OA18526A - Antibodies to Matrix Metalloproteinase 9 - Google Patents

Abstract

The present disclosure provides compositions and methods of use involving binding proteins, e.g., antibodies and antigen-binding fragments thereof, that bind to the matrix metalloproteinase-9 (MMP9) protein (MMP9 is also known as gelatinase-B), wherein the binding proteins comprise an immunoglobulin (Ig) heavy chain (or functional fragment thereof) and an Ig light chain (or functional fragment thereof).

Description

ANTIBODIES TO MATRIX METALLOPROTEINASE 9

CROSS REFERENCE TO RELATED APPLICATIONS [0001] This application claims priority benefit of U.S. provisional application serial no.

61/377,886, filed August 27, 2010, which application is incorporated herein by reference in its entirety.

FIELD

I [0002] This disclosure is in the field of extracellular enzymes, extracellular matrix enzymes, proteases and immunology.

INTRODUCTION [0003] Matrix metalloproteinases (MMPs) are a family of extracellular enzymes involved în forming and remodeling the extracellular matrix. These enzymes contain a conserved catalytic domain in which a zinc atom is coordinated by three histidine residues. Currently, over 20 members of this family are known, organized into a number of groups including collagénases, gelatinases, stromélysine, matrilysins, enamelysins and membrane MMPs.

[0004] MMP2 and MMP9 belong to the gélatinase group of matrix metalloproteinases.

Besîdes containîng signal peptide, propeptide, catalytic, zinc-binding and heamopexin-like domains common to most MMPs, the gelatinases also contain a plurality of fibronectin-like domains and an O-glycosylated domain.

10005] Abnormal activity of certain MMPs has been shown to play a rôle in tumor growth, metastasis, inflammation and vascular disease. See, for example, Hu et al. (2007)

Nature Reviews: Drug Discovery 6:480-498. Because of this, it can be désirable to inhibit the activity of one or more MMPs in certain therapeutic settings. However, the activity of certain other MMPs is often rcquired for normal function. Since most MMP inhibitors are targeted to the conserved catalytic domain and, as a resuit, inhibit a number of different MMPS, their therapeutic use has caused side effects due to the inhibition of essential, non-pathogenically30 related MMPs.

[0006] Despite this problem, it has proven difficult to develop inhibitors that are spécifie to a particular MMP, because inhibition of enzymatic activity generally requires that the inhibitor be targeted to the catalytic domain. Consequently, most inhibitors of matrix metalloproteinase enzymatic activity are likely to react with more than one MMP, due to *

homologies in their catalytic domains. Thus, there remains a need for therapeutic reagents that specifically inhibit the catalytic activity of a single MMP, and that do not react with other

MMPs.

SUMMARY f0007] The présent disclosure provides compositions and methods of use involving binding proteins, e.g., antibodies and antigen-binding fragments thereof, that bind to the matrix metalloproteinase-9 (MMP9) protein (MMP9 is also known as gelatinase-B), wherein the binding proteins comprise an immunoglobulin (Ig) heavy chain (or functional fragment thereof) and an Ig light chain (or functional fragment thereof). The disclosure further provides MMP9 binding proteins that bind specifically to MMP9 and not to other, related matrix metalloproteinases. Such MMP9 binding proteins find use in applications in which it is necessary or désirable to obtain spécifie modulation (e.g., inhibition) of MMP9, e.g., without directly affecting the activity of other matrix metalloproteinases. Thus, in certain embodiments of the présent disclosure an anti-MMP9 antibody is a spécifie inhibitor of the activity of MMP9.

In particular, the MMP9 binding proteins disclosed herein will be useful for inhibition of MMP9 while allowing normal function of other, related matrix metalloproteinases.

[0008] Accordingly, the présent disclosure provides, inter alia:

[0009| 1. A MMP9 binding protein comprising an immunoglobulin heavy chain or functional fragment thereof, and an immunoglobulin light chain or functional fragment thereof, 20 wherein the protein does not bind to a matrix metalloproteinase other than MMP9.

[00101 2. The protein of embodiment 1, wherein the heavy chain comprises a complementarity-determining région (CDR) selected from one or more of SEQ ID NOs: 13-15, and the light chain comprises a CDR selected from one or more of SEQ ID NOs: 16-18.

10011] 3. The protein of embodiment 2, wherein the heavy chain comprises a variable région selected from the group consisting of SEQ ID NOs: 3 or 5-8, and the light chain comprises a variable région selected from the group consisting of SEQ ID NOs: 4 or 9-12. [0012] 4. The protein of embodiment 1, wherein the heavy chain is an IgG.

[0013| 5. The protein of embodiment 1, wherein the light chain is a kappa chain.

[0014] 6. The protein of embodiment 1, wherein the binding of the protein to MMP9 inhibits the enzymatic activity of MMP9.

[0015] 7. The protein of embodiment 6, wherein the inhibition is non-competitive.

[0016] 8. The protein of embodiment 1, wherein the heavy chain is encoded by a polynucleotide having a nucléotide sequence selected from the group consisting of SEQ ID NOs: 19-22 and the light chain is encoded by a polynucleotide having a nucléotide sequence «

selected from the group consisting of SEQ ID NOs: 23-26.

10017]

9. A vector comprising one or more polynucleotides having a nucléotide

sequence selected from the group consisting of SEQ ID NOs: 19-26.

[0018] [0019] [0020] [0021]	10. A cell comprising the vector of embodiment 9. 11. A pharmaceutical composition comprising the protein of embodiment 1. 12. A pharmaceutical composition comprising the vector of embodiment 9. 13. A pharmaceutical composition comprising the cell of embodiment 10. BRIEF DESCRIPTION OF THE DRAWINGS
[0022]	Figure 1 shows the amino acid'sequence of the heavy chain variable région of a

mouse monoclonai anti-MMP9 antibody (AB0041), along with the amino acid sequences of humanized variants of heavy chain (VH1-VH4), aligned to show différences in framework amino acid sequence resulting from humanization. CDRs are shown in italics, and amino acids that are different in the humanized variants, compared to the parent mouse monoclonal, are underlined.

[0023]

Figure 2 shows the amino acid sequence of the light chain variable région of a

mouse monoclonal anti-MMP9 antibody (AB0041), along with the amino acid sequences of humanized variants of this light chain (VH1-VH4), aligned to show différences in framework amino acid sequence resulting from humanization. CDRs are shown in italics, and amino acids that are different in the humanized variants, compared to the parent mouse monoclonal, are underlined.

[0024]	Figure 3 shows a schematic diagram of the MMP9 protein. DETAILED DESCRIPTION
[0025]	Practice of the présent disclosure employs, unless otherwise indicated, standard

methods and conventional techniques in the fields of cell biology, toxicology, molecular biology, biochemistry, cell culture, immunology, oncology, recombinant DNA and related fields as are within the skill of the art. Such techniques are described in the literature and thereby available to those of skill in the ail. See, for example, Alberts, B. et al., “Molecular Biology of the Cell,” 5^th édition, Garland Science, New York, NY, 2008; Voet, D. et al. “Fundamentals of Biochemistry: Life at the Molecular Level,” 3^ édition, John Wiley & Sons, Hoboken, NJ, 2008; Sambrook, J. et al., “Molecular Cloning: A Laboratory Manual,” 3^rd édition, Cold Spring Harbor Laboratory Press, 2001 ; Ausubel, F. et al., “Current Protocols in Molecular Biology,” John Wiley & Sons, New York, 1987 and periodic updates; Freshney, R.I., “Culture of Animal Cells: A Manual of Basic Technique,” 4^Ü1 édition, John Wiley & Sons, Somerset, NJ, 2000; and the sériés “Methods in Enzymology,” Academie Press, San Diego, CA.

[0026] See also, for example, “Current Protocols in Immunology,” (R. Coico, sériés editor), Wiley, last updated August 2010.

MMP9 Binding Proteins [0027] The présent disclosure provides binding proteins, e.g., antibodies and antigenbinding fragments thereof, that bind to the matrix metaIloproteinase-9 (MMP9) protein (MMP9 is also known as gelatinase-B), The binding proteins of the présent disclosure generally comprise an immunoglobulin (Ig) heavy chain (or functional fragment thereof) and an Ig light chain (or fùnctional fragment thereof).

[0028] The disclosure further provides MMP9 binding proteins that bind specifically to

MMP9 and not to other matrix metalloproteinases such as MMPI, MMP2, MMP3, MMP7, MMP9, MMP 10, MMP 12, MMPI3. Such spécifie MMP9 binding proteins are thus generally not significantly or detectably crossreactive with non-MMP9 matrix metalloproteinases. MMP9 binding proteins that specifically bind MMP9 find use in applications in which it is necessary or désirable to obtain spécifie modulation (e.g., inhibition) of MMP9, e.g., without directly affecting the activity of other matrix metalloproteinases.

[0029] In certain embodiments of the présent disclosure an anti-MMP9 antibody is an inhibitor of the activity of MMP9, and can be a spécifie inhibitor of MMP9. In particular, the MMP9 binding proteins disclosed herein will be useful for inhibition of MMP9 while allowing 20 normal function of other, related matrix metalloproteinases. An inhibitor of MMP” or “inhibitor of MMP9 activity can be an antibody or an antigen binding fragment thereof that directly or indirectly inhibits activity of MMP9, including but not limited to enzymatic processing, inhibiting action of MMP9 on it substrate (e.g., by inhibiting substrate binding, substrate cleavage, and the like), and the like.

[0030] The présent disclosure also provides MMP9 binding proteins that specifically bind to non-mouse MMP9, such as human MMP9, Cynomolgus monkey MMP9, and rat MMP9. [0031 ] The présent disclosure also provides MMP9 binding proteins (e.g., antî-MMP9 antibodies and functional fragments thereof) that act as non-competitive inhibitors. A “noncompetitive inhibitor” refers to an inhibitor binds at site away from substrate binding site of an enzyme, and thus can bind the enzyme and effect inhibitory activity regardless of whether or not the enzyme is bound to its substrate, Such non-competitive inhibitors can, for example, provide for a level of inhibition that can be substantially independent of substrate concentration.

[0032] MMP9 binding proteins (e.g., antibodies and functional fragments thereof) of the présent disclosure include those that bind MMP9, particularly human MMP9, and having a heavy chain polypeptide (or functional fragment thereof) that has at least about 80%, 85%, 90%, 95% or more amino acid sequence identity to a heavy chain polypeptide disclosed herein. [0033] MMP9 binding proteins (e.g., antibodies and functional fragments thereof) of the present disclosure include those that bind MMP9, particularly human MMP9, and having a light polypeptide (or functional fragment thereof) that has at least about 80%, 85%, 90%, 95% or more amino acid sequence identity to a heavy chain polypeptide disclosed herein.

[0034] MMP9 binding proteins (e.g., antibodies and functional fragments thereof) of the present disclosure include those that bind MMP9, particularly human MMP9, and hâve a heavy chain polypeptide (or functional fragment thereof) having the complementarity determining régions (“CDRs”) of heavy chain polypeptide and the CDRs of a light chain polypeptide (or functional fragment thereof) as disclosed herein.

10035] “Homology” or “identity” or “similarity” as used herein in the context of nucleic acids and polypeptides refers to the relationship between two polypeptides or two nucleic acid molécules based on an alignment of the amino acid sequences or nucleic acid sequences, respectively. Homology and identity can each be determined by comparing a position in each sequence which may be aligned for purposes of comparison. When an équivalent position in the compared sequences is occupied by the same base or amino acid, then the molécules are identical at that position; when the équivalent site occupied by the same or a similar amino acid residue (e.g., similar in steric and/or electronic nature), then the molécules can be referred to as homologous (similar) at that position. Expression as a percentage of homology/similarity or identity refers to a function of the number of identical or similar amino acids at positions shared by the compared sequences. In comparing two sequences, the absence of residues (amino acids or nucleic acids) or presence of extra residues also decreases the identity and homology/similarity.

[0036] As used herein, “identity” means the percentage of identical nucléotide or amino acid residues at corresponding positions in two or more sequences when the sequences are aligned to maximize sequence matching, i.e., taking into account gaps and insertions. Sequences are generally aligned for maximum correspondence over a designated région, e.g., a région at least about 20, 25, 30, 35, 40, 45, 50, 55, 60, 65 or more amino acids or nucléotides in length, and can be up to the full-length of the reference amino acid or nucléotide. For sequence comparison, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are input into a computer program, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. The sequence comparison algorithm then calculâtes the percent sequence identity for the test sequence(s) relative to the reference sequence, based on the designated program parameters.

[0037] Examples of algorithms that are suitable for determining percent sequence identity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al. (1990) J. Mol. Biol. 215: 403-410 and Altschul et al. (1977) Nucleic Acids Res. 25: 3389-3402, respectively. Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov). Further exemplary algorithms include ClustalW (Higgins D., et al. (1994) Nucleic Acids Res 22: 4673-4680), available at www.ebi.ac.uk/Tools/clustalw/index.html.

[0038] Residue positions which are not identical can differ by conservative amino acid substitutions. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatichydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and méthionine.

[0039] Sequence identity between two nucleic acids can also be described in terms of hybridization of two molécules to each other under stringent conditions. The hybridization conditions are selected following standard methods in the art (see, for example, Sambrook, et al., Molecular Cloning: A Laboratory Manual, Second Edition, (1989) Cold Spring Harbor, N.Y.). An example of stringent hybridization conditions is hybridization at 50°C or higher and 0.1 x SSC (15 mM sodium chloride/1.5 mM sodium citrate). Another example of stringent hybridization conditions is ovemight incubation at 42 °C in a solution: 50 % fomiamide, 5 x SSC (150 mM NaCl, 15 mM trisodium citrate), 50 mM sodium phosphate (pH7.6), 5 x Denhardt’s solution, 10% dextran sulfate, and 20 mg/ml denatured, sheared salmon spenn DNA, followed by washing the filters in 0.1 x SSC at about 65 ^UC. Stringent hybridization conditions are hybridization conditions that are at least as stringent as the above représentative conditions, where conditions are considered to be at least as stringent if they are at least about 80% as stringent, typically at least 90% as stringent as the above spécifie stringent conditions.

[0040] Accordingly, the présent disclosure provides, for example, antibodies or antigen binding fragments thereof, comprising a heavy chain variable région polypeptide having at least 80%, 85%, 90%, 95%, or greater amino acid sequence identity to an amino acid sequence of a heavy chain variable région described herein (e.g., SEQ ID NOS:1 or 5-8), and a variable light chain polypeptide having at least 80%, 85%, 90%, 95%, or greater amino acid sequence identity to an amino acid sequence of a light chain polypeptide as set forth herein (e.g., SEQ ID NOS:2 or 9-12).

[0041] Examples of anti-MMP9 antibodies of the présent dîsclosure are described in more detail below.

Antibodies [0042] MMP9 binding proteins including antibodies and functional fragments thereof.

As used herein, the term antibody means an isolated or recombi'nant polypeptide binding agent 10 that comprises peptide sequences (e.g., variable région sequences) that specîfically bind an antigenic epitope. The term is used in its broadest sense and specîfically covers monoclonal antibodies (including full-length monoclonal antibodies), polyclonal antibodies, human antibodies, humanized antibodies, chimeric antibodies, nanobodies, diabodies, multispecifîc antibodies (e.g., bispecific antibodies), and antibody fragments including but not limited to Fv, 15 scFv, Fab, Fab¹ F(ab')2 and Fab2, so long as they exhibit the desired biologîcal activity. The tenu “human antibody” refers to antibodies containing sequences of human origin, except for possible non-hunian CDR régions, and does not imply that the full structure of an immunoglobulin molécule be présent, only that the antibody has minimal immunogenic effect in a human (i.e., does not induce the production of antibodies to itself).

[0043] An antibody fragment comprises a portion of a full-length antibody, for example, the antigen binding or variable région of a full-length antibody. Such antibody fragments may also be referred to herein as “functional fragments: or “antigen-binding fragments”. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies (Zapata et al. (1995) Protein Eng. 8(10):1057-1062); single-chain antibody molécules; and multispecifîc antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called Fab fragments, each with a single antigen-binding site, and a residual Fc fragment, a désignation reflecting the ability to crystallîze readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen combining sites and is still capable of cross-linking antigen.

[0044] Fv is the minimum antibody fragment which contains a complété antigenrecognition and -binding site. This région consîsts of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. It is in this configuration that the three complementarity-determining régions (CDRs) of each variable domain interact to define an antigen-binding site on the surface of the V_H-V_L dimer. Collectively, the six CDRs confer antigen-binding specificity to the antibody. However, even a single variable domain (or an isoiated Vh or Vl région comprising only three of the six CDRs spécifie for an antigen) has the ability to recognize and bind antigen, although generally at a lower affinity than does the entire F_v fragment.

[0045] The “F_ab” fragment also contains, in addition to heavy and light chain variable régions, the constant domain of the light chain and the first constant domain (CHj) of the heavy chain. Fab fragments were originally observed following papain digestion of an antibody. Fab' fragments differ from Fab fragments in that F(ab') fragments contain several additional residues at the carboxy terminus of the heavy chain CH] domain, including one or more cysteines from the antibody hinge région. F(ab')2 fragments contain two Fab fragments joined, near the hinge 10 région, by disulfide bonds, and were originally observed following pepsin digestion of an antibody. Fab'-SH is the désignation herein for Fab' fragments in which the cysteine residue(s) of the constant domains bear a free thiol group. Other chemical couplings of antibody fragments are also known.

[0046] The light chains of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa and lambda, based on the amino acid sequences of their constant domains. Depending on the amino acid sequence of the constant domain of their heavy chains, immunoglobulins can be assigned to five major classes: IgA, IgD, IgE, IgG, and IgM, and several of these may be further divided into subclasses (isotypes), e.g., IgGl, IgG2, IgG3, lgG4, IgAl, and IgA2.

[0047] Single-chaîn Fv or sFv or scFv antibody fragments comprise the V_H and V_L domains of antibody, wherein these domains arc présent in a single polypeptide chain. Ln some embodiments, the Fv polypeptide further comprises a polypeptide linker between the V_H and V_L domains, which enables the sFv to form the desired structure for antigen binding. For a review of sFv, see Pluckthun, in The Pharmacology of Monoclonal Antibodies, vol. 113 (Rosenburg and 25 Moore eds.) Springer-Verlag, New York, pp. 269-315 (1994).

[0048] The term diabodies refers to small antibody fragments with two antigenbinding sites, which fragments comprise a heavy-chain variable domain (Vu) connected to a light-chain variable domain (V_L) in the same polypeptide chain (Vh-V_l), By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced 30 to pair with the complementary domains of another chain, thereby creatîng two antigen-binding sites. Diabodies are additionally described, for example, in EP 404,097; WO 93/11161 and Hollinger et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448.

[0049] An isoiated antibody is one that has been identified and separated and/or recovered from a component of its natural environment. Components of its natural environment 35 may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutés. In some

I

B embodiments, an isolated antibody is purified (l) to greater than 95% by weight of antibody as determined by the Lowry method, for example, more than 99% by weight, (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internai amino acid sequence, e.g., by use of a spinning cup sequenator, or (3) to homogeneity by gel electrophoresis (e.g., SDSPAGE) under reducing or nonreducing conditions, with détection by Coomassie blue or silver stain. The term isolated antibody includes an antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be présent. In certain embodiments, isolated antibody is prepared by at least one purification step.

[0050] As used herein, “immunoreactive” refers to antibodies or fragments thereof that are spécifie to a sequence of amino acid residues (“binding site” or “epitope”), yet if are crossreactive to other peptides/proteins, are not toxic at the levels at which they are formulated for administration to human use. Epitope refers to that portion of an antigen capable of forming a binding interaction with an antibody or antigen binding fragment thereof. An epitope can be a linear peptide sequence (i.e., “continuons”) or can be composed of noncontiguous amino acid sequences (i.e., “conformationai” or “discontinuons”). The term “preferentially binds” means that the binding agent binds to the binding site with greater affinity than it binds unrelated amino acid sequences.

[0051] Anti-MMP9 antibodies can be described in terms of the CDRs of the heavy and light chains. As used herein, the term CDR or complementarity determining région is intended to mean the non-contiguous antigen combining sites found within the variable région of both heavy and light chain polypeptides. These particular régions hâve been described by Rabat et al., J. Biol. Chem. 252:6609-6616 (1977); Rabat et al., U.S. Dept. ofHealth and Human Services, Sequences of proteins of immunological interest (1991); by Chothia et al., J. Mol. Biol. 196:901-917 (1987); and MacCallum et al., J. Mol. Biol. 262:732-745 (1996), where the définitions include overlapping or subsets of amino acid residues when compared against each other. Nevertheless, application of either définition to refer to a CDR of an antibody or grafted antibodies or variants thereof is intended to be within the scope of the tenu as defined and used herein. The amino acid residues which encompass the CDRs as defined by each of the above cited references are set forth below in Table 1 as a comparison.

Table 1: CDR Définitions

Knbat¹ Chotliia² MacCailum³

VH CDR1	31-35	26-32	30-35
VH CDR2	50-65	53-55	47-58
VH CDR3	95-102	96-101	93-101
VL CDR1	24-34	26-32	30-36
VL CDR2	50-56	50-52	46-55
VL CDR3	89-97	91-96	89-96

¹ Residue numbering follows the nomenclature of Kabat et al., supra ²Residue numbering follows the nomenclature of Chothia et al., supra ³Residue numbering follows the nomenclature of MacCallum et al., supra [0052] As used herein, the term framework when used in reference to an antibody variable région is intended to mean ail amino acid residues outside the CDR régions within the variable région of an antibody. A variable région framework is generally a discontinuous amino acid sequence between about 100-120 amino acids in length but is intended to reference only those amino acids outside of the CDRs. As used herein, the term framework région is intended to mean each domain of the framework that is separated by the CDRs.

10053] ln some embodiments, an antibody is a humanized antibody or a human antibody.

Humanized antibodies include human immununoglobulins (récipient antibody) in which residues from a complementary-determining région (CDR) of the récipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity and capacity. Thus, humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins which contain minimal sequence derived from non-human immunoglobulin. The non-human sequences are located primarily in the variable régions, particularly in the complementarity-determining régions (CDRs). In some embodiments, Fv framework residues of the human immunoglobulin aie replaced by corresponding non-human residues. Humanized antibodies can also comprise residues that are found neither in the récipient antibody nor in the imported CDR or framework sequences. ln certain embodiments, a humanized antibody comprises substantially ail of at least one, and typically two, variable domains, in which ail or substantially ail of the CDRs correspond to those of a non-human immunoglobulin and ail or substantially ail of the framework régions are those of a human immunoglobulin consensus sequence. For the purposes of the présent disclosure, humanized antibodies can also include immunoglobulin fragments, such as Fv, Fab, Fab', F(ab')₂ or other antigen-binding subsequences of antibodies.

[0054] The humanized antibody can also comprise at least a portion of an immunoglobulin constant région (Fc), typically that of a human immunoglobulin. See, for r

t example, Jones et al. (1986) Nature 321:522-525; Riechmann et al. (1988) Nature 332:323329; and Presta (1992) Curr. Op. Struct. Biol. 2:593-596.

[0055] Methods for humanizing non-human antibodies are known in the art. Generally, a humanized antibody has one or more amino acid resîdues introduced into it from a source that is non-human. These non-human amino acid resîdues are often referred to as import or “donor” resîdues, which are typically obtained from an import or “donor” variable domain. For example, humanization can be performed essentially according to the method of Winter and co-workers , by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. See, for example, Jones et al., supra', Riechmann et al., supra and ' Verhoeyen et al. (1988) Science 239:1534-1536. Accordingly, such humanized antibodies include chimeric antibodies (U.S. Patent No. 4,816,567), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a nonhuman species. In certain embodiments, humanized antibodies are human antibodies in which some CDR resîdues and optionally some framework région resîdues are substituted by resîdues from analogous sites in rodent antibodies (e.g., murine monoclonal antibodies).

[0056] Human antibodies can also be produced, for example, by using phage display libraries. Hoogenboom et al. (1991) J. Mol. Biol, 227:381; Marks et al. (1991) J. Mol. Biol. 222:581. Other methods for preparing human monoclonal antibodies are described by Cole et al. (1985) “Monoclonal Antibodies and Cancer Therapy,” Alan R. Liss, p. 77 and Boemer et al. (\99\) J. Immunol. 147:86-95.

[0057J Human antibodies can be made by introducing human immunoglobulin loci into transgenic animais (e.g., mice) in which the endogenous immunoglobulin genes hâve been partially or completely inactivated. Upon immunological challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody répertoire. This approach is described, for example, in U.S. Patent Nos. 5,545,807; 5,545,806; 5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the following scientific publications: Marks et al. (1992) Bio/Technology 10:779-783 (1992); Lonberg et al. (1994) Nature 368: 856-859; Morrison (1994) Nature 368:812-813; Fishwald et al. (1996) Nature Biotechnology 14:845-851; Neuberger (1996) Nature Biotechnology 14:826; and Lonberg et al. (1995) Intern. Rev. Immunol. 13:65-93.

[0058] Antibodies can be affinity matured using known sélection and/or mutagenesis methods as described above. In some embodiments, affinity matured antibodies hâve an affinity which is five times or more, ten times or more, twenty times or more, or thirty times or more than that of the starting antibody (generally murine, rabbit, chicken, humanized or human) from which the matured antibody is prepared.

|0059] An antibody can also be a bispecifîc antibody. Bispecific antibodies are monoclonal, and may be human or humanized antibodies that hâve binding specificities for at least two different antigens. In the présent case, the two different binding specificities can be directed to two different MMPs, or to two different epitopes on a single MMP (e.g., MMP9).

[0060] An antibody as disclosed herein can also be an immunoconjugate. Such immunoconjugates comprise an antibody (e.g., to MMP9) conjugated to a second molécule, such as a reporter An immunoconjugate can also comprise an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, a toxin (e.g., an enzymatically active toxîn of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a fO radioconjugate).

[0061] An antibody that specifically binds to or is spécifie for a particular polypeptide or an epitope on a particular polypeptide is one that binds to that particular polypeptide or epitope without substantially binding to any other polypeptide or polypeptide epitope. ln some embodiments, an antibody of the présent disclosure specifically binds to human MMP9 with a dissociation constant (I<d) equal to or lower than 100 nM, optionally lower than 10 nM, optionally lower than 1 nM, optionally lower than 0.5 nM, optionally lower than 0.1 nM, optionally lower than 0.01 nM, or optionally lower than 0.005 nM; in the form of monoclonal antibody, scFv, Fab, or other form of antibody measured at a température of about 4°C, 25°C, 37°C or 42°C.

10062] ln certain embodiments, an antibody of the présent disclosure binds to one or more processing sites (e.g., sites of proteolytic cleavage) in MMP9, thereby effectively blocking processing of the proenzyme or preproenzyme to the catalytically active enzyme, and thus reducing the proteolytic activity ofthe MMP9.

[0063] In certain embodiments, an antibody according to the présent disclosure binds to

MMP9 with an affinity at least 2 times, at least 5 times, at least 10 times, at least 25 times, at least 50 finies, at least 100 times, at least 500 times, or at least 1000 times greater than its binding affinity for another MMP. Binding affinity can be measured by any method known in the art and can be expressed as, for example, on-rate, off-rate, dissociation constant (KJ, equilibrium constant (K«,) or any term in the art.

10064] In certain embodiments, an antibody according to the présent disclosure is a noncompetitive inhibitor of the catalytic activity of MMP9. ln certain embodiments, an antibody according to the présent disclosure binds within the catalytic domain of MMP9. In additional embodiments, an antibody according to the présent disclosure binds outside the catalytic domain ofMMP9.

[0065] The présent disclosure also contemplâtes antibodies or antigen binding fragments thereof, that compete with anti-MMP9 antibodies or antigen binding fragments thereof described herein for binding to MMP9. Thus, the présent disclosure contemplâtes anti-MMP9 antibodies, and functional fragments thereof, that compete for binding with, for example, an antibody having a heavy chain polypeptide of any ofSEQ ID NOS;1 or 5-8, a light chain polypeptide of SEQ ID NOS:2 or 9-12, or combinations thereof. In one embodiment, the anti-MMP9 antibody, for functional fragment thereof, competes for binding to human MMP9 with the antibody described herein as AB0041.

MMP9 sequence [0066]

The amino acid sequence of human MMP9 protein is as follows:

MSLWQPLVLV LLVLGCCFAA PRQRQSTLVL FPGDLRTNLT DRQLAEEYLY 50

RYGYTRVAEM RGESKSLGPA LLLLQKQLSL PETGELDSAT LKAMRTPRCG 100

VPDLGRFQTF EGDLKWHHHN ITYWIQNYSE DLPRAVIDDA FARAFALWSA 150

VTPLTFTRVY SRDADIVIQF GVAEHGDGYP FDGKDGLLAH AFPPGPGIQG 200 DAHFDDDELW SLGKGVWPT RFGNADGAAC HFPFIFEGRS YSACTTDGRS 250

DGLPWCSTTA NYDTDDRFGF CPSERLYTRD GNADGKPCQF PFIFQGQSYS 300

ACTTDGRSDG YRWCATTANY DRDKLFGFCP TRADSTVMGG NSAGELCVFP 350

FTFLGKEYST CTSEGRGDGR LWCATTSNFD SDKKWGFCPD QGYSLFLVAA 400

HEFGHALGLD HSSVPEALMY PMYRFTEGPP LHKDDVNGIR HLYGPRPEPE 450 PRPPTTTTPQ PTAPPTVCPT GPPTVHPSER PTAGPTGPPS AGPTGPPTAG 500 PSTATTVPLS PVDDACNVNI FDAIAEIGNQ LYLFKDGKYW RFSEGRGSRP 550 QGPFLIADKW PALPRKLDSV FEEPLSKKLF FFSGRQVWVY TGASVLGPRR 600 LDKLGLGADV AQVTGALRSG RGKMLLFSGR RLWRFDVKAQ MVDPRSASEV 650

DRMFPGVPLD THDVFQYREK AYFCQDRFYW RVSSRSELNQ VDQVGYVTYD 700 ILQCPED (SEQ ID NO:27) |0067j Protein domains are shown schematically in Figure 3 and are indicated below:

	Amino Acid #	Feature
30	1-19	Signal Peptide
	38-98	Peptidoglycan Binding Domain
	R98/C99	Propetide cleavage site (dépendent on cleavage enzyme)
	112-445	Zn dépendent metalloproteinase domain
	223-271	Fibronectin type II domain (gelatin binding domain)
35	281-329	Fibronectin type 11 domain (gelatin binding domain)
	340-388	Fibronectin type 11 domain (gelatin binding domain)
	400-411	Zn binding région
	521-565	Hemopexin-lîke domain
	567-608	Hemopexin-like domain
40	613-659	Hemopexin-like domain
	661-704	Hemopexin-like domain

!

t

[0068] The amino acid sequence of mature fulklengtli human MMP9 (which is the amino acid sequence of the propolypeptide of SEQ ID NO:27 without the signal peptide) is:

PRQRQSTLVL FPGDLRTNLT DRQLAEEYLY RYGYTRVAEM RGESKSLGPA LLLLQKQLSL PETGELDSAT LKAMRTPRCG VPDLGRFQTF EGDLKWHHHN ITYWIQNYSE DLPRAVIDDA FARAFALWSA VTPLTFTRVY SRDADIVIQF GVAEHGDGYP FDGKDGLLAH AFPPGPGIQG DAHFDDDELW SLGKGVWPT RFGNADGAAC HFPFIFEGRS YSACTTDGRS DGLPWCSTTA NYDTDDRFGF CPSERLYTRD GNADGKPCQF PFIFQGQSYS ACTTDGRSDG YRWCATTANY DRDKLFGFCP TRADSTVMGG NSAGELCVFP FTFLGKEYST CTSEGRGDGR LWCATTSNFD SDKKWGFCPD QGYSLFLVAA HEFGHALGLD HSSVPEALMY PMYRFTEGPP LHKDDVNGIR HLYGPRPEPE PRPPTTTTPQ PTAPPTVCPT GPPTVHPSER PTAGPTGPPS AGPTGPPTAG PSTATTVPLS PVDDACNVNI FDAIAEIGNQ LYLFKDGKYW RFSEGRGSRP QGPFLIADKW PALPRKLDSV FEEPLSKKLF FFSGRQVWVY TGASVLGPRR LDKLGLGADV AQVTGALRSG RGKMLLFSGR RLWRFDVKAQ MVDPRSASEV DRMFPGVPLD THDVFQYREK AYFCQDRFYW RVSSRSELNQ VDQVGYVTYD ILQCPED (SEQ ID NO:28) where the amino acid sequence of the signal peptide is MSLWQPLVLV LLVLGCCFAA (SEQ ID NO:29).

[0069] The présent disclosure contemplate MMP9 binding proteins that bind any portion of MMP9, e.g., human MMP9, with MMP9 binding proteins that preferentially bind MMP9 relative to other MMPs being of particular interest.

[0070] Anti-MMP9 antibodies, and functional fragments thereof, can be generated accordingly to methods well known in the art. Examples of anti-MMP9 antibodies are provided below.

Mouse monoclonal anti-MMP9 [0071] A mouse monoclonal antibody to human MMP9 was obtained as described in

Example 1. This antibody contains a mouse IgG2b heavy chain and a mouse kappa light chain, and is denoted AB0041.

[0072] The amino acid sequence of the AB0041 heavy chain is as follows:

MAVLVLFLCLVAFPSCVLSQVOLKESGPGLVAPSOSLSITCTVSGFSLLSYGVHW

VRQPPGK.GLEWLGVIWTGGTTNYNSALMSRLSISKDDSKSQVFLKMNSLQTDDTAIYY CARNYYGMOVWGQGYSVYNSSAKTTPPSWPLAPGCGDTTGSSVTLGCLVKGYFPESVTV TWNSGSLSSSVHTFPALLOSGLYTMSSSVTVPSSTWPSQTVTCSVAHPASSTTVDKKLEPSGPJS TINPCPPCKECHKCPAPNLEGGPSVFIFPPMKD VLMISL TPK VTCVVVD VSEDDPD VR1SWF VNNVEVHTAOTOTHREDYNST1R VVSALPIOHQD WMSGKEFKCKVNNKDLPSPIERTISKIKG

L VRAPQVYILPPPAEQLSRKD VSLTCL WGFNPGD1SVEWTSNGHTEENYKDTAPVLDSDGSY FIYSKLDIKTSKWEKTDSFSCNERHEGLKNYYLKKT1SRSPGK (SEQ ID NO:l) [0073] The signal sequence is underlined, and the sequence of the IgG2b constant région is presented italics.

[0074] The amino acid sequence of theAB004l light chain is as follows:

MESQIQVFVFVFLWLSGVDGDIVMTOSHKFMSTSVGDRVSITCKASODVRNTVA

WYQQKTGQSPKLLIYSSSYRNTGVPDRFTGSGSGTDFTFTISSVQAEDLAVYFCQQFIYIT PYTFGGGYKLEIKJIADAAPTPSIFPPSSEOLTSGGASWCFLNNFYPKDINVKWKIDGSERQN GVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIFKSFNRNEC (SEQ ID

NO:2) [0075] The signal sequence is underlined, and the sequence of the kappa constant région is presented in italics.

[0076] The following amino acid sequence comprises the framework régions and complementarity-determining régions (CDRs) of the variable région of the IgG2b heavy chain of 15 AB0041 (with CDRs underlined):

OVQLK.ESGPGLVAPSOSLSITCTVSGFSLLSYGVHWVRQPPGKGLEWLGV1WTGGTTN

YNSALMSRLSISKDDSKSOVFLKMNSLQTDDTAIYYCARYYYGMDYWGOGTSVTVSS (SEQ ID NO:3)

10077] The following amino acid sequence comprises the framework régions and complementarity-determining régions (CDRs) of the variable région of the kappa light chain of

AB0041 (with CDRs underlined):

DIVMTOSHKFMSTSVGDRVSITCKASODVRNTVAWYOQKTGQSPKLLIYSSSYRNTGV

PDRFTGSGSGTDFTFTISSVOAEDLAVYFCOQHYITPYTFGGGTKLEIK (SEQ ID NO:4)

Heavy-chain variants [0078] The amino acid sequences of the variable régions of the AB0041 heavy and light chains were separately modified, by altering framework région sequences in the heavy and light chain variable régions. The effect of these sequence alterations was to deplete the antibody of human T-cell epitopes, thereby reducing or abolishing its immunogenicity in humans (Antitope, 30 B abraham, UK).

|0079] Four heavy-chain variants were constructed, in a human IgG4 heavy chain background containîng a S241P amino acid change that stabilizes the hinge domain (Angal et al.

(1993) Molec. Immunol. 30:105-108), and are denoted VH1, VH2, VH3 and VH4. The amino acid sequences of their framework régions and CDRs are as follows:

VH1

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTG GTTNYNSALMSRLT1SKDDSKSTVYLKMNSLKTEDTAIYYCARYYYGMDYWGQGTSV TVSS (SEQ ID NO:5)

VH2

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTG

GTTNYNSALMSRLTISKDDSK.NTVYLKMNSLKTEDTAIYYCARYYYGMDYWGQGTLV

TVSS (SEQ ID N0:6) ' VH3

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTG

GTTNYNSALMSRFTISKDDSKNTVYLKMNSLKTEDTAIYYCARYYYGMDYWGQGTLV TVSS (SEQ ID NO:7)

VH4

QVQLQESGPGLVKPSETLSLTCTVSGFSLLSYGVHWVRQPPGKGLEWLGVIWTG

GTTNYNSALMSRFTISKDDSKNTLYLKMNSLKTEDTAIYYCARYYYGMDYWGQGTLV

TVSS (SEQ ID NO:8) [0080] Figure 1 shows an alignaient ofthe amino acid sequences ofthe variable régions of the humanized heavy chains and indicates the différences in amino acid sequences in the framework régions among the four variants.

Light-chain variants [0081] Four light-chain variants were constructed, in a human kappa chain background, and are denoted Vkl, Vk2, Vk3 and Vk4. The amino acid sequences of their framework régions and CDRs are as follows:

Vkl

DIVMTQSPSFLSASVGDRVTITCKASQDVRNTVAWYQQKTGKAPKLLIYSSSYR NTGVPDRFTGSGSGTDFTLT1SSLQAEDVAVYFCQQHYITPYTFGGGTKVE1K (SEQ ID NO:9)

Vk2

DIVMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSYR

NTGVPDRFTGSGSGTDFTLTISSLQAEDVAVYFCQQHYITPYTFGGGTKVEIK (SEQ ID NO: 10)

Vk3 DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSYR NTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYFCQQHYITPYTFGGGTKVEIK (SEQ ID NO:ll)

Vk4

DIQMTQSPSSLSASVGDRVTITCKASQDVRNTVAWYQQKPGKAPKLLIYSSSYR NTGVPDRFSGSGSGTDFTLTISSLQAEDVAVYYCQQHYITPYTFGGGTKVEIK (SEQ ID N0:12) [0082] Figure 2 shows an alignment ofthe amino acid sequences of the variable régions of the humanized light chains and indicates the différences in amino acid sequences in the framework régions among the four variants.

[0083] The humanized heavy and light chains are combined in ali possible pair-wise combinations to generate a number of functional humanized anti-MMP9 antibodies.

[0084] Additional heavy chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology to the heavy chain variable région sequences disclosed herein are also provided. Furthermore, additional light chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more homology to the light chain variable région sequences disclosed herein are also provided.

|0085] Additional heavy chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more sequence identity to the heavy chain variable région sequences disclosed herein are also provided. Furthermore, additional light chain variable région amino acid sequences having 75% or more, 80% or more, 90% or more, 95% or more, or 99% or more sequence identity to the light chain variable région sequences disclosed herein are also provided.

Complenientarity-deterniining régions (CDRs) [0086] The CDRs of the heavy chain of an anti-MMP9 antibody as disclosed herein hâve the following amino acid sequences:

CDRl : GFSLLSYGVH (SEQ ID NO:13)

CDR2: VIWTGGTTNYNSALMS (SEQ ID NO:l4)

CDR3:YYYGMDY (SEQIDNO:l5) [0087J The CDRs ofthe light chain of an anti-MMP9 antibody as disclosed herein hâve the following amino acid sequences:

CDRl : KASQDVRNTVA (SEQ ID NO: 16)

CDR2: SSSYRNT (SEQ ID NO:l7)

CDR3: QQHYITPYT (SEQ ID NO:l8)

Nucieic acids encoding anti-MMP9 antibodies [0088] The présent disclosure provides nucieic acids encoding antî-MMP9 antibodies and functional fragments thereof. Accordingly, the présent disclosure provides an isolated polynucleotide (nucieic acid) encoding an antibody or antigen-binding fragment as described herein, vectors contaîning such polynucleotides, and host cells and expression Systems for transcribing and translating such polynucleotides into polypeptides.

[0089] The présent disclosure also contemplâtes constructs in the form of plasmids, vectors, transcription or expression cassettes which comprise at least one polynucleotide as above.

[0090] The présent disclosure also provides a recombinant host cell which comprises one or more constructs as above, as well as methods of production of the antibody or antigen-binding fragments thereof described herein which method comprises expression of nucieic acid encoding a heavy chain polypeptide and a light chain polypeptide (in the same or different host cells, and from the same or different constructs) in a recombination host cell. Expression can be achieved by culturing under appropriate conditions recombinant host cells contaîning the nucieic acid. Following production by expression, an antibody or antigen-binding fragment can be isolated and/or purified using any suitable technique, then used as appropriate.

100911 Systems for cloning and expression of a polypeptide in a variety of different host cells are well known. Suitable host cells include bacteria, mammalian cells, yeast and baculovirus Systems. Mammalian cell lines available in the art for expression of a heterologous polypeptide include Chinese hamster ovary cells, HeLa cells, baby hamster kidney cells, NSO mouse melanoma cells and many others. A common bacterial host is E. coli.

[0092] Suitable vectors can be chosen or constructed, contaîning appropriate regulatory sequences, including operably linked promoter sequences, terminator sequences, polyadenylation sequences, enhancer sequences, marker genes and/or other sequences as appropriate. Vectors can be plasmids, viral e.g. ‘phage, or phagemid, as appropriate. For further details see, for example, Molecular Cloning; a Laboratory Manual: 2nd édition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press. Many known techniques and protocols for manipulation of nucieic acid, for example in préparation of nucieic acid constructs, mutagenesis,

I

I sequencing, introduction of DNA into cells and gene expression, and analysis of proteins, are described in detail in Short Protocols in Molecular Biology, Second Edition, Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures of Sambrook et al. and Ausubel et al. are incorporated herein by reference in their entirety.

[0093] The nucleic acid encoding a polypeptide of interest is integrated into the genome of the host cell or can be maintained as a stable or transient episomal element.

[0094] Any of a wide variety of expression control sequences - sequences that control the expression of a DNA sequence operatively linked to it - can be used in these vectors to express the DNA sequences. For example, a nucleic acid encoding a polypeptide of interest can be operably linked to a promoter, and provided in an expression construct for use in methods of production of recombinant MMP9 proteins or portions thereof.

[0095] Those of skill in the art are aware that nucleic acids encoding the antibody chains disclosed herein can be synthesized using standard knowledge and procedures in molecular biology.

[0096] Examples of nucléotide sequences encoding the heavy and light chain amino acid sequences disclosed herein, are as follows:

VH1: CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGC CCTCCGAGAC

ACTGTCCCTG ACCTGCACCG TGTCCGGCTT CTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCT CCAGGGAAGG GCCTGGAATG GCTGGGCGTG ATCTGGACCG GCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGCT GACCATCTCC AAGGACGACT CCAAGTCCAC CGTGTACCTG AAGATGAACT CCCTGAAAAC CGAGGACACC GCCATCTACT ACTGCGCCCG GTACTACTAC GGCATGGACT ACTGGGGCCA GGGCACCTCC GTGACCGTGT CCTCA (SEQ ID NO: 19) VH2: CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGC CCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTT CTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCT CCAGGCAAAG GCCTGGAATG GCTGGGCGTG ATCTGGACCG GCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGCT GACCATCTCC AAGGACGACT CCAAGAACAC CGTGTACCTG AAGATGAACT CCCTGAAAAC CGAGGACACC GCCATCTACT ACTGCGCCCG GTACTACTAC GGCATGGACT ACTGGGGCCA GGGCACCCTG GTCACCGTGT CCTCA (SEQ ID NO:20) VH3: CAGGTGCAGC TGCAGGAATC CGGCCCTGGC CTGGTCAAGC CCTCCGAGAC ACTGTCCCTG ACCTGCACCG TGTCCGGCTT CTCCCTGCTG TCCTACGGCG TGCACTGGGT CCGACAGCCT CCAGGCAAAG GCCTGGAATG GCTGGGCGTG ATCTGGACCG GCGGCACCAC CAACTACAAC TCCGCCCTGA TGTCCCGGTT CACCATCTCC AAGGACGACT CCAAGAACAC CGTGTACCTG AAGATGAACT

I

	CCCTGAAAAC GGCATGGACT VH4: CCTCCGAGAC	CGAGGACACC GCCATCTACT ACTGCGCCCG ACTGGGGCCA GGGCACCCTG GTCACCGTGT	GTACTACTAC CCTCA (SEQIDNO:21) CTGGTCAAGC CTCCCTGCTG
CAGGTGCAGC TGCAGGAATC ACTGTCCCTG ACCTGCACCG	CGGCCCTGGC TGTCCGGCTT
5	TCCTACGGCG	TGCACTGGGT	CCGACAGCCT	CCAGGCAAAG	GCCTGGAATG
	GCTGGGCGTG	ATCTGGACCG	GCGGCACCAC	CAACTACAAC	TCCGCCCTGA
	TGTCCCGGTT	CACCATCTCC	AAGGACGACT	CCAAGAACAC	CCTGTACCTG
	AAGATGAACT	CCCTGAAAAC	CGAGGACACC	GCCATCTACT	ACTGCGCCCG
	GTACTACTAC	GGCATGGACT	ACTGGGGCCA	GGGCACCCTG	GTCACCGTGT	CCTCA (SEQ
10	ID NO:22)
	Vkl:	GACATCGTGA	TGACCCAGTC	CCCCAGCTTC	CTGTCCGCCT
	CCGTGGGCGA	CAGAGTGACC	ATCACATGCA	AGGCCTCTCA	GGACGTGCGG
	AACACCGTGG	CCTGGTATCA	GCAGAAAACC	GGCAAGGCCC	CCAAGCTGCT
	GATCTACTCC	TCCTCCTACC	GGAACACCGG	CGTGCCCGAC	CGGTTTACCG
15	GCTCTGGCTC	CGGCACCGAC	TTTACCCTGA	CCATCAGCTC	CCTGCAGGCC
	GAGGACGTGG	CCGTGTACTT	CTGCCAGCAG	CACTACATCA	CCCCCTACAC
	CTTCGGCGGA	GGCACCAAGG	TGGAAATAAA	A (SEQ ID NO:23)
	Vk2:	GACATCGTGA	TGACCCAGTC	CCCCTCCAGC	CTGTCCGCCT	CTGTGGGCGA
	CAGAGTGACC	ATCACATGCA	AGGCCTCTCA	GGACGTGCGG	AACACCGTGG
20	CCTGGTATCA	GCAGAAGCCC	GGCAAGGCCC	CCAAGCTGCT	GATCTACTCC
	TCCTCCTACC	GGAACACCGG	CGTGCCCGAC	CGGTTTACCG	GCTCTGGCTC
	CGGCACCGAC	TTTACCCTGA	CCATCAGCTC	CCTGCAGGCC	GAGGACGTGG
	CCGTGTACTT	CTGCCAGCAG	CACTACATCA	CCCCCTACAC	CTTCGGCGGA
	GGCACCAAGG	TGGAAATAAA	A (SEQ ID NO:24)
25	Vk3: GACATCCAGA TGACCCAGTC CCCCTCCAGC CTGTCCGCCT CTGTGGGCGA
	CAGAGTGACC	ATCACATGCA	AGGCCTCCCA	GGACGTGCGG	AACACCGTGG
	CCTGGTATCA	GCAGAAGCCC	GGCAAGGCCC	CCAAGCTGCT	GATCTACTCC
	TCCTCCTACC	GGAACACCGG	CGTGCCCGAC	CGGTTCTCTG	GCTCTGGAAG
	CGGCACCGAC	TTTACCCTGA	CCATCAGCTC	CCTGCAGGCC	GAGGACGTGG
30	CCGTGTACTT	CTGCCAGCAG	CACTACATCA	CCCCCTACAC	CTTCGGCGGA
	GGCACCAAGG	TGGAAATAAA	A (SEQ ID NO:25)
	Vk4: <	GACATCCAGA '	TGACCCAGTC	CCCCTCCAGC	CTGTCCGCCT	CTGTGGGCGA
	CAGAGTGACC	ATCACATGCA	AGGCCTCTCA	GGACGTGCGG	AACACCGTGG
	CCTGGTATCA	GCAGAAGCCC	GGCAAGGCCC	CCAAGCTGCT ♦	GATCTACTCC
35	TCCTCCTACC	GGAACACCGG	CGTGCCCGAC	CGGTTCTCTG	GCTCTGGAAG

«

CGGCACCGAC

TTTACCCTGA

CCATCAGCT.C CCTGCAGGCC GAGGACGTGG

CCGTGTACTA

CTGCCAGCAG

CACTACATCA CCCCCTACAC CTTCGGCGGA

GGCACCAAGG

TGGAAATAAA

A (SEQ IDNO:26) [0097] Because the structure of antibodies, including the juxtaposition of CDRs and framework régions in the variable région, the structure of framework régions and the structure of heavy- and light-chain constant régions, is well-known in the art; it is well within the skill of the art to obtain related nucleic acids that encode anti-MMP-9 antibodies. Accordingly, polynucleotides comprising nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% homology to any of the nucléotide sequences disclosed herein are also provided. Accordingly, polynucleotides comprising nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% and at least 99% identity to any of the nucléotide sequences disclosed herein are also provided.

Pharmaceutical Compositions [0098] MMP9 binding proteins, as well as nucleic acid (e.g., DNA or RNA) encoding

MMP9 binding proteins, can be provided as a pharmaceutical composition, e.g., combined with a pharmaceutically acceptable carrier or excipient. Such pharmaceutical compositions are useful for, for example, administration to a subject in vivo or ex vivo, and for diagnosing and/or treating a subject with the MMP9 binding proteins.

[0099| Pharmaceutically acceptable carriers are physiologically acceptable to the administered patient and retain the therapeutic properties of the antibodies or peptides with which it is administered. Pharmaceutically-acceptable carriers and their formulations are and generally described in, for example, Remington* pharmaceutical Sciences ( 18th Edition, ed. A. Gennaro, Mack Publishing Co., Easton, PA 1990). One exemplary pharmaceutical carrier is physiological saline. Each carrier is “pharmaceutically acceptable” in the sense of being compatible with the other ingrédients of the formulation and not substantially injurious to the patient.

[00100] Pharmaceutical compositions can be formulated to be compatible with a particular route of administration, systemic or local. Thus, pharmaceutical compositions include carriers, diluents, or excipients suitable for administration by various routes.

[00101] Pharmaceutical compositions can include pharmaceutically acceptable additives. Examples of additives include, but are not limited to, a sugar such as mannitol, sorbitol, glucose, xylitol, trehalose, sorbose, sucrose, galactose, dextran, dextrose, fructose, lactose and mixtures thereof. Pharmaceutically acceptable additives can be combined with pharmaceutically ι

acceptable carriers and/or excipients such as dextrose. Additives also include surfactants such as polysorbate 20 or polysorbate 80.

[00102] The formulation and delivery methods will generally be adapted according to the site and the disease to be treated. Exempiary formulations include, but are not limited to, those suitable for parentéral administration, e.g., intravenous, intra-arterial, intramuscular, or subcutaneous administration.

[00103] Pharmaceutical compositions for parentéral delivery include, for example, water, saline, phosphate buffered saline, Hank’s solution, Ringer’s solution, dextrose/saline, and glucose solutions. The formulations can contain auxiliary substances to approximate physiological conditions, such as buffcring agents, tonicity adjusting agents, wetting agents, détergents and the like, Additives can also include additional active ingrédients such as bactericidal agents, or stabilizers. For example, the solution can contain sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, sorbitan monolaurate or triethanolamine oleate. Additional parentéral formulations and methods are described in Bai (1997) J. Neuroimmunol. 80:65 75; Warren (1997) J. Neurol. Sci. 152:31 38; and Tonegawa (1997) J. Exp. Med. 186:507 515. The parentéral préparation can be enclosed in ampules, disposable syringes or multiple dose vials made of glass or plastic.

[00104] Pharmaceutical compositions for intradermal or subcutaneous administration can include a stérile diluent, such as water, saline solution, fixed oils, polyethylene glycols, glycérine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid, glutathione or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acétates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. [00105] Pharmaceutical compositions for injection include aqueous solutions (where water soluble) or dispersions and stérile powders for the extemporaneous préparation of stérile injectable solutions or dispersion. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). The carrier can be a solvent or dispersion medium containing, for example, water, éthanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof. Fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the requîred particle size in the case of dispersion and by the use of surfactants. Antibacterial and antifungal agents include, for example, parabens, chlorobutanol, phénol, ascorbic acid and thimerosal. Isotonie agents, for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride may be included in the composition. The resulting solutions can be packaged for use as is, or >

I «

lyophilized; the lyophilized préparation can later be combined with a stérile solution prior to administration.

[00106] Pharmaceutically acceptable carriers can contain a compound that stabilizes, increases or delays absorption or clearance. Such compounds include, for example, carbohydrates, such as glucose, sucrose, or dextrans; low molecular weight proteins; compositions that reduce the clearance or hydrolysis of peptides; or excipients or other stabilizers and/or buffers. Agents that delay absorption include, for example, aluminum monostearate and gelatin. Détergents can also be used to stabilize or to increase or decrease the absorption of the pharmaceutical composition, including liposomal carriers. To protect from digestion the compound can be complexed with a composition to render it résistant to acidic and enzymatic hydrolysis, or the compound can be complexed in an appropriately résistant carrier such as a liposome. Means of protecting compounds from digestion are known in the art (see, e.g., Fix (1996) Pharm Res. 13:1760 1764; Samanen (1996) J. Phann. Pharmacol. 48:119 135; and U.S. Pat. No. 5,391,377, describing lipid compositions for oral delivery of therapeutic agents).

[00107| Compositions of the présent invention can be combined with other therapeutic moieties or imaging/diagnostic moieties as provided herein. Therapeutic moieties and/or imaging moieties can be provided as a separate composition, or as a conjugated moiety présent on an MMP9 binding protein.

|00108| Formulations for in vivo administration are generally stérile. In one embodiment, the pharmaceutical compositions are formulated to be free of pyrogens such that they are acceptable for administration to human patients.

[00109] Various other pharmaceutical compositions and techniques for their préparation and use will be known to those of skill in the art in light of the présent disclosure. For a detailed listing of suitable pharmacological compositions and associated administrative techniques one can refer to the detailed teachings herein, which can be further supplemented by texts such as Remington: The Science and Practice of Pharmacy 20th Ed. (Lippincott, Williams & Wilkins 2003).

[001101 Pharmaceutical compositions can be formulated based on the physical characteristics of the patient/subject needing treatment, the route of administration, and the like. Such can be packaged in a suitable pharmaceutical package with appropriate labels for the distribution to hospitals and clinics wherein the label is for the indication of treating a disorder as described herein in a subject. Médicaments can be packaged as a single or multiple units. Instructions for the dosage and administration of the pharmaceutical compositions of the présent invention can be included with the pharmaceutical packages and kits described below.

Methods of Use [00111] The MMP9 binding proteins of the présent disclosure can be used in, for example, methods of détection of MMP9 in a sample, methods of treatment (e.g., as in methods of inhibition of angiogenesis), and methods of diagnosis. Examples of methods of use are described below.

Methods of Treatment [00112] Provided herein are methods of treating diseases and disorders associated with

MMP9 activity. Diseases and disorder include, but are not limited to tumors (e.g., primary or metastatic) that express or are disposed in a tissue which expresses MMP9.

[00113] As used herein, “treat” or “treatment” means stasis or a postponement of development of the symptoms associated a disease or disorder described herein. The terms further include ameliorating existing uncontrolled or unwanted symptoms, preventing additional symptoms, and ameliorating or preventing the underlying metabolic causes of symptoms. Thus, the terms dénoté that a bénéficiai resuit has been conferred on a mammalian subject with a disease or symptom, or with the potential to develop such disease or symptom. A response is achieved when the patient expériences partial or total alleviation, or réduction of signs or symptoms of illness, and specifically includes, without limitation, prolongation of survival. The expected progression-free survival times can be measured in months to years, depending on prognostic factors including the number of relapses, stage of disease, and other factors. [00114] The présent disclosure contemplâtes pharmaceutical compositions for use in connection with such methods. Compositions can be suitable for administration locally or systemically by any suitable route.

]00115] In general, MMP9 binding proteins are administered in a therapeutically effective amount, e.g., in an amount to effect inhibition of tumor growth in a subject and/or to inhibit metastasis.

[00116] As used herein, the terni “therapeutically effective amount” or “effective amount” refers to an amount of a therapeutic agent that when administered alone or in combination with 30 another therapeutic agent to a subject is effective to prevent or aineliorate the disease condition or the progression of the disease. A therapeutically effective dose further refers to that amount of the compound sufficient to resuit in amelioration of symptoms, e.g., treatment, healing, prévention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prévention or amelioration of such conditions. When applied to an individual active ingrédient administered alone, a therapeutically effective dose refers to that ingrédient alone.

When applied to a combination, a therapeutically effective dose refers to combined amounts of the active ingrédients that resuit in the therapeutic effect, whether administered in combination, serially or simultaneously. For example, when in vivo administration of an anti-MMP9 antibody is employed, normal dosage amounts can vary from about 10 ng/kg to up to I00 mg/kg of mammal body weight or more per day, preferably about l pg/kg/day to 50 mg/kg/day, optionally about 100 pg/kg/day to 20 mg/kg/day, 500 pg/kg/day to 10 mg/kg/day, or l mg/kg/day to 10 mg/kg/day, depending upon the route of administration.

[001171 The selected dosage regimen will dépend upon a variety of factors including the activity of the MMP9 binding protein, the route of administration, the time of administration, the rate of excrétion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular composition employed, the âge, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors well known in the medical arts.

(00118] A clinician having ordinary skill in the art can readily détermine and prescribe the effective amount (ED50) of the pharmaceutical composition required. For example, the physicien or veterinarian can start doses of the compounds of the invention employed in the pharmaceutical composition at leveis lower than that required in order to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.

100119] As used herein, the terni subject means mammalian subjects. Exemplary subjects include, but are not limited to humans, monkeys, dogs, cats, mice, rats, cows, horses, goats and shcep. In some embodiments, the subject has cancer and can be treated with the agent of the présent invention as described below.

(00120] If needed, for cancer treatments, methods can further include surgical removal of the cancer and/or administration of an anti-cancer agent or treatment in addition to an MMP9 binding protein. Administration of such an anti-cancer agent or treatment can be concurrent with administration of the compositions disclosed herein.

Methods of Détection of MMP9 |00121] The présent disclosure also contemplâtes methods of detecting MMP9 in a subject, e.g., to detect tumor or tumor-associated tissue expressing MMP9. Thus, methods of diagnosing, monitoring, staging or detecting a tumor having MMP9 activity are provided. [00122] Samples from an individual suspected of having a tumor associated with MMP9 expression can be collected and analyzed by detecting the presence or absence of binding of an MMP9 binding protein. This analysis can be performed prior to the initiation of treatment using an MMP9 binding protein as described herein, or can be done as part of monitoring of progress of cancer treatment. Such diagnostic analysis can be performed using any sample, including but not limited to tissud, cells isolated from such tissues, and the like. Tissue samples include, for example, fonnalin-fixed or frozen tissue sections.

100123] Any suitable method for détection and analysis of MMP9 be employed. Various diagnostic assay techniques known in the art can be adapted for such propose, such as compétitive binding assays, direct or indirect sandwich assays and immunoprécipitation assays conducted in either heterogeneous or homogeneous phases.

[00124] MMP9 binding proteins for use in détection methods can be labeled with a détectable moiety. The détectable moiety directly or indirectly produces a détectable signal. For example, the détectable moiety can be any of those described herein such as, for example, a radioisotope, such as 3H, 14C, 32P, 35S, or 1251, a fluorescent or chemiluminescent compound, such as fluorescein isothiocyanate (F1TC), Texas red, cyanin, photocyan, rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase, β-galactosidase or horseradish peroxidase. [00125] Détection can be accomplished by contacting a sample under conditions suitable for MMP9 binding protein binding to MMP9, and assessing the presence (e.g., level) or absence of MMP9 binding protein-MMP9 complexes. A level of MMP9 in the sample in comparison with a level of a reference sample can indicate the presence of a tumor or tumor-associated tissues having MMP9 activity. The reference sample can be a sample taken from the subject at an earlier time point or a sample from another individual.

EXAMPLES

Examplc 1: Préparation of antibodies to human MMP-9.

[00126] The full-length human MMP9 protein without a signal peptide, which is SEQ ID NO. 28 was used to immunize mice. Spleen cells from immunized mice were fused with inyeloma cells to generate a hybridoma library. Monoclonal cultures were prepared and screened to identify cultures expressing an anti-MMP9 monoclonal antibody.

[00127] Antibody (AB004I) was purified from one of the cultures and characterized. The antibody contained an fgG2b heavy chain and a kappa light chain. Characterization included testing for the binding of AB0041 to other human MMPs and to MMP9 proteins from other species, including cynomolgus monkey, rat and mouse. It was found that the AB0041 antibody bound strongly to human and cynomolgus MMP9, that it bound less strongly to rat MMP9, and that it did not bind to murine MMP9 or to many of the human non-MMP matrix metalloproteinases.

[00128] 'Table 2. Cross reactivity of AB0041 and AB0045.

MMP Tested	Dissociation constant (Kd)
AB0045	AB0041
Human MMP 1	>100 nM	>100 nM
Human MMP2	>100 nM	>100 nM
Mouse MMP2	>100 nM	>100 nM
Human MMP3	>100 nM	>100 nM
Human MMP7	>100 nM	>100 nM
Human MMP8	>100 nM	>100 nM
Human MMP9	0.168 ± 0.117 nM	0.133 ±0.030 nM
Cynomolgus monkey MMP9	0.082 ± 0.022 nM	0.145 ± 0.16 nM
Mouse MMP9	>100 nM	>100 nM
Rat MMP9	0.311 ± 0.017 nM	0.332 ± 0.022 nM
Human MMP 10	>100 nM	>100 nM
Human MMP 12	>100 nM	>100 nM
Human MMP 13	>100 nM	>100 nM

[00129] Additional characterization included assaying the binding of the antibody to mouse MMP9 in which certain amino acids were altered to more closely correspond to the human MMP9 sequence. In addition, the human MMP9 protein was mutagenized, and the various mutants tested for their ability to be bound by the antibody, to déterminé amino acids important for antibody binding and thereby define the therapeutic epitope. This analysis identified an arginine residue at position 162 of the MMP9 amino acid sequence (RI62) as important for antibody binding. Other amino acid residues in MMP9 that are important for 10 binding of the AB0041 antibody include El 11, DI 13, and 1198. Recent crystal structure of

MMP9 showed that El 11, DI 13, RI62, and 1198 were grouped near each other around a Ca2+ ion binding pocket of MMP9. Without binding to any spécifie scientific theory, AB0041 may bind to the région on MMP9 wherein these residues are located. Altematively, these MMP9 residues may hâve direct contact with AB0041.

[00130] In an enzymatic assay for MMP9, the AB0041 antibody was found to act as a non-competitive inhibitor.

Example 2: Hunianîzatioii of antibodies to human MMP9 [00131] The amino acid sequences of the heavy chain and light chain of the mouse

AB0041 antibody were altered at certain locations in the framework (i.e., ποη-CDR) portion of their variable régions to generate proteins that are less immunogenic in humans. These amino acid sequence changes were shown in Figures 1 and 2. The cross-reactivity of the humanized antibody (referred to as AB0045) is shown in Table 2 above.

Claims (23)

1. What is claimed is:

   1. A MMP9 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 MMP9.
2. 2. The MMP9 binding protein of claim 1, wherein the MMP9 binding protein binds an epitope of human MMP9 comprising amino acid residues R162, E111, D113, and 1198.

   J*
3. 3. The MMP9 binding protein of claim 1, wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising a heavy chain polypeptide of SEQ. ID. NO: 7 and a light chain polypeptide of SEQ. ID. NO: 12.
4. 4. The MMP9 binding protein of claim 1, wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising a heavy chain polypeptide comprising an amino acid sequence of SEQ ID NOS: 3 or 5-8.
5. 5. The MMP9 binding protein of claim 1, wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising a light chain polypeptide comprising an amino acid sequence of SEQ ID NOS: 4 or 9-12.
6. 6. The MMP9 binding protein of claim 1, wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising a heavy chain polypeptide comprising complementarity-determining régions (CDRs) of SEQ ID NOs: 13-15 and a light chain polypeptide comprising CDRs ofSEQ IDNOs: 16-18.
7. 7. The MMP9 binding protein of claim 6, wherein the heavy chain polypeptide comprises complementarity-determining régions (CDRs) of SEQ ID NOs: 13-15, and the light chain polypeptide comprises CDRs of SEQ ID NOs: 16-18.
8. 8. The MMP9 binding protein ofclaim 1, wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising a heavy chain polypeptide comprising a variable région selected from the group consisting of SEQ ID NOs: 3 or 5-8, and a light chain polypeptide comprising a variable région selected from the group consisting of SEQ ID NOs: 4 or 9-

   12.
9. 9. The MMP9 binding protein of claim 1, wherein the heavy chain polypeptide is an IgG.
10. 10. The MMP9 binding protein ofclaim 1, wherein the binding ofthe MMP9 binding protein to MMP9 inhibits the enzymatic activity of MMP9.
11. 11. The MMP9 binding protein of claim 10, wherein the inhibition is non-competitive.
12. 12. The MMP9 binding protein of claim 1, wherein the MMP9 binding protein competes for binding to human MMP9 with an antibody comprising a heavy chain polypeptide encoded by a polynucleotide having a nucléotide sequence selected from the group consisting of SEQ ID NOs: 19-22 and a light chain polypeptide encoded by a polynucleotide having a nucléotide sequence selected from the group consisting of SEQ ID NOs: 23-26.
13. 13. An isolated nucleic acid comprising a nucléotide sequence encoding:

    a heavy chain polypeptide comprising complementarity-determining régions (CDRs) of SEQ ID NOs: 13-15; and/or a light chain polypeptide comprising CDRs of SEQ ID NOs: 16-18.
14. 14. The isolated nucleic acid of claim 13, wherein the heavy chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:1, 3, and 5-8.
15. 15. The isolated nucleic acid of claim 13, wherein the light chain polypeptide comprises an amino acid sequence selected from the group consisting of SEQ ID NOS:2, 4, and 9-12.
16. 16. The isolated nucleic acid of claim 13 comprising a sequence selected from the group consisting of SEQ ID NOs: 19-26.
17. 17. A vector comprising the isolated nucleic acid of claim 13.
18. 18. A cell comprising the vector of claim 17.
19. 19. A pharmaceutical composition comprising the MMP9 binding protein of claim 1.
20. 20. A pharmaceutical composition comprising the vector of claim 17.
21. 21. A pharmaceutical composition comprising the cell of claim 18.
22. 22. Use ofthe MMP9 binding protein of claim 19 in the manufacture of a pharmaceutical composition for inhibiting MMP9 activity in a subject having a tumor or tumor-associated tissue having MMP9 activity, by administration of said pharmaceutical composition to the subject in an amount effective to inhibit MMP9 activity, wherein MMP9 activity is inhibited in the subject.
23. 23. A method of detecting MMP9 expression in tissue of a patient, the method comprising:

    contacting a tissue sample from the patient with an MMP9 binding protein of claim 1; and detecting the presence or absence of MMP9;

    wherein the presence of MMP9 in the tissue sample indicates the MMP9 is expressed in tissue.