WO2023278491A1 - Mmp-9 antibodies and uses thereof - Google Patents

Abstract

Provided is a method of inhibiting an activity of MMP-9. Also provided is a method for treating a disease, disorder or condition characterized by excess MMP-9 in a mammal.

Description

MMP-9 ANTIBODIES AND USES THEREOF

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63/215,627, filed June 28, 2021, which is incorporated by reference herein in its entirety.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR

DEVELOPMENT

[0002] Not Applicable.

INCORPORATION-BY-REFERENCE OF A SEQUENCE LISTING

[0003] The Sequence Listing, which is a part of the present disclosure, includes a computer readable form and a written sequence listing comprising nucleotide and/or amino acid sequences of the present invention. The sequence listing information recorded in computer readable form is identical to the written sequence listing. The subject matter of the Sequence Listing is incorporated herein by reference in its entirety.

FIELD

[0004] The present application generally relates to antibody technology. More specifically, provided are methods of inhibiting an activity of MMP-9.

INTRODUCTION

[0005] Matrix metallopeptidase 9 (MMP-9), or gelatinase B, is also a zinc endopeptidase important in remodeling ECM. MMP-9 plays a role in tumor growth, metastasis, inflammation including neuroinflammation, axon demyelination or degeneration, myelin degradation, dorsal root ganglion (DRG) neuronal survival, mechanical allodynia, maladaptive neuroplasticity, idiopathic atrial fibrillation, cancer, or aortic aneurysm, vascular disease, diseases characterized by elevated TNF-a or IL-Ib, and pain including chronic pain. See, e.g., US Patent 8,377,443 and references cited therein.

SUMMARY

[0006] Provided is a method of inhibiting an activity of MMP-9. The method comprises contacting the MMP-9 with a binding molecule described in US Patent 8,377,443 that specifically binds to MMP-9; specifically an immunoglobulin heavy chain polypeptide, or functional fragment thereof, and an immunoglobulin light chain polypeptide, or functional fragment thereof, wherein the MMP9 binding protein specifically binds to human MMP9 and wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising heavy chain CDRs of SEQ ID NOs: 1-3 or light chain CDRs of SEQ ID NOs: 4-6 that specifically binds to MMP-9.

[0007] Also provided is a method for treating a disease, disorder or condition characterized by excess MMP-9 in a mammal. The method comprises administering a therapeutically effective amount of a binding molecule described in US Patent 8,377,443 that specifically binds to MMP-9.

[0008] These and other features, aspects and advantages of the present teachings will become better understood with reference to the following description, examples and appended claims.

DRAWINGS

[0009] Those of skill in the art will understand that the drawings, described below, are for illustrative purposes only. The drawings are not intended to limit the scope of the present teachings in any way.

[0010] Figure 1. Sequence of Human MMP9.

[0011] Figure 2. Sequence alignment comparing Human MMP9 to Mus musculus

MMP9.

[0012] Figure 3. Sequence alignment comparing Human MMP9 to Rattus MMP9. [0013] Figure 4. Sequence alignment comparing Mus musculus MMP9 to Rattus

MMP9.

[0014] Figure 5. Inhibition of Catalytic activity of human MMP9 Cat by murine anti-murin MMP9 antibody from Gilead (GS622703). Murine anti-murine MMP9 antibody does not block catalytic activity of HUMAN MMP9 catalytic domain (Enzo Life Sciences, Cat# BML-SE360-0010).

[0015] Figure 6. Inhibition of Catalytic activity of human MMP9 Cat by IgGl- isotype antibody from Gilead (GS645864). IgGl -isotype does not block catalytic activity of HUMAN MMP9 catalytic domain.

[0016] Figure 7. Inhibition of Catalytic activity of murine MMP9 Cat by murine anti-murine MMP9 antibody from Gilead (GS622703). Murine anti-murine MMP9 antibody does not block catalytic activity of MURINE MMP9 catalytic domain (Anaspec, Cat# AS- 55884-10).

[0017] Figure 8. Schematic presentation of the study design. Study days indicate the distance from Post-op analgesia: DO - 2, the rats were administered with buprenorphine (0.05 mg/kg, i.p.), at every 6- 12 hours (max twice daily). Postoperative care: the first week after surgery (D0-D6); included twice-daily checks; detailed description in section 7.3.

Treatments: i.t. intrathecal; i.v. intravenous (tail vein); intra-DRG: direct injection of test articles into lumbar L4 and L5 DRGs, ipsilateral to the PSNL injury. PD: Post-dosing. evF: electronic von Frey test Endpoint sampling: DRGs = ipsilateral L4 and L5; SC = lumbar spinal cord at the level of L4 and L5 DRGs; SN=sciatic nerve distal to the partial nerve ligation.

[0018] Figure 9. Intrathecal injection site indicated.

[0019] Figure 10. Body weight over time. Data are presented as mean ± SEM, n = 5-10 per group. Statistical significances: * p < 0.05, Group 5, 7, 8 and 13 vs. Group 1 (Mixed-effects model (REML), Dunnetfs multiple comparisons test).

[0020] Figure 11. Absolute Mean PWT. Data are presented as mean ± SEM, n = 5- 10 per group. Statistical significances: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, timepoint comparison within group (Mixed-effects model (REML), Uncorrected Fisher's LSD).

[0021] Figure 12. Percentage From Baseline PWT. Data are presented as mean ± SEM, n = 5-10 per group. Statistical significances: * p < 0.05, ** p < 0.01, timepoint comparison within group (Mixed-effects model (REML), Uncorrected Fisher's LSD).

[0022] Figure 13. Percentage from baseline PWT: Injury baseline (D6) vs. the last post-dosing timepoint. Data are presented as mean ± SEM, n = 5-10 per group. Statistical significances: * p < 0.05, ** p < 0.01, *** p < 0.001, timepoint comparison within group (Two-way RM ANOVA, Uncorrected Fisher's LSD).

[0023] Figure 14. Mean PWTs and Timepoint comparison within MMP9-i.v., MMP14-DRG, and the corresponding control antibody -groups, injury baseline (D6) vs. all post-dosing timepoints (D14, D16, and D21). Data are presented as mean ± SEM, n = 5-10 per group. Statistical significances: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, timepoint comparison within group (Two-way RM ANOVA, Uncorrected Fisher's LSD).

[0024] Figure 15. Mean PWTs and Timepoint comparison within MMP14-i.t, MMP9-i.t, and the corresponding control antibody -groups, injury baseline (D6) vs. all post dosing timepoints (D14, D16, and D21). Data are presented as mean ± SEM, n = 5-10 per group. Statistical significances: * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001, timepoint comparison within group (Two-way RM ANOVA, Uncorrected Fisher's LSD).

DETAILED DESCRIPTION

[0025] Abbreviations and Definitions

[0026] To facilitate understanding of the invention, a number of terms and abbreviations as used herein are defined below as follows:

[0027] Binding molecule: As used herein the term "binding molecule" refers to an intact immunoglobulin including single-domain antibodies, including chimeric, or humanized monoclonal antibodies, or to an antigen-binding and/or variable domain comprising fragment of an immunoglobulin that competes with the intact immunoglobulin for specific binding to the binding partner of the immunoglobulin, e.g., MMP-14. Regardless of structure, the antigen-binding fragment binds with the same antigen that is recognized by the intact immunoglobulin. An antigen-binding fragment can comprise a peptide or polypeptide comprising an amino acid sequence of at least ten contiguous amino acid residues, at least 15 contiguous amino acid residues, at least 20 contiguous amino acid residues, at least 25 contiguous amino acid residues, at least 30 contiguous amino acid residues, at least 35 contiguous amino acid residues, at least 40 contiguous amino acid residues, at least 50 contiguous amino acid residues, at least 60 contiguous amino residues, at least 70 contiguous amino acid residues, at least 80 contiguous amino acid residues, at least contiguous 90 amino acid residues, at least 100 contiguous amino acid residues, and at least 130 contiguous amino acid residues of the amino acid sequence of the binding molecule.

[0028] The binding molecule can be a naked or unconjugated binding molecule but can also be part of an immunoconjugate. A naked or unconjugated binding molecule is intended to refer to a binding molecule that is not conjugated, operatively linked or otherwise physically or functionally associated with an effector moiety or tag, such as inter aba a toxic substance, a radioactive substance, a liposome, an enzyme. It will be understood that naked or unconjugated binding molecules do not exclude binding molecules that have been stabilized, multimerized, humanized or in any other way manipulated, other than by the attachment of an effector moiety or tag. Accordingly, all post-translationally modified naked and unconjugated binding molecules are included herein, including where the modifications are made in the natural binding molecule-producing cell environment, by a recombinant binding molecule- producing cell, and are introduced by the hand of man after initial binding molecule preparation. The term naked or unconjugated binding molecule does not exclude the ability of the binding molecule to form functional associations with effector cells and/or molecules after administration to the body, as some of such interactions are necessary in order to exert a biological effect. The lack of associated effector group or tag is, therefore, applied in definition to the naked or unconjugated binding molecule in vitro, not in vivo.

[0029] Specific binding: As used herein, a protein participates in specific binding to MMP-14 by binding to MMP-14 through the noncovalent binding of an antibody heavy and light chain variable domain with MMP-14, where the protein comprises a complementarity determining region 3 (CDR3) of the antibody.

[0030] Conservative amino acid substitutions: As used herein, when referring to mutations in a protein, "conservative amino acid substitutions” are those in which at least one amino acid of the polypeptide encoded by the nucleic acid sequence is substituted with a another amino acid having similar characteristics. Examples of conservative amino acid substitutions are ser for ala, thr, or cys; lys for arg; gin for asn, his, or lys; his for asn; glu for asp or lys; asn for his or gin; asp for glu; pro for gly; leu for ile, phe, met, or val; val for ile or leu; ile for leu, met, or val; arg for lys; met for phe; tyr for phe or trp; thr for ser; trp for tyr; and phe for tyr.

[0031] Complementary determining region (CDR): The term "complementary determining region" as used herein means sequences within a variable region of an antibody that usually contribute to a large extent to the antigen binding site which is complementary in shape and charge distribution to the epitope recognized on the antigen, i.e., MMP-14. The CDR regions can be specific for linear epitopes, discontinuous epitopes, or conformational epitopes of proteins or protein fragments, either as present on the protein in its native conformation or, in some cases, as present on the proteins as denatured, e.g., by solubilization in SDS. Epitopes may also comprise of posttranslational modifications of proteins.

[0032] Functional variant: The term "functional variant," as used herein, refers to a binding molecule or recombinant protein that comprises a nucleotide and/or amino acid sequence that is altered by one or more nucleotides and/or amino acids compared to the nucleotide and/or amino acid sequences of the parent binding molecule and that is still capable of competing for binding to the binding partner, e.g., MMP-14, with the parent binding molecule. In other words, the modifications in the amino acid and/or nucleotide sequence of the parent binding molecule do not significantly affect or alter the binding characteristics of the binding molecule encoded by the nucleotide sequence or containing the amino acid sequence, i.e., the binding molecule is still able to recognize and bind its target. The functional variant may have conservative change including nucleotide and amino acid substitutions, additions and deletions. These modifications can be introduced by standard techniques known in the art, such as site-directed mutagenesis and random PCR-mediated mutagenesis, and may comprise natural as well as non-natural nucleotides and amino acids. Also envisioned is the use of amino acid analogs, e.g. amino acids not DNA or RNA encoded in biological systems, and labels such as fluorescent dyes, radioactive elements, electron dense agents, or any other protein modification, now known or later discovered.

[0033] Recombinant protein: As used herein, a recombinant protein is a protein produced by recombinant DNA technology or chemical synthesis having a specified polypeptide molecule covalently linked to one or more polypeptide molecules which do not naturally link to the specified polypeptide.

[0034] Pharmaceutically acceptable: As used herein, the term “pharmaceutically acceptable” means approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.

[0035] Pharmaceutically acceptable carrier: As used herein, the term “pharmaceutically acceptable carrier” refers to a diluent, adjuvant, excipient, or vehicle with which a compound is administered. Such carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable, or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like, polyethylene glycols, glycerine, propylene glycol, or other synthetic solvents. Water is a preferred carrier when a compound is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol, water, ethanol, and the like. A compound, if desired, can also combine minor amount of wetting or emulsifying agents, or pH buffering agents such as acetates, citrates, or phosphates. Antibacterial agents such as a benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; and agents for the adjustment of tonicity such as sodium chloride or dextrose may also be a carrier. Methods for producing compounds in combination with carriers are known to those of skill in the art.

[0036] A pharmaceutically acceptable carrier may comprise a pharmaceutically acceptable salt, which is includes those salts of a pharmaceutically acceptable compound formed with free amino groups such as those derived from hydrochloric, phosphoric, acetic, oxalic, and tartaric acids, and those formed with free carboxyl groups such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2-ethylamino ethanol, histidine, and procaine. If the compound is basic, salts may be prepared from pharmaceutically acceptable non-toxic acids including inorganic and organic acids. Such acids include acetic, benzene-sulfonic (besylate), benzoic, camphorsulfonic, citric, ethenesulfonic, fumaric, gluconic, glutamic, hydrobromic, hydrochloric, isethionic, lactic, maleic, malic, mandelic, methanesulfonic, mucic, nitric, pamoic, pantothenic, phosphoric, succinic, sulfuric, tartaric, p-toluenesulfonic, and the like. Particularly preferred are besylate, hydrobromic, hydrochloric, phosphoric, and sulfuric acids. If the compound is acidic, salts may be prepared from pharmaceutically acceptable organic and inorganic bases. Suitable organic bases include, but are not limited to, lysine, N,N’- dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethylene diamine, meglumine (N-methyl-glucamine) and procaine. Suitable inorganic bases include, but are not limited to, alkaline and earth-alkaline metals such as aluminum, calcium, lithium, magnesium, potassium, sodium, and zinc. Methods for synthesizing such salts are known to those of skill in the art.

[0037] Polypeptide, protein, and peptide: The terms “polypeptide,” “protein,” and “peptide” are used herein interchangeably to refer to amino acid chains in which the amino acid residues are linked by peptide bonds or modified peptide bonds. The amino acid chains can be of any length of greater than two amino acids. Unless otherwise specified, the terms “polypeptide,” “protein,” and “peptide” also encompass various modified forms thereof.

Such modified forms may be naturally occurring modified forms or chemically modified forms. Examples of modified forms include, but are not limited to, glycosylated forms, phosphorylated forms, myristoylated forms, palmitoylated forms, ribosylated forms, acetylated forms, and the like. Modifications also include intra-molecular crosslinking and covalent attachment of various moieties such as lipids, flavin, biotin, polyethylene glycol or derivatives thereof, and the like. In addition, modifications may also include cyclization, branching and cross-linking. Further, amino acids other than the conventional twenty amino acids encoded by genes may also be included in a polypeptide.

[0038] The term “protein” or “polypeptide” may also encompass a “purified” polypeptide that is substantially separated from other polypeptides in a cell or organism in which the polypeptide naturally occurs (e.g., 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, 100% free of contaminants).

[0039] Primer, probe and oligonucleotide: The terms “primer,” “probe,” and “oligonucleotide” may be used herein interchangeably to refer to a relatively short nucleic acid fragment or sequence. They can be DNA, RNA, or a hybrid thereof, or chemically modified analogs or derivatives thereof. Typically, they are single-stranded. However, they can also be double-stranded having two complementing strands that can be separated apart by denaturation. In certain aspects, they are of a length of from about 8 nucleotides to about 200 nucleotides, preferably from about 12 nucleotides to about 100 nucleotides, and more preferably about 18 to about 50 nucleotides. They can be labeled with detectable markers or modified in any conventional manners for various molecular biological applications.

[0040] Therapeutically effective amount: As used herein, the term “therapeutically effective amount” refers to those amounts that, when administered to a particular subject in view of the nature and severity of that subject’s disease, disorder or condition, will have a desired therapeutic effect, e.g. an amount that will cure, prevent, inhibit, or at least partially arrest or partially prevent a target disease, disorder or condition.

[0041] Single-domain antibody or single-chain Fv (scFv): Single-domain antibodies have been described in, for example, US Pat. Nos. 5,759,808, 5,800,988, 5,840,526, and 5,874,541. Compared with conventional four-chain immunoglobulins of human IgG-type, the single-domain antibodies lack the light chains and CHI domains of conventional human immunoglobulins.

[0042] Methods for obtaining single-domain antibodies that bind to specific antigens or epitopes are described, for example, in WO 2006/040153 and WO 2006/122786; van der Linden et al. (2000); Li et al. (2012); and Diffar et al. (2009). Single-domain antibodies can be “humanized”, and in addition to humanization, sequence optimization may involve one or more conservative changes to improve VHH characteristics. Other modifications of the sequence by mutation, including removal of potential sites for post- translational modifications). A humanized VHH domain can contain one or more fully human framework region sequences and, in certain embodiments, contains an IGHV3 human framework region sequence.

[0043] Diabody: As used herein, a diabody is a dimer of single-chain Fv (scFv) fragment that comprises a heavy chain variable (VH) and light chain variable (VL) region connected by a small peptide linker.

[0044] Single-chain (Fv)2: Two scFv fragments covalently linked to each other.

[0045] Treatment: As used herein, the terms "treat", "treatment", “treating” and the like, refer to relief from or alleviation of pathological processes. In the context of the present invention insofar as it relates to any of the other conditions recited herein below (other than pathological processes mediated by anti-MMP-14 administration), the terms "treat", "treatment", and the like mean to relieve or alleviate at least one symptom associated with such condition, or to slow or reverse the progression of such condition.

[0046] Vector: As used herein, the term “vector” refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked. One type of preferred vector is an episome, i.e., a nucleic acid capable of extra-chromosomal replication. Preferred vectors are those capable of autonomous replication and/expression of nucleic acids to which they are linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as “expression vectors.”

[0047] MMP-9 Antibodies and Uses Thereof

[0048] Antigen-binding proteins include complementarity determining region (CDR, and particularly CDR3) fragments, and polypeptides that contain at least a fragment of an immunoglobulin that is sufficient to confer specific antigen binding to the polypeptide.

The proteins of the present invention may be produced synthetically or by enzymatic or chemical cleavage of intact immunoglobulins or they may be genetically engineered by recombinant DNA techniques. The methods of production are well known in the art and are described in the Examples herein and, for example, in Antibodies: A Laboratory Manual, Second edition (2014). A binding molecule or antigen-binding fragment thereof may have one or more binding sites. If there is more than one binding site, the binding sites may be identical to one another or they may be different.

[0049] Recombinant Proteins

[0050] The recombinant proteins of the present invention can participate in specific binding to MMP-9, as described or derived from binding molecules described in US Patent 8,377,443 that specifically binds to MMP-9 and inhibits enzymatic activity of MMP-9. In various embodiments, the recombinant proteins comprise complementarity-determining regions (CDRs) comprising sequences SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3. In some embodiments, the binding molecule comprises immunoglobulin light chain complementarity-determining regions (CDRs) comprising sequences SEQ ID NO: 4, SEQ ID NO:5, and SEQ ID NO:6. In other embodiments, the binding molecule comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:7, 8, 9, 10 or 11. In additional embodiments, the binding molecule comprises an immunoglobulin light chain variable region comprising SEQ ID NO: 12, 13, 14, 15 or 16. In further embodiments, the binding molecule comprises an Fab heavy chain comprising SEQ ID NO: 17. In additional embodiments, the binding molecule comprises an Fab light chain comprising SEQ ID NO: 18. In still further embodiments, the binding molecule comprises an Fab heavy chain comprising SEQ ID NO: 17 and an Fab light chain comprising SEQ ID NO: 18. By their ability to inhibit MMP-9 enzymatic activity, the recombinant proteins are useful for treating a disease, disorder or condition characterized by excess MMP-9 when administered to an animal having such a disease, disorder or condition.

[0051] Compositions Comprising Recombinant Proteins

[0052] Also provided is a composition comprising any of the recombinant proteins described above, in a physiologically acceptable, non-toxic carrier. In some embodiments, the composition is a pharmaceutical composition where the carrier is a pharmaceutically acceptable carrier.

[0053] Pharmaceutical Compositions

[0054] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art- established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0055] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0056] The identified compositions useful for treating a disease, disorder or condition characterized by excess MMP-9 and can be administered to a subject at therapeutically effective doses for inhibiting MMP-9 enzymatic activity. The compositions of the present invention comprise a therapeutically effective dosage of a binding molecule of the present invention. The subject is preferably an animal, including, but not limited to, mammals, reptiles and avians, more preferably horses, cows, dogs, cats, sheep, pigs, and chickens, and most preferably humans.

[0057] Therapeutically Effective Dosage

[0058] Toxicity and therapeutic efficacy of the compositions of the present invention can be determined by standard pharmaceutical procedures in cell cultures or experimental animals for determining the LD50 (the dose lethal to 50% of the population) and the ED50, (the dose therapeutically effective in 50% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index that can be expressed as the ratio LD50/ED50. Compositions that exhibit large therapeutic indices are preferred. While compositions exhibiting toxic side effects may be used, care should be taken to design a delivery system that targets such compositions to the site affected by the disease, disorder or condition in order to minimize potential damage to unaffected cells and reduce side effects.

[0059] The data obtained from the cell culture assays and animal studies can be used in formulating a range of dosages for use in humans and other mammals. The dosage of such compositions lies preferably within a range of circulating plasma or other bodily fluid concentrations that include the ED50 with little or no toxicity. The dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any composition of the invention, the therapeutically effective dose can be estimated initially from cell culture assays. A dosage may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (the concentration of the test composition that achieves a half-maximal inhibition of symptoms) as determined in cell culture. Such information can be used to more accurately determine useful dosages in humans and other mammals. Composition levels in plasma may be measured, for example, by high performance liquid chromatography.

[0060] The amount of a composition that may be combined with pharmaceutically acceptable carriers to produce a single dosage form will vary depending upon the host treated and the particular mode of administration. It will be appreciated by those skilled in the art that the unit content of a composition contained in an individual dose of each dosage form need not in itself constitute a therapeutically effective amount, as the necessary therapeutically effective amount could be reached by administration of a number of individual doses. The selection of dosage depends upon the dosage form utilized, the condition being treated, and the particular purpose to be achieved according to the determination of those skilled in the art.

[0061] The dosage regime for treating a disease, disorder or condition with the compositions and/or composition combinations of this invention is selected in accordance with a variety of factors, including the type, age, weight, sex, diet and medical condition of the patient, the route of administration, pharmacological considerations such as activity, efficacy, pharmacokinetic and toxicology profiles of the particular composition employed, whether a composition delivery system is utilized and whether the composition is administered as a pro-drug or part of a drug combination. Thus, the dosage regime actually employed may vary widely from subject to subject.

[0062] Formulations and Use

[0063] The compositions of the present invention may be formulated by known methods for administration to a subject using several routes which include, but are not limited to, parenteral, oral, pulmonary, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and ophthalmic routes. The individual compositions may also be administered in combination with one or more additional compositions of the present invention and/or together with other biologically active or biologically inert agents. Such biologically active or inert agents may be in fluid or mechanical communication with the composition(s) or attached to the composition(s) by ionic, covalent, Van der Waals, hydrophobic, hydrophilic or other physical forces. It is preferred that administration is localized in a subject, but administration may also be systemic.

[0064] The compositions may be formulated by any conventional manner using one or more pharmaceutically acceptable carriers and/or excipients. Thus, the compositions and their pharmaceutically acceptable salts and solvates may be specifically formulated for administration, e.g., by inhalation or insufflation (either through the mouth or the nose) or oral, buccal, or parenteral administration. The composition may take the form of charged, neutral and/or other pharmaceutically acceptable salt forms. Examples of pharmaceutically acceptable carriers include, but are not limited to, those described in Remington's Pharmaceutical Sciences (A.R. Gennaro, Ed.), 20th edition, Williams & Wilkins PA, USA (2000).

[0065] The compositions may also take the form of solutions, suspensions, emulsions, tablets, pills, capsules, powders, controlled- or sustained-release formulations and the like. Such compositions will contain a therapeutically effective amount of the composition, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient. The formulation should suit the mode of administration.

[0066] Parenteral and Intravitreal Administration

[0067] The composition or composition combination may be formulated for parenteral or intravitreal administration by injection. Parenteral administration can be made, e.g., by bolus injection or continuous infusion. Formulations for injection may be presented in unit dosage form in ampoules or in multi-dose containers with an optional preservative added. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass, plastic or the like. The composition may take such forms as suspensions, solutions or emulsions in oily or aqueous vehicles, and may contain formulatory agents such as suspending, stabilizing and/or dispersing agents.

[0068] For example, a parenteral preparation may be a sterile injectable solution or suspension in a nontoxic parenterally acceptable diluent or solvent (e.g., as a solution in 1,3- butanediol). Among the acceptable vehicles and solvents that may be employed are water, Ringer’s solution, and isotonic sodium chloride solution. In addition, sterile, fixed oils are conventionally employed as a solvent or suspending medium. For this purpose any bland fixed oil may be employed including synthetic mono- or diglycerides. In addition, fatty acids such as oleic acid may be used in the parenteral preparation.

[0069] Alternatively, the composition may be in powder form for constitution with a suitable vehicle, such as sterile pyrogen-free water, before use. For example, a composition suitable for parenteral administration may comprise a sterile isotonic saline solution containing between 0.1 percent and 90 percent weight per volume of the composition or composition combination. By way of example, a solution may contain from about 5 percent to about 20 percent, more preferably from about 5 percent to about 17 percent, more preferably from about 8 to about 14 percent, and still more preferably about 10 percent of the composition. The solution or powder preparation may also include a solubilizing agent and a local anesthetic such as bgnocaine to ease pain at the site of the injection. Other methods of parenteral delivery of compositions will be known to the skilled artisan and are within the scope of the invention.

[0070] Invention recombinant proteins that inhibit MMP-14 can also be used in combination with other therapeutic modalities. Thus, in addition to the therapies described herein, one may also provide to the subject other therapies known to be efficacious for particular MMP-14 related indications. [0071] Controlled-release (or sustained-release) preparations may be formulated to extend the activity of the recombinant protein and reduce dosage frequency. Controlled- release preparations can also be used to effect the time of onset of action or other characteristics, such as blood levels of the recombinant protein, and consequently affect the occurrence of side effects.

[0072] Controlled-release preparations may be designed to initially release an amount of a recombinant protein that produces the desired therapeutic effect, and gradually and continually release other amounts of the agent to maintain the level of therapeutic effect over an extended period of time. In order to maintain a near-constant level of an recombinant protein in the body, the recombinant protein can be released from the dosage form at a rate that will replace the amount of recombinant protein being metabolized and/or excreted from the body. The controlled-release of recombinant protein may be stimulated by various inducers, e.g., change in pH, change in temperature, enzymes, water, or other physiological conditions or molecules.

[0073] Controlled-release systems may include, for example, an infusion pump which may be used to administer the recombinant proteins in a manner similar to that used for delivering insulin or chemotherapy to specific organs or tumors. Typically, using such a system, the recombinant protein is administered in combination with a biodegradable, biocompatible polymeric implant (see below) that releases the recombinant protein over a controlled period of time at a selected site. Examples of polymeric materials include polyanhydrides, polyorthoesters, polygly colic acid, polylactic acid, polyethylene vinyl acetate, and copolymers and combinations thereof. In addition, a controlled release system can be placed in proximity of a therapeutic target (e.g., a tumor), thus requiring only a fraction of a systemic dosage.

[0074] The recombinant proteins of the invention may be administered by other controlled-release means or delivery devices that are well known to those of ordinary skill in the art. These include, for example, hydropropylmethyl cellulose, other polymer matrices, gels, permeable membranes, osmotic systems, multilayer coatings, microparticles, liposomes, microspheres, or the like, or a combination of any of the above to provide the desired release profile in varying proportions (see below). Other methods of controlled-release delivery of recombinant proteins will be known to the skilled artisan and are within the scope of the invention.

[0075] Recombinant proteins can be administered through a variety of routes well known in the arts. Examples include methods involving direct injection (e.g., systemic or stereotactic), implantation of cells engineered to secrete the factor of interest, drug-releasing biomaterials, implantable matrix devices, implantable pumps, injectable gels and hydrogels, liposomes, micelles (e.g., up to 30 pm), nanospheres (e.g., less than 1 pm), microspheres (e.g., 1-100 pm), reservoir devices, etc.

[0076] Encapsulation

[0077] The recombinant proteins of the present invention can be encapsulated and administered in a variety of carrier delivery systems. Examples of carrier delivery systems include nanoparticles, liposomes, microspheres, hydrogels, polymeric implants, and smart polymeric carriers. Carrier-based systems for biomolecular recombinant protein delivery can: provide for intracellular delivery; tailor binding molecule release rates; increase the proportion of binding molecule that reaches its site of action; improve the transport of the drug to its site of action; allow colocalized deposition with other agents or excipients; improve the stability of the recombinant protein in vivo; prolong the residence time of the recombinant protein at its site of action by reducing clearance; decrease the nonspecific delivery of the recombinant protein to nontarget tissues; decrease irritation caused by the recombinant protein; decrease toxicity due to high initial doses of the recombinant protein; alter the immunogenicity of the recombinant protein; decrease dosage frequency or amount of the recombinant protein; and/or improve shelf life of the recombinant protein.

[0078] The encapsulation of antibodies into nanoparticles (also known as nanocarriers) could lowering dosing of recombinant proteins to promote an overall decrease in toxicity and cost along with potency enhancement (Sousa et al., 2017). Also, the formulation of nanoparticles can be tailored to offer controlled release allowing for the plasma concentration of therapeutic recombinant proteins to be kept within a therapeutic range. Therefore, therapies using antibodies incorporated into nanoparticles are less likely to be inefficient or toxic (Yildirimer et al., 2011). The encapsulation of recombinant proteins also allows delivery of the proteins into the cell if intracellular components (e.g., intracellular enzymes, oncogenic proteins, transcription proteins) are to be targeted (Srinivasan et al., 2013). Furthermore, nanoparticles can shield carried recombinant proteins from lysosomal compartments, therefore, preventing degradation (Perez-Martinez, 2010; Sapra and Allen, 2002).

[0079] Nanocarriers can be functionalized in order to deliver the entrapped recombinant protein directly to therapeutic targets, minimizing undesirable side effects and promoting higher specificity on delivery (Pinto Reis et al., 2006). Functionalization of nanoparticles may be an advantage in protein-based therapy once a drug can be encapsulated in nanoparticles causing a positive and effective synergistic effect on the therapy.

[0080] Polymeric microspheres can be produced using naturally occurring or synthetic polymers and are particulate systems in the size range of 0.1 to 500 pm. Polymeric micelles and polymeromes are polymeric delivery vehicles with similar characteristics to microspheres and can also facilitate encapsulation and delivery of the recombinant proteins described herein. Fabrication, encapsulation, and stabilization of microspheres for a variety of recombinant protein payloads are within the skill of the art (see e.g., Varde & Pack (2004)). Release rate of microspheres can be tailored by type of polymer, polymer molecular weight, copolymer composition, excipients added to the microsphere formulation, and microsphere size. Polymer materials useful for forming microspheres include PLA, PLGA, PLGA coated with DPPC, DPPC, DSPC, EVAc, gelatin, albumin, chitosan, dextran, DL- PLG, SDLMs, PEG (e.g., ProMaxx), sodium hyaluronate, diketopiperazine derivatives (e.g., Technosphere), calcium phosphate-PEG particles, and oligosaccharide derivative DPPG (e.g., Solidose). Encapsulation can be accomplished, for example, using a water/oil single emulsion method, a water-oil-water double emulsion method, or lyophilization. Several commercial encapsulation technologies are available (e.g., ProLease®, Alkerme). Microspheres encapsulating the agents described herein can be administered in a variety of means including parenteral, oral, pulmonary, implantation, and pumping device.

[0081] Polymeric hydrogels, composed of hydrophilic polymers such as collagen, fibrin, and alginate, can also be used for the sustained release of the invention recombinant proteins (see generally, Sakiyama et al. (2001)).

[0082] “Smart” polymeric carriers can be used to administer the invention recombinant proteins (see generally, Stayton et al. (2005); Wu et al. (2005)). Carriers of this type utilize polymers that are hydrophilic and stealth-like at physiological pH but become hydrophobic and membrane-destabilizing after uptake into the virus or endosomal compartment (i.e., acidic stimuli from endosomal pH gradient) where they enhance the release of the cargo molecule into the cytoplasm or virus. Design of the smart polymeric carrier can incorporate pH-sensing functionalities, hydrophobic membrane-destabilizing groups, versatile conjugation and/or complexation elements to allow the binding molecule incorporation, and an optional cell targeting component. As an example, smart polymeric carriers, internalized through receptor mediated endocytosis, can enhance the cytoplasmic delivery of the recombinant proteins described herein. Polymeric carriers include, for example, the family of poly(alkylacrylic acid) polymers, specific examples including poly(methylacrylic acid), poly(ethylacrylic acid) (PEAA), poly(propylacrylic acid) (PPAA), and poly(butylacrylic acid) (PBAA), where the alkyl group progressively increased by one methylene group. Smart polymeric carriers with potent pH-responsive, membrane destabilizing activity can be designed to be below the renal excretion size limit. For example, poly(EAA-co-BA-co-PDSA) and poly(PAA-co-BA-co-PDSA) polymers exhibit high hemolytic/membrane destabilizing activity at the low molecular weights of 9 and 12 kDa, respectively. Various linker chemistries are available to provide degradable conjugation sites for proteins, nucleic acids, and/or targeting moieties. For example, pyridyl disulfide acrylate (PDSA) monomer allow efficient conjugation reactions through disulfide linkages that can be reduced in the cytoplasm after endosomal translocation of the therapeutics.

[0083] Various other delivery systems are known in the art and can be used to administer the recombinant proteins of the invention. Moreover, these and other delivery systems may be combined and/or modified to optimize the administration of the agents of the present invention.

[0084] Emulsions

[0085] The recombinant proteins of the present invention may be prepared and formulated as emulsions. Emulsions are typically heterogenous systems of one liquid dispersed in another in the form of droplets usually exceeding 0.1 pm in diameter (Idson (1988); Rosoff (1988); Block (1988); Higuchi et al (1985)). Emulsions are often biphasic systems comprising two immiscible liquid phases intimately mixed and dispersed with each other. In general, emulsions may be of either the water-in-oil (w/o) or the oil-in-water (o/w) variety. When an aqueous phase is finely divided into and dispersed as minute droplets into a bulk oily phase, the resulting composition is called a water-in-oil (w/o) emulsion. Alternatively, when an oily phase is finely divided into and dispersed as minute droplets into a bulk aqueous phase, the resulting composition is called an oil-in-water (o/w) emulsion. Emulsions may contain additional components in addition to the dispersed phases, and the active drug which may be present as a solution in either the aqueous phase, oily phase or itself as a separate phase. Pharmaceutical excipients such as emulsifiers, stabilizers, dyes, and anti-oxidants may also be present in emulsions as needed. Pharmaceutical emulsions may also be multiple emulsions that are comprised of more than two phases such as, for example, in the case of oil-in-water-in-oil (o/w/o) and water-in-oil-in-water (w/o/w) emulsions. Such complex formulations often provide certain advantages that simple binary emulsions do not. Multiple emulsions in which individual oil droplets of an o/w emulsion enclose small water droplets constitute a w/o/w emulsion. Likewise, a system of oil droplets enclosed in globules of water stabilized in an oily continuous phase provides an o/w/o emulsion.

[0086] Emulsions are characterized by little or no thermodynamic stability. Often, the dispersed or discontinuous phase of the emulsion is well dispersed into the external or continuous phase and maintained in this form through the means of emulsifiers or the viscosity of the formulation. Either of the phases of the emulsion may be a semisolid or a solid, as is the case of emulsion-style ointment bases and creams. Other means of stabilizing emulsions entail the use of emulsifiers that may be incorporated into either phase of the emulsion. Emulsifiers may broadly be classified into four categories: synthetic surfactants, naturally occurring emulsifiers, absorption bases, and finely dispersed solids (Idson (1988)).

[0087] Synthetic surfactants, also known as surface active agents, have found wide applicability in the formulation of emulsions and have been reviewed in the literature (Idson, ; Rieger (1988)). Surfactants are typically amphiphilic and comprise a hydrophilic and a hydrophobic portion. The ratio of the hydrophilic to the hydrophobic nature of the surfactant has been termed the hydrophile/lipophile balance (HLB) and is a valuable tool in categorizing and selecting surfactants in the preparation of formulations. Surfactants may be classified into different classes based on the nature of the hydrophilic group: nonionic, anionic, cationic and amphoteric (Rieger, Id.).

[0088] Naturally occurring emulsifiers used in emulsion formulations include lanolin, beeswax, phosphatides, lecithin and acacia. Absorption bases possess hydrophilic properties such that they can soak up water to form w/o emulsions yet retain their semisolid consistencies, such as anhydrous lanolin and hydrophilic petrolatum. Finely divided solids have also been used as good emulsifiers especially in combination with surfactants and in viscous preparations. These include polar inorganic solids, such as heavy metal hydroxides, nonswelling clays such as bentonite, attapulgite, hectorite, kaolin, montmorillonite, colloidal aluminum silicate and colloidal magnesium aluminum silicate, pigments and nonpolar solids such as carbon or glyceryl tristearate.

[0089] A large variety of non-emulsifying materials are also included in emulsion formulations and contribute to the properties of emulsions. These include fats, oils, waxes, fatty acids, fatty alcohols, fatty esters, humectants, hydrophilic colloids, preservatives and antioxidants (Block, Id.: Idson, Id.).

[0090] Hydrophilic colloids or hydrocolloids include naturally occurring gums and synthetic polymers such as polysaccharides (for example, acacia, agar, alginic acid, carrageenan, guar gum, karaya gum, and tragacanth), cellulose derivatives (for example, carboxymethylcellulose and carboxypropylcellulose), and synthetic polymers (for example, carbomers, cellulose ethers, and carboxyvinyl polymers). These disperse or swell in water to form colloidal solutions that stabilize emulsions by forming strong interfacial films around the dispersed-phase droplets and by increasing the viscosity of the external phase.

[0091] Since emulsions often contain a number of ingredients such as carbohydrates, proteins, sterols and phosphatides that may readily support the growth of microbes, these formulations often incorporate preservatives. Commonly used preservatives included in emulsion formulations include methyl paraben, propyl paraben, quaternary ammonium salts, benzalkonium chloride, esters of p-hydroxybenzoic acid, and boric acid. Antioxidants are also commonly added to emulsion formulations to prevent deterioration of the formulation. Antioxidants used may be free radical scavengers such as tocopherols, alkyl gallates, butylated hydroxyanisole, butylated hydroxytoluene, or reducing agents such as ascorbic acid and sodium metabisulfite, and antioxidant synergists such as citric acid, tartaric acid, and lecithin.

[0092] The application of emulsion formulations via dermatological, oral and parenteral routes and methods for their manufacture have been reviewed in the literature (Idson, Id.). Emulsion formulations for oral delivery have been very widely used because of ease of formulation, as well as efficacy from an absorption and bioavailability standpoint (Rosoff, Id.: Idson, Id.). Mineral-oil base laxatives, oil-soluble vitamins and high fat nutritive preparations are among the materials that have commonly been administered orally as o/w emulsions.

[0093] In one embodiment of the present invention, the binding molecules are formulated as microemulsions. A microemulsion may be defined as a system of water, oil and amphiphile which is a single optically isotropic and thermodynamically stable liquid solution (Rosoff, Id.). Typically, microemulsions are systems that are prepared by first dispersing an oil in an aqueous surfactant solution and then adding a sufficient amount of a fourth component, generally an intermediate chain-length alcohol to form a transparent system. Therefore, microemulsions have also been described as thermodynamically stable, isotropically clear dispersions of two immiscible liquids that are stabilized by interfacial films of surface-active molecules (Leung and Shah (1989)). Microemulsions commonly are prepared via a combination of three to five components that include oil, water, surfactant, cosurfactant and electrolyte. Whether the microemulsion is of the water-in-oil (w/o) or an oil- in-water (o/w) type is dependent on the properties of the oil and surfactant used, and on the structure and geometric packing of the polar heads and hydrocarbon tails of the surfactant molecules (Schott (1985)).

[0094] The phenomenological approach utilizing phase diagrams has been extensively studied and has yielded a comprehensive knowledge, to one skilled in the art, of how to formulate microemulsions (Rosoff, Id.: Block, Id.). Compared to conventional emulsions, microemulsions offer the advantage of solubilizing water-insoluble drugs in a formulation of thermodynamically stable droplets that are formed spontaneously.

[0095] Surfactants used in the preparation of microemulsions include, but are not limited to, ionic surfactants, non-ionic surfactants, Brij 96, polyoxyethylene oleyl ethers, poly glycerol fatty acid esters, tetraglycerol monolaurate (ML310), tetraglycerol monooleate (MO310), hexaglycerol monooleate (PO310), hexaglycerol pentaoleate (PO500), decaglycerol monocaprate (MCA750), decaglycerol monooleate (MO750), decaglycerol sequioleate (SO750), decaglycerol decaoleate (DAO750), alone or in combination with cosurfactants. The cosurfactant, usually a short-chain alcohol such as ethanol, 1 -propanol, and 1 -butanol, serves to increase the interfacial fluidity by penetrating into the surfactant film and consequently creating a disordered film because of the void space generated among surfactant molecules. Microemulsions may, however, be prepared without the use of cosurfactants and alcohol-free self-emulsifying microemulsion systems are known in the art. The aqueous phase may typically be, but is not limited to, water, an aqueous solution of the drug, glycerol, PEG300, PEG400, polyglycerols, propylene glycols, and derivatives of ethylene glycol. The oil phase may include, but is not limited to, materials such as Captex 300, Captex 355, Capmul MCM, fatty acid esters, medium chain (C8-C12) mono, di, and tri glycerides, polyoxyethylated glyceryl fatty acid esters, fatty alcohols, polyglycolized glycerides, saturated polyglycolized C8-C10 glycerides, vegetable oils and silicone oil.

[0096] Microemulsions are particularly of interest from the standpoint of drug solubilization and the enhanced absorption of drugs. Lipid based microemulsions (both o/w and w/o) have been proposed to enhance the oral bioavailability of drugs, including peptides (Constantinides et al. (1994); Ritschel, (1993)). Microemulsions afford advantages of improved drug solubilization, protection of drug from enzymatic hydrolysis, possible enhancement of drug absorption due to surfactant-induced alterations in membrane fluidity and permeability, ease of preparation, ease of oral administration over solid dosage forms, improved clinical potency, and decreased toxicity (Constantinides et al., ; Ho et al.

(1996)). Often microemulsions may form spontaneously when their components are brought together at ambient temperature. This may be particularly advantageous when formulating thermolabile binding molecules. Microemulsions have also been effective in the transdermal delivery of active components in both cosmetic and pharmaceutical applications. It is expected that the microemulsion compositions and formulations of the present invention will facilitate the increased systemic absorption of binding molecules and nucleic acids from the gastrointestinal tract, as well as improve the local cellular uptake of binding molecules and nucleic acids within the gastrointestinal tract, vagina, buccal cavity and other areas of administration.

[0097] Microemulsions of the present invention may also contain additional components and additives such as sorbitan monostearate (Grill 3), Labrasol, and penetration enhancers to improve the properties of the formulation and to enhance the absorption of the binding molecules and nucleic acids of the present invention. Penetration enhancers used in the microemulsions of the present invention may be classified as belonging to one of five broad categories surfactants, fatty acids, bile salts, chelating agents, and non-chelating non surfactants (Lee et al. (1991)). Each of these classes has been discussed above.

[0098] Liposomes

[0099] There are many organized surfactant structures besides microemulsions that have been studied and used for the formulation of drugs. These include monolayers, micelles, bilayers and vesicles. Vesicles, such as liposomes, have attracted great interest because of their specificity and the duration of action they offer from the standpoint of drug delivery. As used in the present invention, the term "liposome" means a vesicle composed of amphiphilic lipids arranged in a spherical bilayer or bilayers.

[0100] Liposomes can be used to administer the recombinant proteins of the present invention. Liposomes are unilamellar or multilamellar vesicles which have a membrane formed from a lipophilic material and an aqueous interior. In these embodiments, the aqueous portion contains the recombinant protein to be delivered. Cationic liposomes possess the advantage of being able to fuse to the cell wall. Non-cationic liposomes, although not able to fuse as efficiently with the cell wall, are taken up by macrophages in vivo.

[0101] The lipid membrane's physical properties, particularly permeability and fluidity, greatly influence the performance of liposomes in vitro and in vivo, and these properties can in principle be specifically tailored by choosing different combinations of lipid mixtures. In general, membrane permeability is minimal to charged ions, whereas uncharged polar molecules can diffuse much faster across the bilayer. Depending on the fluid state of the liposome membrane (gel phase, liquid-crystalline phase, and several other phases of mixed lipids with intermediate fluidity), encapsulated contents may be more permeable with increasing membrane fluidity, assuming homogeneous liposome membranes. In heterogeneous liposome membranes, defined as membranes where lipid lateral phase separation occurs, or when single-component membranes are heated at the glass transition temperature (temperature above which lipids undergo a phase change from solid behavior to a more liquid-like behavior [Tg]), permeability may show an unusual increase. This increase originates from the interface of the membrane heterogeneities. At these interfaces, gain boundaries are formed creating membrane defects due to poor lipid packing with lower effective melting temperatures.

[0102] Because of the flexibility and variety of liposome membranes, and their inherent characteristic to present an interface to the biological milieu that is similar to the immediate surface of cells, liposomes have evolved into major drug delivery carriers that combine several advantages: i) due to their relatively large aqueous core, liposomes exhibit high drug-to-carrier ratios; ii) therapeutic compounds can be encapsulated into the aqueous core or entrapped into the lipophilic membrane without any requirements for chemical modification for attachment to the carrier, that in several cases may interfere with the drug's activity; iii) lipids are not toxic themselves to the doses usually administered, in fact liposomal encapsulation has been used to reduce the toxicity of otherwise toxic drugs; and iv) in immunoliposomes, conjugation of targeting ligands to the liposome surface may result in multivalency and increase in the ligand binding efficacy.

[0103] The drug carrying capacity and release rate of liposomes can depend on the lipid composition, size, charge, drug/lipid ratio, and method of delivery. Conventional liposomes are composed of neutral or anionic lipids (natural or synthetic). Commonly used lipids are lecithins such as (phosphatidylcholines), phosphatidylethanolamines (PE), sphingomyelins, phosphatidylserines, phosphatidylglycerols (PG), and phosphatidylinositols (PI). Liposome encapsulation methods are commonly known in the arts (Galovic et al.

(2002); Wagner et al. (2002)). Targeted liposomes and reactive liposomes can also be used to deliver the recombinant proteins of the invention. Targeted liposomes have targeting ligands, such as lectins, attached to their surface, allowing interaction with specific receptors and/or cell types. Reactive or polymorphic liposomes include a wide range of liposomes, the common property of which is their tendency to change their phase and structure upon a particular interaction (e.g., pH-sensitive liposomes) (see e.g., Lasic (1997)).

[0104] With liposomes for drug delivery applications, size and lamellarity are important parameters and require careful control. Specific and narrow size distributions are necessary, as they influence to a great extent the biodistribution of liposomes. The preparation of unilamellar liposomes is desired to maximize the aqueous encapsulated volume where the drug is entrapped, and to retain control of drug release across the single lipid bilayer membrane. Commonly, lipid suspensions are formed by addition of an aqueous phase to dry lipid layers followed by vortexing. To control size and lamellarity the suspensions are either sonicated or extruded. During sonication, cavitating high-energy ultrasound ruptures and fragments the lipid membranes, which assemble with other lipid fragments in order to minimize water exposure of their hydrophobic edge. When the size of these assembled lipid fragments is large enough to seal with itself at a curvature with less energy than the energy of the exposed hydrophobic regions, then a liposome is formed. Sonicated liposomes are mostly unilamellar. Extrusion through polycarbonate membranes of well-defined pores is another method to control liposome size. Extrusion is performed at temperatures above the Tg of lipids.

[0105] Other liposome preparation methods include reverse phase evaporation, and detergent dialysis. Reverse phase separation introduces a way to develop liposome suspensions with high encapsulated aqueous volumes, and to entrap a large fraction of the aqueous material that needs to be encapsulated. In this process, inverted micelles surrounding the aqueous phase are formed in an excess of an organic solvent. The aqueous phase contains the material for encapsulation by the liposomes. After the organic solvent is slowly removed, a gel-like viscous phase is formed. Upon further removal of the organic solvent, some of the inverted micelles disintegrate, resulting in release of their aqueous phase and the presence of excess phospholipids. In this new polar environment, the excess lipids assemble around the remaining micelles forming closed bilayers (i.e., liposomes).

[0106] Detergent dialysis is another method where upon formation of mixed detergent-lipid micelles, dialysis is used to remove the detergent molecules. This process results in the formation of liposomes, and also in removal of non-encapsulated small molecular weight molecules from the liposome suspension. The rate of detergent removal affects the liposome size distribution. Rapid removal results in smaller liposome sizes. Lipids enriched in detergents have the tendency to fuse when in solution, so as the available time for fusion is decreased with faster detergent dialysis, less time is available for fusion and smaller liposome sizes are formed. This is a mild method that would not easily perturb the stability of even the most sensitive proteins.

[0107] In order to cross intact mammalian skin, lipid vesicles must pass through a series of fine pores, each with a diameter less than 50 nm, under the influence of a suitable transmucosal or transdermal gradient. Therefore, it is desirable to use a liposome which is highly deformable and able to pass through such fine pores.

[0108] Several physical properties that are shown to be of great importance in determining the fate of liposomes in vivo and, consequently, in affecting their efficacy as drug delivery carriers include: liposome size, surface charge, membrane fluidity and PEGylation.

[0109] For drug delivery applications, liposome sizes of ~ 100 nm in diameter are generally used. This is an empirically optimum value between acceptable circulation times and adequate aqueous encapsulated volume that is available for drug loading.

[0110] Further advantages of liposomes include: liposomes obtained from natural phospholipids are biocompatible and biodegradable; liposomes can incorporate a wide range of water and lipid soluble drugs; liposomes can protect encapsulated drugs in their internal compartments from metabolism and degradation (Rosoff, Id.). Important considerations in the preparation of liposome formulations are the lipid surface charge, vesicle size and the aqueous volume of the liposomes.

[0111] Liposomes are useful for the transfer and delivery of active ingredients to the site of action. Because the liposomal membrane is structurally similar to biological membranes, when liposomes are applied to a tissue, the liposomes start to merge with the cellular membranes and as the merging of the liposome and cell progresses, the liposomal contents are emptied into the cell where the active agent may act.

[0112] Liposomal formulations have been the focus of extensive investigation as the mode of delivery for many drugs. Liposomes present several advantages over other formulations. Such advantages include reduced side-effects related to high systemic absorption of the administered drug, increased accumulation of the administered drug at the desired target, and the ability to administer a wide variety of drugs, both hydrophilic and hydrophobic, transdermally. Several reports have detailed the ability of liposomes to deliver agents including high-molecular weight molecules transdermally, including in the human lung.

[0113] Liposomes fall into two broad classes. Cationic liposomes are positively charged liposomes which interact with the negatively charged molecules to form a stable complex. Due to the acidic pH within the endosome, the liposomes are ruptured, releasing their contents into the cell cytoplasm (Wang et al. (1987)).

[0114] Liposomes which are pH-sensitive or negatively -charged, entrap binding molecules rather than complex with it. Since both the DNA and the lipid are similarly charged, repulsion rather than complex formation occurs. Nevertheless, some binding molecule is entrapped within the aqueous interior of these liposomes.

[0115] There are three main structures of antibody-encapsulated liposomes: i) type A liposomes, where the antibodies are directly conjugated on the lipid headgroups resulting in immunoliposomes that exhibit specificity in binding but low blood circulation times; ii) type B liposomes that in addition to antibodies, PEGylated lipids (lipids conjugated to polyethylene glycol [PEG]) are also included on the liposome membrane, resulting in longer circulation times, although with obstruction of specific binding that depends on the extent of steric interference by the polymer chains; and iii) type C liposomes in which the cell targeting antibodies are attached on the free termini of PEGylated lipids (Maruyama, 2002; Sofou et al., 2008).

[0116] One major type of liposomal composition includes phospholipids other than naturally-derived phosphatidylcholine. Neutral liposome compositions, for example, can be formed from dimyristoyl phosphatidylcholine (DMPC) or dipalmitoyl phosphatidylcholine (DPPC). Anionic liposome compositions generally are formed from dimyristoyl phosphatidylglycerol, while anionic fusogenic liposomes are formed primarily from dioleoyl phosphatidylethanolamine (DOPE). Another type of liposomal composition is formed from phosphatidylcholine (PC) such as, for example, soybean PC, and egg PC. Another type is formed from mixtures of phospholipid and/or phosphatidylcholine and/or cholesterol.

[0117] Liposomes also include "sterically stabilized" liposomes, a term which, as used herein, refers to liposomes comprising one or more specialized lipids that, when incorporated into liposomes, result in enhanced circulation lifetimes relative to liposomes lacking such specialized lipids. Examples of sterically stabilized liposomes are those in which part of the vesicle-forming lipid portion of the liposome (A) comprises one or more gly colipids, such as monosialoganglioside Gml, or (B) is derivatized with one or more hydrophilic polymers, such as a polyethylene glycol (PEG) moiety. While not wishing to be bound by any particular theory, it is thought in the art that, at least for sterically stabilized liposomes containing gangliosides, sphingomyelin, or PEG-derivatized lipids, the enhanced circulation half-life of these sterically stabilized liposomes derives from a reduced uptake into cells of the reticuloendothelial system (RES) (Allen et al.(1987); Wu et al. (1993)).

[0118] Various liposomes comprising one or more glycolipids are known in the art. Papahadjopoulos et al. (1987)) reported the ability of monosialoganglioside Gml, galactocerebroside sulfate and phosphatidylinositol to improve blood half-lives of liposomes. These findings were expounded upon by Gabizon et al. (1988). U.S. Pat. No. 4,837,028 and WO 88/04924 disclose liposomes comprising (1) sphingomyelin and (2) the ganglioside Gml or a galactocerebroside sulfate ester. U.S. Pat. No. 5,543,152 discloses liposomes comprising sphingomyelin. Liposomes comprising 1,2-sn-dimyristoylphosphat-idylcholine are disclosed in WO 97/13499.

[0119] Many liposomes comprising lipids derivatized with one or more hydrophilic polymers, and methods of preparation thereof, are known in the art. Sunamoto et al. (1980) described liposomes comprising a nonionic detergent, 2C1215G, that contains a PEG moiety. Ilium et al. (1984) noted that hydrophilic coating of polystyrene particles with polymeric glycols results in significantly enhanced blood half-lives. Synthetic phospholipids modified by the attachment of carboxylic groups of polyalkylene glycols (e.g., PEG) are described by in U.S. Pat. Nos. 4,426,330 and 4,534,899. Klibanov et al. (1990) described experiments demonstrating that liposomes comprising phosphatidylethanolamine (PE) derivatized with PEG or PEG stearate have significant increases in blood circulation half-lives. Blume et al. (1990) extended such observations to other PEG-derivatized phospholipids, e.g., DSPE-PEG, formed from the combination of distearoylphosphatidylethanolamine (DSPE) and PEG. Liposomes having covalently bound PEG moieties on their external surface are described in European Patent No. EP 0445 131 B1 and WO 90/04384. Liposome compositions containing 1-20 mole percent of PE derivatized with PEG, and methods of use thereof, are described in U.S. Pat. Nos. 5,013,556, 5,356,633 and 5,213,804 and European Patent No. EP 0496 813 Bl. Liposomes comprising a number of other lipid-polymer conjugates are disclosed in WO 91/05545 and U.S. Pat. No. 5,225,212 and in WO 94/20073/ Liposomes comprising PEG- modified ceramide lipids are described in WO 96/10391 (Choi et al). U.S. Pat. Nos.

5,540,935 and 5,556,948 describe PEG-containing liposomes that can be further derivatized with functional moieties on their surfaces.

[0120] Transfersomes are yet another type of liposomes and are highly deformable lipid aggregates which are attractive candidates for drug delivery vehicles. Transfersomes are lipid droplets which are so highly deformable that they are easily able to penetrate through pores which are smaller than the droplet. Transfersomes are adaptable to the environment in which they are used, e.g., they are self-optimizing (adaptive to the shape of pores in the dermis), self-repairing, frequently reach their targets without fragmenting, and often self loading. To make transfersomes it is possible to add surface edge-activators, usually surfactants, to a standard liposomal composition. Transfersomes have been used to deliver serum albumin to the skin. The transfersome-mediated delivery of serum albumin has been shown to be as effective as subcutaneous injection of a solution containing serum albumin.

[0121] Surfactants find wide application in formulations such as emulsions (including microemulsions) and liposomes. The most common way of classifying and ranking the properties of the many different types of surfactants, both natural and synthetic, is by the use of the hydrophile/lipophile balance (HLB). The nature of the hydrophilic group (also known as the "head") provides the most useful means for categorizing the different surfactants used in formulations (Rieger, Id.).

[0122] If the surfactant molecule is not ionized, it is classified as a nonionic surfactant. Nonionic surfactants find wide application in pharmaceutical and cosmetic products and are usable over a wide range of pH values. In general, their HLB values range from 2 to about 18 depending on their structure. Nonionic surfactants include nonionic esters such as ethylene glycol esters, propylene glycol esters, glyceryl esters, polyglyceryl esters, sorbitan esters, sucrose esters, and ethoxylated esters. Nonionic alkanolamides and ethers such as fatty alcohol ethoxylates, propoxylated alcohols, and ethoxylated/propoxylated block polymers are also included in this class. The polyoxyethylene surfactants are the most popular members of the nonionic surfactant class.

[0123] If the surfactant molecule carries a negative charge when it is dissolved or dispersed in water, the surfactant is classified as anionic. Anionic surfactants include carboxylates such as soaps, acyl lactylates, acyl amides of amino acids, esters of sulfuric acid such as alkyl sulfates and ethoxylated alkyl sulfates, sulfonates such as alkyl benzene sulfonates, acyl isethionates, acyl taurates and sulfosuccinates, and phosphates. The most important members of the anionic surfactant class are the alkyl sulfates and the soaps.

[0124] If the surfactant molecule carries a positive charge when it is dissolved or dispersed in water, the surfactant is classified as cationic. Cationic surfactants include quaternary ammonium salts and ethoxylated amines. The quaternary ammonium salts are the most used members of this class.

[0125] If the surfactant molecule has the ability to carry either a positive or negative charge, the surfactant is classified as amphoteric. Amphoteric surfactants include acrylic acid derivatives, substituted alkylamides, N-alkylbetaines and phosphatides.

[0126] The use of surfactants in drug products, formulations and in emulsions has been reviewed (Rieger, Id.).

[0127] Carriers

[0128] Certain compositions of the present invention also incorporate carrier compounds in the formulation. As used herein, "carrier compound" or "carrier" can refer to a nucleic acid, or analog thereof, which is inert (i.e., does not possess biological activity per se) but is recognized as a nucleic acid by in vivo processes that reduce the bioavailability of a nucleic acid having biological activity by, for example, degrading the biologically active nucleic acid or promoting its removal from circulation. The coadministration of a nucleic acid and a carrier compound, typically with an excess of the latter substance, can result in a substantial reduction of the amount of nucleic acid recovered in the liver, kidney or other extracirculatory reservoirs, presumably due to competition between the carrier compound and the nucleic acid for a common receptor.

[0129] Excipients

[0130] In contrast to a carrier compound, a "pharmaceutical carrier" or "excipient" is a pharmaceutically acceptable solvent, suspending agent or any other pharmacologically inert vehicle for delivering one or more nucleic acids to an animal. The excipient may be liquid or solid and is selected, with the planned manner of administration in mind, so as to provide for the desired bulk, consistency, etc., when combined with a nucleic acid and the other components of a given pharmaceutical composition. Typical pharmaceutical carriers include, but are not limited to, binding agents (e.g., pregelatinized maize starch, polyvinylpyrrolidone or hydroxypropyl methylcellulose, etc.); fillers (e.g., lactose and other sugars, microcrystalline cellulose, pectin, gelatin, calcium sulfate, ethyl cellulose, polyacrylates or calcium hydrogen phosphate, etc.); lubricants (e.g., magnesium stearate, talc, silica, colloidal silicon dioxide, stearic acid, metallic stearates, hydrogenated vegetable oils, com starch, polyethylene glycols, sodium benzoate, sodium acetate, etc.); disintegrants (e.g., starch, sodium starch glycolate, etc.); and wetting agents (e.g., sodium lauryl sulphate, etc.).

[0131] Pharmaceutically acceptable organic or inorganic excipient suitable for non- parenteral administration which do not deleteriously react with binding molecules can also be used to formulate the compositions of the present invention. Suitable pharmaceutically acceptable carriers include, but are not limited to, water, salt solutions, alcohols, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0132] Suitable pharmaceutically acceptable excipients include, but are not limited to, water, salt solutions, alcohol, polyethylene glycols, gelatin, lactose, amylose, magnesium stearate, talc, silicic acid, viscous paraffin, hydroxymethylcellulose, polyvinylpyrrolidone and the like.

[0133] The compositions of the present invention may additionally contain other adjunct components conventionally found in pharmaceutical compositions, at their art- established usage levels. Thus, for example, the compositions may contain additional, compatible, pharmaceutically-active materials such as, for example, antipruritics, astringents, local anesthetics or anti-inflammatory agents, or may contain additional materials useful in physically formulating various dosage forms of the compositions of the present invention, such as dyes, flavoring agents, preservatives, antioxidants, opacifiers, thickening agents and stabilizers. However, such materials, when added, should not unduly interfere with the biological activities of the components of the compositions of the present invention. The formulations can be sterilized and, if desired, mixed with auxiliary agents, e.g., lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts for influencing osmotic pressure, buffers, colorings, flavorings and/or aromatic substances and the like which do not deleteriously interact with the nucleic acid(s) of the formulation.

[0134] Aqueous suspensions may contain substances which increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and/or dextran. The suspension may also contain stabilizers.

[0135] Other Systems of Administration

[0136] Various other delivery systems are known in the art and can be used to administer the compositions of the invention. Moreover, these and other delivery systems may be combined and/or modified to optimize the administration of the compositions of the present invention.

[0137] Nucleic Acids Encoding Recombinant Proteins

[0138] Additionally provided is a nucleic acid encoding any of the recombinant proteins described above. Techniques to create and use such nucleic acids are well known in the art, and described, for example, in Green and Sambrook, 2012. In some embodiments, the nucleic acid further comprises a promoter.

[0139] Nucleic acid molecules intended to be within the scope of the present invention include variants of the native antibody protein such as those that encode fragments, analogs and derivatives of a native single-domain antibody protein. Such variants may be, e.g., a naturally occurring allelic variant of the native antibody protein, a homolog of the native antibody protein, or a non-naturally occurring variant of the native antibody protein. These variants have a nucleotide sequence that differs from the native antibody protein in one or more bases. For example, the nucleotide sequence of such variants can feature a deletion, addition, or substitution of one or more nucleotides of the native single-domain antibody protein. Nucleic acid insertions are preferably of about 1 to about 10 contiguous nucleotides, and deletions are preferably of about 1 to about 30 contiguous nucleotides.

[0140] In other applications, variant antibody proteins displaying substantial changes in structure can be generated by making nucleotide substitutions that cause less than conservative changes in the encoded polypeptide. Examples of such nucleotide substitutions, are those that cause changes in (a) the structure of the polypeptide backbone; (b) the charge or hydrophobicity of the polypeptide; or (c) the bulk of an amino acid side chain. Nucleotide substitutions generally expected to produce the greatest changes in protein properties are those that cause non-conservative changes in codons. Examples of codon changes that are likely to cause major changes in protein structure are those that cause substitution of (a) a hydrophilic residue, e.g., serine or threonine, for (or by) a hydrophobic residue, e.g., leucine, isoleucine, phenylalanine, valine or alanine; (b) a cysteine or proline for (or by) any other residue; (c) a residue having an electropositive side chain, e.g., lysine, arginine, or histadine, for (or by) an electronegative residue, e.g., glutamine or aspartine; or (d) a residue having a bulky side chain, e.g., phenylalanine, for (or by) one not having a side chain, e.g., glycine.

[0141] Nucleic acids encoding fragments of a recombinant protein within the invention are those that encode, e.g., 2, 3, 4, 5, 10, 25, 50, 100, 130 or more amino acid residues of the peptides provided herein. Shorter oligonucleotides (e.g., those of 6, 7, 8, 9,

10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34,

35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59,

60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 100, 125, and

150 base pairs in length) that encode or hybridize with nucleic acids that encode fragments of a peptide provided herein can be used as probes, primers, or antisense molecules. Longer polynucleotides (e.g., those of 300, 400, 500, 600, 700, 800, 900, 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900 or 2000 base pairs) that encode or hybridize with nucleic acids that encode fragments or variants of the peptides provided in the attached appendices can also be used in various aspects of the invention, including as a binding molecule. Nucleic acids encoding fragments of a binding molecule can be made by enzymatic digestion (e.g., using a restriction enzyme) or chemical degradation of the full length single-domain antibody, mRNA or cDNA, or variants of the foregoing.

[0142] Nucleic acid molecules encoding fusion proteins are also within the scope of the invention. Such nucleic acids can be made by preparing a construct (e.g., an expression vector) that expresses a fusion protein when introduced into a suitable host. For example, such a construct can be made by ligating a first polynucleotide encoding a single-domain antibody, or fragment or variant thereof, fused in frame with a second polynucleotide encoding another protein such that expression of the construct in a suitable expression system yields a fusion protein.

[0143] The nucleic acid molecules of the invention can be modified at a base moiety, sugar moiety, or the phosphate backbone, e.g., to improve stability of the molecule, hybridization, and the like. For example the nucleic acid molecules of the invention can be conjugated to groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989); Lemaitre et al. (1987); PCT Publication No. WO 88/09810), hybridization-triggered cleavage agents (see, e.g., Krol et al. (1988)) or intercalating agents (see, e.g., Zon (1988)).

[0144] An expression cassette comprising the above-described nucleic acid is also provided here, as is a vector comprising the expression cassette. In some embodiments, a phage particle comprising these vectors are provided. A cell comprising these vectors is also provided. The cell can be of any species, e.g., a prokaryote (for example E. coli) or a eukaryote, e.g., human, mouse, rat, etc. The cell can be in cell culture or part of a living multicellular organism, for example a human, a non-human primate, a mouse or a rat.

[0145] Method s of T reatment

[0146] The present invention is also directed to a method of inhibiting an activity of MMP-9. The method comprises contacting the MMP-9 with any of the above-described binding molecules. In these embodiments, the MMP-9 can be inhibited by any amount, e.g., at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, or 100%. In these embodiments, the MMP-9 can be inhibited in vitro, ex vivo, or in vivo, in single cells or in a multicellular organism. In some embodiments when the MMP-9 is to be inhibited in vivo, a cell is transfected with the above-described nucleic acid, expression cassette, or vector, such that the nucleic acid is translated into the recombinant protein in the cell.

[0147] In some embodiments, the MMP-9 is in a subject having a disease, disorder or condition characterized by excess MMP-9, e.g., pain including chronic pain, a disease, disorder or condition characterized by elevated TNF-a or IL-Ib, neuroinflammation, axon demyelination or degeneration, myelin degradation, dorsal root ganglion (DRG) neuronal survival, mechanical allodynia, maladaptive neuroplasticity, idiopathic atrial fibrillation, cancer, or aortic aneurysm. In some specific embodiments, the pain is chronic neuropathic pain. [0148] Thus, the present invention provides a method for treating a disease, disorder or condition characterized by excess MMP-9 activity in a mammal. The method comprises administering a therapeutically effective amount of the above-described binding molecules to the mammal. In some embodiments, the binding molecule is administered to the mammal by administering the above-described nucleic acid, expression cassette, and/or the vector such that the nucleic acid is translated into the recombinant protein in the mammal. These methods can be utilized by any mammal, e.g., human, mouse, rat, cow, pig, dog, cat, etc.

[0149] In these methods, the binding molecule can be administered by any route, including but not limited to parenteral, oral, pulmonary, intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and ophthalmic routes. In some embodiments, the binding molecule is administered to the subject intravenously.

[0150] Preferred embodiments are described in the following examples. Other embodiments within the scope of the claims herein will be apparent to one skilled in the art from consideration of the specification or practice of the invention as disclosed herein. It is intended that the specification, together with the examples, be considered exemplary only, with the scope and spirit of the invention being indicated by the claims, which follow the examples.

[0151] EXAMPLES

[0152] Aspects of the present teachings may be further understood in light of the following examples, which should not be construed as limiting the scope of the present teachings in any way.

[0153] Example 1. Experimental results for inhibitory potency of Gilead antibody against catalytic domains of murine and human MMP9

[0154] Original reagents shipped from Gilead:

[0155] Murine anti-murine MMP9 (GS622703, Loti 1 6.79 mg/ml, 22.7 ml) :

[0156] PBS pH 7.4, 0.01% Tween-20

[0157] IgGl_Isotype control (GS645864 Lot9 8.14 mg/ml, 19.04 ml):

[0158] PBS pH 6.0 (10 mM NaH₂P04, 150 mM NaCl pH 6.0).

[0159] (i) murine anti-murine MMP9 (GS622703): approximately 150 mg at a concentration of 6.79 mg/ml;

[0160] (ii) IgGl Isotype control (GS645864): matching amount at a concentration of 8.14 mg/ml [0161] Dialysis and Concentration:

[0162] Murine anti-murine MMP9 antibody and IgGl Isotype control were dialyzed against Histidine Buffer pH 6.5 (bioPLUS™, Cat# 40125035-2), 150 mM NaCl in Slide-A-Lyzer Dialysis Cassette with 3.5 kDa cut off membrane (Thermo Fisher, Cat# 6610). Next, they were concentrated on Amicon Ultra-4 concentrators (Millipore, Ultracel-lOK,

Cat# UFC801024) on 3000 prm at 4C till 20.00 mg/ml concentrations (Extinction coefficient 1.4).

[0163] Aliquots and Shipment to Charles River Laboratories, Finland. Concentrated samples of Murine anti-murine MMP9 antibody and IgGl Isotype control were than aliquoted for shipping:

[0164] 18 ul aliquots - 10 vials

[0165] 24 ul aliquots - 10 vials

[0166] 180 ul aliquots - 13 vials

[0167] 240 ul aliquots - 6 vials

[0168] 1 mL aliquots - 2 vials

[0169] ALIGNMENTS for MMP9

[0170] Sequences for Human MMP9 and alignments for Human MMP9 vs. Mus musculus, Human vs. Rattus, and Mus musculus vs. Rattus are provided in Figs. 1-4 respectively.

[0171] MMP9 activity measurement and inhibitory assay with fluorogenic peptide and Gilead (GS622703) antibody.

[0172] To determine if Gilead (GS622703) antibody can efficiently block catalytic activity of murine or human MMP9 we measured 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 Gilead (GS622703). Catalytic domains of human or murine MMP9 protease ware purchased in Enzo Life Sciences (Cat# BML-SE360-0010) and Anaspec (Cat# AS-55884-10), correspondently.

[0173] The activity assay with catalytic domains of MMP9 (10 nM) was 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 was 10 mM. To determine the EC50 value of the inhibitory Gilead (GS622703) antibody, MMP9 was pre-incubated for 30 min at 20°C with increasing concentrations of the antibody (0-1,000 nM) (Fig. 5 and Fig. 7). The steady state rate of substrate hydrolysis was continuously monitored at l_ec (excitation wavelength) of 320 nm and l_eih (emission wavelength) of 400 nm by using Varioskan Lux fluorescence spectrophotometer (Thermo Scientific). All assays were performed in triplicate in wells of a 96 well plate. EC50 values were calculated by determining the concentrations of Gilead (GS622703) needed to inhibit 50% of the MMP9 activity against peptidic substrate.

GraphPad Prism was used as fitting software. The same procedure was repeated with Gilead (GS645864) IgGl Isotype control (Fig. 6).

[0174] Example 2. Efficacy of the Test Antibody Administered via Intrathecal, Intravenous, and intra-DRG Infusion, in Rats Subjected to Partial Sciatic Nerve Ligation

[0175] SUMMARY

[0176] Purpose - The objective of this study was to evaluate the effects of ab- MMP9 and ab-MMP14, as well as their combination, on rat tactile allodynia, evoked by the partial sciatic nerve ligation (PSNL).

[0177] Three distinct administration routes were used to evaluate the effects according to the administration routes.

[0178] Methods -The study comprised:

[0179] Prior to DO: Rat pre-handling and baseline electronic vonFrey test

[0180] DO: Surgical induction of the PSNL model

[0181] D0-D6: Post-operative period

[0182] D6: Electronic von Frey (evF) test for injury (allodynia) intensity

[0183] D7 Delivery of the test antibodies (i.t./ i. v./ Intra-DRG)

[0184] D8-D21: Follow-up period of 2 weeks, including evF tests on D8, D14, D16, and D21.

[0185] End-point sampling on D21, after the evF test

[0186] The administration of test antibodies to the animals occurred either via intrathecal, intravenous, or intra-ganglionic routes (intra-ganglionic referring to direct injection to DRGs). Intrathecal and intravenous routes were non-invasive, direct injections, while intra-ganglionic delivery comprised surgical operation, with removal of a small lateral piece of the laminar bone, followed by infusion of the test antibody inside lumbar L4 and L5 dorsal root ganglia (DRG). The infusion was directed to the DRGs ipsilateral to the PSNL surgery (right side).

[0187] Electronic von Frey test was used as the read-out for tactile allodynia evoked by the PSNL model (difference between baseline and D6), and for possible reversal of the evoked mechanical allodynia (difference between pre-dosing evF on D6 vs. post-the dosing timepoints from D7 to D21). Possible acute effects of the test articles on mechanical allodynia were evaluated on D7 (1 day post-dosing), and chronic effects at 2 weeks post dosing (D21). Finally, plasma and CSF samples were collected from 6 rats from each group, along with the ipsilateral DRGs L4 and L5, adjacent piece of spinal cord, and a piece of the ipsilateral sciatic nerve, distal from the ligature.

[0188] Results and Conclusions PSNL surgery evoked robust tactile allodynia, which persisted until the end of the study in-life. Since electronic vonFrey was the only readout of the study at this point, the study outcome is evaluated according to the evF results.

[0189] Comparing the D6 PWT of all groups to their baseline values, a highly significant allodynia increase was observed. After dosing, distinct responses were seen in the treatment groups, of which the most consistent - regarding the corresponding unspecific IgG - administered group and all test timepoints, was the group treated with MMP14-ab via intra- DRG administration route. In addition, MMP9 showed also significant reversion of allodynia, but the size of control antibody groups was too small to evaluate the specificity and reliability of this reversion. Additionally, it would be interesting to perform an identical study with female rats.

[0190] PURPOSE OF THE STUDY

[0191] The objective of this study was to evaluate the effects of ab-MMP9 and ab- MMP14, as well as their combination, on rat tactile allodynia, evoked by the partial sciatic nerve ligation (PSNL) model for neuropathic pain. The PSNL model was first established by Seltzer et al. (1990). The negative control antibody will be ab-IgG.

[0192] In addition, three distinct administration routes were used and compared in order to evaluate the effects according to the administration routes.

[0193] The study comprised:

Prior to DO: Rat pre-handling and baseline tests DO: Surgical induction of the PSNL model D0-D6: Post-operative period D6: Electronic von Frey (evF) test for injury (allodynia) intensity D7 Delivery of the test antibodies (i.t./ i.v./ Intra-DRG)

D8-D21: Follow-up period of 2 weeks, including evF tests on D8, D14, D16, and D21.

End-point sampling on D21, after the evF test [0194] The administration of test antibodies to the animals occurred either via intrathecal, intravenous, or intra-ganglionic routes (intra-ganglionic referring to direct injection to DRGs). Intrathecal and intravenous routes were non-invasive, direct injections, while intra-ganglionic delivery comprised surgical operation, with removal of a small lateral piece of the laminar bone, followed by infusion of the test antibody inside lumbar L4 and L5 dorsal root ganglia (DRG). The infusion was directed to the DRGs ipsilateral to the PSNL surgery (right side).

[0195] Electronic von Frey test was used as the read-out for tactile allodynia evoked by the PSNL model (difference between baseline and D6), and for possible reversal of the evoked mechanical allodynia (difference between pre-dosing evF on D6 vs. post-the dosing timepoints from D7 to D21). Possible acute effects of the test articles on mechanical allodynia were evaluated on D7 (1 day post-dosing), and chronic effects at 2 weeks post dosing (D21). Finally, plasma and CSF samples were collected from 6 rats from each group, along with the ipsilateral DRGs L4 and L5, adjacent piece of spinal cord, and a piece of the ipsilateral sciatic nerve, distal from the ligature.

[0196] MATERIALS [0197] Animals

[0198] All animal experiments were performed as specified in the license authorized by the national Animal Experiment Board of Finland, and according to the guidelines of National Institutes of Health for the care and use of laboratory animals (Bethesda, MD,

USA). Animals were housed at a standard temperature (22 ± 1 °C), in light-controlled environment (lights on from 7 am to 8 pm), and with ad libitum access to food and water. [0199] Species: Rat

[0200] Strain/ Gender: SD/ Male

[0201] Source: Charles River Germany

[0202] Number of Animals Assigned to Study: 150 male [0203] Target Age/weight at the [0204] Upon PSNL surgery: 150 - 225 g [0205] Upon DRG (and other routes) delivery: 200 - 300 g

[0206] Animal Identification

[0207] Study animals were permanently identified by a unique ID number, using ear marks and/or tail numbering.

[0208] Identification was performed according to the Standard Operating Procedures (SOPs) of the Testing Facility.

[0209] Environmental Acclimation and Husbandry

[0210] Following their arrival at the Testing Facility, the animals were acclimatized for a minimum of six (6) days prior to commencing the in-life phase.

[0211] Animal housing: The rats were housed in cages as mixed between the treatment groups. In this study, the rats were not single-housed, unless a brief temporary single-housing was needed due to animal welfare improvement.

[0212] Cages: Polycarbonate cages Mice: Individually ventilated caging systems (IVC) and polycarbonate Type II Long cages (Allentown Inc.)

[0213] Bedding material: Com-o’Cobs (Andersons Ltd) and aspen chips (Tapvei

Ltd)

[0214] Animal enrichment: Environmental enrichment was provided for the animals according to the Government Decree on the Protection of Animals Used for Scientific or Educational Purposes (564/2013). A shelter is provided to all animals: plastic tunnels gnawing material (aspen sticks) and nesting material.

[0215] Chow: Standard chow: Teklad Global 2016 Global 2016 pellet Ad libitum.

[0216] Water: Tap water. Ad libitum.

[0217] Room temperature: 21 ± 1°C

[0218] Veterinary Care

[0219] Veterinary care was available throughout the course of the study. The animals were examined by trained personnel working under supervision of the veterinarian.

[0220] If an animal showed signs of illness or distress, the Study Director and Vet were notified. The Vet gave recommendations either for proper supportive care/ treatment and possibly for changing specific study procedure, or direct euthanasia. These procedures are based on facility SOPs and study -related guidance.

[0221] All such actions were properly documented in the study records. If the condition would not improve by the supportive care/treatment, or if humane endpoint criteria (section 5.6) is reached, the Study Director and Veterinarian have authority and responsibility to act by immediately at her discretion to alleviate suffering and perform euthanasia. The Sponsor representative will be fully informed of any such events without delay.

[0222] General Health Status and Humane End-Points

[0223] Study animals were monitored daily by trained laboratory personnel. In case the general health status of an animal significantly worsens to the degree requiring euthanasia, the animal will be sacrificed by an overdose of CC , and decapitation. Definitions of acceptable general endpoints include: no spontaneous motor activity and inability to drink or eat in a 24-h observation period, massive bleeding, spontaneous severe inflammation, missing anatomy, swelling or tumors (>20 mm), and defective/ lacking righting reflex within a 30-s observation period.

[0224] Animal Assignment to Experimental Groups

[0225] Before study in-life initiation, an animal considered unsuitable for the study was replaced by another obtained within the same shipment and having been maintained under the same environmental conditions.

[0226] The rats for sham operation were randomly selected according to the body weights (BW). In addition, the 12 sham operations were performed on a minimum of 6 distinct surgery days.

[0227] Animals were assigned to treatment groups on D6, by randomization according to the [(Baseline PWT - D6 PWT) / Baseline PWT] and body weights. Animals in poor health or at extremes of the body weight range were not assigned into study groups.

[0228] Test and Reference Articles

[0229] The Sponsor provided documentation of the identity, strength, purity, composition, stability, formulation and storage of the test materials (Annex 1. Test Article Information) along with a Safety Data Sheet, Certificate of Analysis, or equivalent.

[0230] For components provided by external vendors, these information was limited to that provided by the respective vendor.

[0231] Table 4 Identification of the Test - and Reference Articles

[0232] The Test (and Reference) Article(s) and vehicle components were shipped to the address below. A notification email referring to the study number Cl 960120 was requested to be sent to KUO-Formulation@crl.com before shipment delivery from the Sponsor’s premises. The shipment included properly labeled shipping cartons along with information concerning shipment storage conditions.

[0233] Preparation of Formulations and Destiny of the Left-Overs

[0234] Dosing formulations were prepared based on Sponsor instructions ( Annex 1) [0235] Test article formulations, dosage concentrations, instructions for formulation, formulation frequency and storage information for both formulated and un formulated test articles will be performed according to Sponsor’s instructions (Annex 1). [0236] All residual test articles were discarded upon study in-life end.

[0237] EXPERIMENTAL DESIGN

[0238] Animals were treated according to Table 3, n = 10 in each.

[0239] Group 1 : Sham operated control

[0240] Group 2: PSNL, IT, placebo IgGl

[0241] a: 5 animals injected with human IgGl placebo

[0242] b: 5 animals injected with mouse IgG control for MMP-9 antibodies

[0243] Group 3: PSNL, IV, placebo IgGl

[0244] a: 5 animals injected with human IgGl placebo

[0245] b: 5 animals injected with mouse IgG control for MMP-9 antibodies

[0246] Group 4: PSNL, intra-DRG

[0247] a: 5 animals injected with human IgGl placebo

[0248] b: 5 animals injected with mouse IgG control for MMP-9 antibodies

[0249] Group 5 : PSNL, IT, MMP 14 only

[0250] Group 6: PSNL, IV, MMP 14 only

[0251] Group 7: PSNL, intra-DRG, MMP 14 only

[0252] Group 8: PSNL, IT, MMP9 only [0253] Group 9: PSNL, IV, MMP9 only [0254] Group 10: PSNL, intra-DRG, MMP9 only [0255] Group 11 : PSNL, IT, MMP14+MMP9 [0256] Group 12: PSNL, IV, MMP14+MMP9 [0257] Group 13 : P SNL, intra-DRG, MMP 14+MMP9

Table 5. Information for the model induction and test article (TA) administrations to the treatment groups. In addition to the details provided in the table, all groups were subjected to electronic von Frey test (evF) upon baseline, on D6, D8, D14, D16, and D21. Further, on D21, all study animals were euthanized along with endpoint sampling.

*10 mΐ per rat + 65 mΐ empty volume o the injection needle

** - 10 ul/DRG (individual asset) and 5 ul/DRG of MMP-14 mixed with 5 ul/DRG MMP9 in mixed. 10 injections/animal, into 2 DRGs.

***CRL to purchase 15 mg of human IgGl control antibodies, dilute them in the 20 mM Histidine buffer, 150 mM NaCl, pH 6.5.

[0258] METHODS

[0259] The in-life procedures, such as observations, tests, and/or measurements described in this chapter were performed in blinded manner. Blinding occured by coded formulation vials (e.g. alphabets). Coding was defined by the CRL formulation dept/ the Sponsor. The personnel performing either dose administrations or testing were fully blinded.

[0260] Body Weight

[0261] Body weights of the animals were measured upon DO; daily during the post operative recovery period, and on each day of performing the evF test. Terminal body weights were recorded prior to endpoint sampling, and were not collected from animals found dead or euthanized moribund.

[0262] Partial Sciatic Nerve Ligation

[0263] Rats approved to the study according to their baseline tactile allodynia of the right hind paw, were anesthetized first for 2 - 3 min with 5% isoflurane gas, in 70% N20 and 30% 02, with a flow rate of 300 ml/ min. The anesthesia was maintained with 1 - 2% isoflurane gas thereafter. The rat was placed on the surgical pane in prone position, right side towards the surgeon. A homeothermic blanket system with a thermometric probe was used to monitor and maintain rat’s body temperature during the operation. Rectal temperatures upon surgery start and end were recorded.

[0264] Following shaving and disinfecting the skin of the dorsal upper part of the right hind limb a mediolateral incision of 1 - 2 cm was performed onto the skin, immediately caudal to the femur. The incision was emerged from the right side of the spine. The muscle layer was penetrated with blunt scissor opening and sciatic nerve exposed at upper thigh level. A short piece of lifted after which a Silk 6-0 ligature through the midpoint of the nerve approximately 0.5 cm cranial to the furcation of the sural nerve. When completed, a ½ - ½ of the thickness of sciatic nerve was trapped in the ligature. Finally, the muscle layer and the skin were closed, and rats returned to clean cages. The operated rats were under supportive care, as well as close monitoring for any complications, for three days post operation. [0265] Post-Operative Analgesia and Care

[0266] Postoperative care -period occured during the 7 days following the surgery. The period included the following procedures:

[0267] 0.05 mg/kg buprenorphine s.c. were administered until D3, at every 12 hours, starting from the evening of DO.

[0268] Twice-daily careful observation of the general condition and welfare

[0269] Surgical wound and sutures were checked twice a day, until the wound was closed. If needed, the wound area was properly disinfected. The sutures were corrected once according to animal license used at CRL Finland. If the sutures opened 2nd time the animal was euthanized.

[0270] Rehydration with 4 ml of sterile saline i.p. was provided directly after the surgery, and given if needed until D3

[0271] Test Article Administration

[0272] Test article administration was performed by investigators blinded to the treatment. The doses, dosing paradigm, and route of administration for each test article were summarized in tables 3 and 4 above (section 6.1). The dose volume for each animal was based on the body weight on the day of administration.

[0273] Intravenous Delivery

[0274] The animal was subjected to mild anesthesia with isoflurane (maintenance 1.5 - 2 %), and kept on a heating mat throughout the procedure.

[0275] The rat was placed slightly on the other side while sleeping. The needle or cannula was held parallel to the vein and inserted into the vein between the tail flaps. The tip of the needle was inserted a few millimeters into the vein so that the tip of the cannula on top of the needle properly entered the vein. The blood rising into the cannula indicates needle being correctly inserted. The cannula was proceeded a few millimeters into the vein, to prevent loosening during drug administration.

[0276] Air bubbles were avoided by allowing the blood to drain to the end of the cannula before attaching the syringe to the cannula. The intravenous injection off 100 pi was given slowly by free hand.

[0277] At the end of the injection, the normal color of the vessel was restored when the blood circulation returns to normal. When withdrawing the cannula and for a short time thereafter, the injection site was pressed with to prevent leakage. Before returning the rat to the cage, the wound was confirmed not to be leaking.

[0278] Non-Invasive (Free-Hand) Intrathecal Injection

[0279] At a minimum of 30 min prior to the infusion procedure, the rats received analgesics (buprenorphine, 0.05 mg/kg, 1 mL/kg, s.c.). The rat was also slightly anesthetized with isoflurane prior to injection. The area between the L5 and L6 spinous process was indicated as the site for lumbar injection on the skin. The injection site was prepared by shaving and cleaning the intervertebral dorsal side at the lumbar level, followed by disinfecting the skin with 10 % povidone iodine solution (Betadine).

[0280] A total volume of 75 pi (including dead volume of 65 mΐ) of the antibody solution was drawn into a 100-m1 Hamilton-syringe, connected to a sterile disposable 30 G- needle. The injection site was localized by palpating, and injection needle inserted between the L5 and L6 spinous processes (Figure 5).

[0281] Once in contact with the spinal column bone, the syringe angle was reduced to «30°, and the syringe carefully pushed forward into the space. Piercing the dura mater causes a reflexive tail- or limb flick, which is used as a positive indication of the correct injection site.

[0282] The contents of previously loaded syringe were emptied within a time of approximately (determined by CRL mΐ/s). After the injection, the needle was held in place for about 5 s, before beginning to withdraw the needle by careful, slow rotating movement.

[0283] Finally, the rat was monitored for a couple of minutes, to ensure full recovery from the anesthesia, and to confirm that no injuries had occurred during the injection procedure (uses both hind legs, shows no other signs of paralysis).

[0284] Injection into L4 and L5 Dorsal Root Ganglia

[0285] An incision was made on shaved and disinfected skin of the lower back, immediately right of the midline. The lateral side of the L4-L6 vertebrae, and the central part of their transverse branches from the dorsal side were prepared into sight. Next, the spinal nerves L4 and L5, and the vertebral apertures from which the nerves pass, were exposed.

[0286] At this point, the L5 spinal nerve was approximately 2 mm distal to the dorsal root ganglia, beneath the laminar bone. The intervertebral foramen was gently enlarged by small increments so, that eventually an elevation of approximately 1 mm was removed from the crescent part of the laminar bone. One third of the distal portion of the DRG was thereby exposed, and the injection needle inserted into the DRG. [0287] The injection needle was brought as close as possible to the wound, to facilitate access to the DRG but not to press down on the bone below.

[0288] Both DRGs (L4- and L5-DRG) will be infused similarly.

[0289] To inject buffer (endotoxin -free PBS), or any material into the DRG, a 25 pi Hamilton syringe with custom made glass capillary needle was used. The exposed epineurium, or DRG capsule, wee pierced and the needle stopped for 1 min, to allow for DRG membrane to “close” around the needle. The pump was programmed to release the substance at a rate of 0.4 mΐ/min. Once the volume was dispensed, the micropipette will be left in place for an additional a minute to prevent backflow. Afterwards, the needle was slowly pulled out from the DRG. Altogether 2 mΐ of the material were administered into each DRG. To finish, the muscles and skin were closed in two layers using monofilament nylon suture.

[0290] For rehydration, after detaching the anesthesia mask, the animal was provided with 4 ml of sterile saline i.p. and placed into a clean cage lying on a warming pad. The animals were returned to their maintenance room only after full recovery from anesthesia.

[0291] Electronic von Frey Test

[0292] In this study, mechanical sensitivity to touch stimuli was defined at four timepoints by using electronic von Frey (evF) device along with the attached analysis software (Somedic®, Sweden).

[0293] Before subjecting the rats to the baseline evF, they were pre-handled for 2-3 min on two consecutive days, in purpose of decreasing oversensitivity in the test. Pre handling was performed at a maximum of 3 days prior to baseline tests. Baseline evF took place at a minimum of 5 days after their arrival to CR animal facility, and at maximum of 5 days preceding the surgery day (DO).

[0294] Rats displaying inborn oversensitivity were disqualified from the study. Oversensitivity was defined as baseline paw withdrawal threshold (PWT) of < 20 g with 1 mm probe. Following the baseline evF test, the rats were weighed, numbered, and distributed into treatment groups evenly regarding the baseline PWT and body weight.

[0295] Prior to any procedures (handling or tests), the animals were allowed for a 60-min habituation in the room where procedures occured.

[0296] To perform the evF test, the rats were placed in individual von Frey test chambers standing on an elevated steel mesh. The rats were allowed to adapt in the chambers, and the test per se was emerged after they have settled down followed by investigating the chamber and grooming (approximately 15 min). Test was not performed while an animal was grooming, urinating, defecating or sleeping.

[0297] The evF test was performed at the following timepoints:

[0298] Baseline; D6 (pre-dosing); D8, D14, D16, and D21.

[0299] End-Point Sampling

[0300] Tissue samples from the treatment groups were collected on D21 after the PSNL operation. The endpoint samples are summarized in table 3, and a detailed sampling procedures described below the table.

Table 6. Endpoint sampling from the treatment groups and sub-groups

[0301] Five rats from each group were euthanized by deep pentobarbital (Mebunat 180 mg/kg, i.p.) anesthesia, according to the Standard Operating Procedures (SOPs). All these rats were subjected to cerebrospinal fluid (CSF) - and plasma sampling, as well as collection of specific tissues for two distinct purposes thereafter, according to table 6.

[0302] To collect the CSF samples, the rats were subjected to punctures via cistema magna, thereby collecting all available CSF from each rat. The CSF samples were snap- frozen in liquid N2, and stored in -80 °C until shipment.

[0303] Blood samples were collected into K2EDTA -tubes via cardiac punctures. Plasma was isolated by centrifuging 2,500 x g for 10 min at 4°C. Finally, the plasma samples were transferred to clean tubes and stored in -80 °C, until shipped to further analysis.

[0304] Thereafter, to collect samples for IHC. 2 rats from each group were transcardially perfused with 4% PFA in Saline, followed by collecting ipsilateral L4 and L5 DRGs, lumbar spinal cord segment, and common sciatic nerve distal from the ligature.

[0305] The above described samples were collected into mesh tissue cassettes, and allowed to post-fix by immersion in 4% paraformaldehyde in phosphate buffer (0.1 M PB) for an additional 24 h at +4 °C, and stored in 0.01 M PBS at +4 °C until embedding.

[0306] Briefly, the rats were subjected to laminectomy over the length of lumbar segment of the spinal cord, until the end of lumbar spinal nerves. Lumbar DRGs L4 and L5 were collected from the side ipsilateral to PSNL surgery, and spinal cord sample at the level of L4-L5 DRGs. The DRGs were collected by leaving a short stump of spinal nerve included, in order to provide a structure for holding the tissue with forceps during tissue processing. Sciatic nerve was exposed, and a piece dissected at distal from PSNL ligature to the bifurcation of common peroneal and tibial nerves.

[0307] All above described samples - the 2 DRGs, SC, and a piece of sciatic nerve - were collected into mesh tissue cassettes, and allowed to post-fix in 4% PFA-Saline overnight at +4°C. Following post-fix, the samples were embedded in paraffin to obtain paraffin as described in section 7.7.

[0308] Next, three rats per group were perfused with heparinized saline, followed by identical sample collection of the same tissues as for the histopathology.

[0309] The samples were collected into 2-ml round-bottom safe-lock eppendorf tubes containing 5-10 X volumes of RNAlater and stored overnight at +4°C.

[0310] The next day, the supernatant was removed, and samples stored at -20°C until shipped to RNAseq analysis (the samples were not subjected to snap-freezing).

[0311] Tissue Processing, Sample Destination

[0312] The DRGs from two animals were embedded in the same paraffin block.

[0313] Spinal cord- and sciatic nerve -sample from two individual animals were embedded in a single paraffin block, as shown in the Figure 5.

[0314] The paraffin blocks will either be shipped to the Client, or sectioned and analyzed by VRL Finland The RNAlater samples will be shipped to CRL Evereux for RNASeq analysis.

[0315] The paraffin blocks will be molded, as well as likely sectioned and analyzed in CRL Finland, within an upcoming study.

[0316] If samples/specimens will not be shipped, they will be stored at Charles River Finland for a period of 8 weeks after the draft report delivery date. Thereafter, the samples/specimens will be stored at Charles River Finland at additional cost:

[0317] €50 / sample box / month for samples not requiring special storage conditions (RT)

[0318] €100 / sample box / month for samples requiring +4°C, -20°C or -80°C storage conditions [0319] €300 / sample box / month for Cryotank storage

[0320] Sample box size is defined to be maximum 15cm x 15cm x 15cm (3 375 cm3).

[0321] Statistical Analysis

[0322] Prior to further statistical analysis, data quality check and validations will be performed. During that process, potential outliers will be identified and assessed. No outliers will be removed from data without a clear justification for removal (e.g., identified measurement error in the laboratory notes).

[0323] The planned main comparisons of evF test results in this study were:

[0324] All PSNL-operated groups vs. Sham on D6,

[0325] Within-group Follow-up D6 vs. Baseline; D6 vs. D8-D21, and

[0326] D6 - post-dosing test days: Treatment groups MMP9 / MMP14 / MMP9+MMP14 vs. IgGl - treated groups, according to the administration route.

[0327] The results are presented in normalized (% from Baseline) form, in addition to the raw mean PWTs.

[0328] Assumption of normality of each data set will be primarily based on experience (e.g., data within a population is known to be approximately Gaussian) and observations during validation phase. Some biological variables are known to follow lognormal distributions - in these cases the data will be first transformed to logarithms and then parametric statistical test can be used. The same normality assumption will be used for series of experiments, or group of similar readouts from an assay. In addition, the D’Agostino-Pearson omnibus normality test can be used to support the decision whether parametric or nonparametric test will be used. Details how all categorical, ordinal, or other discrete (non-continuous) variables will be analyzed are given further on.

[0329] Statistical analysis of single time point data

[0330] Comparison involving three or more groups were performed using one-way ANOVA followed by Dunnett’s multiple comparisons test (sham vs. PSNL and treatment vs. control). In case of significantly different SDs detected by Brown-Forsythe test, the assumption of equal SDs is rejected and Welch’s ANOVA followed by Dunnett’s T3 multiple comparison test were used. The non-parametric counterpart is the Kruskal-Wallis test followed by Dunn’s multiple comparison test. [0331] Repeated observations of same subjects (longitudinal data):

[0332] In case of longitudinal comparison of three or more groups, similarly, the two-way Mixed-effects model with Geisser-Greenhouse correction, followed by Dunnett’s multiple comparisons was performed. In this case, one family per time point were set. Time, group and the interaction of Time x Group are the fixed effects, and the individual subject is the random effect.

[0333] Data scaling and normalization:

[0334] The evF data were transformed to %change from baseline/, or to ipsi/ contra ratios, depending on the comparisons and analysis means. In addition, the areas under curve for each treatment may be analyzed and compared.

[0335] All values are presented as the mean ± standard error of the mean. In case of two or more comparisons per family, the multiplicity adjusted P values are presented. All statistical analyses were conducted with a significance level of a = 0.05, using GraphPad Prism (Version 8.3, GraphPad Software, Inc., San Diego, CA)

[0336] RESULTS

[0337] Body weight over time

[0338] The mean body weights of the treatment groups developed expectedly. The significant difference between the mean BW of Sham vs. two i.t. and two intra-DRG groups is assessed to be due to more invasive dosing route.

[0339] Electronic von Frey Test

[0340] Paw withdrawal thresholds - i.e. mean PWTs of all treatment groups at all test timepoints are presented in Fig. 7. Despite of sham group, each group displayed significant allodynia induction in baseline vs. D6. In addition, significant reversion of the induced allodynia was displayed by groups 3b, 6, 7 and 9, as well as the combination treatment group 12.

[0341 ] Absolute Mean PWT

[0342] Percentage From Baseline PWT

[0343] In order to obtain proper data for group comparisons, the mean % for baseline-values were calculated to the treatment groups (Fig. 8). In figure 8, only the timepoints after PSNL are shown.In these results too, the i.v. groups are emphasized with significant differences between D6 and post-dosing timepoints.

[0344] Injury baseline (D6) vs. the Last Post-Dosing Timepoint

[0345] To simplify interpretation of the results, the % from baseline values are shown in Fig 9, where only the injury baseline value on D6, and the last post-dosing timepoint are displayed.

[0346] Mean PWTs and Timepoint comparison

[0347] Upon request, line graphs showing the results of just 2 pairs of comparable groups were also drawn. They are presented in Figs 10 and 11.

[0348] CONCLUSIONS

[0349] PSNL surgery evoked robust tactile allodynia, which persisted until the end of the study in-life. Since electronic vonFrey was the only readout of the study at this point, the study outcome is evaluated according to the evF results.

[0350] Comparing the D6 PWT of all groups to their baseline values, a highly significant allodynia increase was observed. After dosing, distinct responses were seen in the treatment groups, of which the most consistent - regarding the corresponding unspecific IgG - administered group and all test timepoints, was the group treated with MMP14-ab via intra- DRG administration route. In addition, MMP9 showed also significant reversion of allodynia, but the size of control antibody groups was too small to evaluate the specificity and reliability of this reversion.

[0351] In view of the above, it will be seen that several objectives of the invention are achieved and other advantages attained.

[0352] As various changes could be made in the above methods and compositions without departing from the scope of the invention, it is intended that all matter contained in the above description and shown in the accompanying drawings shall be interpreted as illustrative and not in a limiting sense.

[0353] All references cited in this specification, including but not limited to patent publications and non-patent literature, and references cited therein, are hereby incorporated by reference. The discussion of the references herein is intended merely to summarize the assertions made by the authors and no admission is made that any reference constitutes prior art. Applicants reserve the right to challenge the accuracy and pertinence of the cited references. [0354] As used herein, in particular embodiments, the terms “about” or “approximately” when preceding a numerical value indicates the value plus or minus a range of 10%. Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range is encompassed within the disclosure. That the upper and lower limits of these smaller ranges can independently be included in the smaller ranges is also encompassed within the disclosure, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the disclosure.

[0355] The indefinite articles “a” and “an,” as used herein in the specification and in the embodiments, unless clearly indicated to the contrary, should be understood to mean “at least one.”

[0356] The phrase “and/or,” as used herein in the specification and in the embodiments, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Multiple elements listed with “and/or” should be construed in the same fashion, i.e., “one or more” of the elements so conjoined. Other elements can optionally be present other than the elements specifically identified by the “and/or” clause, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, a reference to “A and/or B”, when used in conjunction with open-ended language such as “comprising” can refer, in one embodiment, to A only (optionally including elements other than B); in another embodiment, to B only (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0357] As used herein in the specification and in the embodiments, “or” should be understood to have the same meaning as “and/or” as defined above. For example, when separating items in a list, “or” or “and/or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of’ or “exactly one of,” or, when used in the embodiments, “consisting of,” will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives (i.e.

“one or the other but not both”) when preceded by terms of exclusivity, such as “either,” “one of,” “only one of,” or “exactly one of.” “Consisting essentially of,” when used in the embodiments, shall have its ordinary meaning as used in the field of patent law.

[0358] As used herein in the specification and in the embodiments, the phrase “at least one,” in reference to a list of one or more elements, should be understood to mean at least one element selected from any one or more of the elements in the list of elements, but not necessarily including at least one of each and every element specifically listed within the list of elements and not excluding any combinations of elements in the list of elements. This definition also allows that elements can optionally be present other than the elements specifically identified within the list of elements to which the phrase “at least one” refers, whether related or unrelated to those elements specifically identified. Thus, as a non-limiting example, “at least one of A and B” (or, equivalently, “at least one of A or B,” or, equivalently “at least one of A and/or B”) can refer, in one embodiment, to at least one, optionally including more than one, A, with no B present (and optionally including elements other than B); in another embodiment, to at least one, optionally including more than one, B, with no A present (and optionally including elements other than A); in yet another embodiment, to at least one, optionally including more than one, A, and at least one, optionally including more than one, B (and optionally including other elements); etc.

[0359] Other Embodiments

[0360] The detailed description set-forth above is provided to aid those skilled in the art in practicing the present invention. However, the invention described and claimed herein is not to be limited in scope by the specific embodiments herein disclosed because these embodiments are intended as illustration of several aspects of the invention. Any equivalent embodiments are intended to be within the scope of this invention. Indeed, various modifications of the invention in addition to those shown and described herein will become apparent to those skilled in the art from the foregoing description which do not depart from the spirit or scope of the present inventive discovery. Such modifications are also intended to fall within the scope of the appended claims.

[0361] References Cited

[0362] All publications, patents, patent applications and other references cited in this application are incorporated herein by reference in their entirety for all purposes to the same extent as if each individual publication, patent, patent application or other reference was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Citation of a reference herein shall not be construed as an admission that such is prior art to the present invention.

[0363] Specifically intended to be within the scope of the present invention, and incorporated herein by reference in its entirety, is the following publication:

[0364] Allen et al. (1987) FEBS Letters, 223:42.

[0365] Antibodies: A Laboratory Manual, Second edition (2014), Edited by E.A. Greenfield, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0366] Block in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 2, p. 335.

[0367] Blume et al. (1990) Biochimica et Biophysica Acta 1029:91.

[0368] Constantinides et al. (1994) Pharmaceutical Research 11:1385-1390.

[0369] Diffar et al. (2009), African Journal of Biotechnology 8: 2645-2652.

[0370] Gabizon et al. 1988 Proc. Natl. Acad. Sci. U.S.A. 85:6949.

[0371] Galovic et al. (2002) Eur. J. Pharm. Sci. 15:441-448.

[0372] Green, MR and Sambrook, J. (2012) Molecular Cloning: A Laboratory Manual (Fourth Edition), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.

[0373] Griffin et al. (2017) Antimicrob Agents Chemother. 61:e01714-16.

[0374] Haryadi et al. (2014) PLoS ONE 10: eOl 16878.

[0375] Higuchi et al., in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 301.

[0376] Ho et al. (1996) J. Pharm. Sci. 85:138-143.

[0377] https://www.criver.com/products-services/discovery-services/pharmacology- studies/neuroscience-models-assays/pain-studies/neuropathic-pain-model?region=3601

[0378] Idson, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., volume 1, p. 199.

[0379] Ilium et al. (1984) FEBS Lett. 167:79.

[0380] Kbbanov et al. (1990) FEBS Lett. 268:235.

[0381] Krol et al. (1988) BioTechniques 6:958-976.

[0382] Lasic (1997) Liposomes in Gene Delivery, CRC Press, FL)

[0383] Lee et al. (1991) Critical Reviews in Therapeutic Drug Carrier Systems, p. 92.

[0384] Lemaitre et al. (1987) Proc. Natl. Acad. Sci. U.S.A. 84:648-652.

[0385] Letsinger et al. (1989) Proc. Natl. Acad. Sci. U.S.A. 86:6553-6556.

[0386] Leung and Shah, in: Controlled Release of Drugs: Polymers and Aggregate Systems, Rosoff, M., Ed., 1989, VCH Publishers, New York, pages 185-215. [0387] Li et al. (2012), J Biol Chem. 287:13713-13721.

[0388] Maruyama K. (2002) Biosci Rep 22:251-66.

[0389] Mukherjee et al. (2018) J Biol Chem. 293:2815-2828.

[0390] Nakamura et al. (2013) PLoS ONE 8:e73640.

[0391] Papahadjopoulos et al. (1987) Ann. N.Y. Acad. Sci. 507:64.

[0392] Rieger, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Perez-Martinez et al. (2010) Bioessays. 32:589-598.

[0393] Pinto Reis et al. (2006) Nanomedicine. 2:8-21.

[0394] Marcel Dekker, Inc., New York, N.Y., volume 1, p. 285.

[0395] Ritschel (1993) Meth. Find. Exp. Clin. Pharmacol. 13:205.

[0396] Rosoff, in Pharmaceutical Dosage Forms, Lieberman, Rieger and Banker (Eds.), 1988, Marcel Dekker, Inc., New York, N.Y., Volume 1, p. 245.

[0397] Rybakowski (2009) Cardiovasular Psychiatry and Neurology 2009:904836. [0398] Sakiyama et al. (2001) FASEB J. 15:1300-1302.

[0399] Sapra P and Allen TM (2002) Cancer Res. 62:7190-7194.

[0400] Schott (1985), in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, Pa., 1985, p. 271.

[0401] Sofou S. and Sgouros G. (2008) Expert Opinion on Drug Delivery 5:189-

204.

[0402] Sousa et al. (2017) Expert Opinion on Drug Delivery 14:1163-1176.

[0403] Srinivasan et al. (2013) Mol Pharm. 10:3275-3284.

[0404] Stayton et al. (2005) Orthod Craniofacial Res 8:219-225.

[0405] Sunamoto et al. (1980) Bull. Chem. Soc. Jpa, 53:2778.

[0406] van der Linden et al. (2000), Journal of Immunological Methods, 240:185-

195.

[0407] Varde & Pack (2004) Expert Opin. Biol. 4:35-51.

[0408] Wagner et al. (2002) J. Liposome Res. 12:259-270.

[0409] Wang et al. (1987) Biochem. Biophys. Res. Commun. 147:980-985.

[0410] Wu et al. (1993) Cancer Research 53:3765.

[0411] Wu et al. (2005) Nature Biotech 23:1137-1146.

[0412] Yildi rimer et al. (2011) Nano Today. 6:585-607.

[0413] Zhu et al. (2017) Sci Transl Med. 9:eaajl928.

[0414] Zon (1988) Pharm. Res. 5:539-549.

[0415] U.S. Pat. No. 4,426,330. [0416] U.S. Pat. No. 4,534,899.

[0417] U.S. Pat. No. 4,837,028.

[0418] U.S. Pat. No. 5,013,556.

[0419] U.S. Pat. No. 5,213,804.

[0420] U.S. Pat. No. 5,225,212.

[0421] U.S. Pat. No. 5,356,633.

[0422] U.S. Pat. No. 5,540,935.

[0423] U.S. Pat. No. 5,556,948.

[0424] U.S. Pat. No. 5,543,152.

[0425] U.S. Pat. No. 5,759,808.

[0426] U.S. Pat. No. 5,800,988.

[0427] U.S. Pat. No. 5,840,526.

[0428] U.S. Pat. No. 5,874,541.

[0429] U.S. Pat. No. 7,083,782.

[0430] U.S. Pat. No. 8,377,443.

[0431] US Pat. App. Publication 20190185581. [0432] PCT Patent Publication WO 88/04924. [0433] PCT Patent Publication WO 88/09810. [0434] PCT Patent Publication WO 90/04384. [0435] PCT Patent Publication WO 91/05545. [0436] PCT Patent Publication WO 94/20073. [0437] PCT Patent Publication WO 96/10391. [0438] PCT Patent Publication WO 97/13499. [0439] PCT Patent Publication WO 2006/040153. [0440] PCT Patent Publication WO 2006/122786. [0441] European Patent No. EP 0445131.

[0442] European Patent No. EP 0496813. Sequence Listing

SEQ ID NO:l (SEQ ID NO: 13 of 8,377,443)

GFSLLSYGVH

SEQ ID NO:2 (SEQ ID NO: 14 of 8,377,443)

VIWTGGTTNYNSALMS

SEQ ID NO:3 (SEQ ID NO: 15 of 8,377,443)

YYYGMDY

SEQ ID NO:4 (SEQ ID NO: 16 of 8,377,443)

KASQDVRNTVA

SEQ ID NO:5 (SEQ ID NO: 17 of 8,377,443)

SSSYRNT

SEQ ID NO:6 (SEQ ID NO: 18 of 8,377,443)

QQHYITPYT

SEQ ID NO:7 (SEQ ID NO:3 of 8,377,443)

QVQLKESGPGLVAPSQSLSITCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIW

TGGTTNYNSALMSRLSISKDDSKSQVFLKMNSLQTDDTAIYYCARYYYGMDYWGQ

GTSVTVSS

SEQ ID NO:8 (SEQ ID NO:5 of 8,377,443)

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIW

TGGTTNYNSALMSRLTISKDDSKSTVYLKMNSLKTEDTAIYYCARYYYGMDYWGQ

GTSVTVSS

SEQ ID NO:9 (SEQ ID NO:6 of 8,377,443)

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIW

TGGTTNYNSALMSRLTISKDDSKNTVYLKMNSLKTEDTAIYYCARYYYGMDYWGQ

GTLVTVSS SEQ ID NO: 10 (SEQ ID NO:7 of 8,377,443)

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIW T GGTTNYNS ALMS RFTI SKDD SKNTV YLKMNSLKTEDT AI YY C ARYYY GMD YW GQ GTLVTVSS

SEQ ID NO: 11 (SEQ ID NO: 8 of 8,377,443)

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIW T GGTTNYNS ALMS RFTI SKDD S KNTL YLKMNSLKTEDTAI YY C ARY YY GMD YW GQ GTLVTVSS

SEQ ID NO: 12 (SEQ ID NO:4 of 8,377,443)

DIVMTQSHKFMSTS VGDRVSITCKAS QD VRNTV AWYQQKTGQ SPKLLIYS S S YRNT GVPDRFTGS GS GTDFTFTI S S V Q AEDL AV YF C QQHYITP YTF GGGTKLEIK

SEQ ID NO: 13 (SEQ ID NO:9 of 8,377,443)

DIVMTQSPSFLSASVGDRVTITCKASQDVRNTVAWYQQKTGKAPKLLIYSSSY RNTGVPDRFTGSGS GTDFTLTIS SLQ AEDV AVYFCQQHYITPYTF GGGTKVEIK

SEQ ID NO: 14 (SEQ ID NO: 10 of 8,377,443)

DIVMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSY RNTGVPDRFTGSGS GTDFTLTIS SLQ AEDV AVYFCQQHYITPYTF GGGTKVEIK

SEQ ID NO: 15 (SEQ ID NO: 11 of 8,377,443)

DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSY RNT GVPDRF S GS GS GTDFTLTI S S LQ AED V AV YF CQQHYITP YTF GGGTKVEIK

SEQ ID NO: 16 (SEQ ID NO: 12 of 8,377,443)

DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSY RNT GVPDRF S GS GS GTDFTLTI S SLQ AED V AV YY C QQHYITP YTF GGGTKVEIK

SEQ ID NO: 17 (Fab heavy chain of GS-5745)

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIW T GGTTNYNS ALMS RFTI SKDD SKNTV YLKMNSLKTEDT AI YY C ARYYY GMD YW GQ GTLVTVSSASTKGPSVFPLAPCSRSTSESTAALGCLVKDYFPEPVTVSWNSGALTSGV HTFPAVLQSSGLYSLSSVVTVPSSSLGTKTYTCNVDHKPSNTKVDKRVESKYGPPCPP

CPAPEFL

SEQ ID NO: 18 (Fab light chain of GS-5745)

DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSY RNT GVPDRF S GS GS GTDFTLTI S S LQ AED V AV YY C QQHYITP YTF GGGTKVEIKRTV AAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQD SKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC

Claims

CLAIMS What is claimed is:

1. A method of inhibiting an activity of MMP-9, the method comprising contacting the MMP-9 with a binding protein, comprising an immunoglobulin heavy chain polypeptide, or functional fragment thereof, and an immunoglobulin light chain polypeptide, or functional fragment thereof, wherein the MMP9 binding protein specifically binds to human MMP9 and wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising heavy chain CDRs of SEQ ID NOs: 1-3 or light chain CDRs of SEQ ID NOs: 4-6 that specifically binds to MMP-9.

2. The method of claim 1, wherein the binding molecule comprises immunoglobulin heavy chain complementarity-determining regions (CDRs) comprising sequences SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO:3.

3. The method of claim 1 or 2, wherein the binding molecule comprises immunoglobulin light chain complementarity-determining regions (CDRs) comprising sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

4. The method of any one of claims 1-3 wherein the binding molecule comprises an immunoglobulin heavy chain variable region comprising SEQ ID NO:7, 8, 9, 10 or 11.

5. The method of any one of claims 1-4, wherein the binding molecule comprises an immunoglobulin light chain variable region comprising SEQ ID NO: 12, 13, 14, 15 or 16.

6. The method of claim 1 , wherein the binding molecule comprises an Fab heavy chain comprising SEQ ID NO: 17.

7. The method of claim 1, wherein the binding molecule comprises an Fab light chain comprising SEQ ID NO: 18.

8. The method of claim 1 , wherein the binding molecule comprises an Fab heavy chain comprising SEQ ID NO: 17 and an Fab light chain comprising SEQ ID NO: 18.

9. The method of any one of claims 1-8, wherein the MMP-9 is in a subject having a disease, disorder or condition characterized by excess MMP-9.

10. The method of claim 9, wherein the disease, disorder or condition is pain, a disease, disorder or condition characterized by elevated TNF-a or IL-Ib, neuroinflammation, axon demyelination or degeneration, myelin degradation, dorsal root ganglion (DRG) neuronal survival, mechanical allodynia, maladaptive neuroplasticity, idiopathic atrial fibrillation, cancer, or aortic aneurysm.

11. The method of claim 10, wherein the pain is chronic neuropathic pain.

12. The method of any one of claims 9-11, wherein the binding molecule is administered to the subject intravenously.

13. A method for treating a disease, disorder or condition characterized by excess MMP-9 in a mammal, the method comprising administering a therapeutically effective amount of a binding molecule described in US Patent 8,377,443 that specifically binds to MMP-9.

14. The method of claim 13, wherein the binding molecule comprises immunoglobulin heavy chain complementarity-determining regions (CDRs) comprising sequences SEQ ID NO:l, SEQ ID NO:2, and SEQ ID NO: 3; immunoglobulin light chain complementarity-determining regions (CDRs) comprising sequences SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6; an immunoglobulin heavy chain variable region comprising SEQ ID NO:7, 8,

9, 10 or 11; an immunoglobulin light chain variable region comprising SEQ ID NO: 12, 13,

14, 15 or 16; an Fab heavy chain comprising SEQ ID NO: 17; an Fab light chain comprising SEQ ID NO: 18, or any combination thereof.

15. The method of claim 13, wherein the binding molecule comprises an Fab heavy chain comprising SEQ ID NO: 17 and an Fab light chain comprising SEQ ID NO: 18.

16. The method of any one of claims 13-15, wherein the binding molecule is administered to the mammal by administering a nucleic acid encoding the binding molecule such that the nucleic acid is translated into the recombinant protein in the mammal.

17. The method of any one of claims 13-15, wherein the binding molecule is administered to the mammal by administering the binding molecule in a pharmaceutically acceptable excipient.

18. The method of claim 17, wherein the binding molecule is administered intravenously.