intracranial hemorrhage (ICH), comprising: administering to a subject in need thereof a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) inhibitor, wherein the MMP-9 inhibitor is an antibody or antigen-binding fragment thereof that binds MMP-9 and reduces MMP-9 activity.In certain embodiments, the invention provides the isolated antibody or antigen-binding fragment thereof directed against a matrix metalloproteinase 9 (MMP-9) of the present invention for use as a medicament.In certain embodiments, the invention provides the isolated antibody or antigen-binding fragment thereof directed against a matrix metalloproteinase 9 (MMP-9) of the present invention for use in the methods of treatment according to the present invention, in particular in methods of treating stroke according to the present invention or in methods of reducing injury from ischemia or intracranial hemorrhage (ICH) according to the present inventionIn certain embodiments, the invention provides a matrix metalloproteinase 9 (MMP-9) inhibitor, wherein the MMP-9 inhibitor is an antibody or antigen-binding fragment thereof that binds to MMP-9, for use in a method for treating stroke, wherein the MMP-9 inhibitor is administered to a subject in need thereof in a therapeutically effective amount, in particular wherein the MMP-9 inhibitor binds to MMP-9 with at least 5-fold higher affinity than to MMP-2 and / or MMP-3, preferably with at least 10-fold higher affinity7.The present invention further provides a nucleic acid encoding the isolated antibody or antigen-binding fragment thereof directed against a matrix metalloproteinase 9 (MMP-9) or encoding the MMP-9 inhibitor according to the present invention. Further, the present invention provides an expression vector comprising the nucleic acid of the present invention in functional association with an expression control sequence.Furthermore, the present invention provides a host cell comprising a nucleic acid according to the present invention or the expression vector according to the present invention.The present invention provides a method of production of the isolated antibody or antigenbinding fragment thereof or the MMP-9 inhibitor according to the present invention, wherein the method comprises (a) cultivating the host cell of the present invention under conditions allowing expression of the antibody or antigen-binding fragment thereof, and (b) recovering the antibody or antigen-binding fragment thereof.BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figures 1A-1E. Selection of an MMP-9i that inhibits human MMP-9 catalytic activity in a dose-related fashion and binds with high avidity and specificity. Fig. 1 A shows screening to identify an MMP-9i from monoclonal antibody candidates. Fig. I B shows dose-related inhibition of the catalytic activity of human MMP-9 by lead MMP-9i. Fig. 1C shows saturation binding studies of the lead MMP-9i to human MMP-9. Fig. ID shows specific binding of MMP-9i to the full size (—95 kDa) and catalytic subunits (—35 kDa) of human MMP-9. Fig. IE shows that MMP-9i binds specifically to MMP-9.

[0010] Figures 2A-2E. Amino acid sequences of the variable regions of M9i chimeric HC in Fig. 2A, M9i chimeric LC in Fig. 2B, andecaliximab chimerized HC in Fig. 2C, andecaliximab chimerized LC in Fig. 2D, M9i humanized HC and LC in Fig. 2E. The CDR and FR are indicated and provided as SEQ ID NOs: 1-18.

[0011] Figures 3A-3D. M9i potently inhibits the enzymatic activity of different species MMP-9. Trypsin-activated Fig. 3A hMMP9 (51kD), Fig. 3B human MMP9(75kD) and Fig. 3C rat MMP-9 (75 kDa) (1 nM) were mixed with M9iM9i (0.011 to 2000 nM) and DQ-gelatin substrate (2.5 ug / ml) in substrate assay buffer (50 mM Tris-HCl pH 7.6, 150mM NaCl, 5 mM CaC12 and 0.01% Tween 20). Fig. 3D shows that M9i inhibits human MMP-9 gel / ymography.

[0012] Figures 4A-4E. Deletion or inhibition of MMP-9 with M9i reduces disability in Figs. 4A and 4B, brain swelling in Fig.4C, brain cell death or infarction in Figs. 4D and hemorrhage in Fig. E after 2 h brain ischemia with reperfusion.

[0013] Figures 5A-5E. M9i reduces ischemic brain injury, hemorrhage, and neurobehavioral disability after ischemic stroke without reperfusion. Fig. 5A neurobehavioral outcome. Fig. 5B comer test. Fig. 5C brain hemorrhage, Fig. 5D brain swelling and Fig. 5E infarction or brain cell death after intravascular middle cerebral artery occlusion without reperfusion.

[0014] Figures 6A-6D. Effect of M9i on functional neurobehavioral disability and recovery of mice, assessed day 1 and day 3 after experimental ICH, by a Fig. 6A comer test, Fig. 6B cylinder test, and Fig. 6C a neurological deficit score. Mice were administered M9i (45 ug / 100 ul) or control (no agent). The amount of brain hemorrhage in Fig. 6D was measured 72 hrs after ICH.

[0015] Figures 7A-7D. Effect of andecaliximab, another anti-MMP9 monoclonal antibody that reduces MMP-9 activity, on functional neurobehavioral disability and recovers’ of mice, assessed day 1 and day 3 after experimental ICH by a Fig. 7A comer test, Fig. 7B cylinder test, and Fig. 7C a neurological deficit score. Mice were administered andecaliximab (45 ug / 2100 ul) or control (no agent). The amount of brain hemorrhage in Fig. 7D was measured 72 hrs after ICH.DETAILED DESCRIPTIONIn certain embodiments, the invention provides compositions and methods for treating tissue injurs’ before, during or after the onset of ischemia. In certain embodiments, the invention provides a pharmaceutical composition for treatment according to any of thedescribed methods, comprising: a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) inhibitor; and a pharmaceutically acceptable carrier.In certain embodiments, the invention provides an isolated antibody or antigen-binding fragment thereof directed against a matrix metalloproteinase 9 (MMP-9), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a heavy chain CDR1, CDR2, and CDR3 and a light chain variable region having a light chain CDR1, CDR2, and CDR3, wherein the heavy chain CDR1, CDR2, and CDR3 comprises SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively; and wherein the light chain CDR1, CDR2, and CDR3 comprises SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.In certain embodiments, the antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, (a) wherein the heavy chain variable region comprises SEQ ID NO: 4 and the light chain variable region comprises SEQ ID NO: 8; or (b) wherein the heavy chain variable region comprises SEQ ID NO: 17 and the light chain variable region comprises SEQ ID NO: 18.In certain embodiments, the invention provides pharmaceutical compositions comprising an antibody or antigen-binding fragment thereof as described herein.In certain embodiments, the invention provides methods for treating stroke comprising: administering to a subject in need thereof a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) inhibitor, wherein the MMP-9 inhibitor is an antibody or antigen-binding fragment thereof that binds MMP-9, as described herein.In certain embodiments, the invention provides that the subject is a human.In certain embodiments, the invention provides that the MMP-9 inhibitor is administered following ischemic stroke in the subject. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered at least thirty minutes after ischemic stroke in the subject.In certain embodiments, the invention provides that the MMP-9 inhibitor is administered following intracranial hemorrhage (ICH) in the subject. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered at least thirty minutes after ICH in the subject.In certain embodiments, the invention provides that the stroke is caused by trauma, surgery or other mechanical injury.In certain embodiments, the invention provides that the treatment decreases cerebral infarction, ischemic cell death, brain lesion expansion, brain swelling, brain hemorrhage, brain injury, fibrin deposition, inflammation or a combination thereof, in the subject.In certain embodiments, the invention provides that the administration is intravenous, intramuscular, subcutaneous or intranasal.In certain embodiments, the invention provides methods for reducing injury from ischemia or intracranial hemorrhage (ICH) comprising: administering to a subject in need thereof a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) inhibitor, wherein the MMP-9 inhibitor is an antibody or antigen-binding fragment thereof that binds MMP-9.In certain embodiments, the invention provides that the antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof as described herein. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered following ischemic stroke in the subj ect. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered at least thirty' minutes after ischemic stroke in the subject. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered following intracranial hemorrhage (ICH) in the subject. In certain embodiments, the invention provides that the MMP-9 inhibitor is administered at least thirty7minutes after ICH in the subject.In certain embodiments, the invention provides that the stroke is caused by trauma, surgery or other mechanical injury7. In certain embodiments, the invention provides that the treatment decreases cerebral infarction, ischemic cell death, brain lesion expansion,brain swelling, brain hemorrhage, brain injury7, fibrin deposition, inflammation or a combination thereof, in the subject. In certain embodiments, the invention provides that the administration is intravenous, intramuscular, subcutaneous or intranasal.In certain embodiments, the invention provides that the methods are performed before or after the patient receives brain imaging. These methods may be performed before or after the patient receives full clinical assessment. In certain embodiments, the invention provides that the methods are performed whether or not the patient receives reperfusion therapy. In certain embodiments, the invention provides that the methods are performed before or after the patient is treated with a thrombus dissolving drug (such as tissue plasminogen activator, tenecteplase, urinary-type plasminogen activator, plasmin or a derivate of plasmin, an a2 antiplasmin inhibitor, an inhibitor of plasminogen activator inhibitor-1, a TAFI-inhibitor). In certain embodiments, the invention provides that the methods of performed before or after the patient is treated with a thrombus extraction or catheter-based therapy. In certain embodiments, the invention provides that the methods are performed before or after the patient is treated with surgery (open, percutaneous, minimally invasive, etc.)In certain embodiments, the invention provides a method of treating tissue injury comprising administering to a patient in need thereof a treatment effective amount of MMP-9 inhibitor and a plasminogen activator. In embodiments, the administration can be a co-administration of a unitary pharmaceutical composition, or administration of separate pharmaceutical compositions in either order. In embodiments, the invention provides a kit comprising a MMP-9 inhibitor and a plasminogen activator, and instructions for administration for treating tissue injury' after the onset of ischemia. In embodiments, the MMP-9 inhibitor is a monoclonal antibody or antigen-binding fragment thereof, and the plasminogen activator is tissue plasminogen activator (tPA).Monoclonal antibodies that bind to MMP-9, such as human MMP-9, are commercially available, such as from ThermoFisher (5G3, IIA5, 4A3, 2C3), or Sigma Aldrich (9D4.2, 6- 6B, 2H4) and can also be made by methods well-known to those skilled in the art. Andecaliximab (Gilead Sciences) is another well characterized chimeric anti-MMP-9antibody. The present description particularly provides the sequences and CDRs and FRs of advantageous antibodies and equivalents thereof.In embodiments, the MMP-9 inhibitor is an antibody that functionally binds to MMP-9 protein and decreases an activity thereof, such as MMP-9 catalytic activity, ischemic brain injury, hemorrhage, neurobehavioral disability, neural death, brain swelling, infarction, degradation of blood vessel barriers and / or neutrophil inflammation.In certain embodiments, the isolated antibody or antigen-binding fragment thereof directed against a matrix metalloproteinase 9 (MMP-9), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a heavy chain CDR1, CDR2, and CDR3 and a light chain variable region having a light chain CDR1, CDR2, and CDR3.In embodiments, the heavy chain CDR1, CDR2, and CDR3 comprises NY LIE (SEQ ID NO: 1), VITPGSGGTDYNEKFRG (SEQ ID NO: 2), and STIQGAMDY (SEQ ID NO: 3), respectively. See, Fig. 2 A. In embodiments, the light chain CDR1, CDR2, and CDR3 comprises RASQSISNNLH (SEQ ID NO: 5), YASQS1S (SEQ ID NO: 6), and QQSDNWPQYT (SEQ ID NO: 7), respectively. See, Fig. 2B.In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprisesQVQLQQSGAELVRPGTSVKVSCKASGYAFTNYLIEWVKQRPGQGLEWIGVITPGS GGTDYNEKFRGKATLTADKSSSTAYMQFSSLTSDDSAVYFCARSTIQGAMDYWG QGTSVTVSS (SEQ ID NO: 4) and the light chain variable region comprises DIVLTQSPVTLSVTPGNSVSLSCRASQSISNNLHWYQQKSHESPRLLIKYASQSISGI PSRFSGSGSGTDFTLTINSVEAEDFGMYFCQQSDNWPQYTFGGGTKLEIK (SEQ ID NO: 8). See, Figs. 2A-2B.In embodiments, the heavy chain CDR1, CDR2, and CDR3 comprises (SEQ ID NO: 9), (SEQ ID NO: 10), and (SEQ ID NO: 11), respectively. See. Fig. 2C. In embodiments, the light chain CDR1, CDR2, and CDR3 comprises (SEQ ID NO: 13), (SEQ ID NO: 14), and (SEQ ID NO: 15), respectively. See, Fig. 2D.In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region comprises (SEQ ID NO: 12) and the light chain variable region comprises (SEQ ID NO: 16). See, Figs. 2C-2D.

[0016] In certain embodiments, the isolated antibody or antigen-binding fragment thereof comprises a heavy chain variable region and a light chain variable region, wherein the heavy chain variable region comprises the amino acid sequence (SEQ ID NO: 17) and the light chain variable region comprises the amino acid sequence (SEQ ID NO: 18). See. Fig. 2E.

[0017] In certain embodiments, the invention provides a method of treating stoke comprising administering to a subject in need thereof a therapeutically effective amount of composition comprising a means for binding MMP-9. In embodiments, the means for binding MMP-9 is any of the anti-MMP-9 antibodies or fragments thereof as described herein, including CDR and variable region sequences.

[0018] When introducing elements of the present invention or the preferred embodiment(s) thereof, the articles "a", “an”, “the” and “said” are intended to mean that there are one or more of the elements. The terms “comprising”, “including” and “having” are intended to be inclusive and mean that there may be additional elements other than the listed elements.

[0019] It is understood that aspects and embodiments of the invention described herein include “consisting” and / or “consisting essentially of’ aspects and embodiments.

[0020] Throughout this disclosure, various aspects of this invention are presented in a range format. It should be understood that the description in range format is merely for convenience and brevity’ and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub-ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub-ranges such as from 1 to 3, from 1 to 4, from 1 to 5. from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers withinthat range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0021] As used herein, “about’’ will be understood by persons of ordinary skill in the art and will vary’ to some extent depending upon the context in which it is used. If there are uses of the term which are not clear to persons of ordinary skill in the art, given the context in which it is used, “about” will mean up to plus or minus 10% of the particular term.

[0022] As used herein, “antibody” refers to an intact immunoglobulin (Ig) molecule of any isotype, or an immunologically active fragment thereof that can compete with the intact antibody for specific binding to the target antigen, e.g., MMP-9. MMP-9 antibodies are commercially available and well-known, and can be routinely made given the well-known identity of MMP-9 for humans and other mammals. In particular, antibodies can be made using the complementarity’ determining regions (CDRs) disclosed herein. A CDR refers to the region of an immunoglobulin variable domain that recognizes and binds to the target antigen. Each heavy chain and light chain variable domain may comprise up to three CDR sequences, identified as CDR1, CDR2 and CDR3.

[0023] In some instances, the antibody is an immunological fragment of an intact antibody (e.g., a Fab, a Fab', a F(ab')2, or a single-chain Fv fragment scFvs). Antibodies include, but are not limited to, monoclonal antibodies, polyclonal antibodies, bispecific antibodies, minibodies, domain antibodies, synthetic antibodies, chimeric antibodies, humanized antibodies, human antibodies, antibody fusions, and fragments thereof, respectively. An intact antibody will generally comprise at least two full-length heavy chains and two full-length light chains, but in some instances can include fewer chains such as antibodies naturally occurring in camelids which can comprise only heavy chains. Antibodies can be derived solely from a single source, or can be “chimeric,” that is, different portions of the antibody can be derived from two different antibodies. The basic antibody structural unit typically comprises a tetramer. Each such tetramer typically is composed of tw o identical pairs of polypeptide chains, each pair having one full-length “light” (about 25 kDa) and one full-length “heavy” chain (50-70 kDa). The aminoterminal portion of each chain typically includes a variable region of about 100 to 1 10 ormore amino acids that ty pically is responsible for antigen recognition. The carboxyterminal portion of each chain typically defines a constant region that can be responsible for effector function. Human light chains are typically classified as kappa and lambda light chains. Heavy chains are typically classified as mu, delta, gamma, alpha, or epsilon, and define the antibody's isotype as IgM, IgD, IgG, IgA, and IgE, respectively. Certain classes such as IgG and IgM have subclasses as well (e.g. IgGi, IgG2, IgGs, IgG4, and so forth).

[0024] As used herein, “monoclonal antibody” or “MAb” refers to a population of antibodies that are made by identical immune cells that are all clones of a unique parent cell.

[0025] As used herein, “antgen-binding” region refers the part of an antibody molecule that contains the amino acid residues that interact with an antigen and confer on the antibody molecule its specificity and affinity for the antigen. An antigen binding region typically includes one or more “complementary determining regions” (“CDRs”). A “CDR” is an amino acid sequence that contributes to antigen binding specificity and affinity. Certain antigen binding regions also include one or more “framework” regions. “Framework” regions can aid in maintaining the proper conformation of the CDRs to promote binding between the antigen binding region and an antigen. Structurally, framework regions can be located in antibodies between CDRs.

[0026] As used herein, “epitope” refers to a region of an antigen that is bound by an antibody that targets that antigen, and typically includes specific amino acids that directly contact the antibody. Generally, antibodies specific for a particular target antigen will preferentially recognize an epitope on the target antigen in a complex mixture of proteins and / or macromolecules.

[0027] As used herein, “antigen-binding,” refers to the non-covalent interactions of the type which occur between an antibody molecule and an antigen for which the antibody is specific. The strength, or affinity, of immunological binding interactions can be expressed in terms of a dissociation constant (Ka) of the interaction, wherein a smaller Ka represents a greater affinity-. Immunological binding properties of selected antibodies canbe quantified using methods well known in the art. One such method entails measuring the rates of antigen-binding site / antigen complex formation and dissociation wherein those rates depend on the concentrations of the complex partners, the affinity of the interaction, and geometric parameters that equally influence the rate in both directions. Thus, both the “on rate constant” (Kon) and the “off rate constant” (Koff) can be determined by calculation of the concentrations and the actual rates of association and dissociation. The ratio of Koff / Kon enables the cancellation of all parameters not related to affinity, and is equal to the dissociation constant Kd. An antibody of the present disclosure is said to specifically bind its target antigen when the dissociation constant (Kd) is <1 pM. An antibody of the present disclosure specifically binds antigen with “high affinity ” when the Kd is < 5 nM, and with “very high affinity” when the Kd is < 0.5 nM.

[0028] The phrase “specifically binds” or “specific binding” refers to a binding reaction between two molecules that is at least two times the background and more typically more than 10 to 100 times background molecular associations under physiological conditions. When using one or more detectable binding agents that are proteins, specific binding is determinative of the presence of the protein, in a heterogeneous population of proteins and other biologies. Thus, under designated immunoassay conditions, the specified antigen-binding molecule binds to a particular antigenic determinant, thereby identifying its presence. Specific binding to an antigenic determinant under such conditions requires an antigen-binding molecule that is selected for its specificity to that determinant. This selection may be achieved by subtracting out antigen-binding molecules that cross-react with other molecules. A variety of immunoassay formats may be used to select antigen-binding molecules (e. , immunoglobulins) [such that they are specifically immunoreactive with a particular antigen]. For example, solid-phase ELISA immunoassays are routinely used to select antibodies specifically immunoreactive with a protein (see, e.g., Harlow' & Lane, Antibodies, A Laboratory' Manual (1988) for a description of immunoassay formats and conditions that can be used to determine specific immunoreactivity). Methods of determining binding affinity and specificity are also well known in the art (see, for example, Harlow' and Lane, supra); Friefelder, “Physical Biochemistry: Applications to biochemistry' and molecular biology” (W.H. Freeman and Co. 1976).

[0029] Variations in the amino acid sequences of antibodies, and regions thereof, are contemplated as being encompassed by the present disclosure, providing that the variations in the amino acid sequence maintain at least 75%, more preferably at least 80%. 90%, 95%, and most preferably 99%. Certain percentages in between are included, such as 75%, 76%, 77%, 78%, 79% 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, and 99% sequence identity. In particular, conservative amino acid replacements are contemplated. Conservative replacements are those that take place within a family of amino acids that are related in their side chains. Genetically encoded amino acids are generally divided into families: (1) acidic amino acids are aspartate, glutamate; (2) basic amino acids are lysine, arginine, histidine; (3) non-polar amino acids are alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan, and (4) uncharged polar amino acids are glycine, asparagine, glutamine, cysteine, serine, threonine, tyrosine. The hydrophilic amino acids include arginine, asparagine, aspartate, glutamine, glutamate, histidine, lysine, serine, and threonine. The hydrophobic amino acids include alanine, cysteine, isoleucine, leucine, methionine, phenylalanine, proline, tryptophan, tyrosine and valine. Other families of amino acids include (i) serine and threonine, which are the aliphatic-hydroxy family; (ii) asparagine and glutamine, which are the amide containing family; (iii) alanine, valine, leucine and isoleucine, which are the aliphatic family; and (iv) phenylalanine, tryptophan, and tyrosine, which are the aromatic family. For example, it is reasonable to expect that an isolated replacement of a leucine with an isoleucine or valine, an aspartate with a glutamate, a threonine with a serine, or a similar replacement of an amino acid with a structurally related amino acid will not have a major effect on the binding or properties of the resulting molecule, especially if the replacement does not involve an amino acid within a framework site. Whether an amino acid change results in a functional antibody can readily be determined by assaying the specific activity of the antibody derivative. Fragments or analogs of antibodies can be readily prepared by those of ordinary skill in the art. Preferred amino- and carboxy-termini of fragments or analogs occur near boundaries of functional domains.

[0030] Preferred amino acid substitutions are those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for formingprotein complexes, (4) alter binding affinities, and (4) confer or modify other physicochemical or functional properties of such antibodies. Antibodies can include various muteins of a sequence other than the naturally-occurring peptide sequence. For example, single or multiple amino acid substitutions (preferably conservative amino acid substitutions) may be made in the naturally-occurring sequence (preferably in the portion of the polypeptide outside the domain(s) forming intermol ecular contacts. A conservative amino acid substitution should not substantially change the structural characteristics of the parent sequence (e.g., a replacement amino acid should not tend to break a helix that occurs in the parent sequence, or disrupt other types of secondary structure that characterizes the parent sequence).

[0031] As used herein, “substantially pure” means an object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition), and preferably a substantially purified fraction is a composition wherein the object species comprises at least about 50% (on a molar basis) of all macromolecular species present. Generally, a substantially pure composition will comprise more than about 80% of all macromolecular species present in the composition, more preferably more than about 85%, 90%, 95%, and 99%. Most preferably, the object species is purified to essential homogeneity (contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species.

[0032] As used herein, “patient” and “subject” are used interchangeably and include human and veterinary subjects.

[0033] As used herein, “target antigen” refers to a molecule or a portion of a molecule capable of being selectively bound by an antibody. In certain embodiments, a target can have one or more epitopes. In this context, it does not require that the molecule be foreign or that it be capable of inducing an immune response.

[0034] As used herein, “pharmaceutical composition” refers to a pharmaceutically acceptable composition containing chemical compound, composition, agent or drugcapable of inducing a desired therapeutic effect when properly administered to a patient. It does not necessarily require more than one type of ingredient.

[0035] As used herein, “pharmaceutically acceptable carrier” refers to an excipient, diluent, preservative, solubilizer, emulsifier, adjuvant, and / or vehicle with which an active agent is administered. Such carriers may be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents. Antibacterial agents such as benzyl alcohol or methyl parabens: antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compositions in combination with carriers are known to those of skill in the art. In some embodiments, the language “pharmaceutically acceptable carrier” is intended to include any and all solvents, dispersion media, coatings, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known in the art.

[0036] As used herein, “therapeutically effective amount” refers to the amount of a therapeutic agent determined to produce a therapeutic response in a patient. Such therapeutically effective amounts are readily ascertained by one of ordinary skill in the art.

[0037] As used herein, “inhibitor” refers to a composition that decreases the magnitude of at least one activity or function of a molecule when compared to the magnitude of the activity or function observed in the absence of the inhibitor. Certain exemplary' activities and functions of a molecule include, but are not limited to, binding affinity', enzymatic activity, and signal transduction.

[0038] As used herein, “treat” and “treatment” include therapeutic treatments, prophylactic treatments, and applications in which one reduces the risk that a subject will develop a disorder or other risk factor. Treatment does not require the complete curing of a disorder and encompasses embodiments in which one reduces symptoms or underlying risk factors.

[0039] As used herein, “prevent” does not require the 100% elimination of the possibility of an event. Rather, it denotes that the likelihood of the occurrence of the event has been reduced in the presence of the compound or method.

[0040] The MMP-9 inhibitors or pharmaceutical compositions of the disclosure may be administered in a number of ways depending upon whether local or systemic treatment is desired. The compositions are typically suitable for parenteral administration, wherein administration includes any route of administration characterized by physical breaching of a tissue of a subject and administration of the pharmaceutical composition through the breach in the tissue, thus generally resulting in the direct administration into the blood stream, into muscle, or into an internal organ. Parenteral administration thus includes, but is not limited to, administration of a pharmaceutical composition by injection of the composition, by application of the composition through a surgical incision, by application of the composition through a tissue-penetrating non-surgical wound, and the like. In particular, parenteral administration is contemplated to include, but is not limited to. subcutaneous, intraperitoneal, intramuscular, intrastemal, intravenous, intranasal, intratracheal, intraarterial, intrathecal, intraventricular, intraurethral, intracranial, intratumoral, intraocular, intradermal, intrasynovial injection or infusions, and kidney dialytic infusion techniques. In some embodiments, the methods of the present disclosure comprise intravenous administration.

[0041] In some embodiments, the MMP-9 inhibitors or pharmaceutical compositions are administered in combination with other therapies or diagnostic procedures. The term “combination” refers to either a fixed combination in one dosage unit form, or a kit of parts for the combined administration where the composition comprising, and a combination partner (e.g., another drug or “co-agent”) may be administered independently at the same time or separately within time intervals. In some circumstances the combination partners show a cooperative, e g., synergistic effect. The terms “co- administration” or “combined administration” or the like as utilized herein are meant to encompass administration of the selected combination partner to a single subject in need thereof (e.g., a patient), and are intended to include treatment regimens in which the agents are not necessarily administered by the same route of administration or at the same time. The term “pharmaceutical combination” as used herein means a product thatresults from the mixing or combining of more than one active ingredient and includes both fixed and non-fixed combinations of the active ingredients. The term “fixed combination’" means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient simultaneously in the form of a single entity or dosage. The term “non-fixed combination” means that the active ingredients, e.g., a compound and a combination partner, are both administered to a patient as separate entities either simultaneously, concurrently or sequentially with no specific time limits, wherein such administration provides therapeutically effective levels of the two compounds in the body of the patient. The latter also applies to cocktail therapy, e.g., the administration of three or more active ingredients.EXAMPLES

[0042] Figures 1A-1E. Selection of an MMP-9i that inhibits human MMP-9 catalytic activity’ in a dose-related fashion and binds with high avidity and specificity. Fig. 1A shows screening to identify an MMP-9i from monoclonal antibody candidates. Purified human MMP-9 catalytic domain (2 nM, Neobiolab.com) was mixed with purified monoclonal antibodies to human MMP-9 (120 nM), DQ-gelatin (2.5 ug / ml; Invitrogen) in assay buffer (50 rnM Tris HCL, pH 7.6, 150 mM NaCl, 5 mM CaC12, 0.01% Tween 20) in 96 well black microtiter plates (Costar #3915). The cleavage of DQ-gelatin was monitored by the released fluorescence at 37 deg. C (ex. 485, em 530 nm). The percent inhibition of MMP-9 activity was determined by comparison to wells without monoclonal antibody. The data is representative of 3 independent experiments. Fig. IB shows dose-related inhibition of the catalytic activity of human MMP-9 by lead MMP-9i. The activity of MMP-9 (1 nM) was assessed in the presence of various amounts of purified MMP-9i (0- 128 nM) as described in Fig. 1A. The IC50 (0.9 ± 0.07 nM) was determined by non-linear regression, r2=0.996. Fig. 1C shows saturation binding studies of the lead MMP-9i to human MMP-9. Saturation binding of MMP-9i was examined in duplicate in microtiter plate wells coated with human MMP-9 (•, 2 ug / ml). mouse MMP-9 (o, 2 ug / ml). or bovine serum albumin (■, 10 mg / ml). The wells are washed to remove unbound MMP-9 and then non-specific protein binding sites w ere blocked with 1% bovine serum albumin for 1 hr. After washing, purified MMP-9i antibody diluted in 1% BSA was added to the wells in concentrations from 10’8to 10’11molar for 1 hr. The wells were washed. Thengoat-antimouse Fab peroxidase antibody (1 :5000) is added for 1 hr. The wells are washed. (In experiments with humanized MMP-9i we will use goat-antihuman Fab peroxidases instead). Then TMB (peroxidase substrate) is added to the wells and the product formation was monitored continuously at A370. The A370 is plotted vs. concentration of MMP-9i and the data are analyzed with GraphPad Prism to determine the Ka. An experiment is considered successful if the data analysis shows a fit with an r > 0.95. A representative example of a binding study is shown. Fig. ID shows specific binding of MMP-9i to the full size (~95 kDa) and catalytic subunits (~35 kDa) of human MMP-9. Purified recombinant human MMP-9 (1 ug / lane) were subjected to 7.5% SDS-PAGE under reducing conditions. After electroblotting to PVDF membranes the blots were blocked with 3% BSA and then incubated with purified MMP-9i (1 ug / ml) overnight at 4 deg. C. The blots were washed with TBS-tween and then incubated with a fluorescent antimouse secondary antibody (1 :5000, Li-Cor). After washing the blots were developed on a Li-Cor system. The relative migration of standards and the position of MMP-9 proteins are shown. Fig. IE shows that MMP-9i binds specifically to MMP-9. Wells of a microtiter plate were coated with recombinant full length MMP-9. MMP-2. MMP-3 (2 ug / ml) or no MMP for 1 hr at room temperature. After washing, non-specific protein binding sites were blocked with 1% bovine serum albumin for 2 hr. After washing, purified MMP-9i antibody (2 ug / ml) diluted in 1% BSA was added to the wells for 1 hr. The wells were washed. Then goat-antimouse Fab peroxidase antibody (1 :8000) was added for 1 hr. The wells are washed. Then TMB (peroxidase substrate) was added to the wells and the product formation is monitored continuously at A370.

[0043] Figures 2A-2D. Amino acid sequences of the variable regions of Fig. 2A chimeric HC. Fig. 2B chimeric LC, Fig. 2C chimerized HC, and Fig. 2D chimerized LC. The framework (FR) and complementarity determining regions (CDR) are shown. The alignments were performed using using KABAT definitions with the NovoPro tool (novoprolabs.com / tools / cdr). Fig. 2A shows the heavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 1. SEQ ID NO: 2. and SEQ ID NO: 3. respectively for the variable heavy chain sequence of SEQ ID NO: 4 for chimeric M9i. Fig. 2B shows the light chain CDR1, CDR2, and CDR3 of SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively of the variable light chain sequence of SEQ ID NO: 8 for chimeric M9i. Fig. 2C shows theheavy chain CDR1, CDR2, and CDR3 of SEQ ID NO: 9, SEQ ID NO: 10, and SEQ ID NO: 11, respectively of the variable heavy chain sequence of SEQ ID NO: 12 for chimerized andercaliximab. Fig. 2D shows the light chain CDR1. CDR2, and CDR3 of SEQ ID NO: 13, SEQ ID NO: 14, and SEQ ID NO: 15, respectively of the variable light chain sequence of SEQ ID NO: 16 for chimerizedandercaliximab. Fig. 2E shows variable heavy chain sequence of SEQ ID NO: 17 and variable light chain sequence of SEQ ID NO: 18 for humanized M9i.

[0044] Figures 3A-3D. M9i potently inhibits the enzymatic activity of different species MMP-9. Trypsin-activated Fig. 3A hMMP9 (51kD), Fig. 3B human MMP9 (75kD) and Fig. 3C rat MMP-9 (75 kDa) (1 nM) were mixed with M9i (0.011 to 2000 nM) and DQ-gelatin substrate (2.5 ug / ml) in substrate assay buffer (50 mM Tris-HCl pH 7.6, 150mM NaCl, 5 mM CaC12 and 0.01% Tween 20). The cleavage of substrate was monitored at 37 deg. C with excitation at 485 nm and emission at 530 nm, every' 5 min. The amount of inhibition was determined by comparison to wells with MMP-9, dQ- substrate and no M9i and wells with DQ-substrate alone. Data was analyzed by GraphPad Prism. Relative fluorescence was monitored for 20 min in a UV spectrophotometer. Activity' was plotted according relative to the log dose of M9i via a four-parameter fit by Graphpad Prism. Fig. 3D shows that M9i inhibits human MMP-9 gel zymography. Semigradient SDS-PAGE non-reducing gels were made by using 6% (2.5ml), 8% (5ml) and 10% (2.5ml) acrylamide gels containing 0. 1% type A gelatin and 0.1% type B gelatin. Left panel, gel without M9i (left panel) and the second with M9i (right panel). Purified M9i or various amounts of human MMP-9 were added to wells (MMP-9 51 and 75 kDa forms). After electrophoresis the gels and membranes were washed with dH2O and 2.5% Triton- 100X by shaking. They were incubated in enzyme incubation buffer: 14h at 37°C with agitation. The buffer included 50 mM Tris-HCl, pH 7.5 / 5 mM CaCk / lOO mM NaCl, 0.01% Triton X-100 / 0.1 mM ZnCh and 0.2% Brij non-ionic detergent / 0.002% NaN3. The gels were stained with Coomassie Brilliant blue (060322). Gelatinase activity' is indicated by the areas of clearing of blue staining.

[0045] Figures 4A-4E. Deletion or inhibition of MMP-9 with M9i reduces disability in Figs. 4A and 4B, brain swelling in Fig. 4C, brain cell death or infarction in Figs. 4D and hemorrhage in Fig. E after 2 h brain ischemia with reperfusion. Mice wereadministered saline or M9i (15 ug) in 150 ul intravenously at the time of reperfusion. Data represent means ± SE, n= 22. *p<0.05, **p<0.01, ***p<0.001 by Kruskal -Wallis or standard ANOVA, with Dunett’s or Holm-Sidak corrections for multiple comparisons, as appropriate. Experiments and analysis were performed by a blinded operator on congenic C57B16 MMP-9+ / + and MMP-9- / - mice. Animals were anesthetized with 2% isoflurane. A laser-Doppler flow probe was attached to the skull over the territory of the middle cerebral artery (MCA) (2 mm caudal to the bregma and 6 mm lateral to the midline of the skull). A midline incision was made in the neck, and the right common carotid artery (CCA), external carotid artery (ECA), and internal carotid artery (ICA) were isolated from the vagus nerve. The superior thyroid, lingual, and maxillary arteries were cauterized and cut. The CCA was ligated, and 2 closely spaced knots were placed on the distal part of the ECA with silk sutures. The ECA was cut between the knots, and the tied section (or stump) attached proximally to the CCA junction was straightened to allow the filament to enter the ICA and block the MCA or circle of Willis. The ICA and the pterygopalatine artery were cleared and visualized. A microvascular clip was applied temporarily to the ICA proximally to the CCA bifurcation to stop the blood supply, and the ECA stump was incised to insert the filament. Once the tip of the inserted filament (6-0 or 8-0) reached the clip, a knot was tied on the ECA stump to prevent bleeding through the arteriotomy. The clip was then removed permanently, and the filament was carefully advanced up to 11 mm from the carotid artery bifurcation or until resistance was felt. This advancement confirms that the filament was not in the pterygopalatine artery. A drop in relative cerebral blood flow (80%) measured by the Laser Doppler flow meter was considered a successful occlusion. The filament was secured in place. The incision was closed by sewing the wound with 6.0 Vicryl-coated suture (Ethicon). Neurobehavioral assessments were performed 24 hours after stroke onset. Then mice were deeply anesthetized and brains were isolated during euthanization.

[0046] Figures 5A-5E. M9i reduces ischemic brain injury, hemorrhage, and neurobehavioral disability after ischemic stroke without reperfusion. Fig. 5A) neurobehavioral outcome. Fig. 5B comer test. Fig. 5C brain hemorrhage, Fig. 5D brain swelling and Fig. 5E infarction or brain cell death after 24 hours of intravascular middle cerebral artery' occlusion without reperfusion. Mice were administered M9i (30 ugl) orcontrol (no agent) intravenously 30 minutes after onset of ischemia. Mean ± SE are shown, n= 29. *p<0.05, **p<0.01, Mann Whitney test. Stroke was performed as described in Figs. 4A-4E. except that reperfusion did not occur.

[0047] Figures 6A-6D. Effect of M9i on functional neurobehavioral disability and recover}' of mice, assessed day 1 and day 3 after experimental ICH by a Fig. 6A comer test. Fig. 6B cylinder test, and Fig. 6C a neurological deficit score. Mice were administered M9i (45 ug / 100 ul) or control (no agent) 1 hour after ICH. The amount of brain hemorrhage in Fig. 6D was measured 72 hrs after ICH. Means ± SE are shown. n=16. *p<0.05, **p<0.01, pO.001, pO.OOOl.

[0048] Figures 7A-7D. Effect of another MMP-9 antibody (andecaliximab) on functional neurobehavioral disability and recover7of mice, assessed day 1 and day 3 after experimental ICH by a Fig. 7A comer test, Fig. 7B cylinder test, and Fig. 7C a neurological deficit score. Mice were administered M9i (45 ug / 200 ul saline) or control (200 ul saline) 1 hour after ICH. The amount of brain hemorrhage in Fig. 7D was measured 72 hrs after ICH. Means ± SE are shown, n=15. *p<0.05, **p<0.01, pO.OOl, pO.OOOl.

[0049] Intracranial hemorrhage (ICH) was created in isoflurane-anesthetized mice in a stereotactic frame. Experiments and analysis were performed by a blinded operator on congenic C57B16 MMP+ / + and MMP- / - mice. A burr hole was placed and a needle was advanced 1 mm anterior to bregma, 2mm lateral to the midline to a depth of 4 mm from the skull. Mice receive autologous blood (30 ul) over 5 min. through a micro infusion pump. The needle was slowly withdrawn 10 minutes after and the burr hole was closed with bone wax and skin sutured with non-absorbable suture and / or tissue glue.

[0050] All experiments were performed with congenic, C57B16 mice by an operator blinded to the genotype and drug administered. Animals were kept on heating pads. Drugs were provided as a clear fluid (M9i, 45 ug / 100 uL in saline, or 15 ug / 150 ul) in numbered, otherwise identical tubes without an indication of the drug identity. They were provided to the blinded scientist who performed the stroke studies by a second investigator who did not know- the design or goals of the experiment. Drugs wereadministered via the internal jugular vein. Animals were provided thermoregulatory support and recovered post-operatively in a clean cage or area without bedding. They were monitored until ambulatory. Post-operative care for all surgery consisted of volume support with 500 ul of saline injected i.p. to sustain hydration. Softened food or Napa nectar was placed on the floor of the cage. Neurobehavioral assessment was performed daily for 3 days post-ICH, with assessment by a blinded researcher. Then mice were deeply anesthetized and brains were isolated during euthanasia.

[0051] To examine whether specifically MMP-9 contributes to ischemic brain injury in stroke, MAbs were generated against the catalytic domain of MMP-9, which differs from other MMP classes because it is a gelatinase with fibronectin motifs. C57BL / 6J mice (Jackson Laboratory, Bar Harbor, ME) were immunized with recombinant human and mouse MMP-9. Somatic cell fusion of immune splenocytes was performed with SP2 / 0 cells using conventional hybridoma techniques.101Microplate ELISA assays were performed to screen positive clones as described below; Positive clones were subcloned by limiting dilution to create stable monoclonal antibody (mAb) producing hybridomas. All cell cultures were maintained in DMEM medium, supplemented with 5% fetal bovine serum, 2 mmol / L L-glutamine, and 1% penicillin-streptomycin in a humidified 5% CCh / 95% air incubator at 37°C. Mouse isotypes were identified by mouse antibody isotyping kit (Zymed).

[0052] To screen for potential MMP-9i, monoclonal antibodies were purified by affinity chromatography on goat-antimouse agarose columns (goat anti-mouse (H+L) agarose (Invitrogen, Grand Island, NY) as recommended by the manufacturer.

[0053] The enzymatic activity of MMP-9 is typically assayed by a gelatinase assay, which measures the ability of MMP-9 to degrade gelatin peptides. We adapted the DQ-gelatin assays in which quenched fluorescence is relieved by MMP-9 cleavage. This assay is reproducible and sensitive to sub-nanomolar quantities of MMP-9 enzyme. Fig. 1A compares the inhibitory effects of several high affinity MMP-9 mAbs in a screening assay. We selected mAb 7 as the lead MMP-9i for therapeutic development because of its superior potency at inhibiting MMP-9 activity in vitro. In further studies, the lead MMP-9i potently inhibited human MMP-9 catalytic activity in a dose-dependent fashion with anIC50 of 0.9 ± 0.07 nM (Fig. IB). An additional, back-up candidate MMP-9i (mAb 6) was identified that inhibited MMP-9 with slightly less potency than the lead MMP-9i.

[0054] The binding avidity of the lead MMP-9i to MMP-9 versus a control antigen in saturation binding assays (see Fig. 1C) was examined. This MMP-9i bound with high affinity to human MMP-9 with an affinity of 195±47 picomolar. This is sufficient to bind essentially all human MMP-9 in the blood, which is normally present at a concentration of ~ 0.5 to 1 nM.The lead MMP-9i also bound with ~2-fold lower avidity to mouse MMP-9 (Fig. 1C).

[0055] Immunoblotting studies of reduced full-length and the catalytic subunit of MMP-9 showed that the lead MMP-9i bound specifically to the catalytic subunit (Fig. ID). Binding was greater to the non-reduced forms of MMP-9 (not shown) suggesting that the lead MMP-9i prefers a conformation present in the disulfide-linked structure of MMP- 9. The lead MMP-9i bound specifically to MMP-9 and did not bind to MMP-2, the other gelatinase with which it shares the greatest homology or MMP-3 (stromelysin- 1), which may also be activated in ischemic stroke (Fig. IE).

[0056] After testing, antibody sequencing was performed and antibodies were recombinantly produced in CHO cells. M9i antibody was produced as a chimeric and as a humanized antibody. Chimerism was performed by grafting together the variable region of the mouse antibody with the constant region of a human antibody. Humanization was performed with the goal of engineering optimal human consensus sequences for each of the variable heavy and light chain framework regions. This genetic engineering was achieved by first identifying human immunoglobulin germline genes orthologous to the murine heavy and light chain genes that comprise the murine M9i. Through analysis of human germline genes, a human consensus sequence was then designed that constituted a minimal positional template and afforded optimal chain packing residues of enough length to maintain overall 3-D conformation of the critical CDR residues. The template of this human consensus sequence was predicted, based on spacing and topological considerations, to retain the binding properties of the original mouse monoclonal antibody.

[0057] The sequences of the variable regions of humanized M9i (hM9i) and chimeric M9i (cM9i) are shown in Figs. 2A-2D. The humanized M9i (hM9i) and chimeric M9i (cM9i) were expressed in CHO cells using standard methods. They were subsequently purified using immobilized protein A and dialyzed into PBS using sterile technique. Following expression, experiments were performed to examine the binding of both forms of M9i to human MMP-9. Both huM9i (Kd = 61 pM) and chM9i (Kd=50 pM) bound to human MMP-9 with high affinity in saturation binding assays as previously described.

[0008] The ability of M9i to inhibit the catalytic activity of human and rodent MMP-9 in a gelatinase assay was examined. Figs. 3A-3D show that M9i potently inhibited the gelatinase enzymatic activity of APMA-treated human MMP-9 (0.9 nM, 51 kDa, Fig. 2A) and APMA treated full-length human MMP-9 (1.3 nM, 75 kDa, Fig. 2B). M9i also inhibited rat MMP-9, with less potency (IC50 57 nM, Fig. 1C). M9i was a more potent inhibitor of MMP-9 enzymatic activity than small molecule, broad spectrum inhibitors such as SB 3CT, NNGH. EMD inhibitor (not shown). M9i didn't inhibit the amidolysis of a small molecular weight (non-clinical) substrate by MMP-9, unlike broad spectrum small molecule weight inhibitors. M9i potently inhibited the activity of human MMP-9 in a standard gelatinase assay using SDS-PAGE (Fig. ID).

[0059] The effect of MMP-9 inhibition by these M9i was examined in a model of ischemia followed by reperfusion. Mice were subjected to 2 hrs of ischemia with transient middle cerebral artery occlusion followed by reperfusion for 22 hours. At the time of reperfusion mice were given saline (control) or M9i by intravenous infusion. When examined 24 hours after stroke onset, treatment of wild-type mice with M9i markedly reduced neurobehavioral disability vs. wild-type mice treated with saline (Fig. 4A, p<0.001, Fig. 4B, p<0.01). Similarly, MMP-9-deficient (MMP9- / -) also showed significantly less neurobehavioral impairment than wild-type mice treated with saline (Fig. 4A, p<0.001. Fig. 4B, p<0.001). There was no significant difference between wild-type mice treated with M9i or MMP-9- / - mice treated with saline.

[0060] By comparison to WT mice treated with saline, wild-type mice treated with M9i showed significant reductions in brain swelling (Fig. 4C. p<0.05) and brain cell deathor infarction (Fig. 4D. p,0.01). Similarly, MMP-9- / - mice treated with saline showed significant reductions in brain swelling (Fig. 4C, p<0.05) and brain cell death or infarction (Fig. 4D. p,0.01), by comparison to WT mice treated with saline. There was measurable hemorrhage in WT mice treated with saline, but not in WT mice treated with M9i or MMP-9- / - mice treated with saline. There were no significant differences between WT mice treated with M9iM9i or MMP-9- / - mice treated with saline.

[0061] Most patients with ischemic stroke do not receive reperfusion therapy, which means that the pathophysiology of ischemic stroke will be altered. To examine this, ischemic stroke was induced in wild-type, C57B16 mice by a blinded operator through endovascular occlusion without reperfusion. Mice were treated with M9i or no treatment (control) after stroke onset. When examined 24 hours after stroke, mice treated with significantly less neurobehavioral disability than control mice as measured by the neurologic deficit score (5A, p<0.01) and by the comer test (5B, p<0.01). Mice treated with M9i also showed significant reductions in brain hemorrhage (5C, p<0.05), brain swelling (p<0.01) and infarction or brain cell death (p<0.05). by comparison to untreated mice. This shows that even in the absence of reperfusion, M9i was protective, reducing brain cell death, swelling, hemorrhage and disability.

[0062] Whether MMP-9 contributes to disability and hemorrhage expansion in ICH was examined. One day after ICH, control, untreated mice showed a decrease in neurobehavioral function with abnormal comer test, cylinder tests and neurological deficit scores. There was no significant difference between M9i-treated and control mice in the amount of neurobehavioral impairment on day 1. Three days after ICH, control mice showed deterioration of neurobehavioral function by comparison to day 1, with significant decrease in the comer test (p<0.05) and cylinder test (p<0.001). In contrast, by day 3 mice treated with M9i showed significant recovery7and improvement in all neurobehavioral scores by comparison to day 1 post-ICH, including the comer test (Fig. 6A, p<0.0001), cylinder test (Fig. 6B, p<0.0001). and neurologic deficit score (Fig. 6C. pO.OOOl). In every category of neurobehavioral score, mice treated with M9i showed better function on day 3 than control mice (p<0.0001). Consistent with these functional measures of neurologic function and behavior, there was significantly less growth in hemorrhage volume seen on day 3 in the brains of mice treated with M9i than control mice (p<0.0001).Similar to what was observed for M9i for treatment of ICH, another monoclonal antibody also had marked beneficial effects in ICH (Fig. 7), indicating that antibodies that reduce MMP-9 activity are useful for decreasing neurologic deficits, disability and hemorrhage, as well as improving recovery after TCH.References1. Sadaf H. Desai VR, Misra V, Golanov E, Hegde ML, Villapol S, Karmonik C, Regnier-Golanov A, Sayenko D, Homer PJ, Krencik R, Weng YL, Vahidy FS and Britz GW. A contemporary review of therapeutic and regenerative management of intracerebral hemorrhage. Ann Clin Transl Neurol. 2021;8:2211-2221.2. Flaherty ML, Haverbusch M, Sekar P, Kissela B, Kleindorfer D, Moomaw CJ, Sauerbeck L, Schneider A, Broderick JP and Woo D. 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Claims

CLAIMS1. An isolated antibody or antigen-binding fragment thereof directed against a matrix metalloproteinase 9 (MMP-9), wherein the antibody or antigen-binding fragment thereof comprises a heavy chain variable region having a heavy chain CDR1, CDR2, and CDR3 each comprising SEQ ID NO: 1, SEQ ID NO: 2, and SEQ ID NO: 3, respectively, and a light chain variable region having a light chain CDR1, CDR2, and CDR3 each comprising SEQ ID NO: 5, SEQ ID NO: 6, and SEQ ID NO: 7, respectively.

2. The antibody or antigen-binding fragment thereof of claim 1, comprising a heavy chain variable region comprising SEQ ID NO: 4, and a light chain variable region comprising SEQ ID NO: 8.

3. A pharmaceutical composition comprising the antibody or antigen-binding fragment thereof of claim 1.

4. A method for treating stroke comprising: administering to a subject in need thereof a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) inhibitor, wherein the MMP-9 inhibitor is an antibody or antigen-binding fragment thereof that binds MMP-9.

5. The method of claim 4, wherein the antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of claim 1.

6. The method of claim 4, wherein the subj ect is a human.

7. The method of claim 4, wherein the MMP-9 inhibitor is administered following ischemic stroke in the subject.

8. The method of claim 7, wherein the MMP-9 inhibitor is administered at least thirty minutes after ischemic stroke in the subject.

9. The method of claim 4, wherein the MMP-9 inhibitor is administered following intracranial hemorrhage (ICH) in the subject.

10. The method of claim 9, wherein the MMP-9 inhibitor is administered at least thirty minutes after ICH in the subject.

11. The method of claim 4, wherein the stroke is caused by trauma, surgery or other mechanical injury'.

12. The method of claim 4, wherein the treatment decreases cerebral infarction, ischemic cell death, brain lesion expansion, brain swelling, brain hemorrhage, brain injury, fibrin deposition, inflammation or a combination thereof, in the subject.

13. The method of claim 4, wherein the treatment reduces disability, neurobehavioral impairment and / or dependency on caretakers.

14. The method of claim 4, wherein the treatment prevents tissue deterioration and improves recovery from initial disability, neurobehavioral impairment and / or dependency on caretakers.

15. The method of claim 4, wherein the administration is intravenous, intramuscular, subcutaneous or intranasal.

16. A method for reducing injury' from ischemia or intracranial hemorrhage (ICH) comprising: administering to a subject in need thereof a therapeutically effective amount of a matrix metalloproteinase 9 (MMP-9) inhibitor, wherein the MMP-9 inhibitor is an antibody or antigen-binding fragment thereof that binds MMP-9.

17. The method of claim 16, wherein the antibody or antigen-binding fragment thereof is the antibody or antigen-binding fragment thereof of claim 1.

18. The method of claim 16, wherein the subj ect is a human.

19. The method of claim 16, wherein the MMP-9 inhibitor is administered following ischemic stroke in the subject.

20. The method of claim 19, wherein the MMP-9 inhibitor is administered at least thirty minutes after ischemic stroke in the subject.

21. The method of claim 16, wherein the MMP-9 inhibitor is administered following ICH in the subject.

22. The method of claim 21, wherein the MMP-9 inhibitor is administered at least thirty minutes after ICH in the subject.

23. The method of claim 16, wherein the stroke is caused by trauma, surgery or other mechanical injury.

24. The method of claim 16, wherein the treatment decreases cerebral infarction, ischemic cell death, brain lesion expansion, brain swelling, brain hemorrhage, brain injury, fibrin deposition, inflammation, or a combination thereof, in the subject..

25. The method of claim 16, wherein the treatment reduces disability, neurobehavioral impairment and / or dependency on caretakers.

26. The method of claim 16, wherein the treatment prevents tissue deterioration and improves recovery from initial disability, neurobehavioral impairment and / or dependency on caretakers.

27. The method of claim 16, wherein the administration is intravenous, intramuscular, subcutaneous or intranasal.

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