Methods and compositions for MMP-9 inhibition for use with non-genetic heterotopic ossification disorders

Abstract

The present disclosure provides methods of treating nongenetic heterotopic ossification and other MMP mediated conditions. Such methods are directed to inhibiting active MMP or inhibiting activation of MMP in therapeutic applications. Such methods include use of anti-MMP-9 antibodies directed to inhibiting MMP-9. These methods avoid the deleterious effects of treating nongenetic heterotopic ossification and other MMP mediated conditions with broad spectrum MMP inhibitors.

Description

ATTY DKT NO: ASHI-005WOMETHODS AND COMPOSITIONS FOR MMP-9 INHIBITION FOR USE WITH NON-GENETIC HETEROTOPIC OSSIFICATION DISORDERSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] Pursuant to 35 U.S.C. § 119 (e), this application claims priority to the filing dates of United States Provisional Patent Application Serial No. 63 / 703,192, filed October 3, 2024, and United States Provisional Patent Application Serial No. 63 / 795,084, filed April 25, 2025, the disclosure of which applications are incorporated herein by reference in their entirety.INCORPORATION BY REFERENCE OF SEQUENCE LISTING XML FILE

[0002] A Sequence Listing is provided herewith as a Sequence Listing XML, “ASHI- 005WO_SEQ_LISTING”, created on September 29, 2025, and having a size of 10,418 bytes. The contents of the Sequence Listing XML are incorporated herein by reference in their entirety.BACKGROUND

[0003] Matrix metalloproteinases (MMPs) are enzymes involved in extracellular matrix formation and remodeling. All MMPs share a conserved catalytic domain containing a zinc atom coordinated by three histidine residues. Types of MMPs include collagenases, gelatinases, stromelysins, matrilysins, enamelysins and membrane MMPs. Structurally, MMP-9 is a gelatinase and as such, gelatinases have a signal peptide, propeptide, catalytic, zinc-binding and hemopexin-like domains, common to MMPs, as well as a plurality of fibronectin-like domains and an O-glycosylated domain. MMP-9, in particular, has a hemopexin-like domain, catalytic domain, signal peptide, hinge region and propeptide region. The catalytic domain of MMP-9 contains fibronectin type II (FN2) domains, an active site and a zinc-binding region, and the activity of the MMP-9 enzyme depends on zinc. MMPs are capable of degrading numerous extracellular matrix (ECM) components, as well as affecting cell-cell and cell-matrix interactions. MMP-9 is involved in many developmental processes, including ECM degradation, angiogenesis and formation of endochondral bone (for which it appears to play a unique role in the MMP family). Ortega N, et al., Complementary interplay between matrix metalloproteinase-9, vascular endothelial growth factor and osteoclast function drives endochondral bone formation,ATTY DKT NO: ASHI-005WODisease Models & Mechanisms (2010) 3, 224-235. MMP9 in particular is found to be highly elevated in many human diseases, compared to limited expression in healthy tissues. Studies in knockout mice reveal opposing roles for MMP9 and its most closely related MMP, MMP2, the only other gelatinase. While loss of MMP9 confers protection against disease in a variety of different models, loss of MMP2 tends to lack this benefit or even result in disease exacerbation. Substrates of MMP9 include matrix proteins, growth factors and cytokines, and in addition to extracellular matrix remodeling, MMP9 can render growth factors bioavailable (e.g. VEGF, from heparin sulfate proteoglycans) or potentiate the activity of cytokines by cleavage. MMP9 has also been shown to be involved in cell proliferation, migration, invasion and epithelial-mesenchymal transition (EMT) (Farina AR, Mackay AR. Gelatinase B / MMP-9 in Tumour Pathogenesis and Progression. Cancers (Basel). 2014;6:240-296).

[0004] Numerous publications indicate MMPs as suitable targets to treat cardiovascular diseases, autoimmune diseases, and cancer. Despite concerted efforts to target MMPs, development of MMP inhibitors for various indications has been difficult perhaps due to the conserved catalytic domain shared among the different MMPs. Small molecule inhibitors that target the catalytic domain not only affect a specific MMP implicated in pathogenesis, but also essential MMPs needed for normal functioning. For example, a broad-spectrum MMP inhibitor was found to have musculoskeletal toxicity. Another broadspectrum inhibitor, doxycycline, which is approved for the treatment of periodontal disease, appears to inhibit MMP-2 synthesis and activity. Hanemaaijer, R. el al. Inhibition of MMP synthesis by doxycycline and chemically modified tetracyclines (CMTs) in human endothelial cells. Advances in dental research 12, 114-118 (1998).

[0005] Directed therapies are in development, such as andecaliximab (GS-5745), an anti- MMP9 recombinant chimeric, humanized antibody that was evaluated in several clinical trials for a number of conditions including chronic obstructive pulmonary disease, gastric adenocarcinoma, Crohn’s disease, rheumatoid arthritis, cystic fibrosis, various solid tumors and ulcerative colitis. GS-5745 binds MMP9 in the catalytic domain at residue R162.

[0006] Non-genetic heterotopic ossification (HO), also known as non-hereditary HO (NHHO), acquired HO, neurogenic HO, or trauma-induced HO, is a musculoskeletal disorder in which bone forms at an extra-osseous site. Trauma-induced HO is a result of tissue repair gone awry, as ectopic bone forms in soft tissues after severe bums or trauma. It is often associated with extensive tissue injury and inflammation. Development of HO isATTY DKT NO: ASHI-005WO also associated with predisposing, often pro-inflammatory, conditions such as axial spondylarthritis or ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis (DISH). HO has also been described for patients experiencing serious complications as result of infection, such as SarsCoV2 / COVID19.

[0007] Nongenetic HO commonly occurs after trauma or surgery. It occurs in up to 40% of hip arthroplasty cases, 30% of bone fracture or dislocation cases, high-energy extremity trauma, spinal cord injury, trauma, brain injuries, bums, fractures, muscle contusion, joint arthropl as t / replacement, hip surgery / replacement, acetabular surgery / replacement, elbow fracture, fracture of the long bones of the lower leg, combat-related trauma, amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, and nontraumatic myelopathy, and other neurological disorders, and over 90% of severe traumatic amputations. (Meyers C, et al. Heterotopic Ossification: A Comprehensive Review. JBMR Plus. 3(4)(2019):el0172. doi: 10. 1002 / jbm4.10172).

[0008] Heterotopic ossification occurs in one or more tissues selected from the group consisting of: bone, skin, subcutaneous tissue, skeletal muscle, tendons, aponeuroses, facia, fibrous tissue adjacent to joints, fibrotic tissue, walls of blood vessels, ligaments and entheses.

[0009] Patients with certain inflammatory and / or autoimmune diseases can also be predisposed to non-genetic HO. For example, patients with ankylosing spondylitis are at elevated risk with 10 - 15% developing clinically significant HO. These patients are also at high risk for requiring hip replacement surgery and associated HO. Independently, MMP9 complexed with LCN2 has been identified as a risk factor for ankylosing development and progression in patients with ankylosing spondylitis. Tsui FWL, et al., The role of LCN2 and LCN2-MMP9 in spondylitis radiographic development: gender and HLA-B27 status differences, Arthritis Research & Therapy (2022) 24: 164.

[0010] Neurogenic HO is a complication that can result from trauma of the central nervous system and is observed in >20% of patients with traumatic brain injury and / or spinal cord injury. Neurogenic HO can also result from sports injury-associated traumatic brain injury.

[0011] MMP9 could be playing several key roles in non-genetic HO. Chronic, pathologic inflammation and associated wound healing would yield a high local concentration of MMP9, which is strongly induced by a variety of inflammatory stimuli, including hypoxia. While the precise mechanism of nongenetic HO remains to be elucidated, it is clear that osteogenic BMP signaling is required. BMP signaling is matrix regulated, with tethering ofATTY DKT NO: ASHI-005WOBMP ligands (such as BMP2, 4, and others) to extracellular matrix proteins such as heparan sulfate proteoglycans (e.g. perlecan) and fibrillin, which constrains their activity. These proteins are MMP9 substrates. For example, MMP9 generates bioavailable VEGF by release from heparan sulfate proteoglycans. MMP9 could promote BMP signaling via BMPs produced by both macrophages and early progenitors that differentiate into chondrocytes or osteoblasts, and continue to drive chondrocyte or osteoblast activation via provision of bioavailable BMPs. It could then further promote formation of bone via matrix remodeling, angiogenesis (including VEGF), and recruitment of other progenitors. MMP9 could be playing additional roles in the recruitment and or activation of progenitors. Research in mice shows that MMP-9 is involved in osteoblast progenitors passing through the endoneurial barrier into circulation during HO and is significantly more active in tissues within 24 hours of induction of HO. Salisbury E, et al. Sensory nerve induced inflammation contributes to heterotopic ossification. J Cell Biochem 112:2748-2758 (2011); Lazard ZW, et al. Osteoblasts have a neural origin in heterotopic ossification. Clin Orthop Relat Res 473:2790-2806 (2015).

[0012] Current treatment options for nongenetic HO include physical therapy and surgery. Meyers C, et al. Heterotopic Ossification: A Comprehensive Review. JBMR Plus.3(4)(2019):el0172. doi: 10.1002 / jbm4. 10172. Because of a lack of direct comparison trials on the effectiveness of passive range-of-motion exercises, physical therapy is recommended based on clinician preferences. Surgery is recommended after osseous maturation is complete, usually about six months after the onset of nongenetic HO. A challenge of surgical treatment of nongenetic HO is that complete resection may be impossible, and remaining HO lesions may cause recurrence of the condition. Furthermore, surgery risks injury to major neurovascular structures, which nongenetic HO may encase. There are no approved therapies for nongenetic HO. Pharmacological treatments as disodium etidronate have been tested for the treatment of nongenetic HO; however, there is insufficient evidence to recommend the use of pharmacological agents for the treatment of HO. Haran MJ, et al., Pharmacological interventions for treating acute heterotopic ossification (Review), Cochrane Database of Systematic Reviews 2004, Issue 4. Art. No.: CD00332.

[0013] What is needed, therefore, is a therapy or therapies to remedy musculoskeletal diseases or conditions such as nongenetic HO by inhibiting MMP-9, resulting in effective treatment or improved clinical outcomes in subjects with such disorders without the deleterious effects of broad-spectrum MMP inhibitor.ATTY DKT NO: ASHI-005WOSUMMARY OF THE INVENTION

[0014] In various aspects of the invention, disclosed herein are methods of treating, e.g., reducing the likelihood of developing a novel heterotopic ossification lesion, or exacerbating an existing heterotopic ossification lesion such as, e.g., following a trauma or injury, in a subject having or at risk of developing a nongenetic heterotopic ossification condition, i.e. a non-genomically encoded condition. Such a nongenetic heterotopic ossification condition may be referred to herein as an NHHO. The methods of the disclosure comprise administering to a subject an inhibitor of MMP-9 activation or activity. In exemplary embodiments, disclosed herein, are methods of treating or preventing recurrence of a nongenetic heterotopic ossification condition comprising administering to a subject an effective dose of an anti-MMP-9 antibody or an antigen-binding fragment thereof. In some embodiments the antibody comprises the CDR sequences of andecaliximab. In some embodiments the antibody comprises the variable region sequences of andecaliximab. In some embodiments the antibody is andecaliximab. In some embodiments, described herein, are methods of treating one or more musculoskeletal disease or condition comprising administering an inhibitor of MMP-9 activation or activity. Additional embodiments provide methods of treating a connective tissue disorder comprising administering an inhibitor of MMP-9 activation or activity.

[0015] In an embodiment the methods of the disclosure prevent or reduce the progression of at least one symptom of an NHHO. In an embodiment the least one symptom is selected from the group consisting of number of heterotopic ossifications, size of heterotopic ossifications, volume of heterotopic ossifications, growth of heterotopic ossifications, formation of heterotopic ossifications, and formation of new heterotopic ossifications at either adjacent or independent sites. In an embodiment the heterotopic ossification occurs in one or more tissues selected from bone, skin, subcutaneous tissue, skeletal muscle, tendons, fibrous tissue adjacent to joints, fibrotic tissue, walls of blood vessels, entheses, fascia, aponeuroses and ligaments. In an embodiment the one or more lesions of heterotopic ossification are located in the vicinity of the hips and / or shoulders.

[0016] In some embodiments an individual selected for treatment has suffered a traumatic injury prior to treatment. The traumatic injury may include spinal cord injury or traumatic brain injury, and / or disruption of the blood-brain, blood-spinal cord, or peripheral nerve barrier. The treatment may be initially administered within 1 week, 2 weeks, 3 weeks, 4ATTY DKT NO: ASHI-005WO weeks, 1 month, 2 months, 3 months or more after the traumatic injury. The treatment may be initially administered at detection of or suspicion of HO formation by one or more of ultrasound, triple phase bone scan, CT scan, and X ray. The treatment may be administered for a period of time such that the total treatment time is up to about 6 months. Administration may be intravenous, subcutaneous, or a combination thereof. For example, an anti-MMP9 antibody or an antigen-binding fragment thereof may be administered intravenously at a dose of about 400-800 mg every week, 800 mg every 2 weeks, 1200 mg every 3 weeks, 1600 mg every 4 weeks. In another example, an anti-MMP9 antibody or an antigen-binding fragment thereof may be administered subcutaneously at a dose of about 150 mg to 600 mg weekly dose, where the dose is optionally fractionated. In another example an anti-MMP9 antibody or an antigen-binding fragment thereof may be administered intravenously at a dose of about 400-800 mg every week, 800 mg every 2 weeks, 1200 mg every 3 weeks, or 1600 mg every 4 weeks; followed by the anti-MMP9 antibody or an antigen-binding fragment thereof being administered subcutaneously at a dose of about 150 mg to 600 mg weekly dose, where the dose is optionally fractionated.

[0017] In some embodiments an individual selected for treatment has undergone a surgery that predisposes to heterotopic ossification. The subject may be at risk of heterotopic ossification due to presence of existing lesion(s) of heterotopic ossification that are to be surgically excised, wherein there is a risk of formation or re-formation of heterotopic ossification at 1 or more sites. The subject may be at risk of heterotopic ossification due to a first or second joint replacement surgery of the hip, shoulder or knee, and / or surgery of the hip, shoulder, knee or elbow, and / or amputation of one or more limbs or portion(s) of a limb, wherein there is a risk of formation or re-formation of heterotopic ossification at 1 or more sites. The treatment may be administered starting within about 1, 2, 3, or 4 weeks after surgery, and continuing for up to about 6 months after surgery. The treatment may be administered about 1 or 2 months prior to surgery and for about 6 months following surgery. Administration may be intravenous, subcutaneous, or a combination thereof. In an example, anti-MMP-9 antibody or antigen-binding fragment thereof is administered at a dose of 150, 300 or 450 mg by subcutaneous route every week, or twice a week, or 3 times a week, for about 1 or 2 months prior to surgery and for about 6 months following surgery. In another example, an anti-MMP-9 antibody or antigen-binding fragment thereof is administered at a dose of 150, 300 or 450 mg by subcutaneous route every week, or twice aATTY DKT NO: ASHI-005WO week, or 3 times a week with administration starting within about 1 , 2, 3, or 4 weeks after the injury or surgery, and continuing for about 6 months after the injury or surgery.

[0018] In some embodiments an individual selected for treatment suffers from a disease, e.g. an autoimmune or inflammatory disease, associated with a predisposition to heterotopic ossification. In some embodiments the disease is axial spondylarthritis, including ankylosing spondylitis; or diffuse idiopathic skeletal hyperostosis. In an example, an anti- MMP-9 antibody or antigen-binding fragment thereof is administered at a dose of 150, 300 or 450 mg, optionally fractionated, by subcutaneous route every week, or twice a week, or 3 times a week for the duration of the disease condition. In another example, an anti-MMP-9 antibody or antigen-binding fragment thereof is administered at a dose of 150, 300 or 450 mg, optionally fractionated, by subcutaneous route every week, or twice a week, or 3 times a week for about 1 or 2 months followed by a regimen of 150 or 300 mg by subcutaneous route every week, or twice a week, or 3 times a week for the duration of the disease condition.

[0019] In certain embodiments, the disclosed is a method of preventing NHHO in a subject having or at risk of developing a nongenetic heterotopic ossification condition, comprising administering to a human subject an effective amount of an inhibitor of MMP-9 activation or activity. In some embodiments the inhibitor is an antibody that binds to and inhibits MMP9 or an antigen-binding fragment thereof. In some embodiments the antibody comprises the CDR sequences of andecaliximab. In some embodiments the antibody comprises the variable region sequences of andecaliximab. In some embodiments the antibody is andecaliximab.

[0020] In additional embodiments, disclosed is a pharmaceutical composition comprising dosage of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 600 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg or about 1500 mg of an MMP-9 inhibitory antibody, such as a specific anti- MMP9 therapeutic antibody (e.g., andecaliximab), suitable for subcutaneous or intravenous administration to a human subject having a spinal cord injury, trauma, brain injuries, burns, fractures, muscle contusion, joint arthroplasty / replacement, hip surgery / replacement, acetabular surgery / replacement, elbow fracture, fracture of the long bones of the lower leg, combat-related trauma, amputation, neuromuscular blockade used to manage adult respiratory distress syndrome or a nontraumatic myelopathy.ATTY DKT NO: ASHI-005WO

[0021] In some embodiments, a biomarker analysis identifies the subject as exhibiting formation of NHHO by detecting modulation in expression of a one or more biomarkers selected from Prostaglandin E Receptor 2 (PGE2), MMP-9, and / or Bone Morphogenetic Protein 2 (BMP-2) nucleic acid or polypeptide as compared to the expression of the one or more biomarkers in a reference population.

[0022] In further embodiments, disclosed is a dosage regimen comprising administration of an MMP-9 inhibitor (such as andecaliximab) to an individual in need thereof of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 600 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg or about 1600 mg every day, or every 2, 3, 4, 5, 6, 8, 10, 12, 14, 16, 18 days, or every week, twice a week, every 2 weeks, every 3 weeks, every 4 weeks; for a duration of , two weeks, 4 weeks, 6 weeks, 8 weeks, 10 weeks, 1, 2, 3, 4, 5, 6 months, or up to 1 year, or up to 2 years, or as long as indicated by evidence of inflammation or heterotopic ossification in the region of concern. A dosage regimen, e.g. for subcutaneous delivery, may optionally be fractionated into multiple doses.

[0023] In an embodiment, an anti-MMP9 antibody or an antigen-binding fragment thereof is administered intravenously at a dose of about 400-800 mg every week, 800 mg every 2 weeks, 1200 mg every 3 weeks, 1600 mg every 4 weeks. In an embodiment, an anti-MMP9 antibody or an antigen-binding fragment thereof is administered subcutaneously at a dose of about 150 mg to 600, 150 mg to 450 mg weekly dose, where the dose is optionally fractionated into multiple doses.

[0024] In an embodiment, an individual suffering from NHHO or at risk of NHHO is treated by administration of an effective dose of an MMP9 inhibitor for a period of time sufficient to reduce de novo HO. In some embodiments the inhibitor is an antibody that binds to and inhibits MMP9 or an antigen-binding fragment thereof. In some embodiments the antibody comprises the CDR sequences of andecaliximab. In some embodiments the antibody comprises the variable region sequences of andecaliximab. In some embodiments the antibody is andecaliximab. The effective dose may be at least about 150 mg and up to about 1500 mg. The antibody may be delivered subcutaneously. The antibody may be delivered intravenously. The MMP9 inhibitor may be administered once every week for at least 4, 5, 6, 7, 8, 9, 10, I I, 12 weeks or 4, 5, 6, 7, 8 months or greater than 8 months. In some embodiments the individual has suffered a traumatic injury. In some embodiments the individual has undergone a surgery that predisposes to heterotopic ossification. In someATTY DKT NO: ASHI-005WO embodiments, the first administration occurs within 1 week, or within 2, 3, 4, 5, 6, 7, 8 weeks of the traumatic injury or surgery, for example, treatment may commence within a period of from about 10 days to about 30 days following injury or surgery. In some cases, the individual is from about 18 to 89 years of age; and may be from about 18 to 45 years of age. In some embodiments therapy is initiated based on the presence of clinical indicia indicating a suspicion of NHHO development, which indicia include without limitation inflammation consistent with development of NHHO, and / or reduction in range of motion of the affected limb or joint. Such inflammation is typically localized, with sustained swelling, fever, vascularization, etc. An individual may be selected for treatment based on the presence of minimal extraskeletal uptake, e.g. as determined by a triple scan, a triple phase bone scan, an ultrasound, a CT, or an X-ray.

[0025] In some embodiments, the subject is a human. In some embodiments the human is an adult, for example aged 18 years or above, e.g. from about 18 to 80 years of age, 18 to 65 years of age, 18 to 45 years of age, etc. In some embodiments, the subject is under about thirty years of age. In some embodiments, the subject is aged 12 years or above. In some embodiments, the subject is aged 12 years or above, or 18 years or above. In some embodiments, the subject is aged 6-12 years. In some embodiments, the subject is aged 2-5 years. In some embodiments the antibody is administered subcutaneously. In some embodiments the antibody is administered intravenously. In some embodiments, a loading dose is administered, followed by administration of maintenance doses. In some embodiments, the maintenance dose is administered every other week.

[0026] In embodiments where the subject is a pediatric patient, for example under 18 years of age, under 12 years of age, etc., the dose may be appropriately scaled from the adult doses disclosed herein, using methods known in the art. Administration may be subcutaneous, intravenous, etc., and is optionally fractionated into multiple doses. In some embodiments, a loading dose is administered, followed by administration of maintenance doses. As non-limiting examples, in some embodiments, the subject is aged 2-5 years, the loading dose of an MMP-9 inhibitor (such as andecaliximab) is 100 mg, and the maintenance dose is 30 mg, which may be administered every other week. In another embodiment, the subject is aged between 2 and about 5 years and an MMP-9 inhibitor dose of 90 or 100 mg is administered every week. In another embodiment, the subject is aged between 2 and about 5 years and an MMP-9 inhibitor dose of 60 mg is administered every week. In some embodiments, the subject is aged 2-5 years, and a dose of 90 or 100 mg isATTY DKT NO: ASHI-005WO administered every other week. In some embodiments, the subject is aged 2-5 years, and a dose of 30 mg is administered every other week. In some embodiments, the subject is aged 2-5 years, and a dose of 90 or 100 mg is administered every other week.

[0027] In some embodiments, the subject is aged 6-11 years, the loading dose is 150 to 300 mg, and the maintenance dose is 50 to 150 mg. In an embodiment, the subject is aged 6-11 years, and a dose of 100 mg is administered every week. In an embodiment, the subject is aged between about 6 to about 11 years and an MMP-9 inhibitor dose of 150 mg is administered every week. In another embodiment, the subject is aged between about 6 to about 1 1 years and an MMP-9 inhibitor dose of 200 mg is administered every week. In an embodiment, the subject is aged 6-11 years, and a dose of 300 mg is administered every week.

[0028] In an embodiment, the subject is greater than 12 years of age, e.g. from about 12 to about 18 years of age, or an adult, and an antibody MMP9 inhibitor is administered at an initial dose of from about 800 mg to about 1600 mg. For example, 400-800 mg may be delivered weekly, 800 mg may be delivered over about 2 weeks, 1200 mg may be delivered over about 3 weeks, 1600 mg may be delivered over about 4 weeks. In another embodiment the subject is greater than 12 years of age and an antibody MMP9 inhibitor is administered subcutaneously at a maintenance dose of 150 to 300 mg every 10 to 18 days.

[0029] In additional embodiments, disclosed is a pharmaceutical composition comprising an MMP-9 inhibitor, in a dosage determined by the presence and / or level in the human subject in need thereof of at least one biomarker selected from PGE2, MMP-9, and BMP-2.

[0030] In additional embodiments, the administration of an MMP9 inhibitor is determined by the presence of an inflammatory signal, as determined by the first phase of a triple phase bone scan, X-ray, computed tomography, the second phase of a triple phase bone scan, or via ultrasound.

[0031] In additional embodiments, the administration of an MMP9 inhibitor is determined by existing risk factors associated with the individual experiencing an injury, surgery or proinflammatory disease.

[0032] In further embodiments, disclosed is a method of preventing or reducing the severity and / or duration of a condition associated with a nonhereditary heterotopic ossification (NHHO) comprising administering to a human subject an effective amount of an inhibitor of MMP-9 activation or activity.ATTY DKT NO: ASHI-005WOBRIEF DESCRIPTION OF THE DRAWINGS

[0033] FIG 1. Shows the quantification of soft tissue HO (in (iCT) present in both the WT and MMP-9 KO mice, and a sample image of soft tissue HO in both WT and KO mice. This figure demonstrates significant inhibition of NHHO demonstrated with an MMP-9 blockade, as HO originating in soft tissue was significantly reduced in MMP-9 KO mice compared to wild-type littermate controls.

[0034] FIG. 2 describes the total measured heterotopic bone (in pCT) present in both the WT and KO MMP-9 mice. HO is measured in soft tissue and exostoses attached to calcaneum, and depicts that HO was significantly reduced in MMP-9 KO mice compared to wild-type littermate controls.DETAILED DESCRIPTIONDEFINITIONS

[0035] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art. Further, unless otherwise required by context, singular terms shall include the plural and plural terms shall include the singular. Generally, nomenclatures used in connection with, and techniques of, biochemistry, enzymology, molecular and cellular biology, microbiology, genetics and protein and nucleic acid chemistry and hybridization described herein are those well-known and commonly used in the art.

[0036] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0037] All publications, patents and other references mentioned herein are hereby incorporated by reference in their entirety.

[0038] The following terms, unless otherwise indicated, shall be understood to have the following meanings:

[0039] Throughout this specification and claims, the word “comprise” or variations such as “comprises” or “comprising”, will be understood to imply the inclusion of a stated integer or group of integers but not the exclusion of any other integer or group of integers.ATTY DKT NO: ASHI-005WO

[0040] The term “dosing” refers to the administration of one or more compositions (e.g., an anti-MMP-9 antibody) in a subject with the associated MMP-9 disorder.

[0041] The term “MMP-9 mediated disorder” generally refers to disease or conditions in which MMP-9 activity or expression is implicated or associated with one or more diseases, or conditions.

[0020] The term “combination” therapy refers to the administration of one or more therapeutic compositions, e.g., a JAK inhibitor with an MMP-9 inhibitor or binding protein; or a BTK inhibitor with an MMP-9 inhibitor or binding protein; or a BMP receptor kinase inhibitor with an MMP-9 inhibitor or binding protein; or an MMP-9 inhibitor or binding protein with sarcatnib; or an MMP-9 inhibitor or binding protein with a TNF inhibitor; or an inhibitor of another MMP, including, but not limited to, MMP-2, 7, 14, and / or 16, with a MMP-9 inhibitor or binding protein; or an inhibitor of the BMP signaling pathway, or an anti-TNF agent.

[0042] The term “polynucleotide” or “nucleic acid molecule” or “nucleotide sequence” refers to a polymeric form of nucleotides of at least 10 bases in length. The term includes DNA molecules (e.g., cDNA or genomic or synthetic DNA) and RNA molecules (e.g., mRNA or synthetic RNA), as well as analogs of DNA or RNA containing non-natural nucleotide analogs, non-native intemucleoside bonds, or both. The nucleic acid can be in any topological conformation. For instance, the nucleic acid can be single-stranded, doublestranded, triple-stranded, quadruplexed, partially double-stranded, branched, hairpinned, circular, or in a padlocked conformation.

[0043] Unless otherwise indicated, and as an example for all sequences described herein under the general format “SEQ ID NO:”, “nucleic acid comprising SEQ ID NO:1” refers to a nucleic acid, at least a portion of which has either (i) the sequence of SEQ ID NO:1, or (ii) a sequence complementary to SEQ ID NO:1. The choice between the two is dictated by the context. For instance, if the nucleic acid is used as a probe, the choice between the two is dictated by the requirement that the probe be complementary to the desired target.

[0044] An “isolated” RNA, DNA or a mixed polymer is one which is substantially separated from other cellular components that naturally accompany the native polynucleotide in its natural host cell, e.g., ribosomes, polymerases and genomic sequences with which it is naturally associated.

[0045] The term “recombinant” refers to a biomolecule, e.g., a gene or protein, that (1) has been removed from its naturally occurring environment, (2) is not associated with all or aATTY DKT NO: ASHI-005WO portion of a polynucleotide in which the gene is found in nature, (3) is operatively linked to a polynucleotide which it is not linked to in nature, or (4) does not occur in nature. The term “recombinant” can be used in reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs that are biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0046] As used herein, an endogenous nucleic acid sequence in the genome of an organism (or the encoded protein product of that sequence) is deemed “recombinant” herein if a heterologous sequence is placed adjacent to the endogenous nucleic acid sequence, such that the expression of this endogenous nucleic acid sequence is altered. In this context, a heterologous sequence is a sequence that is not naturally adjacent to the endogenous nucleic acid sequence, whether or not the heterologous sequence is itself endogenous (originating from the same host cell or progeny thereof) or exogenous (originating from a different host cell or progeny thereof). By way of example, a promoter sequence can be substituted (e.g., by homologous recombination) for the native promoter of a gene in the genome of a host cell, such that this gene has an altered expression pattern. This gene would now become “recombinant” because it is separated from at least some of the sequences that naturally flank it.

[0047] A nucleic acid is also considered “recombinant” or “engineered” if it contains any modifications that do not naturally occur to the corresponding nucleic acid in a genome. For instance, an endogenous coding sequence is considered “recombinant” or “engineered” if it contains an insertion, deletion or a point mutation introduced artificially, e.g., by human intervention. A “recombinant” or “engineered” nucleic acid also includes a nucleic acid integrated into a host cell chromosome at a heterologous site and a nucleic acid construct present as an episome.

[0048] As used herein, the term “modification,” with reference to a nucleic acid sequence, refers to a nucleic acid sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of nucleotide compared to a reference nucleic acid sequence. As used herein, the term “modification,” with reference to an amino acid sequence refers to an amino acid sequence that comprises at least one substitution, alteration, inversion, addition, or deletion of an amino acid residue compared to a reference nucleic acid sequence.ATTY DKT NO: ASHI-005WO

[0049] As used herein, the phrase “degenerate variant” of a reference nucleic acid sequence encompasses nucleic acid sequences that can be translated, according to the standard genetic code, to provide an amino acid sequence identical to that translated from the reference nucleic acid sequence. The term “degenerate oligonucleotide” or “degenerate primer” is used to signify an oligonucleotide capable of hybridizing with target nucleic acid sequences that are not necessarily identical in sequence but that are homologous to one another within one or more particular segments.

[0050] The term “percent sequence identity” or “identical” in the context of nucleic acid sequences refers to the residues in the two sequences which are the same when aligned for maximum correspondence. The length of sequence identity comparison may be over a stretch of at least about nine nucleotides, usually at least about 20 nucleotides, more usually at least about 24 nucleotides, typically at least about 28 nucleotides, more typically at least about 32 nucleotides, and preferably at least about 36 or more nucleotides. There are a number of different algorithms known in the art which can be used to measure nucleotide sequence identity. For instance, polynucleotide sequences can be compared using FASTA, Gap or Bestfit, which are programs in Wisconsin Package Version 10.0, Genetics Computer Group (GCG), Madison, Wis. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (hereby incorporated by reference in its entirety). For instance, percent sequence identity between nucleic acid sequences can be determined using FASTA with its default parameters (a word size of 6 and the NOPAM factor for the scoring matrix) or using Gap with its default parameters as provided in GCG Version 6.1, herein incorporated by reference. Alternatively, sequences can be compared using the computer program, BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0051] The term “substantial homology” or “substantial similarity,” when referring to a nucleic acid or fragment thereof, indicates that, when optimally aligned with appropriate nucleotide insertions or deletions with another nucleic acid (or its complementary strand), there is nucleotide sequence identity in at least about 76%, 80%, 85%, preferably at least about 90%, and more preferably at least about 95%, 96%, 97%, 98% or 99% of theATTY DKT NO: ASHI-005WO nucleotide bases, as measured by any well-known algorithm of sequence identity, such as FASTA, BLAST or Gap, as discussed above.

[0052] Alternatively, substantial homology or similarity exists when a nucleic acid or fragment thereof hybridizes to another nucleic acid, to a strand of another nucleic acid, or to the complementary strand thereof, under stringent hybridization conditions. “Stringent hybridization conditions” and “stringent wash conditions” in the context of nucleic acid hybridization experiments depend upon a number of different physical parameters. Nucleic acid hybridization will be affected by such conditions as salt concentration, temperature, solvents, the base composition of the hybridizing species, length of the complementary regions, and the number of nucleotide base mismatches between the hybridizing nucleic acids, as will be readily appreciated by those skilled in the art. One having ordinary skill in the art knows how to vary these parameters to achieve a particular stringency of hybridization.

[0053] In general, “stringent hybridization” is performed at about 25°C below the thermal melting point I for the specific DNA hybrid under a particular set of conditions. “Stringent washing” is performed at temperatures about 5 °C lower than the Tm for the specific DNA hybrid under a particular set of conditions. The Tm is the temperature at which 50% of the target sequence hybridizes to a perfectly matched probe. See Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1989), page 9.51, hereby incorporated by reference. For purposes herein, “stringent conditions” are defined for solution phase hybridization as aqueous hybridization (i.e., free of formamide) in 6xSSC (where 20xSSC contains 3.0 M NaCl and 0.3 M sodium citrate), 1% SDS at 65°C for 8-12 hours, followed by two washes in 0.2xSSC, 0.1% SDS at 65°C for 20 minutes. It will be appreciated by the skilled worker that hybridization at 65°C will occur at different rates depending on a number of factors including the length and percent identity of the sequences which are hybridizing.

[0054] The nucleic acids (also referred to as polynucleotides) may include both sense and antisense strands of RNA, cDNA, genomic DNA, and synthetic forms and mixed polymers of the above. They may be modified chemically or biochemically or may contain nonnatural or derivatized nucleotide bases, as will be readily appreciated by those of skill in the art. Such modifications include, for example, labels, methylation, substitution of one or more of the naturally occurring nucleotides with an analog, internucleotide modifications such as uncharged linkages (e.g., methyl phosphonates, phosphotriesters,ATTY DKT NO: ASHI-005WO phosphoramidates, carbamates, etc.), charged linkages (e.g., phosphorothioates, phosphorodithioates, etc.), pendent moieties (e.g., polypeptides), intercalators (e.g., acridine, psoralen, etc.), chelators, alkylators, and modified linkages (e.g., alpha anomeric nucleic acids, etc.) Also included are synthetic molecules that mimic polynucleotides in their ability to bind to a designated sequence via hydrogen bonding and other chemical interactions. Such molecules are known in the art and include, for example, those in which peptide linkages substitute for phosphate linkages in the backbone of the molecule. Other modifications can include, for example, analogs in which the ribose ring contains a bridging moiety or other structure such as the modifications found in “locked” nucleic acids.

[0055] The term “mutated” when applied to nucleic acid sequences means that nucleotides in a nucleic acid sequence may be inserted, deleted or changed compared to a reference nucleic acid sequence. A single alteration may be made at a locus (a point mutation) or multiple nucleotides may be inserted, deleted or changed at a single locus. In addition, one or more alterations may be made at any number of loci within a nucleic acid sequence. A nucleic acid sequence may be mutated by any method known in the art including but not limited to mutagenesis techniques such as “error-prone PCR” (a process for performing PCR under conditions where the copying fidelity of the DNA polymerase is low, such that a high rate of point mutations is obtained along the entire length of the PCR product; see, e.g., Leung et al., Technique, 1:11-15 (1989) and Caldwell and Joyce, PCR Methods Applic. 2:28-33 (1992)); and “oligonucleotide-directed mutagenesis” (a process which enables the generation of site-specific mutations in any cloned DNA segment of interest; see, e.g., Reidhaar-Olson and Sauer, Science 241:53-57 (1988)).

[0056] The term “attenuate” as used herein generally refers to a functional deletion, including a mutation, partial or complete deletion, insertion, or other variation made to a gene sequence or a sequence controlling the transcription of a gene sequence, which reduces or inhibits production of the gene product, or renders the gene product nonfunctional. In some instances, a functional deletion is described as a knockout mutation. Attenuation also includes amino acid sequence changes by altering the nucleic acid sequence, placing the gene under the control of a less active promoter, down-regulation, expressing interfering RNA, ribozymes or antisense sequences that target the gene of interest, or through any other technique known in the art. In one example, the sensitivity of a particular enzyme to feedback inhibition or inhibition caused by a composition that is not a product or a reactant (non-pathway specific feedback) is lessened such that the enzymeATTY DKT NO: ASHI-005WO activity is not impacted by the presence of a compound. Tn other instances, an enzyme that has been altered to be less active can be referred to as attenuated.

[0057] The term “vector” as used herein 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 generally refers to a circular double stranded DNA loop into which additional DNA segments may be ligated, but also includes linear double- stranded molecules such as those resulting from amplification by the polymerase chain reaction (PCR) or from treatment of a circular plasmid with a restriction enzyme. Other vectors include cosmids, bacterial artificial chromosomes (BAC) and yeast artificial chromosomes (YAC). Another type of vector is a viral vector, wherein additional DNA segments may be ligated into the viral genome (discussed in more detail below). Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., vectors having an origin of replication which functions in the host cell). Other vectors can be integrated into the genome of a host cell upon introduction into the host cell and are thereby replicated along with the host genome. Moreover, certain preferred 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”).

[0058] The term “operatively linked” or “operably linked” expression control sequences refers to a linkage in which the expression control sequence is contiguous with the gene of interest to control the gene of interest, as well as expression control sequences that act in trans or at a distance to control the gene of interest.

[0059] The term “expression control sequence” as used herein refers to polynucleotide sequences which are necessary to affect the expression of coding sequences to which they are operatively linked. Expression control sequences are sequences which control the transcription, post-transcriptional events and translation of nucleic acid sequences. Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency e.g., ribosome binding sites); sequences that enhance protein stability; and when desired, sequences that enhance protein secretion. The nature of such control sequences differs depending upon the host organism; in prokaryotes, such control sequences generally include promoter, ribosomal binding site, and transcription termination sequence. The term “control sequences” is intended to include, at a minimum, allATTY DKT NO: ASHI-005WO components whose presence is essential for expression, and can also include additional components whose presence is advantageous, for example, leader sequences and fusion partner sequences.

[0060] The term “recombinant host cell” (or simply “host cell”), as used herein, is intended to refer to a cell into which a recombinant vector has been introduced. It should be understood that such terms are 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 cell, but are still included within the scope of the term “host cell” as used herein. A recombinant host cell may be an isolated cell or cell line grown in culture or may be a cell which resides in a living tissue or organism.

[0061] The term “peptide” as used herein refers to a short polypeptide, e.g., one that is typically less than about 50 amino acids long and more typically less than about 30 amino acids long. The term as used herein encompasses analogs and mimetics that mimic structural and thus biological function.

[0062] The term “polypeptide” encompasses both naturally-occurring and non-naturally- occurring proteins, and fragments, mutants, derivatives and analogs thereof. A polypeptide may be monomeric or polymeric. Further, a polypeptide may comprise a number of different domains each of which has one or more distinct activities.

[0063] The term “isolated protein” or “isolated polypeptide” is a protein or polypeptide that by virtue of its origin or source of derivation (1) is not associated with naturally associated components that accompany it in its native state, (2) exists in a purity not found in nature, where purity can be adjudged with respect to the presence of other cellular material (e.g., is free of other proteins from the same species) (3) is expressed by a cell from a different species, or (4) does not occur in nature (e.g., it is a fragment of a polypeptide found in nature or it includes amino acid analogs or derivatives not found in nature or linkages other than standard peptide bonds). Thus, a polypeptide that is chemically synthesized or synthesized in a cellular system different from the cell from which it naturally originates will be “isolated” from its naturally associated components. A polypeptide or protein may also be rendered substantially free of naturally associated components by isolation, using protein purification techniques well known in the art. As thus defined, “isolated” does not necessarily require that the protein, polypeptide, peptide or oligopeptide so described has been physically removed from its native environment.ATTY DKT NO: ASHI-005WO

[0064] The term “polypeptide fragment” as used herein refers to a polypeptide that has a deletion, e.g., an amino-terminal and / or carboxy-terminal deletion compared to a full-length polypeptide. In a preferred embodiment, the polypeptide fragment is a contiguous sequence in which the amino acid sequence of the fragment is identical to the corresponding positions in the naturally-occurring sequence. Fragments typically are at least 5, 6, 7, 8, 9 or 10 amino acids long, preferably at least 12, 14, 16 or 18 amino acids long, more preferably at least 20 amino acids long, more preferably at least 25, 30, 35, 40 or 45, amino acids, even more preferably at least 50 or 60 amino acids long, and even more preferably at least 70 amino acids long.

[0065] A “modified derivative” refers to polypeptides or fragments thereof that are substantially homologous in primary structural sequence, but which include, e.g., in vivo or in vitro chemical and biochemical modifications or which incorporate amino acids that are not found in the native polypeptide. Such modifications include, for example, acetylation, carboxylation, phosphorylation, glycosylation, ubiquitination, labeling, e.g., with radionuclides, and various enzymatic modifications, as will be readily appreciated by those skilled in the art. A variety of methods for labeling polypeptides and of substituents or labels useful for such purposes are well known in the art, and include radioactive isotopes such as1251,32P,35S, and3H, ligands which bind to labeled antiligands (e.g., antibodies), fluorophores, chemiluminescent agents, enzymes, and antiligands which can serve as specific binding pair members for a labeled ligand. The choice of label depends on the sensitivity required, ease of conjugation with the primer, stability requirements, and available instrumentation. Methods for labeling polypeptides are well known in the art. See, e.g., Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992, and Supplements to 2002) (hereby incorporated by reference).

[0066] The term “fusion protein” refers to a polypeptide comprising a polypeptide or fragment coupled to heterologous amino acid sequences. Fusion proteins are useful because they can be constructed to contain two or more desired functional elements from two or more different proteins. A fusion protein comprises at least 10 contiguous amino acids from a polypeptide of interest, more preferably at least 20 or 30 amino acids, even more preferably at least 40, 50 or 60 amino acids, yet more preferably at least 75, 100 or 125 amino acids. Fusions that include the entirety of the proteins of the present invention have particular utility. The heterologous polypeptide included within the fusion protein of the present invention is at least 6 amino acids in length, often at least 8 amino acids in length,ATTY DKT NO: ASHI-005WO and usefully at least 15, 20, and 25 amino acids in length. Fusions that include larger polypeptides, such as an IgG Fc region, and even entire proteins, such as the green fluorescent protein (“GFP”) chromophore-containing proteins, have particular utility. Fusion proteins can be produced recombinantly by constructing a nucleic acid sequence which encodes the polypeptide or a fragment thereof in frame with a nucleic acid sequence encoding a different protein or peptide and then expressing the fusion protein.Alternatively, a fusion protein can be produced chemically by crosslinking the polypeptide or a fragment thereof to another protein.

[0067] The term “non-peptide analog” refers to a compound with properties that are analogous to those of a reference polypeptide. A non-peptide compound may also be termed a “peptide mimetic” or a “peptidomimetic.” See, e.g. , Jones, Amino Acid and Peptide Synthesis, Oxford University Press (1992); Jung, Combinatorial Peptide and Nonpeptide Libraries: A Handbook, John Wiley (1997); Bodanszky et al., Peptide Chemistry— A Practical Textbook, Springer Verlag (1993); Synthetic Peptides: A Users Guide, (Grant, ed., W. H. Freeman and Co., 1992); Evans et al., J. Med. Chem. 30:1229 (1987); Fauchere, J. Adv. Drug Res. 15:29 (1986); Veber and Freidinger, Trends Neurosci., 8:392-396 (1985); and references sited in each of the above, which are incorporated herein by reference. Such compounds are often developed with the aid of computerized molecular modeling. Peptide mimetics that are structurally similar to useful peptides of the present invention may be used to produce an equivalent effect and are therefore envisioned to be part of the present invention.

[0068] A “polypeptide mutant” or “mutein” refers to a polypeptide whose sequence contains an insertion, duplication, deletion, rearrangement or substitution of one or more amino acids compared to the amino acid sequence of a native or wild-type protein. A mutein may have one or more amino acid point substitutions, in which a single amino acid at a position has been changed to another amino acid, one or more insertions and / or deletions, in which one or more amino acids are inserted or deleted, respectively, in the sequence of the naturally-occurring protein, and / or truncations of the amino acid sequence at either or both the amino or carboxy termini. A mutein may have the same but preferably has a different biological activity compared to the naturally-occurring protein. A mutein has at least 85% overall sequence homology to its wild-type counterpart. Even more preferred are muteins having at least 90%, 95%, 96%, 97%, 98%, 99% or higher overall sequence homology to the wild-type protein.ATTY DKT NO: ASHI-005WO

[0069] Sequence homology may be measured by any common sequence analysis algorithm, such as Gap or Bestfit.

[0070] Amino acid substitutions can include those which: (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) alter binding affinity for forming protein complexes, (4) alter binding affinity or enzymatic activity, and (5) confer or modify other physicochemical or functional properties of such analogs.

[0071] As used herein, the twenty conventional amino acids and their abbreviations follow conventional usage. See Immtmology-A Synthesis (Golub and Gren eds., Sinauer Associates, Sunderland, Mass., 2nded. 1991), which is incorporated herein by reference. Stereoisomers (e.g., D-amino acids) of the twenty conventional amino acids, unnatural amino acids such as a-, a-disubstituted amino acids, N-alkyl amino acids, and other unconventional amino acids may also be suitable components for polypeptides of the present invention. Examples of unconventional amino acids include: 4-hydroxyproline, y- carboxyglutamate, e-N,N,N-trimethyllysine, s-N-acetyllysine, O-phosphoserine, N- acetylserine, N-formylmethionine, 3 -methylhistidine, 5-hydroxylysine, N-methylarginine, and other similar amino acids and imino acids e.g. , 4-hydroxyproline). In the polypeptide notation used herein, the left-hand end corresponds to the amino terminal end and the righthand end corresponds to the carboxy-terminal end, in accordance with standard usage and convention.

[0072] A protein has “homology” or is “homologous” to a second protein if the nucleic acid sequence that encodes the protein has a similar sequence to the nucleic acid sequence that encodes the second protein. Alternatively, a protein has homology to a second protein if the two proteins have similar amino acid sequences. (Thus, the term “homologous proteins” is defined to mean that the two proteins have similar amino acid sequences.) As used herein, homology between two regions of amino acid sequence (especially with respect to predicted structural similarities) is interpreted as implying similarity in function.

[0073] When “homologous” is used in reference to proteins or peptides, it is recognized that residue positions that are not identical often differ by conservative amino acid substitutions. A “conservative amino acid substitution” is one in which an amino acid residue is substituted by another amino acid residue having a side chain (R group) with similar chemical properties e.g., charge or hydrophobicity). In general, a conservative amino acid substitution will not substantially change the functional properties of a protein. In cases where two or more amino acid sequences differ from each other by conservativeATTY DKT NO: ASHI-005WO substitutions, the percent sequence identity or degree of homology may be adjusted upwards to correct for the conservative nature of the substitution. The means for making this adjustment are well known to those of skill in the art. See, e.g., Pearson, 1994, Methods Mol. Biol. 24:307-31 and 25:365-89 (herein incorporated by reference).

[0074] The following six groups each contain amino acids that are conservative substitutions for one another: 1) Serine (S), Threonine (T); 2) Aspartic Acid (D), Glutamic Al (E); 3) Asparagine (N), Glutamine (Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L), Methionine (M), Alanine (A), Valine (V), and 6) Phenylalanine (F), Tyrosine (Y), Tryptophan (W).

[0075] Sequence homology for polypeptides, which is also referred to as percent sequence identity, is typically measured using sequence analysis software. See, e.g., the Sequence Analysis Software Package of the Genetics Computer Group (GCG), University of Wisconsin Biotechnology Center, 910 University Avenue, Madison, Wis. 53705. Protein analysis software matches similar sequences using a measure of homology assigned to various substitutions, deletions and other modifications, including conservative amino acid substitutions. For instance, GCG contains programs such as “Gap” and “Bestfit” which can be used with default parameters to determine sequence homology or sequence identity between closely related polypeptides, such as homologous polypeptides from different species of organisms or between a wild-type protein and a mutein thereof. See, e.g., GCG Version 6.1.

[0076] A preferred algorithm when comparing a particular polypeptide sequence to a database containing a large number of sequences from different organisms is the computer program BLAST (Altschul et al., J. Mol. Biol. 215:403-410 (1990); Gish and States, Nature Genet. 3:266-272 (1993); Madden et al., Meth. Enzymol. 266:131-141 (1996); Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997); Zhang and Madden, Genome Res. 7:649-656 (1997)), especially blastp or tblastn (Altschul et al., Nucleic Acids Res. 25:3389-3402 (1997)).

[0077] Preferred parameters for BLASTp are: Expectation value: 10 (default); Filter: seg (default); Cost to open a gap: 11 (default); Cost to extend a gap: 1 (default); Max. alignments: 100 (default); Word size: 11 (default); No. of descriptions: 100 (default); Penalty Matrix: BLOWSUM62.

[0078] The length of polypeptide sequences compared for homology will generally be at least about 16 amino acid residues, usually at least about 20 residues, more usually at leastATTY DKT NO: ASHI-005WO about 24 residues, typically at least about 28 residues, and preferably more than about 35 residues. When searching a database containing sequences from a large number of different organisms, it is preferable to compare amino acid sequences. Database searching using amino acid sequences can be measured by algorithms other than blastp known in the art. For instance, polypeptide sequences can be compared using FASTA, a program in GCG Version 6.1. FASTA provides alignments and percent sequence identity of the regions of the best overlap between the query and search sequences. Pearson, Methods Enzymol. 183:63-98 (1990) (incorporated by reference herein). For example, percent sequence identity between amino acid sequences can be determined using FASTA with its default parameters (a word size of 2 and the PAM250 scoring matrix), as provided in GCG Version 6.1 , herein incorporated by reference.

[0079] “Specific binding” refers to the ability of two molecules to bind to each other in preference to binding to other molecules in the environment. Typically, “specific binding” discriminates over adventitious binding in a reaction by at least two-fold, more typically by at least 10-fold, often at least 100-fold. Typically, the affinity or avidity of a specific binding reaction, as quantified by a dissociation constant, is about 10'7M or stronger (e.g., about 10'8M, 10'9M or even stronger).

[0080] The term “region” as used herein refers to a physically contiguous portion of the primary structure of a biomolecule. In the case of proteins, a region is defined by a contiguous portion of the amino acid sequence of that protein.

[0081] The term “domain” as used herein refers to a structure of a biomolecule that contributes to a known or suspected function of the biomolecule. Domains may be coextensive with regions or portions thereof; domains may also include distinct, noncontiguous regions of a biomolecule. Examples of protein domains include, but are not limited to, an Ig domain, an extracellular domain, a transmembrane domain, and a cytoplasmic domain.

[0082] As used herein, the term “molecule” means any compound, including, but not limited to, a small molecule, peptide, protein, sugar, nucleotide, nucleic acid, lipid, etc., and such a compound can be natural or synthetic.

[0083] As used herein, the terms “NHHO” or “non-hereditary heterotopic ossification” or “NGHO” or “non-genetic heterotopic ossification” are used interchangeably and include a non-genetic condition of progressive formation of ectopic bone in soft tissues, often following trauma. Spinal cord and traumatic brain injuries, injuries associated with war orATTY DKT NO: ASHI-005WO gunshots, and severe burns can give rise to the formation of extraskeletal bone in joints, muscles or tendons. Nongenetic heterotopic ossification can occur with essentially any musculoskeletal trauma, spinal cord injury, central nervous system injury, head injury, cerebrovascular accident, sickle cell anemia, hemophilia, tetanus, poliomyelitis, multiple sclerosis, toxic epidermal necrolysis, pandemic viral infections, e.g. COVID-19, bums, and accidents, for example car accidents, gunshot, head trauma, sporting accidents such as incurred in skiing, football, and the like. Examples of musculoskeletal trauma include, but are not limited to, hip, knee, shoulder, or elbow arthroplasty; fractures; joint dislocations; or soft-tissue trauma, with the musculus quadriceps femoris and musculus brachialis. Surgical removal or resection often leads to the recurrence of HO, as does surgical intervention in some pre-disposing conditions involving chronic inflammation and bone formation, such as axial spondyloarthritis. NHHO can occur nearly anywhere in the body, but the most common areas include locations that are susceptible to trauma, such as the elbow, thigh, pelvis, and shoulder. NHHO can cause pain around the ossification site, loss of joint mobility and subsequently loss of function, as discussed in Myers C, et al., (2019). JBMR Plus, 3(4): el0172. Examples of musculoskeletal trauma include, but are not limited to, hip, knee, shoulder, or elbow arthroplasty; fractures; joint dislocations; or soft-tissue trauma, with the musculus quadriceps femoris and musculus brachialis. Nongenetic heterotopic ossification can also be associated with fever, swelling, and erythema (e.g., local, patchy reddening of the skin). As a non-limiting example, NHHO is associated with one or more disorders or conditions selected from the group of spinal cord injury, trauma, brain injuries, bums, fractures, muscle contusion, joint arthroplasty / replacement, hip surgery / replacement, acetabular surgery / replacement, elbow fracture, fracture of the long bones of the lower leg, combat-related trauma, amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, and nontraumatic myelopathy. In some embodiments an individual selected for treatment has suffered a traumatic injury prior to treatment. In some embodiments an individual selected for treatment has been surgically treated by surgery prior to treatment with the methods of the disclosure, or is selected for prophylactic therapy prior to surgery. In some embodiments an individual selected for treatment suffers from an autoimmune disease associated with a predisposition to heterotopic ossification, e.g. axial spondylarthritis, diffuse idiopathic skeletal hyperostosis autoimmune disease, ankylosing spondylitis, Guillain-Barre Syndrome (GBS), Anti-NMDA Receptor Encephalitis, dermatomyositis, systemic sclerosis, inflammatory arthritis, etc. Axial spondylarthritis suchATTY DKT NO: ASHI-005WO as ankylosing spondylitis and diffuse idiopathic skeletal hyperostosis are of particular interest.

[0084] As used herein, the terms “triple bone scan” or a “three-phase bone scan” are used interchangeably and refer to the diagnostic procedure that includes a series of images that can show early bone disease, infection, or fractures. These conditions can be imaged with the scan before they can be seen on standard X-rays in many cases. This scan uses a small amount of radioactive material as a tracer that is absorbed into the bones and detected by a camera to result in an image.

[0085] As used herein, the term “prevention” refers to the treatment of at-risk patients before the symptoms of NHHO arise, to further prevent condition development.

[0086] As used herein, the term “treatment” (as well as “treat” or “treating”) refers to partial or complete alleviation, amelioration, mitigation, prevention, reduction in risk of onset, relief, inhibition, delay in onset of, reduction in severity of, reduction in frequency or incidence of one or more causes, features, and / or symptoms of or associated with a particular disease, disorder, and / or condition. With respect to a nongenetic heterotopic ossification condition, in some embodiments, treatment encompasses methods of reducing the likelihood of developing a novel heterotopic lesion, or exacerbating an existing heterotopic ossification lesion such as, e.g., following a trauma or injury, in a subject having a nongenetic heterotopic ossification condition, by administering to the subject an effective dose of an inhibitor of MMP-9 activation or activity.

[0087] As used herein, the term “tenotomy” refers to the surgical act which involves the division or severance of a tendon.

[0088] The term “ad libitum" as used herein, refer to having access to something as often or as necessary as desired. As a non-limiting example, in this application, the mice used in these experimental conditions have “ad libitum'', or free access and availability to food and water.

[0089] The term “musculoskeletal disease or condition” or “MSD” refers to an injury, condition and / or pain in a subject's bones, joints, ligaments, muscles, nerves, tendons, and structures that support limbs, neck, and back.

[0090] The terms “antibody” and “immunoglobulin” are used interchangeably in the broadest sense and include monoclonal antibodies (e.g., full length or intact monoclonal antibodies), polyclonal antibodies, monovalent antibodies, multivalent antibodies, multispecific antibodies (e.g., bispecific antibodies so long as they exhibit the desiredATTY DKT NO: ASHI-005WO biological activity) and antibody fragments as described herein. Bispecific antibodies are monoclonal and may be human or humanized antibodies that have binding specificities for at least two different antigens. In the present case, the two different binding specificities can be directed to two different MMPs, or to two different epitopes on a single MMP (e.g., MMP-9).

[0091] An antibody as disclosed herein can also be an immunoconjugate. Such immunoconjugates comprise an antibody (e.g., to MMP-9) conjugated to a second molecule, such as a reporter. An immunoconjugate can also comprise an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0092] An antibody that “specifically binds to” or is “specific for” a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope without substantially binding to any other polypeptide or polypeptide epitope. In some embodiments, an antibody of the present disclosure specifically binds to human MMP-9 with a dissociation constant (Kd) equal to or lower than 100 nM, optionally lower than 10 nM, optionally lower than 1 nM, optionally lower than 0.5 nM, optionally lower than 0.1 nM, optionally lower than 0.01 nM, or optionally lower than 0.005 nM; in the form of monoclonal antibody, scFv, Fab, or other form of antibody measured at a temperature of about 4° C., 25° C., 37° C. or 42° C.

[0093] An antibody can be human, humanized and / or affinity matured.

[0094] The term “recombinant human antibody”, as used herein, is intended to include all human antibodies that are prepared, expressed, created or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant, combinatorial human antibody library, antibodies isolated from an animal (e.g., a mouse) that is transgenic for human immunoglobulin genes (see e.g., Taylor, L. D., et al. (1992) Nucl. Acids Res. 20:6287- 6295) or antibodies prepared, expressed, created or isolated by any other means that involves splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. In certain embodiments, however, such recombinant human antibodies are subjected to in vitro mutagenesis (or, when an animal transgenic for human Ig sequences is used, in vivo somatic mutagenesis) and thus the amino acidATTY DKT NO: ASHI-005WO sequences of the VH and VL regions of the recombinant antibodies are sequences that, while derived from and related to human germline VH and VL sequences, may not naturally exist within the human antibody germline repertoire in vivo.

[0095] An “isolated antibody”, as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (e.g., an isolated antibody that specifically binds MMP-9 is substantially free of antibodies that specifically bind antigens other than MMP-9). An isolated antibody that specifically binds MMP-9 may, however, have cross-reactivity to other antigens, such as MMP-9 molecules from other species. Moreover, an isolated antibody may be substantially free of other cellular material and / or chemicals.

[0096] An “antibody fragment” comprises a portion of a full-length antibody, for example, the antigen binding or variable region of a full-length antibody. Such antibody fragments may also be referred to herein as “functional fragments” or “antigen-binding fragments”. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 8(10): 1057-1062); single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0097] “Fv” is the minimum antibody fragment which contains a complete antigenrecognition and -binding site. This region consists of a dimer of one heavy- and one lightchain variable domain in tight, non-covalent association. It is in this configuration that the three complementarity-determining regions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the VH-VL dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or an isolated VH or VL region comprising only three of the six CDRs specific for an antigen) has the ability to recognize and bind antigen, although generally at a lower affinity than does the entire Fv fragment.

[0098] The “Fab” fragment also contains, in addition to heavy and light chain variable regions, the constant domain of the light chain and the first constant domain (CHI) of the heavy chain. Fab fragments were originally observed following papain digestion of an antibody. Fab' fragments differ from Fab fragments in that F(ab') fragments contain severalATTY DKT NO: ASHI-005WO additional residues at the carboxy terminus of the heavy chain CHI domain, including one or more cysteines from the antibody hinge region. F(ab')2 fragments contain two Fab fragments joined, near the hinge region, by disulfide bonds, and were originally observed following pepsin digestion of an antibody. Fab'-SH is the designation herein for Fab' fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Other chemical couplings of antibody fragments are also known.

[0099] The “light chains” of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, IgG4, IgAl, and IgA2.

[0100] The terms “Single-chain Fv” or “sFv” or “scFv” antibody fragments comprise the VH and VL domains of antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the VH and VL domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113 (Rosenburg and Moore eds.) Springer- Verlag, New York, pp. 269-315 (1994).

[0101] The term “diabodies” refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy-chain variable domain (VH) connected to a light-chain variable domain (VL) in the same polypeptide chain (VH-VL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain, thereby creating two antigen-binding sites. Diabodies are additionally described, for example, in EP 404,097; WO 93 / 11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444- 6448.

[0102] The term “humanized” forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. For the most part, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) such as mouse, rat, rabbit or nonhuman primate having the desired specificity, affinity, andATTY DKT NO: ASHI-005WO capacity. In some instances, framework region (FR) residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient antibody or in the donor antibody. These modifications are made to further refine antibody performance. In general, the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also the following review articles and references cited therein: Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1 : 105-115 (1998); Harris, Biochem. Soc. Transactions 23: 1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994).

[0103] Human antibodies can also be produced, for example, by using phage display libraries. Hoogenboom et al. (1991) J. Mol. Biol, 227:381; Marks et al. (1991) J. Mol. Biol. 222:581. Other methods for preparing human monoclonal antibodies are described by Cole et al. (1985) “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, p. 77 and Boemer et al. (1991) J. Immunol. 147:86-95.

[0104] The term “affinity matured” antibody is one with one or more alterations in one or more CDRs thereof which result in an improvement in the affinity of the antibody for antigen, compared to a parent antibody which does not possess those alteration(s). Preferred affinity matured antibodies will have nanomolar or even picomolar affinities for the target antigen. Affinity-matured antibodies are produced by procedures known in the art. Marks et al. Bio / Technology 10:779-783 (1992) describes affinity maturation by VH and VL domain shuffling. Random mutagenesis of CDR and / or framework residues is described by: Barbas et al. Proc Nat. Acad. Sci, USA 91 :3809-3813 (1994); Schier et al. Gene 169:147-155 (1995); Yelton et al. J. Immunol. 155:1994-2004 (1995); Jackson et al., J. Immunol. 154(7):3310-9 (1995); and Hawkins et al, J. Mol. Biol. 226:889-896 (1992).

[0105] As used herein, the term “immunoadhesin” designates antibody-like molecules which combine the “binding domain” of a heterologous protein (an “adhesin”, e.g. a receptor, ligand or enzyme) with the effector component of immunoglobulin constant domains. Structurally, the immunoadhesins comprise a fusion of the adhesin amino acidATTY DKT NO: ASHI-005WO sequence with the desired binding specificity which is other than the antigen recognition and binding site (antigen combining site) of an antibody (i.e. is “heterologous”) and an immunoglobulin constant domain sequence. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgGl, IgG2, IgG3, or IgG4 subtypes, IgA, IgE, IgD or IgM.

[0106] The term “Fc region”, as used herein, generally refers to a dimer complex comprising the C-terminal polypeptide sequences of an immunoglobulin heavy chain, wherein a C-terminal polypeptide sequence is that which is obtainable by papain digestion of an intact antibody. The Fc region may comprise native or variant Fc sequences.Although the boundaries of the Fc sequence of an immunoglobulin heavy chain might vary, the human IgG heavy chain Fc sequence is usually defined to stretch from an amino acid residue at about position Cys226, or from about position Pro230, to the carboxyl terminus of the Fc sequence. The Fc sequence of an immunoglobulin generally comprises two constant domains, a CH2 domain and a CH3 domain, and optionally comprises a CH4 domain. By “Fc polypeptide” herein is meant one of the polypeptides that make up an Fc region. An Fc polypeptide may be obtained from any suitable immunoglobulin, such as IgGi, IgG . IgG?, or IgG4subtypes, IgA, IgE, IgD or IgM. In some embodiments, an Fc polypeptide comprises part or all of a wild-type hinge sequence (generally at its N terminus). In some embodiments, an Fc polypeptide does not comprise a functional or wild type hinge sequence.

[0107] As used herein, the term “framework” when used in reference to an antibody variable region is intended to mean all amino acid residues outside the CDR regions within the variable region of an antibody. A variable region framework is generally a discontinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs. As used herein, the term “framework region” is intended to mean each domain of the framework that is separated by the CDRs.

[0108] The term “pharmaceutical formulation” refers to preparations of the active ingredients with one or more additional pharmaceutical agents, which can be found, for example, in in Remington: The Science and Practice of Pharmacy 21st ed., Lippincott Williams & Wilkins, 2005 (incorporated herein by reference).ATTY DKT NO: ASHI-005WO

[0109] “Pharmaceutically acceptable” excipients (vehicles, additives) are those which can reasonably be administered to a subject to provide an effective dose of the active ingredient employed.

[0110] The phrase “pharmaceutically acceptable carrier” is art recognized and includes a pharmaceutically acceptable material, composition or vehicle, suitable for administration to mammals. The carriers include liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient or subject.

[0111] A “stable” formulation is one in which the molecule, for example, the antibody composition essentially retains its physical stability and / or chemical stability and / or biological activity upon storage. Various analytical techniques for measuring protein stability are available in the art and are reviewed in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10: 29-90 (1993), for example. Stability can be measured at a selected temperature for a selected time period. Preferably, the formulation is stable at room temperature (about 30°C.) or at 40°C for at least 1 month and / or stable at about 2-8° C for at least 1 year or for at least 2 years. Furthermore, the formulation is preferably stable following freezing (to, e.g., -70°C.) and thawing of the formulation, hereinafter referred to as a “freeze / thaw cycle.”

[0112] As used herein in reference to antibody compositions, an antibody “retains its physical stability” in a pharmaceutical formulation if it shows substantially no signs of aggregation, precipitation and / or denaturation upon visual examination of color and / or clarity, or as measured by UV light scattering or by size exclusion chromatography.

[0113] As used herein in reference to antibody compositions, an antibody “retains its chemical stability” in a pharmaceutical formulation, if the chemical stability at a given time is such that the antibody is considered to still retain its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying chemically altered forms of the antibody. Chemical alteration may involve size modification (e.g. clipping) which can be evaluated using size exclusion chromatography, SDS-PAGE and / or matrix-assisted laser desorption ionization / time-of-flight mass spectrometry (MALDI / TOF MS), for example.ATTY DKT NO: ASHI-005WOOther types of chemical alteration include charge alteration (e.g. occurring as a result of deamidation) which can be evaluated by ion-exchange chromatography, for example.

[0114] As used herein in reference to antibody compositions, an antibody “retains its biological activity” in a pharmaceutical formulation, if the antibody in a pharmaceutical formulation is biologically active for its intended purpose. For example, biological activity is retained if the biological activity of the antibody in the pharmaceutical formulation is within about 30%, about 20%, or about 10% (within the errors of the assay) of the biological activity exhibited at the time the pharmaceutical formulation was prepared (e.g., as determined in an antigen binding assay).

[0115] As used herein, “buffer” refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. The buffer of this invention has a pH in the range from about 4 to about 8; preferably from about 4.5 to about 7; and most preferably has a pH in the range from about 5.0 to about 6.5. Examples of buffers that will control the pH in this range include acetate (e.g. sodium acetate), succinate (such as sodium succinate), gluconate, histidine, citrate and other organic acid buffers.

[0116] A “preservative” is a compound which can be included in the formulation to essentially reduce bacterial action therein, thus facilitating the production of a multi-use formulation, for example. Examples of potential preservatives include octadecyldimethylbenzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkylbenzyldimethylammonium chlorides in which the alkyl groups are long-chain compounds), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butyl and benzyl alcohol, alkyl parabens such as methyl or propyl paraben, catechol, resorcinol, cyclohexanol, 3-pentanol, and m-cresol.

[0117] As used herein “treatment” refers to treating, reducing, attenuating ameliorating one or more disease or conditions and / or symptoms. Treatment also includes prevention of one or more disease or conditions and / or symptoms as well as prophylactic measures. A “treatment” thus refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented.

[0118] The phrases “parenteral administration” and “administered parenterally” as used herein means modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal,ATTY DKT NO: ASHI-005WO intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal and intrastemal injection and infusion.

[0119] The phrases “systemic administration,” “administered systemically,” “peripheral administration” and “administered peripherally” as used herein mean the administration of a compound, drug or other material other than directly into the central nervous system, such that it enters the patient's system and, thus, is subject to metabolism and other like processes, for example, subcutaneous administration.

[0120] As used herein, the term “therapeutically effective amount” or “effective amount” refers to an amount of a therapeutic composition that when administered alone or in combination with another therapeutic composition to a subject is effective to prevent or ameliorate the disease condition or the progression of the NHHO. The term also includes a prophylactically effective amount at dosages and for periods of time necessary, to achieve the desired prophylactic result. Typically, since a prophylactic dose is used in subjects prior to or at an earlier stage of disease, the prophylactically effective amount may be less than the therapeutically effective amount.

[0121] A “disorder” is any condition that would benefit from treatment with the antibody. This includes chronic and acute disorders or diseases including those pathological conditions which predisposes the subject to the disorder in question.

[0122] Actual dosage levels of the active ingredients (e.g., antibody, chemical inhibitor, nucleic acids) in the pharmaceutical formulation of this invention may be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.

[0123] The selected dosage level will depend upon a variety of factors including the activity of the antibody found in the formulation, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular compound employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

[0124] A physician or veterinarian having ordinary skill in the art can readily determine and prescribe the effective amount of the pharmaceutical composition of the present invention required. For example, the physician or veterinarian could start doses ofATTY DKT NO: ASHI-005WO the compounds of the invention employed in the pharmaceutical formulation at levels lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

[0125] The term “surface plasmon resonance”, as used herein, refers to an optical phenomenon that allows for the analysis of real-time biospecific interactions by detection of alterations in protein concentrations within a biosensor matrix, for example using the BIAcore system (Pharmacia Biosensor AB, Uppsala, Sweden and Piscataway, N.J.). For further descriptions, see Jonsson, U., et al. (1993) Ann. Biol. Clin. 51:19-26; Jonsson, U., et al. (1991) Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit. 8: 125- 131 ; and Johnsson, B., et al. (1991) Anal. Biochem. 198:268-277. “The term "Koff", as used herein, is intended to refer to the off-rate constant for dissociation of an antibody from the antibody / antigen complex. The term "Kci", as used herein, is intended to refer to the dissociation constant of a particular antibody-antigen interaction.

[0126] The term, “ECso ” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% activation or enhancement of a biological process, or component of a process. For example, ECso can refer to the concentration of agonist that provokes a response halfway between the baseline and maximum response in an appropriate assay of the target activity.

[0127] The term, “IC50,” is intended to refer to the concentration of a substance (e.g., a compound or a drug) that is required for 50% inhibition of a biological process, or component of a process. For example, IC50 refers to the half maximal (50%) inhibitory concentration (IC) of a substance as determined in a suitable assay.

[0128] As used herein in reference to small molecules, the term “derivative” refers to a compound having a structure derived from the structure of a parent compound (e.g., a compound disclosed herein) and whose structure is sufficiently similar to those disclosed herein and based upon that similarity, would be expected by one skilled in the art to exhibit the same or similar activities and utilities as the claimed compounds, or to induce, as a precursor, the same or similar activities and utilities as the claimed compounds. Exemplary derivatives include salts, esters, amides, salts of esters or amides, and N-oxides of a parent compound.

[0129] As used herein in reference to small molecules, the term “substituted” is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched,ATTY DKT NO: ASHI-005WO carbocyclic and heterocyclic, and aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described below. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this disclosure, the heteroatoms, such as nitrogen, can have hydrogen substituents and / or any permissible substituents of organic compounds described herein which satisfy the valences of the heteroatoms. This disclosure is not intended to be limited in any manner by the permissible substituents of organic compounds. Also, the terms “substitution” or “substituted with” include the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., a compound that does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc. It is also contemplated that, in certain aspects, unless expressly indicated to the contrary, individual substituents can be further optionally substituted (i.e., further substituted or unsubstituted).

[0130] Certain materials, compounds, compositions, and components disclosed herein can be obtained commercially or readily synthesized using techniques generally known to those of skill in the art. For example, the starting materials and reagents used in preparing the disclosed compounds and compositions are either available from commercial suppliers such as Aldrich Chemical Co., (Milwaukee, Wis.), Acres Organics (Morris Plains, N.J.), Fisher Scientific (Pittsburgh, Pa.), or Sigma (St. Louis, Mo.) or are prepared by methods known to those skilled in the art following procedures set forth in references such as Fieser and Fieser's Reagents for Organic Synthesis, Volumes 1-17 (John Wiley and Sons, 1 ’91); Rodd's Chemistry of Carbon Compounds, Volumes 1-5 and Suppiementals (Elsevier Science Publishers, 1989); Organic Reactions, Volumes 1-40 (John Wiley and Sons, 19’ 1); March's Advanced Organic Chemistry, (John Wiley andSons, 4th Edition); and Larock’s Comprehensive Organic Transformations (VCH Publishers Inc., 1989).

[0131] As used herein, the phrase “inhibitory nucleic acid” refers to any nucleic acid that, when administered interacts with a target gene, results in inhibition of the expression or activity of that target gene, e.g., MMP-9. A nucleic acid molecule that inhibits, i.e., an inhibitory nucleic acid may be DNA, or an inhibitory RNA (e.g., siRNA, miRNA, antisense RNA, shRNA, IncRNA, pre-miRNA, or mRNA), wherein the RNA is single stranded, double stranded, or contains both single stranded and double stranded regions. In some embodiments, an inhibitory nucleic acid is an antisense oligonucleotide (ASO). The ASOATTY DKT NO: ASHI-005WO can be a single-stranded or double-stranded DNA, RNA, or DNA / RNA hybrid. See, e.g., Antisense Oligodeoxynucleotides and Antisense RNA: Novel Pharmacological and Therapeutic Agents, CRC Press, Boca Raton, Fla., 1997.

[0132] It is understood that the compositions disclosed herein have certain desired functions, e.g., inhibit MMP-9 activity or activation to treat musculoskeletal disease or conditions or disease, e.g., non-genetic heterotopic ossification. Disclosed herein are methods and compositions of a number of therapeutic modalities for performing the disclosed functions, and it is understood that there are a variety of structures that can perform the same or similar function that are related to the methods and compositions, which are anticipated to achieve the same or similar result.

[0133] As used herein, the term “heterotopic ossification” refers to the abnormal formation of bone in soft tissue where bone typically does not exist.

[0134] As used herein, the term “MMP-9 activation” refers to cleavage of the inactive pro-form of MMP-9 to produce the active form of MMP-9.

[0135] As used herein, the term “MMP-9 activity” refers to the enzymatic activity of the active form of MMP-9.

[0136] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this present invention pertains. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can also be used in the practice of the present invention and will be apparent to those of skill in the art. All publications and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. The materials, methods, and examples are illustrative only and not intended to be limiting.

[0137] Provided are various aspects of the invention as described in further detail.

[0138] In various aspects of the present invention, provided are methods and compositions for treating a subject by administering one or more inhibitors of MMP-9. In certain embodiments, the methods described herein are used to treat one or more MMP-9- mediated disorders including one or more musculoskeletal diseases.INDICATIONS

[0139] MSD encompasses a group of degenerative diseases and conditions and inflammatory diseases and conditions. Body parts of a subject that may be associated withATTY DKT NO: ASHI-005WOMSDs include upper and lower back, neck, shoulders, and extremities (arms, legs, feet, and hands). MSD includes bone and bone-marrow diseases, such as achondroplasia, acromegaly, bone callus, bone demineralization, bone fracture, bone marrow disease, bone marrow neoplasm, dyskeratosis congenita, leukemia (e.g., hairy cell leukemia, lymphocytic leukemia, myeloid leukemia, Philadelphia chromosome-positive leukemia, plasma cell leukemia, stem cell leukemia), systemic mastocytosis, myelodysplastic syndromes, paroxysmal nocturnal hemoglobinuria, myeloid sarcoma, myeloproliferative disorders, multiple myeloma, polycythemia vera, pearson marrow-pancreas syndrome, bone neoplasm, bone marrow neoplasm, Ewing sarcoma, osteochondroma, osteoclastoma, osteosarcoma, chondrosarcoma, brachydactyly, Camurati-Engelmann syndrome, Craniosynostosis, Crouzon craniofacial dysostosis, dwarfism, achondroplasia, bloom syndrome, Cockayne syndrome, Ellis-van Creveld syndrome, Seckel syndrome, spondyloepiphyseal dysplasia, spondyloepiphyseal dysplasia congenita, Werner syndrome, hyperostosis, osteophyte, Klippel-Trenaunay-Weber syndrome, Marfan syndrome, McCune-Albright syndrome, osteitis, osteoarthritis, osteochondritis, osteochondrodysplasia, Kashin-Beck disease, Leri-Weill dyschondrosteosis, osteochondrosis, osteodystrophy, osteogenesis imperfecta, osteolysis, Gorham-Stout syndrome, osteomalacia, osteomyelitis, osteonecrosis, osteopenia, osteopetrosis, osteoporosis, osteosclerosis, otospondylomegaepiphyseal dysplasia, pachydermoperiostosis, Paget disease of bone, Polydactyly, Meckel syndrome, rickets, Rothmund-Thomson syndrome, Sotos syndrome, spondyloepiphyseal dysplasia, spondyloepiphyseal dysplasia congenita, syndactyly, Apert syndrome, syndactyly type II, or Werner syndrome. MSD also includes cartilage disease, such as cartilage neoplasm, osteochondritis, osteochondrodysplasia, Kashin-Beck disease, or Leri-Weill dyschondrosteosis. MSD also includes hernia, such as intervertebral disk hernia. MSD also includes joint disease, such as arthralgia, arthritis (e.g., gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome), Lyme disease, osteoarthritis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty syndrome, synovitis, Blau syndrome, nail-patella syndrome, spondyloarthropathy, reactive arthritis, Stickler syndrome, synovial membrane disease, synovitis, or Blau syndrome. MSD includes Langer- Giedion syndrome. MSD includes muscle disease, such as Barth syndrome, mitochondrial encephalomyopathy, MELAS syndrome, MERRF syndrome, MNGIE syndrome, mitochondrial myopathy, Kearns-Sayre syndrome, myalgia, fibromyalgia,ATTY DKT NO: ASHI-005WO polymyalgia rheumatica, myoma, myositis, dermatomyositis, neuromuscular disease, Kearns-Sayre syndrome, muscular dystrophy, myasthenia, congenital myasthenic syndrome, Lambert-Eaton myasthenic syndrome, myasthenia gravis, myotonia, myotonia congenita, spinal muscular atrophy, tetany, ophthalmoplegia, or rhabdomyolysis. MSD includes Proteus syndrome. MSD includes rheumatic diseases, such as arthritis (e.g., gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan lyme disease)), osteoarthritis, psoriatic arthritis, reactive arthritis, rheumatic fever, rheumatoid arthritis, Felty syndrome, synovitis, Blau syndrome, gout (e.g., Kelley-Seegmiller syndrome, Lesch-Nyhan syndrome), polymyalgia rheumatica, rheumatic fever, rheumatic heart disease, or Sjogren syndrome. MSD includes Schwartz- Jampel syndrome. MSD includes a skeleton disease, such as Leri- Weill dyschondrosteosis, skeleton malformations, Melnick-Needles syndrome, pachydermoperiostosis, Rieger syndrome, spinal column disease, intervertebral disk hernia, scoliosis, spina bifida, spondylitis, ankylosing spondylitis, spondyloarthropathy, reactive arthritis, spondyloepiphyseal dysplasia, spondyloepiphyseal dysplasia congenita, or spondylosis.

[0140] MSD also includes cancer metastasis-related bone loss, renal osteodystrophy, treatment-related bone loss, diet-related bone loss, bone loss associated with the treatment of obesity, low gravity-related bone loss, or immobility-related bone loss.

[0141] Certain embodiments disclose a method of treating vascular aneurysms, blood vessel dissection, or other disorders of vascular connective tissue in a subject. The method includes administering to a subject with or at risk of developing an aneurysm, blood vessel dissection, or other disorders of vascular connective tissue an effective amount of one or more therapeutic compositions as disclosed. In some embodiments, the aneurysm, blood vessel dissection, or other disorders of vascular connective tissue is associated with Marfan Syndrome. Ikonomidis, J. et al., Expression of Matrix Metalloproteinases and Endogenous Inhibitors Within Ascending Aortic Aneurysms of Patients With Marfan Syndrome, Circulation, 2006; 114: 365-370. LeMaire, S.A. et al., Matrix Metalloproteinase Levels Are Elevated in Patients with Marfan Syndrome and Chronic Descending Thoracic Aortic Dissection, Journal ofSurg. Res., 2010, 158(2): 171. Segura AM, et al., Immunohistochemistry of matrix metalloproteinases and their inhibitors in thoracic aortic aneurysms and aortic valves of patients with Marfan's syndrome, Circulation, 1998; 98(19 Suppl):II331 -7; discussion II337-8. Benke K, et al., The role of transforming growth factorbeta in Marfan syndrome, Cardiol J. 2013;20(3):227-34.ATTY DKT NO: ASHI-005WO

[0142] In other embodiments, a method is provided of treating aneurysms associated with bicuspid aortic valve syndrome. The method includes administering to a subject with or at risk of developing an aneurysm an effective amount of one or more therapeutic compositions as disclosed. Fedak, P. et al., Vascular matrix remodeling in patients with bicuspid aortic valve malformations: implications for aortic dilatation, Journal of Thoracic and Cardiovascular Surgery’, 2003, 126(3): 797-805. Cione E, Piegari E, Gallelli G, et al., Expression of MMP-2, MMP-9, and NGAL in Tissue and Serum of Patients with Vascular Aneurysms and Their Modulation by Statin Treatment: A Pilot Study, Biomolecules, 2020; 10(3):359. Ramella M, et al., Endothelial MMP-9 drives the inflammatory response in abdominal aortic aneurysm (AAA), Am J Transl Res, 2017;9(12):5485-5495. Li, T., Jiang, B., Li, X. et al., Serum matrix metalloproteinase-9 is a valuable biomarker for identification of abdominal and thoracic aortic aneurysm: a case-control study, BMC Cardiovasc Disord, 2018; 18, 202. Ikonomidis, J. et al., Effects of Deletion of the Matrix Metalloproteinase 9 Gene on Development of Murine Thoracic Aortic Aneurysms, Circulation, 2005; 112: 242- 248. Hisato Takagi, Hideaki Manabe, Norikazu Kawai, Shin-nosuke Goto, Takuya Umemoto, Circulating matrix metalloproteinase-9 concentrations and abdominal aortic aneurysm presence: a meta-analysis, Interactive Cardiovascular and Thoracic Surgery, 2009, 9(3): 437-440.

[0143] In certain embodiments, a method is disclosed of treating calcific aortic valve disease. The method includes administering to a subject with or at risk of developing calcific aortic valve disease an effective amount of one or more therapeutic compositions as disclosed. Towler, D., Molecular and Cellular Aspects of Calcific Aortic Valve Disease, Circ. Res., 2013; 1 13(2): 198-208.

[0144] In further aspects of the invention, methods and compositions of the invention reduce BMP signaling in the context of inflammation and heterotopic ossification in soft tissues, joints, muscles, ligaments, tendons, bones and other sites of injury, inflammation of surgical intervention. In some embodiments of the invention, the method reduces pathologic BMP signaling without impacting homeostatic BMP signaling.NHHO DIAGNOSIS AND CONDITIONS

[0145] Acquired heterotopic ossification can occur with essentially any musculoskeletal trauma, accidents, spinal cord injury, traumatic brain injury, central nervous system injury, head injury, cerebrovascular accident, sickle cell anemia,ATTY DKT NO: ASHI-005WO hemophilia, tetanus, poliomyelitis, multiple sclerosis, toxic epidermal necrolysis and bums. Heterotopic ossification occurs most commonly after joint arthroplasty, spinal cord injury, traumatic brain injury, blast trauma, elbow and acetabular fractures, and thermal injury (Ranganathan K, et al., (2015). J Bone Joint Surg Am, 97(17)). Heterotopic ossification can also occur after severe infection and prolonged recovery, for example, with pandemic viruses, such as COVID 19. Examples of musculoskeletal trauma include, but are not limited to, hip, knee, shoulder, or elbow arthroplasty; fractures; joint dislocations; or soft- tissue trauma, with the musculus quadriceps femoris and musculus brachialis. Acquired heterotopic ossification can also be associated with fever, swelling, and erythema (e.g., local, patchy reddening of the skin). In one embodiment, neurogenic heterotopic ossification is not associated with local trauma.

[0146] A triple bone scan or a three-phase bone scan is used in diagnostic procedure that includes a series of images that can show early inflammation, vascularization, bone formation, bone disease, infection, or fractures. These conditions, especially the inflammatory and vascular phases that precede heterotopic ossification, can be imaged with the scan before they can be seen on standard X-rays in many cases. This scan uses a small amount of radioactive material as a tracer that is absorbed into the bones and detected by a camera to result in an image. This tracer particularly informs the third phase of the triple phase bone scan, where heterotopic bone is already forming.

[0147] As disclosed in WO2016130897, NHHO typically occurs between about 1 week and 12 weeks following an injury although it can arise at later timepoints such as months after injury. Heterotopic ossification can be reliably diagnosed by computed tomography, bone scintigraphy and ultrasonography. Two to six weeks later, the abnormal bone formation has progressed to the point that it is detectable by radiography. Bony maturation typically occurs within six months although this time frame is highly variable and maturation of at least portions of new bone can occur at much earlier timepoints. Conventional treatment usually involves non-steroidal anti-inflammatory drugs (indomethecin, rofecoxib), or bisphosphonate (etidronate, pamidronate), Coumadin / warfarin, salicylates, and / or local radiation can also be administered. Often, surgery is the only option for treatment. However, there is a significant risk of recurrence of HO with surgery.

[0148] In some embodiments, the MMP-9 inhibitor is an anti-MMP-9 antibody that inhibits MMP9, and is administered via intravenous or subcutaneous administration at aATTY DKT NO: ASHI-005WO dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1500 mg, about 1600 mg. In some embodiments the antibody comprises the CDR sequences of andecaliximab. In some embodiments the antibody comprises the variable region sequences of andecaliximab. In some embodiments the antibody is andecaliximab.

[0149] In further embodiments, disclosed is a dosage regimen comprising administration of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg, about 600 mg every 1, 2, 3, or 4 days, for example delivered subcutaneously.

[0150] Where the subject is a pediatric patient, the dosage may be scaled from an adult dose by using standard methodology. Medication dosing for pediatric patients is described to use either the following methods for medication dosing: age-based dosing, allometric scaling, body surface area-based dosing, and weight-based dosing. Weight-based dosing is the most commonly used method for calculating recommended medication doses in pediatric clinical practice. For example, Clark's rule is an equation used to calculate pediatric medication dosage based on the known weight of a patient and a known adult dose of medication to be used. Clark's rule equation is defined as the weight of the patient in pounds divided by the average standard weight of 150 pounds (68 kg) multiplied by the adult dose of a drug to obtain the pediatric medication dose. Other equations that utilize pediatric weight to calculate medication dosing include Salisbury's rule, Penna’s rule, and the Body Surface Area rule. Other methods besides Clark's rule that utilize pediatric age to calculate radiopharmaceutical medication dosages used in nuclear imaging include Young's rule, Webster's rule, and Fried's rule.

[0151] In some embodiments, the subject is a human. In some embodiments the subject is an adult. In some embodiments, the subject is under about thirty years of age. In some embodiments, the subject is aged 12 or above, for example 12 to 18 years. In some embodiments, the subject is aged 18 or above. In some embodiments, the subject is aged 6- 12. In some embodiments, the subject is aged 2-12.

[0152] In embodiments where the subject is a pediatric patient, for example under 18 years of age, under 12 years of age, etc., the dose may be appropriately scaled from the adult doses disclosed herein, using methods known in the art. Administration may be subcutaneous, intravenous, etc., and is optionally fractionated into multiple doses. In someATTY DKT NO: ASHI-005WO embodiments, a loading dose is administered, followed by administration of maintenance doses. As non-limiting examples, in some embodiments, the subject is aged 2-5 years, the loading dose of an MMP-9 inhibitor (such as andecaliximab) is 100 mg, and the maintenance dose is 30 mg, which may be administered every other week. In another embodiment, the subject is aged between 2 and about 5 years and an MMP-9 inhibitor dose of 90 or 100 mg is administered every week. In another embodiment, the subject is aged between 2 and about 5 years and an MMP-9 inhibitor dose of 60 mg is administered every week. In some embodiments, the subject is aged 2-5 years, and a dose of 90 or 100 mg is administered every other week. In some embodiments, the subject is aged 2-5 years, and a dose of 30 mg is administered every other week. In some embodiments, the subject is aged 2-5 years, and a dose of 90 or 100 mg is administered every other week.

[0153] In some embodiments, the subject is aged 6-11 years, the loading dose is 150 to 300 mg, and the maintenance dose is 50 to 150 mg. In an embodiment, the subject is aged 6-11 years, and a dose of 100 mg is administered every week. In an embodiment, the subject is aged between about 6 to about 11 years and an MMP-9 inhibitor dose of 150 mg is administered every week. In another embodiment, the subject is aged between about 6 to about 11 years and an MMP-9 inhibitor dose of 200 mg is administered every week. In an embodiment, the subject is aged 6-11 years, and a dose of 300 mg is administered every week.

[0154] In an embodiment, the subject is greater than 12 years of age, e.g. from about 12 to about 18 years of age, or an adult, and an antibody MMP9 inhibitor is administered at an initial dose of from about 800 mg to about 1600 mg. For example, 400-800 mg may be delivered weekly, 800 mg may be delivered over about 2 weeks, 1200 mg may be delivered over about 3 weeks, 1600 mg may be delivered over about 4 weeks. In another embodiment the subject is greater than 12 years of age and an antibody MMP9 inhibitor is administered subcutaneously at a maintenance dose of 150 to 300 mg every 10 to 18 days.

[0155] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 2 days of the of the NHHO-inductive event.

[0156] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 3 days of the of the NHHO-inductive event.

[0157] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 4 days of the of the NHHO-inductive event.ATTY DKT NO: ASHI-005WO

[0158] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 5 days of the of the NHHO-inductive event.

[0159] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 6 days of the of the NHHO-inductive event.

[0160] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 7 days of the of the NHHO-inductive event.

[0161] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 8 days of the of the NHHO-inductive event.

[0162] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 9 days of the of the NHHO-inductive event.

[0163] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 10 days of the of the NHHO-inductive event.

[0164] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 11 days of the of the NHHO-inductive event.

[0165] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 12 days of the of the NHHO-inductive event.

[0166] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 13 days of the of the NHHO-inductive event.

[0167] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 2 weeks of the of the NHHO-inductive event.

[0168] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 3 weeks of the of the NHHO-inductive event.

[0169] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 1 month of the of the NHHO-inductive event.

[0170] In some embodiments, the occurrence of NHHO or a symptom of NHHO is identified with a triple bone scan within 2 months of the of the NHHO-inductive event.

[0171] In additional embodiments, disclosed is a pharmaceutical composition comprises a unit dose of an MMP9 inhibitor, suitable for delivery of a dose of 75 mg administered every week, a dose of 25 mg administered every week, a dose of 50 or 45 mg administered every other week, a dose of 15 mg administered every other week, a dose of 75 mg administered every other week, or a dose of 45 or 50 mg administered every other week of an MMP-9 inhibitor suitable for intravenous administration to a human subject having a spinal cord injury, trauma, brain injuries, bums, fractures, muscle contusion, jointATTY DKT NO: ASHI-005WO arthroplasty / replacement, hip surgery / replacement, acetabular surgery / replacement, elbow fracture, fracture of the long bones of the lower leg, combat-related trauma, amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, or a nontraumatic myelopathy.

[0172] In additional embodiments, disclosed is a pharmaceutical composition including an MMP-9 inhibitor suitable for subcutaneous administration to a human subject having a spinal cord injury, administered every week for at least 4, 5, 6, 7, 8, 9, 10, 11, 12 weeks or 4, 5, 6, 7, 8 months or greater than 8 months. In some embodiments, the first administration occurs within 1 week, or within 2, 3, 4, 5, 6, 7, 8 weeks of spinal cord injury.

[0173] In further embodiments, disclosed is a method of preventing or reducing the severity and / or duration of a condition associated with a nonhereditary heterotopic ossification (NHHO) comprising administering to a human subject an effective amount of an inhibitor of MMP-9 activation or activity.

[0174] The efficacy of a given treatment for a disorder comprising abnormal bone growth as described herein can be determined by the skilled clinician. However, a treatment is considered "effective treatment," as the term is used herein, if any one or all of the signs or symptoms of the disease or disorder is / are altered in a beneficial manner (e.g., reduced ossification, reduction in the number of new sites of ossification, reduction in anticipated new sites of ossification, prevention of ossification from an inflammatory lesion, regression of abnormal bone growths, reduced pain, increased range of motion etc.), other clinically accepted symptoms or markers of disease are improved, or even ameliorated, e.g., by at least 10% following treatment with an agent. Efficacy can also be measured by the failure of an individual to worsen as assessed by stabilization of the disease or disorder, hospitalization or need for medical interventions (i.e., progression of the disease is halted or at least slowed). Methods of measuring these indicators are known to those of skill in the art and / or described herein. Treatment includes any treatment of a disease in an individual or an animal (some non-limiting examples include a human, or a mammal) and includes: (1) inhibiting the disease, e.g., arresting, slowing the progression of abnormal bone growth, or preventing new bone growth at susceptible sites; or (2) relieving the disease, e.g., causing regression of symptoms; and (3) preventing or reducing the likelihood of the development of a disease (e.g., ossification following trauma). As provided herein, increasing range of motion (RoM) in a subject may include passive rangeATTY DKT NO: ASHI-005WO of motion, active range of motion, or both, and increasing RoM upon administration of an effective treatment may occur independent of treating HO.THERAPEUTIC USE FOR ANTI-MMP-9 ANTIBODIES

[0175] WO / 2017 / 177179 discloses various anti-MMP-9 antibodies. The antibodies show anti-MMP-9 activity; however, there appears to be no known successful therapies including administration of such antibodies used to treat musculoskeletal disease or conditions or disease e.g., nongenetic heterotopic ossification. Accordingly, provided herein are methods for treating musculoskeletal disease or conditions or disease, e.g., nongenetic heterotopic ossification or other MMP-9 mediated disorders by administering an antibody or antibody fragment to inhibit MMP-9 activation or enzymatic activity.

[0176] In one embodiment, the inhibitor of MMP-9 comprises an anti-MMP-9 antibody, wherein the anti-MMP-9 antibody binds to (i) an MMP-9 pro-form and inhibits activation of the pro-form or (ii) an MMP-9 active form and inhibits activity of the active form and is used as a method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders.

[0177] In another embodiment, the anti-MMP-9 antibody binds to the MMP-9 proform to inhibit MMP-9 activation and is used as a method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders.

[0178] In certain embodiments, the anti-MMP-9 antibody binds allosterically to the active form of MMP-9 to inhibit MMP-9 activity and is used as a method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders.

[0179] In various embodiments, therapeutic antibodies for administration are characterized as binding to one or more processing sites (e.g., sites of proteolytic cleavage) in MMP-9, thereby effectively blocking processing of the proenzyme or pre-proenzyme to the catalytically active enzyme and thus reducing the proteolytic activity of the MMP-9.

[0180] In certain embodiments, therapeutic antibodies for administration are characterized as binding to MMP-9 with an affinity at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times greater than its binding affinity for another MMP (e.g., a non-MMP-9). Binding affinity can be measured by any method known in the art and can be expressed as, for example, on-rate, off-rate, dissociation constant (Kd), equilibrium constant (Keq) or anyATTY DKT NO: ASHI-005WO term in the art. Various examples of such affinity-matured antibodies are within the scope of the invention.

[0181] In certain embodiments, therapeutic antibodies for administration are characterized as non-competitive inhibitor of the catalytic activity of MMP-9. In certain embodiments, an antibody according to the present disclosure binds within the catalytic domain of MMP-9. In additional embodiments, such therapeutic antibodies are characterized as binding outside the catalytic domain of MMP-9.

[0182] Additional antibodies or antibody fragments are contemplated within the scope of the invention that compete with anti-MMP9 antibodies or antibody fragments thereof described herein for binding to MMP-9. For instance, anti-MMP9 antibodies, and functional fragments thereof, compete for binding with, for example, an antibody having a heavy chain polypeptide, a light chain polypeptide, or combinations thereof. In one embodiment, administration of AB0041, AB0046 or antibody fragments thereof is contemplated for the treatment of nongenetic heterotopic ossification or other MMP-9 mediated disorders. In further embodiments, the method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders comprises administration of one or more anti-MMP-9 antibodies encoded by the one or more sequences listed in Table 1.

[0183] SDS3 is a targeting antibody that binds to the Zn2+active site and surface epitopes of activated MMP-9, rather than mimicking the endogenous MMP-9 inhibitors, tissue inhibitor of metalloproteinases (TIMPs). In mice, SDS3 bound and inhibited MMP-9 with a KD of 200 nM and Ki of 1 pM, respectively, providing both prophylactic and therapeutic benefits in a model of dextran sodium sulfate-induced colitis. (Sela-Passwell N, Kikkeri R, Dym O, Rozenberg H, Margalit R, Arad-Yellin R, et al. Antibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential. Nat Med. (2011) 18: 143-7). Accordingly, in preferred embodiments, the method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders comprises administration of humanized SDS3 antibodies. In various other embodiments, the method provides for the administration of similar antibodies or antibody fragments that bind to the Zn2+active site and surface epitopes of activated MMP-9.

[0184] Similar to SDS3, SDS4 is a targeting antibody that binds to the Zn2+active site and surface epitopes of activated MMP-9, rather than mimicking TIMPS. In mice, SDS3 bound and inhibited MMP-9 with a KD of 20 nM and Ki of 54 nM, respectively. (Sela-Passwell N, Kikkeri R, Dym O, Rozenberg H, Margalit R, Arad-Yellin R, et al.ATTY DKT NO: ASHI-005WOAntibodies targeting the catalytic zinc complex of activated matrix metalloproteinases show therapeutic potential. Nat Med. (2011) 18:143-7). Accordingly, in preferred embodiments, the method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders comprises administration of humanized SDS4 antibodies. In various other embodiments, the method provides for the administration of similar antibodies or antibody fragments that bind to the Zn2+active site and surface epitopes of activated MMP-9.

[0185] Mouse REGA-3G12 binds MMP-9 between Trpl 16 to Lys214, which is located in the catalytic domain separate from the Zn2+binding site. (Martens E, Leyssen A, Van Aelst 1, Fiten P, Piccard H, Hu J, et al. A monoclonal antibody inhibits gelatinase B / MMP-9 by selective binding to part of the catalytic domain and not to the fibronectin or zinc binding domains. Biochim Biophys Acta. (2007) 1770:178-86). Mouse REGA-3G12 binds MMP-9 with a KD of 2.1 nM. (Paemen L, Martens E, Masure S, Opdenakker G. Monoclonal antibodies specific for natural human neutrophil gelatinase B used for affinity purification, quantitation by two-site ELISA and inhibition of enzymatic activity. Eur J Biochem. (1995) 234:759-65). In rhesus monkeys, REGA-3G12 prevented the mobilization of hematopoietic progenitor cells in response to interleukin- 8. (Hu J, Van den Steen PE, Houde M, Ilenchuk TT, Opdenakker G. Inhibitors of gelatinase B / matrix metalloproteinase-9 activity comparison of a peptidomimetic and polyhistidine with singlechain derivatives of a neutralizing monoclonal antibody. Biochem Pharmacol. (2004) 67:1001-9). Accordingly, in preferred embodiments, the method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders comprises administration of humanized REGA-3G12 antibodies or antibody fragments that bind MMP-9 between Trpl 16 to Lys214, or that bind in the catalytic domain separate from the Zn2+binding site.

[0186] WO / 2017 / 177179 discloses anti-MMP-9 antibodies, such as AB0045(andecaliximab). Andecaliximab binds the catalytic domain of MMP-9 with additional points of contact shielding the site of physiological activation in the prodomain, where it inhibits pro-MMP-9 activation and non-competitively inhibits MMP-9 activity. Andecaliximab binds to MMP-9 with a KD of 2.0-6.6 nM and to pro-MMP-9 with a KD of 0.008-0.043 nM. Andecaliximab inhibits activation of MMP-9 from human pro-MMP-9 with an IC50 of 8.2 pM. Clinical trials with andecaliximab have shown the antibody to be safe and well-tolerated. (Sandborn WJ, Bhandari BR, Fogel R, Onken J, Yen E, Zhao X, et al. Randomised clinical trial: a phase 1 , dose-ranging study of the anti-matrix metalloproteinase-9 monoclonal antibody GS-5745 versus placebo for ulcerative colitis.ATTY DKT NO: ASHI-005WOAliment Pharmacol Ther. (2016) 44:157-69; Sandborn WJ, Bhandari BR, Randall C, Younes ZH, Romanczyk T, Xin Y, et al. Andecaliximab [anti-matrix metalloproteinase-9] induction therapy for ulcerative colitis: a randomised, double-blind, placebo-controlled, phase 2 / 3 study in patients with moderate to severe disease. J Crohns Colitis. (2018) 12: 1021-9; Gossage DL, Cieslarova B, Ap S, Zheng H, Xin Y, Lal P, et al. Phase lb Study of the safety, pharmacokinetics, and disease-related outcomes of the matrix metalloproteinase-9 inhibitor andecaliximab in patients with rheumatoid arthritis. Clin. Ther. (2018) 40: 156-65. el55). Accordingly, in certain embodiments, the method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders comprises administration of humanized AB0041, also known as GS-5745 or andecaliximab or antibody fragments that bind between the pro-domain and catalytic domain of MMP-9, distal to the active site, in which pro-MMP-9 activation is inhibited and MMP-9 activity is non-competitively inhibited. In some embodiments, the ability of humanized AB0041, also known as GS-5745 or andecaliximab, antibody fragments to bind between the pro-domain and catalytic domain of MMP-9, distal to the active site, so that pro-MMP-9 activation is inhibited and MMP-9 activity is non-competitively inhibited contributes to the therapeutic effect by preventing activation of MMP-9, a massive quantity of which is induced and secreted due to the trauma, injury, or hypoxia, including from neutrophils. Humanized AB0041, also known as GS-5745 or andecaliximab, antibody fragments bind to the MMP9- LCN2 and MMP9-LCN2-TIMP1 complexes. For one of the epitopes on human MMP-9 that humanized AB0041, also known as GS-5745 or andecaliximab, binds, R162 is the key residue. In mice, AB0046 binds the same epitope, with proline being the key residue in murine MMP-9.

[0187] Provided herein are amino acid sequences of andecaliximab for use in the methods of the disclosure. In some embodiments of the disclosure an MMP9 inhibitor is an antibody that comprises the CDR sequences of andecaliximab. The heavy chain variable region (VH) of andecaliximab comprises a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:5, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:7, and the light chain variable region of andecaliximab comprises a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 8, an LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 10. InATTY DKT NO: ASHI-005WO some embodiments the antibody comprises the variable region sequences of andecaliximab, which are variable heavy chain of SEQ ID NO:3 and variable light chain of SEQ ID NO:4. In some embodiments the antibody comprises a heavy chain sequence of SEQ ID NO: 1 and a light chain sequence of SEQ ID NO:2.

[0188] In some embodiments, the protein inhibitor of MMP-9 includes an antibody against MMP-9, or an antigen binding fragment thereof. Examples of antibody against MMP-9 include anti-Ac-MMP-9 antibody (4A3), anti-MMP-9 antibody (E-l 1), anti-MMP- 9 antibody (2C3), and anti-MMP-9 antibody (6-6B) by Santa Cruz Biotechnology, MMP9 monoclonal antibody (5G3), MMP9 Recombinant Rabbit Monoclonal Antibody (JA80-73), and MMP9 Monoclonal Antibody (5C3) by Invitrogen, and the like.

[0189] In certain embodiments, the antibodies used as a method for treating nongenetic heterotopic ossification or other MMP-9 mediated disorders comprise antibodies disclosed in WO / 2017 / 177179, PCT / US2012 / 027160, PCT / US2016 / 067036, PCT / US2016 / 054780, WO / 2016 / 023979A 1 , WQ / 2008 / 102359A1, WQ / 2002 / 066057, WO / 2006 / 037513, WO / 2009 / 111508, WO / 2011 / 028883, WO / 2012 / 154654, US / 2010 / 0098659, WO / 2010 / 048455, WO / 2012 / 048291, WO / 2023 / 278491 and Marshall, Derek C et al. “Selective Allosteric Inhibition of MMP9 Is Efficacious in Preclinical Models of Ulcerative Colitis and Colorectal Cancer.” PloS one vol. 10,5 e0127063. 11 May. 2015, doi:10.1371 / journal.pone.0127063.

[0190] In certain embodiments, the antibodies of the invention can be derivatized or linked to another functional molecule. For example, the antibody is functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody (e.g., a bispecific antibody or a diabody), a detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0191] In various embodiments, administration of the preferred amounts or therapeutically effective amounts of the anti-MMP-9 antibody compositions of the invention may vary according to factors such as the disease state, age, sex, and weight of the individual, and the ability of the antibody or antibody portion to elicit a desired response or change in biomarkers in the individual. A therapeutically effective amount is also one in which any toxic or detrimental effects of the antibody or antibody portion are outweighed by the therapeutically beneficial effects. Dosage regimens may be adjusted toATTY DKT NO: ASHI-005WO provide the optimum desired response (e.g., a therapeutic or prophylactic response) or change in biomarkers (e.g. levels of MMP-9 in blood, body fluids or tissues). For example, a single bolus may be administered, several divided doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation.

[0192] In various embodiments, the antibody compositions of the invention are used in a variety of settings. For example, an antibody of the invention is administered as a therapeutic antibody. In such embodiments, the antibody of the invention can exert its therapeutic effect by any of a variety of mechanisms. For example, the antibody of the invention may be an agonist antibody. In another example, the antibody of the invention may be an antagonistic antibody. In yet another example, an antibody of the invention may be a blocking antibody. In another example, an antibody of the invention is a neutralizing antibody.

[0193] It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the mammalian subjects to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier. Additional routes of administration are contemplated which include but are not limited to subcutaneous, intravenous administration, intravitreal administration, intramuscular administration, inhalation, intranasal administration, topical administration, transdermal administration, ophthalmic administration, optic administration, intraabdominal, intra-thoracic or intratumoral administration, or by local injection into areas of heterotopic ossification or soft tissues including gums.

[0194] The specification for the dosage unit forms of the invention is dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic or prophylactic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.

[0195] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is selected from 10- 1500 mg, more preferably 200-800 mg and even more preferably about 40 mg. As disclosed in US 8,377,443, additional non-limiting ranges include 10 ng / kg to up to 100ATTY DKT NO: ASHI-005WO mg / kg of mammal body weight or more per day administered to a subject. A dosage may be selected from 1 pg / kg / day, 100 pg / kg / day, 500 pg / kg / day, 1 mg / kg / day, 10 mg / kg / day or 20 mg / kg / day. It is noted that dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0196] In some embodiments, the therapeutically effective amount of an antibody of the invention is selected from 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg, or 1500 mg by intravenous administration. In some embodiments, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg or 1500 mg of the antibody is given by intravenous administration weekly or biweekly. In some embodiments, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg or 1500 mg of the antibody is given by intravenous administration once every 3 weeks. In some embodiments, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1200 mg or 1500 mg of the antibody is given by intravenous administration monthly.

[0197] In some embodiments, the antibody is given at an initial loading dose by intravenous administration, with a dosing interval of 1 or 2 weeks for a period of 1 or 2 or 3 or 4 week or 1 or 2 months, then a maintenance dose given weekly via subcutaneous administration. In some embodiments, the initial loading dose is 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000, 1200 or 1500 mg of the antibody administered intravenously. In some embodiments, the maintenance dose is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, 400 mg or 600 mg administered subcutaneously on a weekly schedule or every other week schedule. The maintenance schedule could also involve administering the antibody 2, 3 or more times per week to achieve a desired weekly dose. For example, 150 mg twice per week (300 mg per week) or 150 mg three times per week (450 mg) or 150 mg four times per week (600 mg).

[0198] In some embodiments, the initial loading dose and maintenance dose are both administered subcutaneously. In some embodiments, the initial loading dose is 150mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, or 1000 mg of the antibody administered subcutaneously, optionally fractioned into multiple doses. In some embodiments, the maintenance dose is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg,ATTY DKT NO: ASHI-005WO350 mg, or 400 mg administered subcutaneously on a weekly schedule or every other week schedule.

[0199] Human antibodies can be made by introducing human immunoglobulin loci into transgenic animals (e.g., mice) in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon immunological challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al. (1992) Bio / Technology 10:779-783 (1992); Lonberg et al. (1994) Nature 368: 856-859; Morrison (1994) Nature 368:812-813; Fishwald et al. (1996) Nature Biotechnology 14:845-851 ;Neuberger (1996) Nature Biotechnology 14:826; and Lonberg et al. (1995) Intern. Rev. Immunol. 13:65-93.Methods of TreatmentSelection of Subjects

[0200] Disclosed herein, in some embodiments, are methods of treating a subject identified as having a non-genetic heterotopic ossification (NHHO), comprising administering to the subject a composition, such as a pharmaceutical composition comprising an MMP-9 inhibitor e.g. , anti-MMP-9 antibody or antigen-binding fragment thereof, inhibitory RNA, small molecule, inhibitory polypeptide, or a recombinant gene knock-out or knock-down system, such as a CRISPR-Cas system). In some embodiments, the disclosure provides a use of an MMP-9 inhibitor as described herein for the manufacture of a medicament. In some embodiments, the disclosure provides the use of an antibody as described herein for the treatment of NHHO.

[0201] In some embodiments, the subject is a mammal, such as a human. In some embodiments, the subject is an adult. In some embodiments the subject is an adult human, for example an adult human from about 18 to 80 years of age; and adult human from about 18 to 65 years of age, an adult human from about 18 to about 45 years of age. In some embodiments the subject is under about thirty years of age. In some embodiments, the subject is aged 12 or above. In some embodiments, the subject is aged 18 or above. In some embodiments, the subject is aged 6-12. In some embodiments, the subject is aged 2-12.ATTY DKT NO: ASHI-005WO

[0202] The clinical signs of heterotopic ossification are pain, decreased range of motion, swelling or warmth in the joint area, increased spasticity, and fever. There is an absence of effective treatment, and heterotopic ossification can occur for unknown cause. (Lisa Harvey BAppSc, GradDipAppSc(exSpSc), MAppSc, PhD, in Management of Spinal Cord Injuries, 2008).

[0203] In certain aspects of the invention, methods and compositions of the invention reduce BMP levels and / or BMP signaling in the subject. In further aspects of the invention, methods and compositions of the invention reduce BMP signaling in the context of inflammation and heterotopic ossification in soft tissues, joints, muscles, tendons, ligaments, bones and other sites of injury or inflammation. In some embodiments of the invention, the method reduces pathologic BMP signaling without impacting homeostatic BMP signaling.

[0204] In certain aspects of the invention, methods and compositions of the invention reduce pain, particularly neuropathic pain, in the subject. In further aspects of the invention, methods and compositions of the invention reduce pain in the context of inflammation and heterotopic ossification in soft tissues, joints, muscles, ligaments, bones and other sites of injury or inflammation. In some embodiments of the invention, the method reduces neuropathic pain without impacting heterotopic ossification.

[0205] In certain aspects of the invention, methods and compositions of the invention restore spinal cord function in a subject having a spinal cord injury. In some embodiments of the invention, the method restores spinal cord function in a subject without impacting heterotopic ossification.

[0206] In certain aspects of the invention, methods and compositions of the invention increase wound healing in the subject. In further aspects of the invention, methods and compositions of the invention increase or improve wound healing in the context of inflammation in soft tissues, joints, muscles, ligaments, bones and other sites of injury or inflammation. In some embodiments of the invention, the method increases wound healing without impacting heterotopic ossification.Route of Administration

[0207] MMP-9 inhibitors suitable for use with the disclosed methods are formulated for any suitable route of administration to a subject including, but not limited to injection (e.g., intravenous injection), in some embodiments. Injection includes, e.g., subcutaneous,ATTY DKT NO: ASHI-005WO peritoneal, intravenous injection, or intramuscular injection. In some embodiments, the antibodies of the disclosure are formulated for subcutaneous administration. In some embodiments, the antibodies of the disclosure are formulated for peritoneal administration. In some embodiments, the antibodies of the disclosure are formulated for intravenous administration (e.g., intravenous injection or infusion). In some embodiments, the antibodies of the disclosure are formulated for subcutaneous administration. In some embodiments, administration is in one, two, three, four, five, six, seven, or more injection sites. In some embodiments, administration is in one injection site. In some embodiments, administration is in two injection sites. In some embodiments, administration is in three injection sites. In some embodiments, administration is in four injection sites. In some embodiments, administration is in five injection sites. In some embodiments, administration is in six injection sites.

[0208] For in vivo applications, contacting occurs, e.g., via administration of a composition (e.g., a composition comprising an MMP-9 inhibitor) to a subject by any suitable means. An MMP-9 inhibitor disclosed herein, in some embodiments, is administered, e.g., either systemically or locally, including via parenteral, subcutaneous, intraperitoneal, intrapulmonary, and intranasal administration, and, if desired for local treatment, injection directly into the site of heterotopic ossification. Parenteral routes include, e.g., intravenous, intraarterial, intraperitoneal, epidural, intramuscular, and intrathecal administration. Such administration, in some embodiments, is as a bolus, continuous infusion, or pulse infusion. In some embodiments, compositions are administered by injection depending in part on whether the administration is brief or chronic. Other modes of administration methods are contemplated, including topical, particularly transdermal, transmucosal, rectal, oral, or local administration e.g., through a catheter placed close to the desired site.Dosing

[0209] MMP-9 inhibitors suitable for use with the disclosed methods and compositions comprising the same may be administered in a manner compatible with the dosage formulation and in a therapeutically effective amount. The quantity to be administered depends on the subject to be treated, in some embodiments. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each subject. Suitable regimes for initial administration, but are typifiedATTY DKT NO: ASHI-005WO by an initial administration followed by repeated doses at one-hour intervals or longer by a subsequent administration. Alternatively, continuous administration that is sufficient to maintain concentrations in the blood are contemplated.

[0210] The amounts of the active ingredients (e.g., an MMP-9 inhibitor) in the compositions, the composition formulation, and the mode of administration, are among the factors that are varied to provide an amount of the active ingredient that is effective to achieve the desired therapeutic response for each subject, without being unduly toxic to the subject. The selected dosage level will depend upon a variety of factors including the activity of the particular compound employed, the route of administration, the time of administration, the rate of excretion or metabolism of the particular compound being employed, the duration of the treatment, other drugs, compounds and / or materials used in combination with the particular composition employed, the age, sex, weight, condition, general health, diet and prior medical history of the subject being treated, and like factors well known in the medical arts.

[0211] In some embodiments, an MMP-9 inhibitor suitable for use with the disclosed methods or a pharmaceutical composition comprising the same is administered to a subject in various dosing amounts and over various time frames. Additionally, the dose(s) of an MMP-9 inhibitor is administered, in some embodiments, twice a week, weekly, every two weeks, every three weeks, every 4 weeks, every 6 weeks, every 8 weeks, every 12 weeks, or any combination of weeks therein. Dosing cycles are also contemplated, such as, e.g. , administering the MMP-9 inhibitor once or twice a week for 4 weeks, followed by two weeks without therapy. Additional dosing cycles including, e.g., different combinations of the doses and weekly cycles described herein are also contemplated within the disclosure.

[0212] Therapeutically effective amounts of an MMP-9 inhibitor or a composition comprising the same, in some embodiments, vary and depend on the severity of the disease, the subject’s weight, and general state of the subject being treated. Administration is, in some embodiments, daily, on alternating days, weekly, twice a month, monthly, or more or less frequently, as necessary depending on the response of the disorder or condition and the subject’s tolerance to the therapy. In some embodiments, maintenance dosages over a longer period of time, such as 4, 5, 6, 7, 8, 10, or 12 weeks or longer, are needed until a desired suppression of disorder symptoms occurs, and dosages are adjusted as necessary. The progress of this therapy is easily monitored by conventional techniques and assays.ATTY DKT NO: ASHI-005WO

[0213] A physician having ordinary skill in the art, in some cases, readily determines and prescribes the effective amount (ED50) of the composition required. For example, the physician could start doses of the active agents employed in the composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. Alternatively, a dose remains constant in some embodiments.

[0214] An MMP-9 inhibitor suitable for use with the disclosed methods and compositions comprising the same may be presented in unit dosage forms to facilitate accurate dosing. The term “unit dose” or “unit dosage forms” refers to physically discrete units suitable as unitary dosages for human subjects and other mammals, each unit containing a predetermined quantity of active material calculated to produce the desired therapeutic effect, in association with a suitable pharmaceutical excipient. In some embodiments, the dosage forms described herein can be administered as a unit dose. Typical unit dosage forms include prefilled, premeasured ampules or syringes of the liquid compositions or pills, tablets, capsules or the like in the case of solid compositions.

[0215] The dosage of an MMP-9 inhibitor or compositions comprising the same can vary depending on multiple factors, such as, e.g., the pharmacodynamic properties of the compound, the mode of administration, age, health, or weight of the recipient, the nature and extent of the symptoms, frequency of the treatment, the type of concurrent treatment, if any, and the clearance rate of the therapeutic agent in the animal to be treated. One of skill in the art can determine the appropriate dosage based on the above factors. An MMP-9 inhibitor or compositions comprising the same may be administered initially in a suitable dosage that may be adjusted as required, depending on the clinical response. It is noted that dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0216] In some embodiments, the antibody is given at an initial loading dose by intravenous administration, then a maintenance dose given weekly via subcutaneous administration. In some embodiments, the initial loading dose and maintenance dose are both administered subcutaneously.ATTY DKT NO: ASHI-005WO

[0217] An exemplary, non-limiting range for a therapeutically or prophylactically effective amount of an antibody or antibody portion of the invention is selected from 100- 1500 mg, more preferably 200-800 mg and may be about 150, about 300, about 400 mg, about 500 mg, or about 800 mg. As disclosed in US 8,377,443, additional non-limiting ranges include 10 ng / kg to up to 100 mg / kg of mammal body weight or more per day administered to a subject. The dosage may be selected from 1 pg / kg / day, 100 pg / kg / day, 500 pg / kg / day, 1 mg / kg / day, 10 mg / kg / day or 20 mg / kg / day. It is noted that dosage values may vary with the type and severity of the condition to be alleviated. It is further understood that for any particular subject, specific dosage regimens should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions, and that dosage ranges set forth herein are exemplary only and are not intended to limit the scope or practice of the claimed composition.

[0218] In some embodiments, the antibody is given at an initial loading dose by intravenous administration, then a maintenance dose given weekly via subcutaneous administration. In some embodiments, the initial loading dose is 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000, 1200, 1500 mg of the antibody administered intravenously. In some embodiments, the maintenance dose is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg administered subcutaneously on a weekly schedule or every other week schedule.

[0219] In some embodiments, the initial loading dose and maintenance dose are both administered subcutaneously. In some embodiments, the initial loading dose is 150 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000, 1200, or 1500 mg of the antibody administered subcutaneously. In some embodiments, the maintenance dose is 50 mg, 100 mg, 150 mg, 200 mg, 250 mg, 300 mg, 350 mg, or 400 mg administered subcutaneously on a weekly schedule or every other week schedule.

[0220] In some embodiments, the subject is between about 2 and about 5 years of age and the antibody is administered subcutaneously with a dose of between 10 mg and 150 mg or as appropriately scaled from an adult dose. Preferably, the antibody is administered subcutaneously with a dose of 15mg to 50mg. In some embodiments, the dose is 10 mg. In some embodiments, the dose is 15 mg. In some embodiments, the dose is 20 mg. In some embodiments, the dose is 25 mg. In some embodiments, the dose is 30 mg. In some embodiments, the dose is 35 mg. In some embodiments, the dose is 40 mg. In someATTY DKT NO: ASHI-005WO embodiments, the dose is 45 mg. In some embodiments, the dose is 50 mg. In some embodiments, the dose is administered every week or every other week.

[0221] In some embodiments, a loading dose, or up to three loading doses, is followed one week later or two weeks later by a maintenance dose of administered every week or every other week, where the loading dose is greater than the maintenance dose. In other embodiments, the loading dose is not administered. In other embodiments, the loading dose is replaced by administration of another therapeutic modality.

[0222] In some embodiments, the subject is between about 6 and about 11 years of age and the antibody is administered subcutaneously with a dose of 25mg to 150mg. Preferably, the antibody is administered subcutaneously with a dose of 25mg to 75mg. In some embodiments, the dose is 25 mg. In some embodiments, the dose is 30 mg. In some embodiments, the dose is 35 mg. In some embodiments, the dose is 40 mg. In some embodiments, the dose is 45 mg. In some embodiments, the dose is 50 mg. In some embodiments, the dose is 55 mg. In some embodiments, the dose is 60 mg. In some embodiments, the dose is 65 mg. In some embodiments, the dose is 70 mg. In some embodiments, the dose is 75 mg. In some embodiments, the dose is administered every week or every other week.

[0223] In preferred embodiments, the subject is of age greater than about 12 years and an MMP-9 inhibitor (such as andecaliximab) dose of 150 to 1500 mg is administered every week. In another preferred embodiment, the subject is of age greater than about 12 years and an MMP-9 inhibitor dose of 50 mg to 300 mg is administered every week. In another preferred embodiment, the subject is aged between about 6 to about 11 years and an MMP- 9 inhibitor dose of 75 mg is administered every week. In another preferred embodiment, the subject is aged between about 6 to about 11 years and an MMP-9 inhibitor dose of 25 mg is administered every week. In another preferred embodiment, the subject is aged between 2 and about 5 years and an MMP-9 inhibitor dose of 45 or 50 mg is administered every week. In another preferred embodiment, the subject is aged between 2 and about 5 years and an MMP-9 inhibitor dose of 15 mg is administered every week.

[0224] In preferred embodiments, the subject is aged between about 6 and about 11 years and an MMP-9 inhibitor (such as andecaliximab) dose of 75 mg is administered every week. In some embodiments, the subject is aged between about 6 and about 11 years and a dose of 25 mg is administered every week. In some embodiments, the subject is aged between about 2 and about 5 years and a dose of 50 or 45 mg is administered every otherATTY DKT NO: ASHI-005WO week. In some embodiments, the subject is aged 2-5 years, and a dose of 15 mg is administered every other week. In some embodiments, the subject is aged 6-11 years, and a dose of 75 mg is administered every other week. In some embodiments, the subject is aged 2-5 years, and a dose of 45 or 50 mg is administered every other week.

[0225] In some embodiments, there are two dosing regimens. In some embodiments, a higher dose gives complete target coverage across the entire age population. In other embodiments, the lower dose gives good coverage and mitigates potential side effects regarding open growth plates in children.

[0226] In other embodiments, the dosing is adjusted by age group.

[0227] In some embodiments, the antibody is formulated as a depot or sustained release formulation and the dosing interval frequency is reduced by a factor of about two or three or four or greater than four. Sustained release formulations are described in, e.g., US 10,000,562. Suitable examples of sustained-release preparations include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films, or microcapsules. Examples of sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and y-ethyl-L-glutamate, non-degradable ethylene- vinyl acetate, degradable lactic acid- glycolic acid copolymers such as the LUPRON DEPOT™ (injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolide acetate), and poly-D-(-)-3- hydroxybutyric acid. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon-(rhlFN-), interleukin-2, and MN rpg 120. Johnson et al., Nat. Med. 2: 795-799 (1996); Yasuda et al., Biomed. Ther. 27: 1221-1223 (1993); Hora et al., Bio / Technology 8: 755-758 (1990); Cleland, “Design and Production of Single Immunization Vaccines Using Polylactide Polyglycolide Microsphere Systems,” in Vaccine Design: The Subunit and Adjuvant Approach, Powell and Newman, eds., (Plenum Press: New York, 1995), pp. 439-462; WO 97 / 03692; WO 96 / 40072; WO 96 / 07399; and U.S. Pat. No. 5,654,010.Anti-MMP-9 Antibodies

[0228] Disclosed herein, in some embodiments, are anti-MMP-9 antibodies or antigen-binding fragments thereof, such as inhibitory anti-MMP-9 antibodies or antigenbinding fragments thereof. For example, WO / 2017 / 177179 discloses various anti-MMP-9ATTY DKT NO: ASHI-005WO antibodies. The antibodies show anti-MMP-9 activity; however, there appears to be no known successful therapies including administration of such antibodies used to treat heterotopic ossification such as NHHO. Accordingly, provided herein are methods for treating NHHO by administering an anti-MMP-9 antibody or antigen-binding fragment thereof to inhibit MMP-9. In some embodiments, the anti-MMP-9 antibody or antigenbinding fragment thereof inhibits MMP-9 activation or enzymatic activity.

[0229] In some embodiments, the inhibitor of MMP-9 comprises an anti-MPP-9 antibody or antigen-binding fragment thereof, wherein the anti-MPP-9 antibody or antigenbinding fragment thereof binds to (i) an MMP-9 pro-form and inhibits activation of the preform and / or (ii) an MMP-9 active form and inhibits activity of the active form and is used as a method for treating HO (e.g., NHHO).

[0230] In some embodiments, the anti-MMP-9 antibody or antigen- binding fragment thereof binds to the MMP-9 pro-form to inhibit MMP-9 activation and is used as a method for treating HO (e.g., NHHO).

[0231] In some embodiments, the anti-MMP-9 antibody or antigen-binding fragment thereof binds allosterically to the active form of MMP-9 to inhibit MMP-9 activity and is used as a method for HO (e.g., NHHO).

[0232] In some embodiments, therapeutic antibodies for administration are characterized as binding to one or more processing sites (e.g., sites of proteolytic cleavage) in MMP-9, thereby effectively blocking processing of the proenzyme or preproenzyme to the catalytically active enzyme and thus reducing the proteolytic activity of the MMP-9.

[0233] In some embodiments, therapeutic antibodies for administration are characterized as binding to MMP-9 with an affinity at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 times, at least 100 times, at least 500 times, or at least 1000 times greater than its binding affinity for another MMP. Binding affinity can be measured by any method known in the art and can be expressed as, for example, on-rate, off-rate, dissociation constant (Kd), equilibrium constant (Keq) or any term in the art.Various examples of such affinity-matured antibodies are within the scope of the invention.

[0234] In some embodiments, therapeutic antibodies for administration are characterized as non-competitive inhibitor of the catalytic activity of MMP-9. In some embodiments, an anti-MMP-9 antibody or antigen-binding fragment thereof suitable for use with the methods of the present disclosure binds within the catalytic domain of MMP-9. In some embodiments, an anti-MMP-9 antibody or antigen-binding fragment thereof suitableATTY DKT NO: ASHI-005WO for use with the methods of the present disclosure binds outside the catalytic domain of MMP-9.

[0235] Additional antibodies or antigen-binding fragments thereof are contemplated within the scope of the invention that compete with anti-MMP-9 antibodies or antigenbinding fragments thereof described herein for binding to MMP-9. For instance, anti-MMP- 9 antibodies, and functional fragments thereof, that compete for binding with, for example, an antibody having a heavy chain polypeptide, a light chain polypeptide, or combinations thereof. In some embodiments, a method for treating HO (e.g., NHHO) comprises administration of one or more anti-MMP-9 antibodies or antigen-binding fragments comprising one or more amino acid sequences listed in Table 2.

[0236] WO / 2017 / 177179 discloses anti-MMP-9 antibodies, such as AB0045(andecaliximab). Andecaliximab binds the catalytic domain of MMP-9 with additional points of contact shielding the site of physiological activation in the prodomain, where it inhibits pro-MMP-9 activation and non-competitively inhibits MMP-9 activity.Andecaliximab binds to MMP-9 with a KD of 2.0-6.6 nM and to pro-MMP-9 with a KD of 0.008-0.043 nM. Andecaliximab inhibits activation of MMP-9 from human pro-MMP-9 with an IC50 of 8.2 pM. Clinical trials with andecaliximab have shown the antibody to be safe and well-tolerated. Accordingly, in some embodiments, a method of treating HO e.g., NHHO) comprises administration of andecaliximab or antigen-binding fragments thereof to a subject. In a surgical orthotopic xenograft model of colorectal carcinoma, the AB0046 antibody (a murine surrogate of andecaliximab) treatment resulted in reduced tumor growth and metastasis.

[0237] In some embodiments, the antibodies used in a method for treating NHHO comprise antibodies disclosed in WO / 2017 / 177179, PCT / US2012 / 027160,PCT / US2016 / 067036, PCT / US2016 / 054780, WO / 2016 / 023979A1, WO / 2008 / 102359A1, WO / 2002 / 066057, WO / 2006 / 037513, WO / 2009 / 111508, WO / 2011 / 028883,WO / 2012 / 154654, US / 2010 / 0098659, WO / 2010 / 048455, or WO / 2012 / 048291 , all of which are herein incorporated by reference in their entirety.

[0238] In some embodiments, antibodies or antigen-binding fragments thereof suitable for use with the methods of the disclosure can be derivatized or linked to another functional molecule. For example, the antibody is functionally linked (by chemical coupling, genetic fusion, noncovalent association or otherwise) to one or more other molecular entities, such as another antibody e.g., a bispecific antibody or a diabody), aATTY DKT NO: ASHI-005WO detectable agent, a cytotoxic agent, a pharmaceutical agent, and / or a protein or peptide that can mediate association of the antibody with another molecule (such as a streptavidin core region or a polyhistidine tag).

[0239] In some embodiments, an antibody or antigen-binding fragment thereof suitable for use with the disclosed methods is used in a variety of settings. For example, an antibody or antigen-binding fragment thereof suitable for use with the disclosed methods is administered as a therapeutic agent. In such embodiments, an antibody or antigen-binding fragment thereof can exert its therapeutic effect by any of a variety of mechanisms. For example, an antibody or antigen-binding fragment thereof suitable for use with the disclosed methods may be an antagonist antibody. In yet another example, an antibody or antigen-binding fragment thereof suitable for use with the disclosed methods may be a blocking antibody. In some embodiments, an antibody or antigen-binding fragment thereof suitable for use with the disclosed methods is a neutralizing antibody.

[0240] In certain embodiments, the anti-MMP-9 antibodies is andecaliximab or an antigen-binding fragment thereof. Andecaliximab binds MMP-9 at an arginine residue at position 162 of the MMP-9 amino acid sequence (R162). US / 2012 / 0135004. Additional MMP-9 residues that are bound by andecaliximab are El 11, DI 13, and 1198. R162, El 11, DI 13, and 1198 are near a Ca2+ ion binding pocket on MMP-9. US / 2012 / 0135004. Accordingly, in some embodiments, administration of an andecaliximab or an antigenbinding fragment thereof results in binding of andecaliximab to these residues.

[0241] In certain embodiments, an anti-MMP-9 antibody suitable for use with the disclosed methods comprises a heavy chain comprising an amino acid having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:1 and a light chain comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:2. The variable regions may comprise an amino acid having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:3 and a light chain comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% sequence identity to the amino acid sequence of SEQ ID NO:4.ATTY DKT NO: ASHI-005WODiagnostic Use of Antibodies

[0242] In certain preferred embodiments, the antibodies are used to detect subjects having certain musculoskeletal diseases or conditions or disorders, or other MMP-9 mediated disorders, using a conventional immunoassay, such as an enzyme-linked immunosorbent assays (ELISA), a radioimmunoassay or tissue immunohistochemistry. The method comprises contacting a biological sample with the antibody of the invention wherein the antibody detects one or more disorders. Preferably, the antibody is labeled with a detectable marker such as fluorescent labels. Certain preferred examples of suitable enzymes include horseradish peroxidase, alkaline phosphatase, -galactosidase, or acetylcholinesterase; examples of suitable prosthetic group complexes include streptavidin / biotin and avidin / biotin; examples of suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; an example of a luminescent material includes luminol; and examples of suitable radioactive material include125I,131I,35S or3H. In alternative embodiments, MMP-9 can be assayed in biological fluids by one of many known techniques in the art.COMBINATIONS WITH OTHER DRUGS

[0243] In some embodiments, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with a hypoxia-inducible factor- la (Hif-la) inhibitor. Without being bound by theory, treatment with a Hif-la inhibitor is thought to reduce nongenetic heterotopic ossification or other MMP-9 mediated disorders by preventing Hif- la-mediated upregulation of signaling from BMP-2. In the BMP-2 pathway, a ligand such as BMP2 activates the receptor(s) of BMP-2, causing phosphorylation of SMAD 1 / 5 / 8, which leads to gene transcription, cell differentiation, and cell proliferation resulting in increased osteogenic factors. Thus, blocking this pathway with a Hif-la inhibitor could reduce nongenetic heterotopic ossification or other MMP-9-mediated disorders. Additionally, Hif- la leads to increased expression of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (BFGF), platelet-derived growth factor (PDGF), and angiopoietin- 2, all of which are needed for endothelial cell motility, recruitment, and proliferation. (Ranganathan, K., Loder, S., Agarwal, S., Wong, V.W., Forsberg, J., David, T.A., Wang,ATTY DKT NO: ASHI-005WOS., James, A.W., and Levi, B., J Bone Joint Surg Am. 2015 Jul 1 ; 97(13): 1 101-1 1 1 1 ). Hif- la also contributes to nongenetic heterotopic ossification or other MMP-9 mediated disorders by regulating sex-determining region Y-box 9, which is a necessary cartilage precursor. (Ranganathan, K., Loder, S., Agarwal, S., Wong, V.W., Forsberg, J., David,T.A., Wang, S., James, A.W., and Levi, B., J Bone Joint Surg Am. 2015 Jul 1 ; 97(13):1101-1111). In some embodiments, the method of nongenetic heterotopic ossification or other MMP-9 mediated disorders by administering an MMP-9 inhibitor is combined with the Hif- la inhibitor applied locally. In some embodiments, the Hif-la inhibitor is imatinib, rapamycin, or a siRNA-based therapy.

[0244] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with a small molecule inhibitor of bone morphogenetic protein (BMP) signaling.

[0245] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with an antibody against BMP9.

[0246] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with an antibody against BMP10.

[0247] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with momelotinib.

[0248] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with BMP pathway inhibitors, such as small molecule kinase inhibitors (eg., sarcatinib).

[0249] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with palovarotene. Without being bound by theory, the mechanism by which palovarotene is thought to inhibit osteochondromas, chondrosarcomas, osteosarcomas and hereditary multiple exostoses is through the RAR-y receptor expressed on chondrogenic cells and chondrocytes, which is a transcriptional repressor that will inhibit osteogenesis. (Shimono K, Tung WE, Macolino C, Chi AH, Didizian JH, Mundy C,ATTY DKT NO: ASHI-005WOChandraratna RA, Mishina Y, Enomoto-Iwamoto M, Pacifici M, Iwamoto M. Potent inhibition of osteochondromas, osteosarcomas and hereditary multiple exostoses by nuclear retinoic acid receptor-y agonists. Nat Med. 2011. April; 17(4): 454-60. Epub 2011 Apr 3). Administering palovarotene, a RAR-y receptor agonist, prevents mesenchymal stem cell differentiation into chondrocytes, thereby preventing chondrogenesis and endochondral ossification. (Shimono K, Tung WE, Macolino C, Chi AH, Didizian JH, Mundy C, Chandraratna RA, Mishina Y, Enomoto-Iwamoto M, Pacifici M, Iwamoto M. Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-y agonists. Nat Med. 2011. April;17(4):454-60. Epub 201 1 Apr 3). Palovarotene has shown promise in a mouse model of fibrodysplasia ossificans progressiva and is currently in phase 3 clinical trials. (Shimono K, Tung WE, Macolino C, Chi AH, Didizian JH, Mundy C, Chandraratna RA, Mishina Y, Enomoto-Iwamoto M, Pacifici M, Iwamoto M. Potent inhibition of heterotopic ossification by nuclear retinoic acid receptor-y agonists. Nat Med. 2011. April; 17(4) :454- 60. Epub 2011 Apr 3;).

[0250] In another embodiment, the method further comprises administration of an additional active agent or supportive therapy for treating heterotopic ossification, selected from the group consisting of isotretinoin, etidronate with oral corticosteroids, perhexiline maleate, ALK2 small-molecule inhibitors, palovarotene, retinoic acid receptor gamma agonists, retinoic acid receptor alpha agonists, activin antibodies, allele-specific RNA interference of ALK2, bisphosphonates, radiation therapy anti-inflammatory agents, and conservative treatments such as passive range of motion exercises or other mobilization techniques.

[0251] In another embodimen t, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with imatinib.

[0252] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with momelotinib.

[0253] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with chromolyn.ATTY DKT NO: ASHI-005WO

[0254] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with apyrase.

[0255] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with a corticosteroid.

[0256] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with a broad-spectrum antibiotic, such as tetracycline antibiotics, including minocycline, doxycycline, sarecycline, or lymecycline.

[0257] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with an ALK2 inhibitor or antibody.

[0258] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with an ALK3 inhibitor or antibody.

[0259] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with an ALK6 inhibitor or antibody.

[0260] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with an Activin-A antibody or inhibitor.

[0261] In another embodiment, the method of treating nongenetic heterotopic ossification or other MMP-9 mediated disorders with an MMP-9 inhibitor is administered in combination with another MMP inhibitor, including, but not limited to, MMP-2, MMP- 7, MMP- 13, MMP- 14, and / or MMP- 16 inhibitor.

[0262] As disclosed in WO2023192880, non-limiting examples of non-antibody protein or peptide inhibitors of MMP-9 include proteins from the tissue inhibitors of metalloproteinase (TIMP) family such as TIMP-1, TIMP-3 and the like, FFAGLDD peptide, FFAGLDD TFA, cyclic CTTHWGFTLC, cyclic CTTHWGFTLC TFA, and the like.

[0263] Non-limiting examples of small molecules that inhibit MMP-9 include actinonin, ageladine A TFA, apigenin-7-glucuronide, ARP 100, astragaloside IV, BR351,ATTY DKT NO: ASHI-005WO chlorhexidine dihydrochloride, cipemastat, CMC2.24, CP-471474, CP-544439, cyclic CTTHWGFTLC, cyclic CTTHWGFTLC TFA, FSL-1 TFA, ginkgolide C, ilomastat (also referred to as GM6001), JNJ0966, luteolin 7-0-glucuronide, marimastat, MMP-2 / MMP-9 Inhibitor I, MMP-2 / MMP-9 Inhibitor II, MMP3 inhibitor 1, MMP-9-IN-1, MMP-9 Inhibitor I, MMP-9 Inhibitor II, MMP- 9 / MMP-13 inhibitor I, MMP Inhibitor II, MMP13- IN-3, MMPI-1154, morroniside, ND-336, NNGH, (R)-ND-336, PF-00356231 hydrochloride, PD-166793, prinomastat, prinomastat hydrochloride, salvianolic acid A, S 3304, SB-3CT, SM-7368, tanomastat, tetracycline derivatives such as doxycycline, incyclinide, and minocycline, UK 356618, UK-370106, XL-784, and the like. MMP-9 inhibitors are described in, for example, Fields (Cells. 2019 Sep; 8(9): 984.)TARGET BIOLOGY

[0264] MMP-9 is a matrix metalloproteinase that breaks down extracellular matrix proteins and recruits cytokines and chemokines involved in tissue remodeling.(Yabluchanskiy, A., Ma, Y., Iyer, R.P., Hall, M.E., and Lindsey, M. L., Physiology (Bethesda). 2013 Nov; 28(6): 391-403). Neutrophils, macrophages, fibroblasts, and endothelial cells are among the cell types that secrete MMP-9. (Vandooren J., Van den Steen P.E., Opdenakker G. Biochemistry and molecular biology of gelatinase B or matrix metalloproteinase-9 (MMP-9): The next decade. Crit. Rev. Biochem. Mol. Biol. 2013;48:222-272. doi: 10.3109 / 10409238.2013.770819). Secreted MMP-9 exists in a pro- form, which requires other proteases to cleave it, thereby converting it into the active form. (Huang, H., Sensors (Basel). 2018 Oct; 18(10): 3249). MMP-9 latency is maintained by the interaction of one cysteine (Cys99) residue in the propeptide of pro-MMP-9 with the catalytic zinc ion of MMP-9. (Huang, H., Sensors (Basel). 2018 Oct; 18(10): 3249). When this interaction is disrupted by proteolytic cleavage, reactive oxygen species, or nitric oxide, MMP-9 is activated. (Huang, H., Sensors (Basel). 2018 Oct; 18(10): 3249). In some embodiments, the methods of the invention provide one or more MMP-9 inhibitors, which when administered protects the Cys99 interaction with the catalytic zinc ion of MMP-9, thereby preventing MMP-9 activation.

[0265] Activated MMP-9 recognizes cleavage sites with the sequence Pro-X-X-Hy- (Ser / Thr) at P3 position through P2' position, where X is any residue; Hy is a hydrophobic residue; and the cleavage site is between Pl -Pl '. (Kridel S.J., Chen E., Kotra L.P., Howard E.W., Mobashery S., Smith J.W. Substrate hydrolysis by matrix metalloproteinase-9. J.ATTY DKT NO: ASHI-005WOBiol. Chem. 2001 ;276:20572-20578. doi: 10.1074 / jbc. Ml 00900200). In some embodiments, the methods of the invention provide one or more MMP-9 inhibitors, which prevent recognition of these cleavage sites.

[0266] In various aspects, the methods of the invention are used as therapies to treat disorders or health conditions related to the biological effects associated with MMP-9. Such diseases or health conditions include but are not limited to nongenetic heterotopic ossification and other MMP-9 mediated diseases, including chronic inflammatory autoimmune disease and diseases associated with apoptotic, necrotic, aberrant or oncogenic mammalian cells, among others. (Davis EL, et al., Location-dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9, J Orthop Res, 2016, 34(11):1894-1904; Ortega N, et al., Complementary interplay between matrix metalloproteinase-9, vascular endothelial growth factor and osteoclast function drives endochondral bone formation, Dis Model Meeh., 2010, 3(3-4):224-35.)

[0267] In some embodiments an inflammatory disease, e.g. an inflammatory autoimmune disease, that is associated with nongenetic heterotopic ossification is treated by the methods of the disclosure. Such diseases include, without limitation, axial spondylarthritis, diffuse idiopathic skeletal hyperostosis autoimmune disease, ankylosing spondylitis, Guillain-Barre Syndrome (GBS), Anti-NMDA Receptor Encephalitis, dermatomyositis, systemic sclerosis, inflammatory arthritis, etc.

[0268] In some embodiments a condition for treatment is axial spondyloarthritis (axSpA), which is an inflammatory disease continuum that ranges from nonradiographic axSpA (nr-axSpA) to radiographic axSpA (r-axSpA), also known as ankylosing spondylitis (AS). Heterotopic ossification is a complication in the late stage of AS. The condition is defined by axial joint involvement, often sacroiliitis, but peripheral arthritis and extra- articular involvement (uveitis, inflammatory bowel disease [IBD], enthesitis, and psoriasis), which are shared with other types of spondyloarthritis (SpA), are quite common. Patients with nr-axSpA vs AS are distinguished by the absence vs presence of definitive sacroiliitis on plain radiographs. Patients present with symptoms common to other conditions, which can confound diagnosis, particularly in the absence of radiographic sacroiliitis (as in nr- axSpA). AS may be classified using the modified New York criteria, which require radiographically definitive sacroiliitis. In 2009, the Assessment of SpondyloArthritis international Society (ASAS) produced a classification of axSpA that includes patients with r-axSpA (also called AS) and nr-axSpA. Although nr-axSpA is currently classified as aATTY DKT NO: ASHI-005WO separate condition, “axSpA” may become a universally accepted unitary term embracing both r- and nr-axSpA.

[0269] In some embodiments a condition for treatment is diffuse idiopathic skeletal hyperostosis (DISH), which is a progressive non-inflammatory bone and entheses disease. The hallmark of this disease is the ossification of the ligaments and entheses in the prevertebral and prediscal regions of the spine. The hyperostotic calcification typically affects the anterolateral ligaments of the thoracic vertebrae. However, the cervical and lumbar vertebrae may also be affected. The diagnosis of DISH requires the radiological presence of free flowing ossification of the anterior longitudinal ligaments of the spine over at least four consecutive levels. Peripheral involvement is not uncommon, but it is hard to ascribe to DISH in the absence of typical spinal changes. The aetiology of DISH is unknown, but several authors have demonstrated its association with diabetes mellitus and advanced age. Studies conducted in different population settings have shown that the incidence of DISH varies from 2.9% in an Asian population to between 15% and 25% in the Caucasian population.

[0270] In various preferred embodiments, administration of an MMP-9 inhibitor as a remedy to treat MMP-9 mediated disorders or health conditions involves an anti-MMP-9 antibody or antibody fragment. GS-5745 or humanized AB0041 or andecaliximab is shown to bind MMP-9 at an arginine residue at position 162 of the MMP9 amino acid sequence (R162). US / 2012 / 0135004. Additional MMP-9 residues that bind GS-5745 or humanized AB0041 or andecaliximab are El 11, DI 13, and 1198. US / 2012 / 0135004. R162, El 11, DI 13, and 1198 are near a Ca2+ion binding pocket on MMP-9. US / 2012 / 0135004. Accordingly, in various embodiments, additional aspects of the invention contemplate administration of an MMP-9 inhibitor such as, for instance, anti-MMP-9 antibody GS-5745 or humanized AB0041 or andecaliximab or antibody fragments that bind to these residues.

[0271] Heterotopic ossification in mice involves MMP-9, which opens the bloodnerve barrier in mouse peripheral nerves. Rodenberg E, et al. Matrix metalloproteinase-9 is a diagnostic marker of heterotopic ossification in a murine model. Tissue Eng Part A 17:2487-2496 (2011). Opening the blood-nerve barrier is necessary for heterotopic ossification because it allows osteoblast progenitors to enter into endoneurial vessels and exit the nerve through the circulation. Salisbury E, et al. Sensory nerve induced inflammation contributes to heterotopic ossification. J Cell Biochem 112:2748-2758ATTY DKT NO: ASHI-005WO(2011); Lazard ZW, et al. Osteoblasts have a neural origin in heterotopic ossification. Clin Orthop Relat Res 473:2790-2806 (2015). MMP-9 may mediate opening of the endoneurial vessels through binding and regulation of claudin 1. Hackel D, et al. Transient opening of the perineurial barrier for analgesic drug delivery. Proc Natl Acad Sci USA 109:E2018- E2027 (2012).

[0272] MMP-9 activity is elevated in mice within 24 hours following the induction of heterotopic ossification. Davis EL, et al. Location-dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9. J Orthop Res. 34(11): 1894- 1904 (2016)). Treatment of heterotopic ossification in mice with the MMP-9 inhibitor minocycline results in significantly reduced (p<0.057) bone formation compared to mice treated with the vehicle. Minocycline treatment may reduce heterotopic ossification in mice by inhibiting activation of MMP-9. The activated form of MMP-9 was absent from minocycline treated mice, which had a percentage of cells expressing active MMP-9 / total MMP-9 of 4.1% ± 0.56 S.E.M. compared to 95.5% ± 1.75 S.E.M for vehicle treated mice. Davis EL, et al. Location-dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9. J Orthop Res. 34(11): 1894-1904 (2016). As disclosed by Davis EL, the knockout of MMP-9 decreases the permeability of vasculature and fibrinogen deposition, demonstrating that a decrease in fibrinogen deposition, associated with a lack of active MMP-9, resulted in suppression of HO (Davis EL, et al., (2017). Bone, 109:22-27). These results suggest a knockout MMP-9 study may lead to a favorable result in the treatment and prevention of heterotopic ossification.

[0273] MMP-9 may play a role in heterotopic ossification in humans as well. Tissues from patients undergoing active bone formation show positive staining for both active MMP9 and total MMP9 protein, with the majority of the protein being activated. Davis EL, et al. Location-dependent heterotopic ossification in the rat model: The role of activated matrix metalloproteinase 9. J Orthop Res. 34(11): 1894-1904 (2016). In humans, the entire bone may be of neural origin, so MMP-9 inhibition could be more effective in treating heterotopic ossification in humans than in mice.

[0274] Table 1. Animal study designATTY DKT NO: ASHI-005WOKITS

[0275] In various aspects, the invention provides kits comprising one or more components that comprise, but are not limited to, MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids, as disclosed herein, one or more pharmaceutically acceptable carrier and / or a therapeutic agent. In certain embodiments, the MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids and / or the therapeutic agent can be formulated as a substantially pure composition or in combination with a pharmaceutically acceptable carrier, in a pharmaceutical composition.

[0276] In alternative aspects, the MMP-9 inhibitors such as antibodies or antigenbinding fragments, small molecules, peptides, nucleic acids are provided as components of a diagnostic or theranostic kit including MMP9 imaging kits incorporating radioisotopes such as gallium, copper, indium or similar imaging agents.

[0277] In various aspects, the kit includes MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids thereof of the invention or a pharmaceutical composition thereof in one container (e.g., in a sterile glass or plastic vial) and a pharmaceutical composition thereof and / or a therapeutic agent in another container (e.g., in a sterile glass or plastic vial).

[0278] In another embodiment, the kit comprises a combination of the invention, including MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids thereof of the invention with a pharmaceutically acceptable carrier, optionally in combination with one or more therapeutic agents formulated together, optionally, in a pharmaceutical composition, in a single, common container.

[0279] If the kit includes a pharmaceutical composition for parenteral administration to a subject, the kit can include a device for performing such administration. For example,ATTY DKT NO: ASHI-005WO the kit can include one or more hypodermic needles or other injection devices or autoinjector devices known in the art.

[0280] Optional components of the kit include a package insert including information concerning the pharmaceutical compositions and dosage forms of MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids in the kit. Generally, such information aids patients and physicians in using the enclosed pharmaceutical compositions and dosage forms effectively and safely. For example, the following information regarding a combination of the invention may be supplied in the insert: pharmacokinetics, pharmacodynamics, clinical studies, efficacy parameters, indications and usage, contraindications, warnings, precautions, adverse reactions, overdosage, proper dosage and administration, how supplied, proper storage conditions, references, manufacturer / distributor information and patent information.

[0281] Preferred use for the kits comprising the anti-MMP-9 antibody or antigenbinding fragment thereof of the present invention includes applications for detecting nongenetic heterotopic ossification conditions.DETECTION KITS AND THERAPEUTIC KITS

[0282] MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids thereof of the invention can be packaged as reagents in predetermined amounts with instructions for performing the diagnostic or detection assay. In certain embodiments, where the antibody or fragment is labeled with an enzyme, the kit will include substrates and cofactors required by the enzyme (e.g., a substrate precursor which provides the detectable chromophore or fluorophore). Other additives may be included such as stabilizers, buffers (e.g., a block buffer or lysis buffer) and the like. The relative amounts of the various reagents may be varied widely to provide for concentrations in solution of the reagents which substantially optimize the sensitivity of the assay. Depending on the desired use, the reagents may be provided as dry powders, usually lyophilized, including excipients which on dissolution will provide a reagent solution having the appropriate concentration.

[0283] Also provided are diagnostic or detection reagents and kits comprising one or more such reagents for use in a variety of detection assays, including for example, immunoassays such as ELISA (sandwich-type or competitive format). The kit's components may be pre-attached to a solid support or may be applied to the surface of aATTY DKT NO: ASHI-005WO solid support when the kit is used. In some embodiments of the invention, the signal generating means may come pre- associated with an antibody or fragment of the invention or may require combination with one or more components, e.g., buffers, antibody-enzyme conjugates, enzyme substrates, or the like, prior to use. Kits may also include additional reagents, e.g., blocking reagents for reducing nonspecific binding to the solid phase surface, washing reagents, enzyme substrates, and the like. The solid phase surface may be in the form of a tube, a bead, a microtiter plate, a microsphere, or other materials suitable for immobilizing proteins, peptides, or polypeptides. In particular aspects, an enzyme that catalyzes the formation of a chemiluminescent or chromogenic product or the reduction of a chemiluminescent or chromogenic substrate is a component of the signal generating means. Such enzymes are well known in the art. Kits may comprise any of the capture agents and detection reagents described herein. The kit can also include instructions for carrying out the methods of the invention.

[0284] In certain preferred aspects, the kit comprises one or more labels describing the contents of the container and providing indications and / or instructions regarding use of the contents of the container to treat one or more MMP-9 associated disorders as described herein using MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids thereof of the invention.

[0285] In one aspect, the kit comprises treatment for MMP-9 related disorders including nongenetic heterotopic ossification by administering one or more MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids thereof of the invention. The kit may optionally further include a syringe for parenteral, e.g., intravenous, or subcutaneous administration. Preferably, the kit comprises anti-MMP-9 antibody or antigen-binding fragment thereof and a label attached to or packaged with the container describing use of the antibody or fragment. In yet another aspect, the kit comprises a prophylactic agent or further therapeutic agent and a label attached to or packaged with the container describing use of the prophylactic agent or further therapeutic agent with the anti-MMP-9 antibody or fragment. In certain embodiments, an anti-MMP-9 antibody and prophylactic agent or further therapeutic agent are in separate vials or are combined together in the same pharmaceutical composition.

[0286] As discussed above in the combination therapy section, concurrent administration of two therapeutic agents does not require that the agents he administered at the same time or by the same route, as long as there is an overlap in the time period duringATTY DKT NO: ASHI-005WO which the agents are exerting their therapeutic effect. Simultaneous or sequential administration is contemplated, as is administration on different days or weeks.

[0287] The therapeutic and detection kits disclosed herein may also be prepared with one or more MMP-9 inhibitors such as antibodies or antigen-binding fragments, small molecules, peptides, nucleic acids disclosed herein and instructions for using the composition as a detection reagent or therapeutic agent. Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection and / or therapeutic composition(s) may be placed, and preferably suitably aliquoted. Where a second therapeutic agent is also provided, the kit may also contain a second distinct container into which this second detection and / or therapeutic composition may be placed. Alternatively, a plurality of compounds may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container. The kits disclosed herein will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorogenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the detection or therapeutic composition itself or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and / or convenient packaging and delivery. A device or apparatus for carrying out the detection or monitoring methods described herein is also provided. Such an apparatus may include a chamber or tube into which sample can be input, a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex. The flow of sample may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied) or active (e.g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure), or by aATTY DKT NO: ASHI-005WO combination of active and passive forces. In further embodiments, also provided is a processor, a computer readable memory, and a routine stored on the computer readable memory and adapted to be executed on the processor to perform any of the methods described herein. Examples of suitable computing systems, environments, and / or configurations include personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, or any other systems known in the art.

[0288] The therapeutic and detection kits disclosed herein may also be prepared as a combination treatment with one or more MMP-9 inhibitors, such as antibodies or antigenbinding fragments, small molecules, peptides, nucleic acids disclosed herein, and another MMP inhibitor, including, but not limited to, MMP-2, MMP-7, MMP-13, MMP-14, and / or MMP-16, along with instructions for using the composition as a detection reagent or therapeutic agent. Containers for use in such kits may typically comprise at least one vial, test tube, flask, bottle, syringe or other suitable container, into which one or more of the detection and / or therapeutic composition(s) may be placed, and preferably suitably aliquoted. Where a second therapeutic agent is also provided, the kit may also contain a second distinct container into which this second detection and / or therapeutic composition may be placed. Alternatively, a plurality of compounds may be prepared in a single pharmaceutical composition, and may be packaged in a single container means, such as a vial, flask, syringe, bottle, or other suitable single container. The kits disclosed herein will also typically include a means for containing the vial(s) in close confinement for commercial sale, such as, e.g., injection or blow-molded plastic containers into which the desired vial(s) are retained. Where a radiolabel, chromogenic, fluorogenic, or other type of detectable label or detecting means is included within the kit, the labeling agent may be provided either in the same container as the detection or therapeutic composition itself or may alternatively be placed in a second distinct container means into which this second composition may be placed and suitably aliquoted. Alternatively, the detection reagent and the label may be prepared in a single container means, and in most cases, the kit will also typically include a means for containing the vial(s) in close confinement for commercial sale and / or convenient packaging and delivery. A device or apparatus for carrying out the detection or monitoring methods described herein is also provided. Such an apparatus mayATTY DKT NO: ASHI-005WO include a chamber or tube into which sample can be input, a fluid handling system optionally including valves or pumps to direct flow of the sample through the device, optionally filters to separate plasma or serum from blood, mixing chambers for the addition of capture agents or detection reagents, and optionally a detection device for detecting the amount of detectable label bound to the capture agent immunocomplex. The flow of sample may be passive (e.g., by capillary, hydrostatic, or other forces that do not require further manipulation of the device once sample is applied) or active (e.g., by application of force generated via mechanical pumps, electroosmotic pumps, centrifugal force, or increased air pressure), or by a combination of active and passive forces. In further embodiments, also provided is a processor, a computer readable memory, and a routine stored on the computer readable memory and adapted to be executed on the processor to perform any of the methods described herein. Examples of suitable computing systems, environments, and / or configurations include personal computers, server computers, hand-held or laptop devices, multiprocessor systems, microprocessor-based systems, set top boxes, programmable consumer electronics, network PCs, minicomputers, mainframe computers, distributed computing environments that include any of the above systems or devices, or any other systems known in the art.EQUIVALENTS

[0289] It will be readily apparent to those skilled in the art that other suitable modifications and adaptions of the methods of the invention described herein are obvious and may be made using suitable equivalents without departing from the scope of the disclosure or the embodiments. Having now described certain compounds and methods in detail, the same will be more clearly understood by reference to the following examples, which are introduced for illustration only and not intended to be limiting.EXAMPLES

[0290] The present invention is further described by the following examples, which are not intended to be limiting in any way.EXAMPLE 1 : In vitro assay for antibody binding to MMP-9

[0291] MMP-9 levels and gene expression levels are measured by ELISA and reverse transcription-quantitative polymerase chain reaction. ELISA kit for MMP-9 is available from Cusabio Biotech Co., Ltd. (Wuhan, China). Anti-MMP-9 antibodies (cat. no.ATTY DKT NO: ASHI-005WO abl 19906) are available from Abeam (Cambridge, MA, USA). In mice, the anti-MMP-9 antibody administered is AB0046. Serum MMP-9 expression levels in mice are identified at each time point. Blood samples from the mice are stored at room temperature for 2 h and centrifuged at 4°C for 15 min at 1,000 x g. The supernatant is used to measure the MMP-9, expression levels following the protocol in the ELISA kit.EXAMPLE 2: In vitro assay for antibody inhibiting activation and activity of MMP-9

[0292] To determine if the antibody can efficiently block catalytic activity of murine or human MMP9, the ability of MMP9 to cleave the (7- methoxycoumarin-4-yl)acetyl-Pro- Leu-Gly-Leu-(3-[2,4-dinitrophenyl]-L-2,3- diaminopropionyl)-Ala-Arg-NH2 fluorescent substrate (R&D Systems, ES001) at the presence of different concentrations of antibody are measured. Catalytic domains of human or murine MMP9 protease are purchased in Enzo Life Sciences (Cat# BML-SE360-0010) and Anaspec (Cat# AS-55884-10), correspondently.

[0293] In mice, the anti-MMP-9 antibody administered is AB0046. In humans, the anti-MMP-9 antibody administered is AB0041. The activity assay with catalytic domains of MMP9 (10 nM) is performed in 0.2 ml of 50 mM Hepes buffer, pH 7.5 containing 100 mM NaCl, 10 mM CaCh. 10 mM ZnCh, 0.5 mM MgCh and 0.005% Brij 35 at 37°C. The concentration of Mca-PLGL-Dpa- AR-NH2 fluorescent substrate is 10 mM. To determine the EC50 value of the inhibitory antibody, MMP-9 is pre-incubated for 30 min at 20°C with increasing concentrations of the antibody (0-1,000 nM). The steady state rate of substrate hydrolysis is continuously monitored at lec(excitation wavelength) of 320 nm and leih (emission wavelength) of 400 nm by using Varioskan Lux fluorescence spectrophotometer (Thermo Scientific). All assays are performed in triplicate in wells of a 96 well plate. EC50 values are calculated by determining the concentrations of antibody needed to inhibit 50% of the MMP9 activity against peptidic substrate.

[0294] GraphPad Prism was used as fitting software. The same procedure was repeated with IgGl Isotype control.EXAMPLE 3 : In Vivo Evaluation of Anti-MMP-9 Antibody

[0295] Initial treatment with a loading dose of anti-MMP9 antibody (e.g. 30 mg / kg AB0046) is made prior to HO induction to address the existing pool of MMP-9 and promote generation of more predictable pharmacokinetic properties thereafter. FollowingATTY DKT NO: ASHI-005WO induction of HO, mice are dosed twice a week with 15 mg / kg AB0046 (or control IgG) for 9-12 weeks. The effect of anti-MMP-9 antibody on bone formation in the mouse model is evaluated using whole-mount skeletal preparation. At end of treatment, the degree of HO formation is quantified using longitudinal micro CT (computed tomography) scans focused on the hind limbs at the site of injury. After the mice are euthanized by CO2 inhalation, soft tissues encompassing the site of new bone formation were isolated from the rear hind limb and flash frozen to enable additional histopathological analysis.

[0296] In micro CT analysis, scans are taken 9 weeks post-injury using 80 peak kilovoltage (kVp), 80 mA, and 1,100-ms exposure. Images are reconstructed, and HO volume formation is analyzed using a calibrated imaging protocol. Calculation of cortical thickness is performed in MicroView. Briefly, the fusepoint of the tibia and fibula in the uninjured right leg is selected as a reference landmark. A region encompassing only the proximal tibia is defined as the region of interest. The mean cortical thickness of the given region is then determined automatically with an 1 ,800-Hounsfield unit threshold cutoff.

[0297] The quantity of HO is reported for each treatment group. In IgG control- treated mice, it is anticipated that animals show HO in the injured limb. In IgG control treated mice, the amount of HO is expected to be significantly greater than in anti-MMP-9 antibody treated mice. Results are expected to show that anti-MMP-9 antibody treatment reduces HO in mice.EXAMPLE 4: Mouse Model for NHHO formation

[0298] To evaluate the effect of matrix metalloproteinase 9 (MMP9) gene knockdown, mice are burned and induced with tenotomy to form non-hereditary heterotopic ossification. Male mice are housed in a group of 4 animals in a cage with clean bedding before surgery, and are individually housed post-surgery. The three mice strains include B6.FVB(Cg)-Mmp9tmlTvu / J as strain 1, B6FVBF1 / J (wild type) as strain 2, and C57BL / 6J as strain 3, as seen in Table 2. Each cage is clearly labeled with a color-coded cage card indicating study, group, and animal numbers. Animals are maintained and monitored for good health in accordance with test facility SOPs, and at the discretion of the laboratory animal veterinarian. Food and water are provided ad libitum for the mice in this study. Environmental controls for the animal room are set to maintain a temperature of 22 + °C, humidity of 30-70% RH, and a 12-hour light / 12-hour dark cycle.ATTY DKT NO: ASHI-005WO

[0299] This protocol involves creating a 30% total body surface area partial thickness contact burn on the dorsal skin, as well as division of the Achilles tendon at its midpoint, as described in the Peterson literature (Peterson JR, et al, (2015). J. Vis. Exp., (102): e52880). Relying solely on a traumatic injury to induce HO at a predictable location provides an isolated time-course study of endochondral heterotopic bone formation from intrinsic physiologic processes and environment only.

[0300] To induce heterotopic ossification, bum injury and tenotomy is performed on day 0. Animals are anesthetized using inhalation anesthesia. Buprenorphine at 0. 1 mg / kg is administered subcutaneously before surgery initiation. The mouse dorsum (2 cm x 3 cm) and left hind paw (from heel to the knee) are shaved. Achilles tenotomy is performed on the left Achilles tendon.

[0301] A longitudinal incision is made along the medial aspect of the left Achilles tendon. The incision is extended so the Achilles tendon can be easily visualized; approximately 0.5 cm. Achilles tenotomy is performed with sharp dissection of the tendon at the midpoint with sharp tissue scissors. One blade of the tissue scissor is inserted in the tissue plane beneath the tendon and dissected along the plane until the blade is at the tendon midpoint. The scissor blades are closed to sever the tendon sharply.

[0302] Following the tenotomy surgery, a dorsal partial thickness burn injury is performed with an aluminum block weighing 35 g and measurements of approximately 2 cm x 2 cm x 3 cm which are heated to 60 °C on water bath. To produce partial thickness burn injury, hot aluminum block is kept on shaved skin for 17 seconds. After drying, a Tegaderm dressing is applied on the bum injury to avoid any infection. After surgery, buprenorphine is administered every five hours for the next five days for pain management.

[0303] Body weight is measured once on pre-study and then followed by twice a week. Detailed clinical observation occurs every two-weeks pre-study and then weekly.Animals are observed for cage-side observation daily from day 1 and mortality / morbidity is checked twice daily. On the terminal day 62, gross examination is performed. A tail snip is individually collected from all study animals, and using PCR techniques, the snips are placed into polypropylene tubes and frozen immediately over dry ice. Samples are stored in a freezer set to maintain -80 °C for possible future analysis. Hind limbs are collected, skin removed, pinned on paraffin blocks, fixed in 10% neutral buffered formalin for 48 hours and then transferred to 70% ethanol. Samples are shipped in ethanol. After the completion of the experiments, animals are euthanized as per SOP-BIO-IPH- 126-03 and are disposedATTY DKT NO: ASHI-005WO as per SOP-BIO-AH-1 17-04. See FIG. 2 for the HO quantification of soft tissue for both WT and MMP-9 KO mice.EXAMPLE 5: NHHO Mice Imaging Protocol for pC Acquisition and Analysis

[0304] To image the results of the mouse model for NHHO formation, the hind limbs are taped securely to the bed to prevent motion artifact. The air, water, and hydroxyapatite containing phantom is included beneath the mouse for image calibration. The Bone Analysis software is opened and a region of interest (ROI) is defined to encompass the hind limb from the hip joint proximally to the tip of the hind paw distally. The image is obtained with 80 kV, 500 MA and 1,300 msec exposure, 48 um voxel size parameters.

[0305] The image is then calibrated to Hounsfield units (HU) by drawing a ROI in each of the three phantom chambers and inputting the average density into the appropriate fields in the software. Using the "grabber" tool in the software, the image is re-oriented so the tibia of the left hind limb is parallel along the Z axis to allow the clearest anatomical view for the delineation of orthotopic cortical bone structures and HO formation. Beginning at the knee, the image slices are scrolled distally until HO is encountered. Using the manual spline tool, an ROI is made around the ectopic bone on every 5 slice continuing distally through the paw or until HO has been surpassed. Using the extrapolate tool, the ROTs are extended and stitched together into one ROI that contains all the HO. A 3D ROI is made, and the bone volume is calculated by setting the lower and upper threshold values that best show the bone window. The same fixed threshold values are used for all scans. See FIG. 1 for the total HO formed plot. The total HO includes both HO formed in soft tissue and ectopic bone on the calcaneum that results from limb dragging by the mice during recovery. Quantification of this ectopic bone includes some normal bone of the calcaneum as well, as the boundary is not clearly defined. As this ectopic bone does not represent soft tissue HO, a second analysis excluding this ectopic bone provides a more accurate assessment of formation of HO.EXAMPLE 6: Evaluation of the Impact of MMP9 Blockade in a Burn-Tenotomy mouse model of NHHO

[0306] The burn-tenotomy model of NHHO, as described by Peterson et al (Peterson et al Direct Mouse Trauma / Burn Model of Heterotopic Ossification J. Vis. Exp 2015), has been used to identify and characterize factors involved in driving formation of heterotopicATTY DKT NO: ASHI-005WO bone. Tn this model, mice are subjected to a severe bum over a large surface area and a surgical procedure (tenotomy), thus modeling both systemic inflammation and local injury. These studies have highlighted the critical role of canonical BMP signaling, via several ligand and receptor pairs, to drive aberrant differentiation of progenitor cells and formation of heterotopic ossification in extraskeletal soft tissue (Agarwal et al Strategic Targeting of Multiple BMP Receptors Prevents Trauma-Induced Heterotopic Ossification. Mol Ther. 2017; Strong et al BMP Ligand Trap ALK3-Fc Attenuates Osteogenesis and Heterotopic Ossification in Blast-Related Lower Extremity Trauma. Stem Cells Dev. 2021). This is notably distinct from the driver of genetic forms of heterotopic ossification such as fibrodysplasia ossificans progressiva (FOP), where activin A promotes noncanonical signaling via mutated BMP receptor ACVR1. Consistent with this understanding, no efficacy was observed with activin A blockade in the burn-tenotomy mouse model of NHHO, where the BMP receptors are wild type and not mutated (Hwang et al Activin A does not drive post-traumatic heterotopic ossification Bone 2020).

[0307] MMP9 knockout mice were used for this study as clinical antibody andecaliximab demonstrates the capacity in rats to fully recapitulate the known MMP9- deficient phenotype reported in mice and humans, thus achieving a “knockout” effect. Andecaliximab does not cross-react with mouse MMP9, and murine surrogate AB0046 is a weaker antibody that does not fully recapitulate the mechanism of action of andecaliximab.Test System

[0308] Mice were housed in a group of 4 animals per cage with clean bedding before surgery and were individually housed post-surgery. Each cage was clearly labeled with a color-coded cage card indicating study, group, and animal numbers. Animals were maintained and monitored for good health in accordance with test facility SOPs and at the discretion of the laboratory animal veterinarian. Normal rodent diet was provided ad libitum. Water was available ad libitum. Periodic analysis of the water was performed, andATTY DKT NO: ASHI-005WO the results are archived at the test facility. Environmental controls for the animal room were set to maintain a temperature of 22 ± 3°C, humidity of 30-70% RH, and a 12-hour light / 12- hour dark cycle.

[0309] Bum injury and Achilles tenotomy were performed on day 1. Animals were anesthetized using inhalation anesthesia. Buprenorphine at 0.1 mg / kg was administered subcutaneously before surgery. Mouse dorsum (2cm x 3cm) and left hind paw (from heel to the knee) were shaved. Achilles tenotomy was performed on the left Achilles tendon. A longitudinal incision was made along the medial aspect of the left Achilles tendon. The incision was extended so the Achilles tendon can be easily visualized; approximately 0.5 cm. Achilles tenotomy was performed with sharp dissection of the tendon at the midpoint with sharp tissue scissors.

[0310] Following the tenotomy surgery, a dorsal partial thickness burn injury was performed with an aluminum block weighing 35 g and measurements of approximately 2cm x 2cm x 3cm which was heated to 60°C on water bath. To produce partial thickness bum injury, hot aluminum block was kept on shaved skin for 17 seconds. The bum site was dried with gauze and a Tegaderm dressing was applied to avoid any infection. Animals were housed individually post-surgery and buprenorphine was administered every five hours for the next five days for pain management. The experimental design was reviewed and approved by an Institutional Use and Animal Care Committee. Clinical observation was performed daily. Body weight was measured daily on days 1 to 5 and then twice weekly.Experimental design

[0311] On terminal day (day 62), gross examination was performed. Tail snip was collected from all study animals and stored in a freezer set to maintain -80°C for possible future analysis. Left hind limbs were collected, skin removed, pinned on parafilm blocs, fixed in neutral buffered formalin for 48 hours and then transferred to 70% ethanol.Samples were shipped to Charles River Laboratories for micro-computed tomography (micro-CT) analysis.ATTY DKT NO: ASHI-005WO

[0312] Ex-vivo quantitation of heterotopic ossification was assessed in all animals (Group 1 to 3). Injured hindlimbs from all animals were scanned using a high-resolution micro-CT system (Scanco Medical AG micro-CT 100) and analyzed using the 3-D morphometry, in order to determine the volume of heterotopic bone. Cutoffs for background and identification of region of interest (ROI) were defined as described above in Example 5. Two data sets of data were provided: 1) total HO volume (BV, mm3) including the exostoses attached to the calcaneum and some normal calcaneal bone which results from limb dragging post-surgery, and the HO observed in the soft tissues; 2) total HO volume excluding exostoses attached to the calcaneum (BV, mm3), which represents the clinically relevant HO in terms of location, mechanism of action and impact.

[0313] Data were analyzed and visualized using Prism software (GraphPad, vlO.4.1). For HO volume assessment by micro-CT, the unpaired t test with Welch’s correction was performed. Summary of P value designations are as follows; *<0.05. **<0.01, ***<0.001, ****<0.0001. Data are presented as mean ± standard deviation (SD).Results

[0314] There were no unscheduled deaths during the course of this study and body weight was similar between groups. There was no significant difference between WT and MMP9 KO mice on the incidence and severity of clinical signs observed (such as erythema and edema) during the course of this study indicating that MMP9 knock down doesn’t impact the healing process following burn / tenotomy injury in mice.

[0315] HO was observed in hindlimb soft tissues and emerging from the calcaneum following bum / tenotomy injury in WT and MMP9 KO mice (Figure 1). Manual contouring of the HO attached to the calcaneum was performed to exclude as much normal bone as possible. Significant inhibition of HO, total HO and soft tissue HO, was demonstrated with MMP9 blockade (Figure 1 and Figure 2).ATTY DKT NO: ASHI-005WOTabulated Summary of HO VolumeaTotal HO includes soft tissue HO and calcaneal exostoses (which includes some normal calcaneal bone).bFour animals were excluded from the analysis because the nails used to fix their hindlimb on a parafilm block were located in the region of interest and created substantial residual signal upon micro-CT even after removal.HO=heterotopic ossification; KO=knockout; WT=wild type.

[0316] Mice deficient in MMP9 where significantly protected against the formation of soft tissue HO following administration of a burn injury and tenotomy (p< 0.0001), as well as against total measured bone (soft tissue HO plus exostoses, p< 0.0001). In addition, mice deficient in MMP9 recovered similarly from the burn injury and tenotomy to control mice, indicating that absence of MMP9 did not impair healing. These data support the potential therapeutic benefit of MMP9 inhibition in NHHO.TABLE 2. SEQUENCESATTY DKT NO: ASHI-005WONumbered ClausesClause 1. A method of preventing or reducing a nonhereditary heterotopic ossification (NHHO) comprising administering to a human subject an effective amount of an inhibitor of MMP-9 activation or activity.Clause 2. The method of clause 1, wherein the heterotopic ossification is associated with one or more disorders or conditions selected from the group consisting of: spinal cord injury, trauma, brain injuries, bums, fractures, muscle contusion, joint arthroplasty / replacement, hip surgery / replacement, acetabular surgery / replacement, elbow fracture, fracture of the long bones of the lower leg, combat-related trauma, amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, nontraumatic myelopathy, viral infection, autoimmune or inflammatory diseases, axial spondylarthritis and diffuse idiopathic skeletal hyperostosis.Clause 3. The method of clause 1, wherein the heterotopic ossification occurs in one or more tissues selected from the group consisting of: bone, skin, subcutaneous tissue, skeletal muscle, tendons, ligaments, fibrosis tissue adjacent to joints, walls of blood vessels, and ligaments.Clause 4. The method of clause 1, wherein the method further comprises administration of an additional active agent or supportive therapy for treating heterotopic ossification, selected from the group consisting of isotretinoin, etidronate with oral corticosteroids, perhexiline maleate, ALK2 small-molecule inhibitors, palovarotene, retinoic acid receptor gamma agonists, retinoic acid receptor alpha agonists, activin antibodies, allele- specific RNA interference of ALK2, bisphosphonates, radiation therapy anti-inflammatory agents, and conservative treatments such as passive range of motion exercises or other mobilization techniques.ATTY DKT NO: ASHI-005WOClause 5. The method of clause 1 , wherein a triple hone scan of the subject demonstrates the formation of a NHHO or the potential to form HO due to detection of inflammation on the first phase of the triple phase bone scan, or wherein ultrasound of the subject demonstrates the potential to form HO based on tissue inflammation.Clause 6. The method of clause 1, wherein a biomarker analysis demonstrates an elevated risk of formation of NHHO, wherein the biomarker is selected from a level of a PGE2, MMP- 9 and / or BMP-2 nucleic acid or polypeptide.Clause 7. The method of clause 1, wherein certain genetic and other predisposing factors identify a patient with traumatic injury who is at high risk of developing NHHO.Clause 7A. The method of any one of the preceding clauses, wherein the inhibitor of MMP-9 activation or activity is selected from the group consisting of an anti-MMP-9 antibody, an siRNA agent directed against MMP-9, a peptide MMP-9 inhibitor, a small molecule inhibitor of MMP-9Clause 8. A pharmaceutical composition comprising dosage of 150, 300, 400, 500, 600 or 800 mg of andecalixiamb suitable for subcutaneous or intravenous administration to a human subject having a spinal cord injury, trauma, brain injuries, bums, fractures, muscle contusion, joint arthroplasty / replacement, hip surgery / replacement, acetabular surgery / replacement, elbow fracture, fracture of the long bones of the lower leg, combat-related trauma, amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, nontraumatic myelopathy, viral infection, autoimmune or inflammatory diseases, axial spondylarthritis and diffuse idiopathic skeletal hyperostosis.Clause 9. A dosage regimen comprising administration of 10, 25, 50, 75, 100, 150, 300, 400, 500, 600 or 800 mg of andecalixiamb every 1, 2, 3, or 4 weeks for up to 3, 6, 9, 12 or 24 months.Clause 10. A pharmaceutical composition comprising andecliximab, in a dosage determined by the presence and / or level in a human subject in need thereof of at least one biomarker selected from PGE2, MMP-9, BMP-2.Clause 11. A method of preventing or reducing the severity and / or duration of a condition associated with a nonhereditary heterotopic ossification (NHHO) comprising administering to a human subject an effective amount of an inhibitor of MMP-9 activation or activity.Clause 12. The method of clause 11, wherein the heterotopic ossification is associated with one or more disorders or conditions selected from the group consisting of: spinal cord injury, trauma, brain injuries, bums, fractures, muscle contusion, joint arthroplasty / replacement, hipATTY DKT NO: ASHI-005WO surgery / replacement, acetabular surgery / replacement, elbow fracture, fracture of the long bones of the lower leg, combat-related trauma, amputation, neuromuscular blockade used to manage adult respiratory distress syndrome, nontraumatic myelopathy, viral infection, autoimmune or inflammatory diseases, axial spondylarthritis and diffuse idiopathic skeletal hyperostosis.Clause 13. The method of clause 11, wherein the condition is characterized by an endochondral ossification process, an intramembranous ossification process, a combination of both these processes, or a dystrophic calcification leading to ossification.Clause 14. The method of clause 1 1, wherein the inhibitor of MMP-9 activation or activity comprises an anti-MMP-9 antibody, wherein the anti-MMP-9 antibody binds to (i) an MMP- 9 pro-form and inhibits activation of the pro-form and / or (ii) an MMP-9 active form and inhibits activity of the active form.Clause 15. The method of clause 7, wherein the anti-MMP-9 antibody binds to the MMP-9 pro-form to inhibit MMP-9 activation.Clause 15 A. The method of clause 7, wherein the anti-MMP-9 antibody binds allosterically to the active form of MMP-9 to inhibit MMP-9 activity.Clause 16. The method of clause 7, wherein the anti-MMP-9 antibody is encoded by one or more polypeptide sequences listed in Table 1.Clause 17. The method of any one of clauses 1-7 and 11-16, wherein the inhibitor of MMP-9 activation or activity is administered systemically.Clause 18. The method of any one of clauses 1-7 and 11-16, wherein the inhibitor of MMP-9 activation or activity is administered locally.Clause 19. The method of any one of the clauses 1-7 and 11-16, wherein the inhibitor of MMP-9 activation or activity is administered via intravenous, intraarterial, intramuscular, subcutaneous, intraperitoneal, intra-thoracic, systemic parenteral administration and direct injection or by administration into an area of heterotopic ossification, a tissue or an organ. Clause 20. The method of clause 19, wherein the anti-MMP-9 antibody is administered via intravenous administration at a dose of about 400 mg, 500 mg, 600 mg, about 700 mg, about 800 mg, about 900 mg, or about 1000 mg.Clause 21. The method of clause 19, wherein the anti-MMP-9 antibody is administered via subcutaneous administration at a dose of about 50 mg, about 100 mg, about 150 mg, about 200 mg, about 250 mg, about 300 mg, about 400 mg or about 600 mg.ATTY DKT NO: ASHI-005WOClause 22. The method of any one of clauses 1 -7 and 11-21 , wherein the inhibitor of MMP-9 activation or activity is administered in combination with one or more drugs selected from the group consisting of hypoxia inducible factor- la (Hif-la) inhibitor, small molecule inhibitor of bone morphogenetic protein (BMP) signaling, antibody against BMP9, antibody against BMP 10, antibody against TGF-B, momelotinib, palovarotene, imatinib, chromolyn, imatinib, a corticosteroid, apyrase, rapamycin, a kinase inhibitor, an anti-inflammatory agent, a nonsteroidal anti-inflammatory agent, an ALK- 2 inhibitor, or an inhibitor of a BMP receptor. Clause 23. The method of any one of clauses 1-7 and 11-22, wherein the inhibitor of MMP-9 activation or activity is administered in combination with a second MMP inhibitor selected from an MMP-2 inhibitor, an MMP-7 inhibitor, an MMP- 13 inhibitor, an MMP- 14 inhibitor, or an MMP- 16 inhibitor.Clause 24. The method of any one clauses 1-7 and 11-23, wherein the inhibitor of MMP-9 activation or activity is administered in a subject for the treatment of one or more musculoskeletal conditions.Clause 25. The method of any one of clauses 1-7 and 11-24, further comprising ameliorating one or more musculoskeletal diseases or conditions.Clause 26. The method of any one of clauses 1-7 and 11-25, wherein the inhibitor of MMP-9 activation or activity results in a reduction in number of nongenetic heterotopic ossifications, size of nongenetic heterotopic ossifications, growth of nongenetic heterotopic ossifications, or formation of nongenetic heterotopic ossifications.Clause 27. A method for reducing non-hereditary heterotopic ossification in an individual following a spinal cord injury, the method comprising: administering an effective dose of an antibody that inhibits MMP9, for a period of time sufficient to reduce heterotopic ossification.Clause 28. The method of clause 27, wherein the antibody is andecaliximab.Clause 29. The method of clause 27 or 28, wherein the effective dose is from about 150 to 800 mg.Clause 30. The method of any of clauses 27-29, wherein the antibody is delivered subcutaneously.Clause 31. The method of any of clauses 27-30, wherein the antibody administration commences within about 10 days to about 1 month of the spinal cord injury.Clause 32. The method of any of clauses 27- 1 , wherein the individual is an adult human, optionally from about 18 to 45 years of age.

Claims

ATTY DKT NO: ASHI-005WOWe claim:

1. A method of preventing or reducing the progression of at least one symptom of a non-genetic heterotopic ossification (NHHO) condition, comprising: administering to a human subject having experienced a traumatic injury a pharmaceutical composition comprising an anti-MMP-9 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:5, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO:8, an LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 10, at a dose schedule effective to prevent or reduce at least one symptom selected from the group consisting of number of heterotopic ossifications, size of heterotopic ossifications, volume of heterotopic ossifications, growth of heterotopic ossifications, formation of heterotopic ossifications, and formation of new heterotopic ossifications at either adjacent or independent sites.

2. The method of claim 1, wherein the anti-MMP9 antibody or an antigen-binding fragment thereof is administered intravenously at a dose of about 400-800 mg every week, about 800 mg every 2 weeks, about 1200 mg every 3 weeks, or about 1600 mg every 4 weeks.

3. The method of claim 1 or claim 2, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is administered subcutaneously at a dose of about 150 mg to about 600 mg weekly dose, where the dose is optionally fractionated.

4. The method of any of claims 1-3, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is administered intravenously at a dose of about 400-800 mg every week, about 800 mg every 2 weeks, about 1200 mg every 3 weeks, or about 1600 mg every 4 weeks; followed by the anti-MMP9 antibody or an antigen-binding fragment thereof being administered subcutaneously at a dose of about 150 mg to about 600 mg weekly dose, where the dose is optionally fractionated.ATTY DKT NO: ASHI-005WO5. The method of any of claims 1 -4, wherein the traumatic injury includes spinal cord injury or traumatic brain injury, and / or disruption of the blood-brain, blood-spinal cord, or peripheral nerve barrier.

6. The method of any of claims 1-5, wherein the heterotopic ossification occurs in one or more tissues selected from bone, skin, subcutaneous tissue, skeletal muscle, tendons, fibrous tissue adjacent to joints, fibrotic tissue, walls of blood vessels, entheses, fascia, aponeuroses and ligaments.

7. The method of any of claims 1-6, wherein one or more lesions of heterotopic ossification are located in the vicinity of the hips and / or shoulders.

8. A method of any of claims 1-7, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is initially administered within 1 week, 2 weeks, 3 weeks, 4 weeks, 1 month, 2 months, or 3 months, of the traumatic injury.

9. The method of any of claims 1-7, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is initially administered at detection of or suspicion of HO formation or potential HO formation by one or more of ultrasound, triple phase bone scan, CT scan, and X ray.

10. The method of any of claims 1-9, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is administered for a period of time such that the total treatment time is up to about 6 months.

11. A method of preventing or reducing the progression of at least one symptom of a non-genetic heterotopic ossification (NHHO) condition, comprising: administering to a human subject at risk of heterotopic ossification due to surgery a pharmaceutical composition comprising an anti-MMP-9 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:5, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable regionATTY DKT NO: ASHI-005WO comprising a light chain complementarity determining region 1 (LCDR1 ) comprising the amino acid sequence of SEQ ID NO:8, an LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and an LCDR3 comprising the amino acid sequence of SEQ ID NO: 10, at a dose schedule effective to prevent or reduce the at least one symptom selected from the group consisting of number of heterotopic ossifications, size of heterotopic ossifications, volume of heterotopic ossifications, growth of heterotopic ossifications, formation of heterotopic ossifications, and formation of new heterotopic ossifications at either adjacent or independent sites.

12. The method of claim 11, wherein the human subject is at risk of heterotopic ossification due to presence of existing lesion(s) of heterotopic ossification that are to be surgically excised, wherein there is a risk of formation or re-formation of heterotopic ossification at 1 or more sites.

13. The method of claim 11, wherein the human subject is at risk of heterotopic ossification due to a first or second joint replacement surgery of the hip, shoulder or knee, and / or surgery of the hip, shoulder, knee or elbow, and / or amputation of one or more limbs or portion(s) of a limb, wherein there is a risk of formation or re-formation of heterotopic ossification at 1 or more sites.

14. The method of any of claims 11-13, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is administered intravenously at a dose of about 400-800 mg every week, about 800 mg every 2 weeks, about 1200 mg every 3 weeks, about 1600 mg every 4 weeks.

15. The method of any of claims 11-14, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is administered subcutaneously at a dose of about 150 mg to 600 mg weekly dose, where the dose is optionally fractionated.

16. The of any of claims 11-15, wherein the anti-MMP9 antibody or an antigen-binding fragment thereof is administered intravenously at a dose of about 400-800 mg every week, about 800 mg every 2 weeks, about 1200 mg every 3 weeks, or about 1600 mg every 4 weeks; followed by the anti-MMP9 antibody or an antigen-binding fragment thereof beingATTY DKT NO: ASHI-005WO administered subcutaneously at a dose of about 150 mg to about 600 mg weekly dose, where the dose is optionally fractionated.

17. The method of any of claims 11-16, wherein the anti-MMP-9 antibody or antigenbinding fragment thereof is administered starting within about 1, 2, 3, or 4 weeks after surgery, and continuing for up to about 6 months after surgery.

18. The method of any of claims 11-16, wherein the anti-MMP-9 antibody or antigenbinding fragment thereof is administered starting 1, 2, 3, or 4 weeks prior to surgery, and continuing for up to about 6 months after surgery.

19. The method of any of claims 11-18, wherein the anti-MMP9 antibody or an antigenbinding fragment thereof is administered for a period of time such that the total treatment time is up to about 6 months.

20. A method of preventing or reducing the progression of at least one symptom of a non- genetic heterotopic ossification (NHHO) condition, comprising: administering to a human subject at risk of heterotopic ossification due to a disease where there is a predisposition to heterotopic ossification, a pharmaceutical composition comprising an anti-MMP-9 antibody or an antigen-binding fragment thereof comprising a heavy chain variable region (VH) comprising a heavy chain complementarity determining region 1 (HCDR1) comprising the amino acid sequence of SEQ ID NO:5, an HCDR2 comprising the amino acid sequence of SEQ ID NO:6, and an HCDR3 comprising the amino acid sequence of SEQ ID NO:7, and a light chain variable region comprising a light chain complementarity determining region 1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 8, an LCDR2 comprising the amino acid sequence of SEQ ID NO:9, and an LCDR3 comprising the amino acid sequence of SEQ ID NOTO, at a dose schedule effective to prevent or reduce the at least one symptom selected from the group consisting of number of heterotopic ossifications, size of heterotopic ossifications, volume of heterotopic ossifications, growth of heterotopic ossifications, formation of heterotopic ossifications, and formation of new heterotopic ossifications at either adjacent or independent sites.ATTY DKT NO: ASHI-005WO21. The method of claim 20, wherein the disease is axial spondylarthritis, including ankylosing spondylitis; or diffuse idiopathic skeletal hyperostosis.

22. The method of claim 20 or 21, wherein the anti-MMP-9 antibody or antigen-binding fragment thereof is administered at a dose of about 150, about 300, about 450 mg, or about 600 mg, optionally fractionated, by subcutaneous route every week, or twice a week, or 3 times a week for the duration of the disease condition.

23. The method of any of claims 1-22, wherein the human subject is an adult.

24. The method of any of claims 1-23, wherein the anti-MMP-9 antibody or an antigenbinding fragment thereof comprises the variable region sequence of SEQ ID NO:3 and SEQ ID NON.

25. The method of any of claims 1-24, wherein the antibody comprises a heavy chain of SEQ ID NO: 1 and a light chain of SEQ ID NO:2.

Images