Variants of tissue inhibitor of metalloproteinase-3 (TIMP-3), compositions and methods

Abstract

To provide TIMP-3 in a form exhibiting advantageous production characteristics, purification characteristics and pharmacokinetic / pharmacodynamic characteristics.SOLUTION: TIMP-3 muteins, variants, and derivatives, nucleic acids encoding them, and methods of making and using them are disclosed.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit of U.S. Provisional Patent Application No. 61 / 782,613, filed March 14, 2013; U.S. Provisional Patent Application No. 61 / 798,160, filed March 15, 2013; U.S. Provisional Patent Application No. 61 / 802,988, filed March 18, 2013; and U.S. Provisional Patent Application No. 61 / 940,673, filed February 17, 2014, which are incorporated by reference in their entireties.

[0002] Sequence Listing Reference This application is submitted with a Sequence Listing in electronic format, which is provided as file A-1827WOPCT_SL31314, created on March 11, 2014, and is 306 KB in size. The information in electronic format regarding the Sequence Listing is incorporated herein by reference in its entirety.

[0003] The present invention relates generally to metalloproteinase inhibitors. Specifically, the present invention relates to tissue inhibitor of metalloproteinase 3 ("TIMP-3") and novel and useful variants, muteins, and derivatives thereof. [Background technology]

[0004] Connective tissue and articular cartilage are maintained in a dynamic equilibrium by the opposing actions of extracellular matrix synthesis and degradation. Matrix degradation is primarily driven by the enzymatic action of metalloproteinases, including matrix metalloproteinases (MMPs) and a disintegrin metalloproteinase with thrombospondin motifs (ADAMTS). While these enzymes are important in many natural processes (including development, morphogenesis, bone remodeling, wound healing, and angiogenesis), dysregulation of these enzymes, leading to elevated levels, is thought to play a detrimental role in connective tissue destructive diseases, including rheumatoid arthritis and osteoarthritis, as well as cancer and cardiovascular pathologies.

[0005] Endogenous metalloproteinase inhibitors include plasma α2-macroglobulin and tissue inhibitors of metalloproteinases (TIMPs), of which four are known to be encoded in the human genome. TIMP-3 inhibits all major cartilage-degrading metalloproteinases, and multiple studies have shown that it protects cartilage. Addition of this protein to cartilage implants prevents cytokine-induced degradation, and intra-articular injection reduces cartilage damage in a rat medial meniscus tear model of osteoarthritis.

[0006] Dysregulation of MMPs also occurs in congestive heart failure and is thought to contribute to many proinflammatory processes. However, the development of TIMP-3 as a therapeutic inhibitor of MMP activity has been hampered by challenges in recombinant protein production and the short half-life of recombinant forms of TIMP-3. Thus, there is a need in the art for forms of TIMP-3 that exhibit advantageous production, purification, and pharmacokinetic / pharmacodynamic properties. [Brief explanation of the drawings]

[0007] [Figure 1] A sequence comparison of native full-length human TIMP-3 with a mutant form of full-length human TIMP-3 is shown, in which certain amino acids in the sequence are replaced with the letter "X." The signal sequence is underlined and can therefore be replaced with other signal sequences as described herein. [Figure 2] 1 shows a sequence comparison of native full-length human TIMP-3 with mutant TIMP-3s, in which the indicated amino acid substitutions have been made. The signal sequence is present and is underlined in the native full-length TIMP-3 sequence to maintain consistency in numbering, and therefore can be replaced with other signal sequences as described herein. [Figure 3]A two-dimensional polypeptide map is shown, with amino acids aligned to identify the amino acid residues comprising the N-region (residues 23-143) and C-region (residues 144-211) of TIMP-3, as well as the cysteine ​​positions that form disulfide bonds. [Figure 4] A sequence comparison of native human N-terminal TIMP-3 and mutant forms of human N-terminal TIMP-3 is shown, with the letter "X" replacing certain amino acids in the sequence. The signal sequence is underlined, and therefore, it can be replaced with other signal sequences as described herein. Certain substitutions are contemplated in the mature N-terminal TIMP-3 and are represented herein as "n#m," where "n" represents the amino acid found in the native N-terminal region of TIMP-3, "#" represents the amino acid residue number, and "m" represents the substituted amino acid ("-" indicates that the amino acid has been deleted). Thus, for example, "K45I" indicates that the lysine (K) at amino acid 45 has been replaced with an isoleucine (I). A deletion of M67 is indicated by M67-. A substitution of residues 71-77 with a pair of glycines is indicated by Y70-GG-H78. Exemplary mutant forms of human TIMP-3 include the following mutations (alone or in combination): R43F, K45I, K45T, K53T, E54Y, K65T, M67-, K68T, K68I, Y70-GG-H78, H78W. Specific combinations of mutations include: K45I, K53T, E54Y, K65T, M67-, K68T; K45I, K53T, E54Y, M67-, K68I; K45I, K53T, K65T, M67-, K68T; K45I, K53T, M67-, K68I; K45I, K53T, M67-, K68I, H78W; K45I, K65T, K68I; K45I , K65T, M67-, K68T; K45I, K65T, M67-, K68T, H78W; K45I, M67-, K68I, H78W; K45I, M67-, K68T; K45T, K65T, M67-, K68I; K45T, K65T, M67-, K68T; K53T, E54Y; K53T, H78W; R43F, K45I, K65T, K68I. [Figure 5]A sequence comparison of native human N-terminal TIMP3 and mutant forms of human N-terminal TIMP3 is shown, in which certain amino acids in the sequence have been replaced with the letter "X." The signal sequence is underlined, and therefore can be replaced with other signal sequences as described herein. Certain substitutions are contemplated in the mature N-terminal TIMP-3 and are represented herein as "n#m," where "n" represents the amino acid found in the native N-terminal region of TIMP-3, "#" represents the amino acid residue number, and "m" represents the substituted amino acid ("-" indicates that the amino acid has been deleted). Thus, for example, "K45E" indicates that the lysine (K) at amino acid 45 has been replaced with glutamine (E). Mutant forms of human TIMP-3 exemplified herein contain the following mutations (alone or in combination): T25G; T25H; T25K; T25P; T25R; T25S; T25W; C26A; S27V; S27A; P28A; P28D; P28L; P28S; S29I; H30A; P31A; Q32A; F35A; C36A; N37A; D39A;V41A;I42A;R43A;R43E;R43T;K45E;V46A;G48A;G48S;K49S;K49E;L51E;L51T;K53D;E54S;P56N;L60I;V61Q;T63E;T74E;H78D;H78E;Q80E;G116T;C118A;N119D;C143A; and 144N-. Specific combinations of mutations include S27V S29I; V46A G48S K49E L51E K53D E54S P56N L60I V61Q G116T N119D; C26A C118A; C36A C143A; K45E K49E; K45E K49S; K45E Q80E; K45E T63E; K45E T63E H78E; K45E T63E H78E Q80E; L51T T74E H78D; R43E T74E H78D Q80E; R43T T74E H78D Q80E; T63E H78D; T63E H78E; T63E H78E; T63E H78E Q80E; T63E T74E H78D; T63E Examples include T74E H78E; T74E H78D Q80E; T74E H78E Q80E; Y70-GG-78H. [Figure 6]Figure 1 shows a sequence comparison of native TIMP-2 and native TIMP-3. Amino acid identity at corresponding residues is indicated by an asterisk below the sequence. Summary of the Invention

[0008] In one embodiment, the present invention provides an isolated TIMP-3 mutein having a mature region that is at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2 and having at least one mutation, wherein the mutation is K45E; K45N; K45S; V47T; K49N; K49E; K49S; K50N; L51T; L51N; V52T; K53T; P56N; F57N; G58T; T63E; T63N; K65T; K65N; M67T; K68S; T74E; K75N; P77T ;H78D;H78E;H78N;Q80E;Q80T;K94N;E96T;E96N;V97N;N98T;K99T;D110N;K112T;Q126N;K133S;R138T;R138N;H140T;T158N;K160T;T166N;M168T;G173T;H181N;A183T;R186N;R186Q, R186E, K188T;K188Q, K188E, P201N;K203T;I205F, I205Y, A208G, A208V, and A208Y.

[0009] In another embodiment of the invention, there is provided an isolated TIMP-3 mutein having a mature region at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO: 2, and having the mutation F57N and at least one additional mutation, which is a substitution of one or more of the K residues of TIMP-3. In a further aspect of the invention, the additional mutation introduces an N-linked glycosylation site into the amino acid sequence.

[0010] Also embodied in the present invention is an isolated TIMP-3 mutein having a mature region at least 90% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO: 2, which mutein has at least one mutation that introduces at least one N-linked glycosylation site into the amino acid sequence. In additional embodiments, the TIMP-3 mutein has 2, 3, 4, 5, or 6 N-linked glycosylation sites; in still further embodiments, the number of N-linked glycosylation sites introduced is 7, 8, 9, or 10.

[0011] In one embodiment of the invention, N-linked glycosylation sites are introduced at a region of the TIMP-3 amino acid sequence selected from the group consisting of: a region comprising amino acids 48-54; a region comprising amino acids 93-100; a region comprising amino acids 121-125; a region comprising amino acids 143-152; a region comprising amino acids 156-164; a region comprising amino acids 183-191; and combinations thereof. In additional embodiments, the TIMP-3 mutein has 2, 3, 4, or 5 N-linked glycosylation sites; in yet further embodiments, the number of N-linked glycosylation sites introduced is 6, 7, 8, 9, or 10.

[0012] Also provided is an isolated TIMP-3 mutant protein having a mature region at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO: 2, wherein the mutant protein has at least one mutation selected from the group consisting of: (a) one or more mutations in the TIMP-3 charge patch that cause a change in the properties of the exposed positive charge patch on the TIMP-3 surface that mimic the TIMP-2 charge surface; (b) one or more mutations that reduce susceptibility to proteolytic cleavage; (c) one or more mutations that cause a decrease in the interaction of the TIMP-3 mutant protein with the scavenger receptor LRP-1; (d) one or more mutations that cause a decrease in the interaction of the TIMP-3 mutant protein with heparin or extracellular matrix components; (e) the addition of one or more cysteinyl residues to the native TIMP-3 sequence; (f) improved pharmacokinetic and / or pharmacodynamic properties; and (g) a combination of the mutations set forth in (a) to (f). In one embodiment, one or more mutations are introduced in a region of the TIMP-3 amino acid sequence selected from the group consisting of: a region including amino acids 48-54; a region including amino acids 93-100; a region including amino acids 121-125; a region including amino acids 143-152; a region including amino acids 156-164; a region including amino acids 183-191; and combinations thereof.

[0013] One aspect of the present invention is an isolated nucleic acid encoding any one of the TIMP-3 mutant proteins described above. Other aspects of the present invention are an expression vector comprising such an isolated nucleic acid; an isolated host cell transformed or transfected with the expression vector; and a method for producing a recombinant TIMP-3 mutant protein, the method comprising culturing the transformed or transfected host cell under conditions promoting expression of the TIMP-3 mutant protein and recovering the TIMP-3 mutant protein.

[0014] Also provided are compositions comprising the TIMP-3 mutant proteins described herein and methods for treating conditions in which matrix metalloproteinases (MMPs) and / or other proteinases that are inhibited or can be inhibited by TIMP-3 act as causative or exacerbating factors, the methods comprising administering to an individual suffering from such a condition an amount of such a composition sufficient to treat the condition.

[0015] In one embodiment, the condition is selected from the group consisting of inflammatory conditions, osteoarthritis, myocardial ischemia, reperfusion injury, and progression to congestive heart failure. In another embodiment, the condition is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, and celiac disease), psoriasis, myocarditis, including viral myocarditis, inflammation associated with atherosclerosis, and arthritic conditions, including rheumatoid arthritis and psoriatic arthritis.

[0016] In further embodiments, the condition is selected from the group consisting of: dystrophic epidermolysis bullosa, osteoarthritis, Reiter's syndrome, pseudogout, rheumatoid arthritis including juvenile rheumatoid arthritis, ankylosing spondylitis, scleroderma, periodontal disease, corneal ulcers, epidermal ulcers, ulcers including gastric ulcers, post-surgical wound healing, restenosis, emphysema, Paget's disease of bone, osteoporosis, scleroderma, pressure atrophy of bone or tissue such as pressure ulcers, cholesteatoma, wound healing abnormalities, rheumatoid arthritis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome. , SEA syndrome (seronegative, enthesopathy, arthropathy syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myolitis, polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, psoriasis invertica, pustular psoriasis, erythrodermic psoriasis, dermatitis, at dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic or collagenous colitis, eosinophilic gastroenteritis, or pouchitis occurring after proctocolectomy and ileoanal anastomosis, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, multiple sclerosis (MS), asthma (extrinsic and intrinsic asthma), and related chronic inflammatory conditions or responses of the airways. hypersensitivity, including hypersensitivity), chronic obstructive pulmonary disease (COPD, i.e., chronic bronchitis, emphysema), acute respiratory distress syndrome (ARDS), respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, acute lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic pneumonia, bronchitis, allergic bronchitis bronchiectasis, tuberculosis, hypersensitivity pneumonitis, occupational asthma, asthma-like disorders, sarcoid, reactive airway disease (or dysfunction) syndrome, byssinosis, interstitial lung disease, hypereosinophilic syndrome, rhinitis, sinusitis, and pulmonary parasitosis, airway hyperresponsiveness associated with virally induced conditions (e.g.,Respiratory syncytial virus (RSV), parainfluenza virus (PIV), rhinovirus (RV), and adenovirus), Guillain-Barré disease, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease (GVHD), cerebral ischemia, traumatic brain injury, multiple sclerosis, neuropathy, myopathy, spinal cord injury, and amyotrophic lateral sclerosis (ALS). DETAILED DESCRIPTION OF THE INVENTION

[0017] The present invention provides compositions, kits, and methods related to TIMP-3 polypeptides, variants, derivatives, or muteins. Also provided are nucleic acids and derivatives and fragments thereof (which include sequences of nucleotides encoding all or part of such TIMP-3 polypeptides, variants, derivatives, or muteins, e.g., nucleic acids encoding all or part of such TIMP-3 polypeptides, variants, derivatives, or muteins); plasmids and vectors containing such nucleic acids; and cells or cell lines containing such nucleic acids and / or vectors and plasmids. For example, provided methods include methods for making, identifying, and isolating TIMP-3 polypeptides, variants, derivatives, or muteins exhibiting desirable properties.

[0018] There are many conditions in which increasing endogenous TIMP-3 in mammals or increasing the level of TIMP-3 in specific tissues would be beneficial. Accordingly, also provided herein are methods for making compositions, such as pharmaceutical compositions, containing TIMP-3 polypeptides, variants, derivatives, or mutant proteins, and methods for administering compositions containing TIMP-3 polypeptides, variants, derivatives, or mutant proteins to subjects, for example, subjects suffering from conditions in which dysregulation of matrix metalloproteinase activity causes excessive or inappropriate tissue repair.

[0019] Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. Furthermore, unless otherwise required by context, singular terms shall include pluralities and plural terms shall include the singular. Generally, the terminology used in connection with and techniques of cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those commonly used and known in the art. The methods and techniques of the present invention are generally carried out according to conventional methods well known in the art, unless otherwise indicated, and as described in the various general and more specialized references cited and discussed throughout this specification. See, e.g., Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1989), and Ausubel et al., Current Protocols in Molecular Biology, Greene Publishing Associates (1992), and Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1990), which are incorporated herein by reference. Enzymatic reactions and purification techniques are performed according to manufacturer's specifications, as commonly practiced in the art, or as described herein. The terminology used in connection with, and the experimental methods and techniques of, analytical chemistry, synthetic organic chemistry, and medicinal and pharmaceutical chemistry described herein are those commonly used and well known in the art. Standard techniques may be used for chemical syntheses, chemical analyses, pharmaceutical preparation, formulation, and delivery, and treatment of patients.

[0020] Unless otherwise specified, the following terms shall be understood to have the following meanings:

[0021] The term "isolated," used to describe a molecule (e.g., the molecule is a polypeptide, polynucleotide, or antibody), indicates that the molecule, in terms of its origin or source, is (1) free from association with naturally associated components that accompany it in the natural state; (2) substantially free from other molecules from the same species; (3) expressed by cells from a different species; or (4) not occurring at all but through human intervention. Thus, a molecule is "isolated" from its naturally associated components if it is chemically synthesized or synthesized in a cellular system different from the cell in which it naturally occurs. A molecule may also be rendered substantially free of naturally associated components by isolation using purification techniques well known in the art. The purity or homogeneity of a molecule may be assayed by several means well known in the art. For example, the purity of a polypeptide sample may be assayed by visualization of the polypeptide by polyacrylamide gel electrophoresis and staining of the gel, using techniques well known in the art. For certain purposes, higher resolution may be obtained by using HPLC or other purification means well known in the art.

[0022] The terms "peptide," "polypeptide," and "protein" each refer to a molecule comprising two or more amino acid residues joined together by peptide bonds. These terms encompass, for example, naturally occurring and artificial proteins, protein fragments, polypeptide analogs of protein sequences (such as muteins, variants, and fusion proteins), and post-translationally or otherwise covalently or non-covalently modified proteins. A peptide, polypeptide, or protein may be monomeric or polymeric.

[0023] As used herein, the term "polypeptide fragment" refers to a polypeptide that has an amino-terminal and / or carboxy-terminal deletion when compared to the corresponding full-length protein. Fragments may be, for example, at least 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 50, 70, 80, 90, 100, 150, or 200 amino acids in length. Fragments may also be, for example, up to 1,000, 750, 500, 250, 200, 175, 150, 125, 100, 90, 80, 70, 60, 50, 40, 30, 20, 15, 14, 13, 12, 11, or 10 amino acids in length. The fragment may further comprise one or more additional amino acids at either or both ends, for example, a stretch of amino acids derived from a different naturally occurring protein (e.g., an Fc or leucine zipper region) or an artificial amino acid sequence (e.g., an artificial linker sequence or tag protein).

[0024] A "variant" or "mutein" of a polypeptide (e.g., a TIMP-3 variant or mutein) includes an amino acid sequence in which one or more amino acid residues have been inserted, deleted, and / or substituted, relative to another polypeptide sequence. Variants of the present invention include fusion proteins.

[0025] A "conservative amino acid substitution" is one that does not substantially alter the structural characteristics of the parent sequence (e.g., the substituting amino acid does not tend to disrupt helices occurring in the parent sequence or other types of secondary structure that characterize the parent sequence or are necessary for its function). Art-recognized examples of polypeptide secondary and tertiary structure are described in Proteins, Structures and Molecular Principles (Creighton, Ed., W.H. Freeman and Company, New York (1984)); Introduction to Protein Structure (C. Branden and J. Tooze, eds., Garland Publishing, New York, NY (1991)); and Thornton et al. Nature 354:105 (1991), each of which is incorporated herein by reference.

[0026] One way to demonstrate the similarity of a variant or mutein to the native protein is by comparing the percent identity between two (or more) polypeptide sequences or encoding nucleic acid sequences. The "percent identity" of two polynucleotides or two polypeptide sequences is determined by comparing the sequences using the GAP computer program (part of the GCG Wisconsin Package, version 10.3 (Accelrys, San Diego, CA)) with its default parameters.

[0027] A "derivative" of a polypeptide is a polypeptide (e.g., a TIMP-3 polypeptide, variant, or mutein) that has been chemically modified, for example, by conjugation to another chemical moiety (e.g., polyethylene glycol or albumin, such as human serum albumin), phosphorylation, and / or glycosylation.

[0028] Polynucleotide and polypeptide sequences are depicted using standard one- or three-letter abbreviations. Unless otherwise noted, each polypeptide sequence has its amino terminus on the left and its carboxy terminus on the right; each single-stranded nucleic acid sequence and the top strand of each double-stranded nucleic acid sequence has its 5' terminus on the left and its 3' terminus on the right. A particular polypeptide or polynucleotide sequence can also be described by explaining how it differs from a reference sequence. For example, amino acid substitutions are represented herein as "n#m," where "n" represents the amino acid found in the native, full-length polypeptide, "#" represents the amino acid residue number, and "m" represents the substituted amino acid.

[0029] The terms "polynucleotide," "oligonucleotide," and "nucleic acid" are used interchangeably throughout this specification and include DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of DNA or RNA generated using nucleotide analogs (e.g., peptide nucleic acids and non-natural nucleotide analogs), and hybrids thereof. Nucleic acid molecules can be single-stranded or double-stranded. In one embodiment, a nucleic acid molecule of the invention comprises a contiguous open reading frame encoding a TIMP-3 polypeptide, fragment, variant, derivative, or mutein of the invention.

[0030] Two single-stranded polynucleotides are "complementary" to each other if their sequences can be arranged in an antiparallel orientation, with each nucleotide of one polynucleotide facing a complementary nucleotide in the other polynucleotide, without introducing gaps and without unpaired nucleotides at the 5' or 3' end of each sequence. A polynucleotide is "complementary" to another polynucleotide if the two polynucleotides can hybridize to each other under moderately stringent conditions. Thus, a polynucleotide can be complementary to another polynucleotide even if it is not its complementary partner.

[0031] A "vector" is a nucleic acid that can be used to introduce another nucleic acid linked to it into a cell. One type of vector is a "plasmid," which refers to a linear or circular double-stranded DNA molecule into which additional nucleic acid segments can be ligated. Another type of vector is a viral vector (e.g., replication defective retroviruses, adenoviruses, and adeno-associated viruses), in which additional DNA segments can be incorporated into the viral genome. Some vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors containing a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell and are thereby replicated along with the host genome. An "expression vector" is a type of vector capable of directing the expression of a selected polynucleotide.

[0032] A nucleotide sequence is "operably linked" to a regulatory sequence if the regulatory sequence affects the expression (e.g., level, timing, location of expression) of the nucleotide sequence. A "regulatory sequence" is a nucleic acid that affects the expression (e.g., level, timing, location of expression) of a nucleic acid to which it is operably linked. A regulatory sequence can, for example, exert its effect directly on the regulated nucleic acid or through the action of one or more other molecules (e.g., polypeptides that bind to the regulatory sequence and / or nucleic acid). Examples of regulatory sequences include promoters, enhancers, and other expression control elements (e.g., polyadenylation signals). Further examples of regulatory sequences are described, for example, in Goeddel, 1990, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA, and Baron et al., 1995, Nucleic Acids Res. 23:3605-06.

[0033] Native extracellular proteins typically contain a "signal sequence," which directs the protein into the cellular pathway so that the protein is secreted, but which is not present in the mature protein. Signal sequences, which may also be referred to as "signal peptides" or "leader peptides," are enzymatically cleaved from extracellular proteins. Proteins so processed (i.e., with the signal sequence removed) are often referred to as "mature" proteins. Polynucleotides encoding proteins or polypeptides of the invention may encode native signal sequences or heterologous signal sequences, many of which are known in the art.

[0034] As will be appreciated by those skilled in the art, recombinant proteins or polypeptides according to the present embodiments can be expressed in cell lines, including mammalian cell lines. A sequence encoding a particular protein can be used to transform a suitable mammalian host cell. Transformation can be by any known method for introducing a polynucleotide into a host cell, including, for example, packaging the polynucleotide into a virus (or viral vector) and transducing the host cell with the virus (or vector), or by transfection procedures known in the art, such as those exemplified in U.S. Pat. Nos. 4,399,216, 4,912,040, 4,740,461, and 4,959,455 (which are hereby incorporated by reference). The transformation procedure used will depend on the host being transformed. Methods for introducing heterologous polynucleotides into mammalian cells are well known in the art and include, for example, dextran-mediated transfection, calcium phosphate precipitation, polybrene-mediated transfection, protoplast fusion, electroporation, encapsulation of polynucleotides in liposomes, and direct microinjection of DNA into the nucleus.

[0035] A "host cell" is a cell that can be used to express a nucleic acid, e.g., a nucleic acid of the invention. The host cell can be a prokaryote, e.g., E. coli, or a eukaryote, e.g., a unicellular eukaryote (e.g., yeast or other fungus), a plant cell (e.g., a tobacco or tomato plant cell), an animal cell (e.g., a human cell, a monkey cell, a hamster cell, a rat cell, a mouse cell, or an insect cell), or a hybridoma. Examples of host cells include the monkey kidney-derived COS-7 line (ATCC CRL 1651) (see Gluzman et al., 1981, Cell 23:175), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells or Veggie CHO, and their derivatives and related cell lines that grow in serum-free medium (see Rasmussen et al., 1998, Cytotechnology 28:31), or the DHFR-deficient CHO line DX-B11 (see Urlaub et al., 1980, Proc. Natl. Acad. Sci. USA 77:4216-20), HeLa cells, the BHK (ATCC CRL10) cell line, the CV1 / EBNA cell line derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) (see McMahan et al., 1998, Cytotechnology 28:31). al., 1991, EMBO J. 10:2821), human embryonic kidney cells such as 293, 293EBNA, or MSR293, human epidermal A431 cells, human Colo205 cells, other transformed primate cell lines, normal diploid cells, cell lines derived from in vitro culture of primary tissue, primary explants, HL-60, U937, HaK, or Jurkat cells.

[0036] Typically, host cells are cultured cells that can be transformed or transfected with a nucleic acid encoding a polypeptide, which can then be expressed in the host cell. In "transient transfection," a nucleic acid is introduced into the host cell by one of several methods known in the art, and the recombinant protein is expressed for a finite period, typically up to about four days, before the nucleic acid is lost or degraded, for example, when the host cell undergoes mitosis. If "stable transfection" is desired, the nucleic acid encoding the polypeptide may be introduced into the host cell along with a nucleic acid encoding a selectable marker. The use of a selectable marker allows one skilled in the art to select transfected host cells in which the nucleic acid encoding the polypeptide has been integrated into the host cell genome so that the nucleic acid can be maintained throughout mitosis and expressed by progeny cells.

[0037] The phrase "recombinant host cell" can be used to refer to a host cell that has been transformed or transfected with a nucleic acid to be expressed. A host cell can also be a cell that contains a nucleic acid but does not express the nucleic acid at a desired level unless a regulatory sequence is introduced into the host cell so that the regulatory sequence is operably linked to the nucleic acid. It should be understood that the term host cell refers not only to the particular subject cell but also to the progeny or potential progeny of such a cell. Because some variations due, for example, to mutation or environmental influences, may occur in successive generations, such progeny may not actually be identical to the parent cell but are still included within the scope of the term as used herein.

[0038] As used herein, "TIMP-3 DNA," "DNA encoding TIMP-3," and similar terms refer to a selected nucleic acid encoding TIMP-3, and the TIMP-3 expressed therefrom can be either native TIMP-3 or a TIMP-3 variant or mutein described herein. Similarly, "TIMP-3," "TIMP-3 protein," and "TIMP-3 polypeptide" are used to refer to either native TIMP-3 protein or a TIMP-3 protein containing one or more mutations (i.e., a TIMP-3 polypeptide, variant, derivative, or mutein). Particular muteins of TIMP-3 may be represented by one or more mutations; for example, "K45N TIMP-3" or a "K45N TIMP-3 polypeptide" refers to a polypeptide in which the lysine (K) at amino acid 45 of native TIMP-3 has been replaced with asparagine (N).

[0039] The term "native TIMP-3" as used herein refers to wild-type TIMP-3. TIMP-3 is expressed by various mammalian cells or tissues and is present in the extracellular matrix; TIMP-3 expressed in this manner is also referred to herein as "endogenous" TIMP-3. The amino acid sequence of TIMP-3 and the nucleic acid sequence of DNA encoding TIMP-3 are disclosed in U.S. Patent No. 6,562,596, issued May 13, 2003, the disclosure of which is incorporated herein by reference. The amino acid numbering system used in U.S. Patent No. 6,562,596 designates the amino acids of the signal (or leader) peptide with negative numbers, and the mature protein (i.e., the protein with the signal or leader peptide removed) is designated amino acids 1 to 188. The numbering system used herein refers to TIMP-3 by designating the first amino acid of the native leader peptide as #1; thus, full-length TIMP-3 includes amino acids 1 to 211, and the mature form is designated amino acids 24 to 211. Those skilled in the art will readily appreciate the differences in amino acid numbering that may result from the use of these different numbering systems, and thus can readily apply the numbering system used herein to, for example, a TIMP-3 polypeptide, in which the first amino acid of the mature form is designated as 1. Thus, for example, K45N designated herein would be designated as K22N using the numbering system of U.S. Patent No. 6,562,596.

[0040] TIMP-3 is formed from two regions: an N-terminal region encompassing amino acids 24-143 of TIMP-3 (i.e., approximately two-thirds of the molecule) and a C-terminal region encompassing amino acids 144-211. Figure 3 shows the two-dimensional polypeptide sequence of TIMP-3, highlighting the complex nature of the disulfide bonds that facilitate the formation of the secondary and tertiary structure of TIMP-3. The N-terminal region of TIMP-3, often referred to as "N-TIMP-3," has been shown to exhibit at least some of the biological activity of TIMP-3; therefore, the TIMP-3 variants, derivatives, and muteins described herein encompass variants, derivatives, and muteins of TIMP-3 fragments comprising the N-terminal region.

[0041] The native TIMP-3 protein poses several challenges when used as a therapeutic molecule. For example, when TIMP-3 protein is obtained using standard mammalian expression techniques, the mammalian expression titers are too low to produce sufficient amounts of TIMP-3 at a scale appropriate for therapeutic use. Furthermore, TIMP-3 binds to the extracellular matrix, necessitating the inclusion of heparin (or a similar agent that reduces TIMP-3 binding to the extracellular matrix) in the cell culture medium. Conjugation to the low-density lipoprotein receptor-related protein 1 (LRP1) scavenger protein further complicates the challenge of secreting recombinant TIMP-3 into the medium at levels that would allow the development of a production-scale process. Microbial production of full-length TIMP-3 in prokaryotic cells has proven difficult due to incorrect protein folding.

[0042] Accordingly, the TIMP-3 variants or muteins of the present invention have been modified to overcome one or more of these challenges. Polypeptides of the present invention include those modified in some way or for some reason, for example, to (1) reduce susceptibility to proteolysis, (2) reduce susceptibility to oxidation, (3) reduce the need for agents that inhibit TIMP-3 binding to the extracellular matrix in cell culture, (4) change the binding affinity to other components, e.g., scavenger receptors such as LRP-1, (5) confer or modify other physicochemical or functional properties, including pharmacokinetics and / or pharmacodynamics, or (6) facilitate expression and / or purification of the recombinant protein. Analogs include muteins of the polypeptide. For example, single or multiple amino acid substitutions (e.g., conservative amino acid substitutions) may be made in the native sequence (e.g., in portions of the polypeptide outside the regions that form intermolecular contacts). A consensus sequence can be used to select amino acid residues for substitution, and those skilled in the art will recognize that additional amino acid residues may also be substituted.

[0043] In one aspect of the present invention, TIMP-3 mutant proteins or variants are provided that exhibit increased expression levels relative to those observed for native TIMP-3; in another aspect of the present invention, the increased expression occurs in a mammalian cell expression system. Expression levels may be determined by any suitable method that allows quantitative or semi-quantitative analysis of the amount of recombinant TIMP-3 (native, mutant, or mutein) in cell culture supernatants, i.e., conditioned medium (CM). In one embodiment, samples or CM are assessed by Western blot; in another embodiment, CM samples are assessed by standard human TIMP-3 ELISA.

[0044] In one embodiment, the increased expression is observed in a transient expression system, and in another embodiment, the increased expression is observed in a stable transfection system. One embodiment provides a TIMP-3 mutein or mutant for which the observed expression is 2-fold (2x) that observed for native TIMP-3, and another embodiment provides a TIMP-3 mutein or mutant for which the observed expression is 5-fold (5x) that observed for native TIMP-3. Further embodiments encompass TIMP-3 muteins or mutants that exhibit a 3-fold (3x), 4-fold (4x), or 6-fold (6x) increase in expression. In one embodiment, the expression of the TIMP-3 mutein or mutant is 10-fold (10x) that observed for native TIMP-3, and in another embodiment, the observed expression is 10-fold or more, e.g., 20-fold (20x) or more, that observed for native TIMP-3.

[0045] In another aspect of the present invention, TIMP-3 muteins (or variants) are provided that exhibit a reduced requirement for the addition of heparin (or another agent that inhibits TIMP-3 binding to the extracellular matrix) to cell culture media. The reduction in the amount of heparin (or other agent) can be described semiquantitatively, i.e., the reduction may be partial, moderate, substantial, or complete. In another embodiment, the reduction is expressed as a percentage; for example, the amount of heparin (or similar agent) may be reduced by 10%, 20%, 30%, 40%, 50%, or more (e.g., 60%, 70%, 80%, 90%, or 100%).

[0046] In one embodiment, a TIMP-3 mutant or mutein containing an inserted glycosylation site is provided. As is known in the art, glycosylation patterns can depend on both the protein sequence (e.g., the presence or absence of specific glycosylated amino acid residues, as discussed below) and the host cell or organism in which the protein is produced. Specific expression systems are discussed below. The presence, absence, or degree of glycosylation can be determined by any method known to those skilled in the art, including semi-qualitative measurements of molecular weight (MW) observed by Western blot or changes in molecular weight obtained from Coomassie-stained SDS-PAGE gels, while quantitative measurements can be made using mass spectrophotometric techniques to observe changes in MW corresponding to the addition of asparagine-linked glycosylation or by observing changes in mass upon removal of asparagine-linked glycosylation with an enzyme such as peptide-N-glycosidase F (PNGase-F; Sigma-Aldrich, St. Louis, MO).

[0047] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine (NXS) and asparagine-X-threonine (NXT), where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine can also be used.

[0048] Addition of glycosylation sites to an antigen-binding protein is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). Alterations may also be made by adding or substituting one or more serine or threonine residues to the starting sequence (for O-linked glycosylation sites). For simplicity, the protein amino acid sequence is preferably altered through alterations at the DNA level, specifically by mutating the DNA encoding the target polypeptide at preselected bases to generate codons that translate into the desired amino acids.

[0049] Thus, N-linked glycosylation sites may be added by altering the codon for a single amino acid. For example, NXz (where z can be any amino acid) can be altered to encode NXT (or NXS), or a codon encoding yXT / S can be altered to encode NXT / S. Alternatively, codons encoding two amino acids can be altered simultaneously to introduce N-linked glycosylation sites (e.g., a codon for yXz can be altered to encode NXT / S). In this manner, between 1 and 10 N-linked glycosylation sites can be inserted.

[0050] In addition to inserting N-linked glycosylation sites into TIMP-3, any glycosylation sites present in native TIMP-3 can be modified, e.g., to stabilize the structure of the molecule. Thus, for example, A at residue 208 can be replaced with a different residue, such as Y, V, or G. Additional mutations at the "NXT" sites at residues 206-208 include substituting F for I at residue 205 or substituting I for Y at residue 205, in combination with one of the above-mentioned substitutions at residue 208.

[0051] In another embodiment, regions that are susceptible to or readily prone to proteolytic cleavage are identified and mutated. Another aspect of the invention provides TIMP-3 muteins or variants that exhibit reduced interaction with the scavenger receptor LRP-1. In one embodiment, such muteins are generated by identifying and mutating lysine residues hypothesized to be important in the interaction between TIMP-3 and LRP-1.

[0052] Moreover, it is recognized that a TIMP-3 mutein or variant may exhibit more than one of these properties (e.g., an inserted glycosylation site may reduce the need for heparin in cell culture media, reduce interaction with LRP-1, and increase resistance to proteolysis). Additional embodiments include TIMP-3 muteins or variants with two or more mutations such that the combination of mutations results in two or more of the above-mentioned properties or effects.

[0053] Desirable TIMP-3 mutant proteins can be identified in several ways. The first method uses in silico analysis to promote charge rebalance between TIMP-3 and the related metalloproteinase inhibitor TIMP-2 (the latter has been observed to exhibit a favorable mammalian expression profile). In one embodiment of the present invention, the exposed positively charged patches on the TIMP-3 surface are redistributed to mimic the TIMP-2 charge surface. In another embodiment, the charge difference between TIMP-2 and TIMP-3 is masked by the insertion of glycosylation sites. Glycosylation insertions can also be useful for improving expression (see, e.g., "Enhancing the Secretion of Recombinant Proteins by Engineering N-Glycosylation Sites," Liu Y. et al., Amer Inst Chem Eng 2009, pg. 1468).

[0054] Thus, in another embodiment, a subset of solvent-exposed sites revealed by computational analysis are screened for N-glycosylation likelihood. For methods involving the insertion of glycosylation sites, sites that can be mutated to promote potential N-linked glycosylation are selected, for example, by identifying residues that can be mutated to form a canonical NxT glycosylation site (where N is asparagine, x is any amino acid, and T is threonine), for which N-glycosylation prediction tools are useful. In a further embodiment, structure-based methods are used to identify solvent-exposed amino acids (those with side chain exposures of 20 Å or less). 2 An additional embodiment involves mutating the LRP1 interacting lysine on TIMP-3 based on the crystal structure of LRP1 / RAP (receptor associated protein) where the interacting RAP lysine was mapped to TIMP-3.

[0055] Additional combinations are contemplated herein. For example, the F57N mutation can be made in combination with a mutation at a lysine residue, where the lysine residue is any lysine in TIMP-3. In one embodiment, a single lysine is mutated, and in another embodiment, two, three, four, or five lysine residues are mutated. In certain embodiments, the lysine residues at amino acids 45 and / or 133 can be mutated. In another example, the F57N mutation introduces a single N-linked glycosylation site, and this mutation can be made with additional mutations that introduce additional glycosylation sites or other mutations designed to affect one or more of the above-mentioned properties of TIMP-3. TIMP-3 muteins or variants containing one introduced N-linked glycosylation site, two, three, or four N-linked glycosylation sites, and TIMP-3 muteins or variants containing five or more N-linked glycosylation sites are contemplated herein.

[0056] Specific mutations are shown in Figures 1 and 2. Figure 1 shows a sequence comparison of native full-length human TIMP-3 with mutant forms of full-length human TIMP-3, in which specific amino acids within the sequence have been replaced with the letter "X." The signal sequence is underlined, and therefore can be replaced with other signal sequences as described herein. Certain substitutions are contemplated in the mature form of TIMP-3 and are represented herein as "n#m," where "n" represents the amino acid found in native full-length TIMP-3, "#" represents the amino acid residue number, and "m" represents the substituted amino acid. Thus, for example, "K45N" indicates that the lysine (K) at amino acid 45 has been replaced with asparagine (N). Exemplary mutant forms of human TIMP-3 include the following mutations (alone or in combination): K45N; K45S; V47T; K50N; V52T; P56N; F57N; G58T; T63E; T63N; K65T; T74E; H78E; H78E; H78N; Q80T; K94N; E96T; D110N; K112T; Q126N; R138T; and G173T. Combinations of these mutations are also contemplated and can include 2 to 10 (i.e., 2, 3, 4, 5, 6, 7, 8, 9, or 10) of the above substitutions.

[0057] Specific combinations of mutations include K45E, K49S; K45E, K49E; K45E, T63E; K45E, Q80E; K45E, T63E, H78E; T63E, H78E, Q80E; K45E, T63E, H78E, Q80E; L51T, T74E, H78D; T74E, H78E, Q80E; T74E, H78D, Q80E; K45 N, V47T; K49N, L51T; K75N, P77T; K45E, K49N, L51T, T63E; E96N, N98T; V97N, K99T; R138N, H140 T;T158N, K160T;T166N, M168T;H181N, A183T;R186N, K188T;P201N, K203T;A208Y;A208V;T63E , T74E, H78E; T63E, T74E, H78D; K65N, M67T; K45N, V47T, T63E, T74E, H78E; K49N, L51T, T63E, T 74E, H78E; K49N, L51T, T74E, H78E; K49N, L51T; K50N, V52T; L51N, K53T; T63N, K65T; H78N, Q80T ;K94N, E96T;D110N, K112T;Q126N;R138T;G173T;F57N;P56N, G58T;P56N, G58T;T63N, K65T;K45S, F57N;K49S, F57N;K68S, F57N;K133S, F57N;K45S, K133S, F57N; and K49S, K68S, F57N.

[0058] Additional combinations include: K45S, F57N, D110N, K112T; K45S, F57N, H78N, Q80T, D110N, K112T; K45S, F57N, H78N, Q80T, D110N, K112T, Q126N; K45S, F57N, H78N, Q80T, K94N, E96T Q126N;K45S, F57N, H78N, Q80T, Q126N, G173T;K45S, F57N, T63N, K65T;K45S, F57N, T63N, K65T, K94N, E96T;K45S, F57N, T63N, K6 5T, K94N, E96T, G173T; K45S, F57N, T63N, K65T, R138T, G173T; K45N, V47T, F57N, T63N, K65T, R138T, G173T; K45S, F57N, T63N, K6 5T, K94N, E96T, R138T; K45N, V47T, F57N, T63N, K65T, K94N, E96T, R138T; K45S, F57N, Q126N, R138T, G173T; P56N, G58T, T63N, K65T, K94N, E96T, Q126N, G173T; P56N, G58T, T63N, K65T, D110N, K112T, Q126N, G173T; and K45S, F57N, Q126N, R138T, G173T.

[0059] Additional mutations include K49S, K50N / V52T, K53E, V97N / K99T, R186N / K188T; K50N / V52T, V97N / K99T, R186N / K188T; K49E, K53E, K188Q; K50N / V52T, R186N / K188T; K50N / V52T, F57N, R186N / K188T; K45S, K50N / V52T, F57N, R186N / K188T; K50N / V52T, F57N, T63N / K65T, R186N / K188T; K45S, K50N / V52T, F57N R186N / K188T; K45S, K49S, K50N / V52T, F57N R186N / K188T; K49S, K50N / V52T, F57N, V97N / K99T, R186N / K188T; and K45S, K50N / V52T, F57N, V97N / K99T, R186N / K188T.

[0060] Figure 2 shows a sequence comparison of native full-length human TIMP-3 with mutant TIMP-3s, in which certain amino acid substitutions have been made to make the sequence more similar to that of TIMP-2. The signal sequence is present and is underlined in the native full-length TIMP-3 sequence to maintain consistency in numbering; therefore, it can be replaced with other signal sequences as described herein. In the sequences of the TIMP-3 mutants, specific amino acids are replaced with "X" to indicate possible substitutions in the mature form of TIMP-3. These substitutions include H, K, P, R, S, or W at residue 25; A at residue 27; D, L, or S at residue 28; N at residue 32; T at residue 39; T, F, A, or N at residue 43; I or T at residue 45; D at residue 46; S at residue 48; S at residue 49; T at residue 51; N at residue 63; N at residue 67; I at residue 68; D or W at residue 78; T at residue 96; N at residue 202; and S at residue 207. Substitutions may be made alone or in combination. Thus, using the symbology described for Figure 1, one variant exemplified in Figure 2 is A27T, I68K. Additional combinations are contemplated and may include 2 to 10 of the above substitutions. Furthermore, the substitutions described in Figure 2 can be combined with those described in Figure 1, e.g., A27T, P56N, G58T.

[0061] Lee et al. (J. Biol. Chem. 282:6887; 2007) disclose a study that purported to identify an extracellular matrix-binding motif in TIMP-3. When they failed to identify known heparin-binding sequences in TIMP-3, they identified 11 lysine and arginine residues whose positions suggested that the side chains of these basic amino acid residues would be exposed on the surface of TIMP-3 with a high density. These residues were K26, K27, K30, K71, K76, R100, K123, K125, K137, R163, and K165 (in the numbering system used herein, these residues would be numbered K49, K50, K53, K94, K99, R123, K146, K148, K160, R186, and K188). Accordingly, additional TIMP-3 mutant proteins include those shown below. These mutant proteins appear to exhibit partial or complete heparin independence. In addition to mutations in surface-exposed basic amino acid side chains, certain mutations also introduce N-linked glycosylation sites into the TIMP-3 mutant proteins (i.e., K94N / E96T).

[0062] Muteins engineered to reduce heparin independence include K49E, K50E, K53E, K99E, R186Q, K188Q; K49E, K50E, K53E, F57N, K99E, R186Q, K188Q; K45S, K50E, K53E, F57N, K99E, R186Q, K188Q; K49S, K50N / V52T, K99E, K188Q; K50N / V52T, K99E, K188Q; K50N / V52T, K94N / E96T, K188Q; K50N / V52T, K94N / E96T, K188Q; These include G173T; K50N / V52T, R186N / K188T; K50N / V52T, K94N / E96T, R186N, K188T; K50N / V52T, F57N, K94N / E96T, R186N / K188T; K45S, K50N / V52T, F57N, K94N / E96T, R186N / K188T; K50N / V52T, T63N / K65T, K94N / E96T, R186N / K188T; K45S, K50N / V52T, T63N / K65T, K94N / E96T, R186N / K188T. According to the present invention, some of these mutant proteins may exhibit multiple advantageous properties. For example, some muteins contain inserted N-linked glycosylation sites, while others contain mutations that improve expression in mammalian cell lines.

[0063] A TIMP-3 variant, mutein, or derivative will have an amino acid sequence substantially similar to that of native TIMP-3. In one embodiment, the TIMP-3 variant, mutein, or derivative will be at least 85% identical to native TIMP-3; in another embodiment, the TIMP-3 variant, mutein, or derivative will be at least 90% identical to native TIMP-3; in another embodiment, the TIMP-3 variant, mutein, or derivative will be at least 95% identical to native TIMP-3. In further embodiments, the TIMP-3 variant, mutein, or derivative will be at least 96%, 97%, 98%, or 99% identical to native TIMP-3. As used herein, percent identity refers to a comparison of the mature, full-length variant, mutein, or derivative to the mature, full-length native TIMP-3, i.e., TIMP-3 lacking the signal peptide (amino acids 24-211 of TIMP-3). Those skilled in the art will readily appreciate that similar comparisons can be made between mutants, muteins or derivatives of the N-terminal region of TIMP-3 and the N-terminal region of native TIMP-3.

[0064] Similarity can also be expressed by the number of amino acids that differ between a mutant or variant and native TIMP-3. For example, a TIMP-3 variant or variant may differ from native TIMP-3 by one, two, three, four, five, six, seven, eight, nine, or ten amino acids. A variant or variant that differs from native TIMP-3 by 10 amino acids would be about 95% identical to native TIMP-3. In a further embodiment, a TIMP-3 variant or variant differs from native mature TIMP-3 by 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids.

[0065] Additional modifications can be made to the nucleic acid encoding a TIMP-3 polypeptide (whether native, mutein, variant, or derivative) to facilitate expression, e.g., the signal peptide of native TIMP-3 can be replaced with a different signal peptide.

[0066] Other derivatives of TIMP-3 polypeptides within the scope of the present invention include covalent or aggregative conjugates of TIMP-3 polypeptides or fragments thereof with other proteins or polypeptides, such as by expression of recombinant fusion proteins containing heterologous polypeptides fused to the N- or C-terminus of the TIMP-3 polypeptide. For example, the conjugated peptide may be a heterologous signal (or leader) peptide, such as the yeast α-factor leader or an epitope tag. While heterologous signal peptides may differ in length from the native TIMP-3 signal peptide, those skilled in the art will appreciate that alignment of the N-terminal cysteine ​​residue of TIMP-3 polypeptides produced using heterologous signal peptides allows accurate identification of the position of the mutant protein relative to the amino acid sequence of mature TIMP-3.

[0067] Fusion proteins containing TIMP-3 polypeptides may contain an added peptide (e.g., poly-His) to facilitate purification or identification of the TIMP-3 polypeptide. Another tag peptide is the FLAG® peptide described in Hopp et al., Bio / Technology 6:1204, 1988, and U.S. Pat. No. 5,011,912. The FLAG® peptide is highly antigenic and provides an epitope that is reversibly bound by a specific monoclonal antibody (mAb), allowing for rapid assay and easy purification of expressed recombinant proteins. Reagents useful for preparing fusion proteins in which the FLAG® peptide is fused to a given polypeptide are commercially available (Sigma, St. Louis, MO).

[0068] Covalent modifications are also considered derivatives of TIMP-3 polypeptides and are included within the scope of the present invention, and are generally, but not necessarily, made post-translationally. For example, several types of covalent modifications of antigen-binding proteins are introduced into the molecule by reacting specific amino acid residues of the antigen-binding protein with organic derivatizing agents capable of reacting with selected side chains or the N- or C-terminal residues.

[0069] Cysteinyl residues are most commonly reacted with α-haloacetates (and corresponding amines), such as chloroacetic acid or chloroacetamide, to yield carboxymethyl or carboxyamidomethyl derivatives. Cysteinyl residues have also been derivatized by reaction with bromotrifluoroacetone, α-bromo-β-(5-imidozoyl)propionic acid, chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide, methyl 2-pyridyl disulfide, p-chloromercuric benzoate, 2-chloromercuri-4-nitrophenol, or chloro-7-nitrobenzo-2-oxa-1,3-diazole. Thus, in one embodiment of the invention, cysteinyl residues are added to the native TIMP-3 sequence, e.g., by altering selected codons to encode Cys. Such Cys substitutions can be made in regions in TIMP-3 that are shown to be important for expression, folding, or other properties described herein.

[0070] The number of carbohydrate moieties on the proteins of the invention can be increased by chemical or enzymatic coupling of glycosides to the protein. These procedures are advantageous in that they do not require production of the protein in a host cell with glycosylation capabilities for N- and O-linked glycosylation. Depending on the mode of attachment used, sugars may be added to (a) arginine and histidine, (b) free carboxyl groups, (c) free sulfhydryl groups, such as the free sulfhydryl groups of cysteine, (d) free hydroxyl groups, such as the free hydroxyl groups of serine, threonine, or hydroxyproline, (e) aromatic residues, such as the aromatic residues of phenylalanine, tyrosine, or tryptophan, or (f) the amide group of glutamine. These methods are described in WO 87 / 05330, published September 11, 1987, and in Aplin and Wriston, 1981, CRC Crit. Rev. Biochem., pp. 259-306.

[0071] Removal of carbohydrate moieties present on the starting recombinant protein may be accomplished chemically or enzymatically. Chemical deglycosylation requires exposure of the protein to the compound trifluoromethanesulfonic acid or an equivalent compound. This treatment results in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalactosamine), while leaving the polypeptide intact. Chemical deglycosylation is described in Hakimuddin et al., 1987, Arch. Biochem. Biophys. 259:52, and Edge et al., 1981, Anal. Biochem. 118:131. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of various endo- and exoglycosidases, such as those described in Thotakura et al., 1987, Meth. Enzymol. 138:350. Glycosylation at potential glycosylation sites may be prevented by the use of the compound tunicamycin as described in Duskin et al., 1982, J. Biol. Chem. 257:3105. Tunicamycin prevents the formation of protein-N-glycosidic bonds.

[0072] Another type of covalent modification of antigen-binding proteins involves conjugating the protein to various nonproteinaceous polymers, for example, various polyols, such as, but not limited to, polyethylene glycol, polypropylene glycol, or polyoxyalkylenes, by methods set forth in U.S. Patent Nos. 4,640,835; 4,496,689; 4,301,144; 4,670,417; 4,791,192; or 4,179,337. Additionally, as is known in the art, amino acid substitutions may be made at various positions within the protein to facilitate the addition of polymers, such as PEG.

[0073] Expression of TIMP-3 polypeptide Any expression system known in the art can be used to produce the recombinant polypeptides of the present invention. Generally, host cells are transformed with a recombinant expression vector containing DNA encoding the desired TIMP-3 polypeptide (including TIMP-3 mutant proteins or variants). Host cells that can be used include prokaryotes, yeast, or higher eukaryotic cells. Prokaryotes include gram-negative or gram-positive bacteria, such as Escherichia coli or Bacillus. Higher eukaryotic cells include insect cells and established cell lines of mammalian origin. Examples of suitable mammalian host cell lines include the monkey kidney-derived COS-7 line (ATCC CRL 1651) (Gluzman et al., 1981, Cell 23:175), L cells, 293 cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, BHK (ATCC CRL 10) cell line, and the CVI / EBNA cell line, derived from the African green monkey kidney-derived cell line CVI (ATCC CCL 70) as described in McMahan et al., 1991, EMBO J. 10:2821. Cloning and expression vectors suitable for use with bacterial, fungal, yeast, and mammalian cell hosts are described in Pouwels et al. (Cloning Vectors: A Laboratory Manual, Elsevier, New York, 1985).

[0074] Mammalian cell expression can offer advantages for the production of TIMP-3 polypeptides in that it facilitates folding and adopting a higher order structure that closely resembles the production of native TIMP-3. Numerous mammalian cell expression systems are known in the art and / or commercially available, including, for example, Gibco® Freedom® CHO-S® (a product designed to facilitate all aspects of recombinant protein cloning and expression in Chinese hamster ovary (CHO)-derived suspension cultures; ProBioGen, Life Technologies; Carlsbad, CA), GS Gene Expression System™ (a transfection system designed to provide for the generation of high-yielding, stable, cGMP-compliant mammalian cell lines; Lonza Biologics, Slough, UK), PER.C6® technology (a suite of tools designed to facilitate large-scale production of recombinant proteins using a serially divided cell line derived from a single immortalized human cell; Crucell Leiden, The Netherlands), or immortalized amniotic cells such as CAP and CAP-T (human cell-based expression systems for the expression and production of complex proteins; Cevec, Cologne, Germany).

[0075] Additional cell expression systems include, for example, the Selexis SUREtechnology Platform™ (a technology platform applicable to a variety of cell lines to induce developmental cell lines to produce recombinant proteins; Selexis Inc., Switzerland); the ProFection® Mammalian Transfection Systems (a transfection system that provides high-efficiency transfection of cells for the production of recombinant proteins; Promega, Madison, WI); the Expi293™ Expression System (a high-density mammalian transient protein expression system, Life Technologies, Grand Island, NY); and the MaxCyte® VLX™ and STX™ Transient Transfection Systems (scalable transfection systems used in the production of recombinant proteins, including antibodies; MaxCyte, Gaithersburg, MD). Those of skill in the art will also be aware of other expression systems, such as the technology first described in Wigler et al. (Cell 1979:777), as well as additional technology described, for example, on the website of the National Research Council of Canada.

[0076] A variety of vessels suitable for culturing transformed cells and producing recombinant proteins are known in the art. These include 24-deep well plates, 250 ml and 1 L shake flasks, and various bioreactors of various sizes, e.g., 2 L, 5 L, 10 L, 30 L, 100 L, 1000 L, 10,000 L, and larger bioreactors. Other suitable vessels for cell culture are known in the art and can also be used as described herein.

[0077] Cell culture medium formulations are well known in the art, and typically, media provide essential and non-essential amino acids, vitamins, energy sources, lipids, and trace elements, as well as buffers and salts, required by cells for minimal growth and / or survival. Culture media may also contain supplemental components that enhance growth and / or survival beyond the minimum rate, such as, but not limited to, hormones and / or other growth factors, specific ions (such as sodium, chloride, calcium, magnesium, and phosphate), buffers, vitamins, nucleosides or nucleotides, trace elements (inorganic compounds usually present at very low final concentrations), amino acids, lipids, and / or glucose or other energy sources; cell cycle inhibitors may also be added to the culture medium, as described herein. In certain embodiments, the medium is advantageously formulated to an optimal pH and salt concentration for cell survival and growth. In certain embodiments, the medium is a feed medium that is added after the initial stage of cell culture. In certain embodiments, the cell culture medium is a mixture of a starting nutrient solution and any feed medium that is added after the initial stage of cell culture.

[0078] A variety of tissue culture media, including defined media, are commercially available; for example, any one or combination of the following cell culture media, among others, can be used: RPMI-1640 medium, RPMI-1641 medium, Dulbecco's Modified Eagle's Medium (DMEM), Minimum Essential Medium Eagle, F-12K medium, Ham's F12 medium, Iscove's Modified Dulbecco's Medium, McCoy's 5A medium, Leibovitz's L-15 medium, and serum-free media such as EX-CELL™ 300 series (JRH Biosciences, Lenexa, Kansas). Serum-free versions of such media are also commercially available. Depending on the needs of the cells being cultured and / or the desired cell culture parameters, additional or more concentrated components, such as amino acids, salts, sugars, vitamins, hormones, growth factors, buffers, antibiotics, lipids, trace elements, and the like, may be added to the cell culture media.

[0079] The transformed cells can be cultured under conditions that promote polypeptide expression, and the polypeptide can be recovered by conventional protein purification techniques. One such purification technique involves the use of affinity chromatography and other methods known in the art. One method for isolating parent TIMP-3 or TIMP-3 mutant proteins from mammalian supernatants is to use TIMP-3 fused to a carboxy-terminal 6x-histidine tag in combination with a 6x-histidine affinity Ni-Sepharose resin (e.g., immobilized metal affinity chromatography (IMAC); general techniques are known in the art, and reagents and examples for such techniques are reviewed by QIAGEN, Germantown, MD, and GE Healthcare, Pittsburgh, PA). Cation exchange chromatography (e.g., SP-HP Sepharose®, GE Healthcare) can be used to further isolate TIMP-3 after IMAC elution, or alternatively, TIMP-3 can be captured from mammalian supernatants without IMAC (elution of TIMP-3 and its mutant proteins occurs using a sodium chloride gradient at neutral pH). Size exclusion chromatography (e.g., Superdex 200®, GE Healthcare (example mobile phase: 10 mM NaHPO, 1.8 mM KHPO, 137 mM NaCl, 2.7 mM KCl)) is a common means that can be used to further isolate TIMP-3 or its muteins (in combination with IMAC methods or ion exchange chromatography). These and other methods are known in the art; see, for example, Protein Purification: Principles: High Resolution Methods, and Applications, Third Edition (2012, John Wiley and Sons; Hoboken, NJ).

[0080] The amount of polypeptide (native TIMP-3 or TIMP-3 mutant or variant) can be determined by any suitable quantitative or semi-quantitative method that allows for the analysis of the amount of recombinant TIMP-3 (native, mutant, or variant) in cell culture supernatant, i.e., conditioned medium (CM). Suitable qualitative or semi-quantitative methods include Western blot and Coomassie-stained SDS-PAGE gel. Quantitative measurement methods include, for example, human TIMP-3 ELISA (R&D Systems Inc., Minneapolis, MN), or ForteBio Octet® (Pall ForteBio Corp, Menlo Park, CA), which captures TIMP-3 via antibody, or enzyme immunoassays such as direct UV (ultraviolet) absorbance (280 nm) measurement of purified TIMP-3.

[0081] In this way, the effect of a particular mutation in TIMP-3 can be assessed by comparing the amount of recombinant mutant protein produced with the amount of native protein produced under similar culture conditions. TIMP-3 mutant proteins or variants can be expressed at 1-, 2-, 3-, 4-, 5-, 10-, or more times the level observed for native TIMP-3. If desired, the specific productivity of a particular transformed or transfected cell line can be determined to allow for comparison or specific productivity for various forms of TIMP-3. Specific productivity, or qP, is expressed in picograms of recombinant protein per cell per day (pg / c / d) and can be readily determined by applying art-known methods for quantifying cells in culture and the above-described methods for quantifying recombinant protein.

[0082] Uses of TIMP-3 Polypeptides TIMP-3 polypeptides, variants, muteins, or derivatives can be used, for example, in assays or in the treatment of any condition in which increased levels of TIMP-3 activity are desired (i.e., conditions caused or exacerbated by matrix metalloproteinases (MMPs) and / or other proteinases that are inhibited or can be inhibited by TIMP-3), including, but not limited to, inflammatory conditions, osteoarthritis, and other conditions in which excessive or inappropriate MMP activity occurs (e.g., myocardial ischemia, reperfusion injury, and progression to congestive heart failure). Inflammatory conditions include asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, and celiac disease), psoriasis, myocarditis, including viral myocarditis, inflammation associated with atherosclerosis, and arthritic conditions, including rheumatoid arthritis, psoriatic arthritis, and the like.

[0083] The TIMP-3 polypeptide, mutant mutein, or derivative compositions described herein modify pathogenesis and provide beneficial treatments for diseases or conditions characterized by matrix degradation and / or inflammation, i.e., conditions in which metalloproteinases play a detrimental role. The compositions may be used alone or in combination with one or more agents used in the treatment of such conditions. Thus, the TIMP-3 polypeptide, mutant mutein, or derivative compositions of the present invention may be useful in the treatment of any disorder in which excessive matrix loss is caused by metalloproteinase activity. The TIMP-3 mutant muteins or derivative compositions of the present invention are useful, alone or in combination with other drugs, in the treatment of various disorders associated with the overproduction of collagenases, aggrecanases, or other matrix-degrading or pro-inflammatory enzymes, such as dystrophic epidermolysis bullosa, osteoarthritis, Reiter's syndrome, pseudogout, rheumatoid arthritis, including juvenile rheumatoid arthritis, ankylosing spondylitis, scleroderma, periodontal disease, ulcers, including corneal ulcers, epidermal ulcers, and gastric ulcers, postoperative wound healing, and restenosis. Other conditions that may be contributed to by excessive collagen and / or proteoglycan degradation and therefore amenable to the use of TIMP-3 polypeptides, mutant muteins, or derivative compositions include emphysema, Paget's disease of bone, osteoporosis, scleroderma, pressure atrophy of bone or tissue, such as pressure ulcers, cholesteatoma, and wound healing disorders. Additional conditions that result directly or indirectly from decreased levels of TIMP-3 or increased levels of metalloproteases (e.g., in myocardial ischemia-reperfusion injury and in the progression to congestive heart failure) can also be treated using the compositions described herein, either alone or in combination with other drugs commonly used to treat individuals suffering from such conditions. TIMP-3 polypeptide, variant, mutein, or derivative compositions can additionally be applied as adjuncts to other wound healing promoters, for example, to regulate collagen turnover during the healing process.

[0084] Many metalloproteinases also exhibit pro-inflammatory activity, and accordingly, additional embodiments include methods of treating inflammatory and / or autoimmune disorders, including, but not limited to, cartilage inflammation and / or bone degradation, arthritis, rheumatoid arthritis, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathic syndrome), dermatomyositis, psoriatic arthritis, scleroderma, systemic lupus erythematosus, vasculitis, myelitis, and the like. Related conditions include: psoriasis, polymyalgia, dermatomyalgia, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, psoriasis invertis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE), myasthenia gravis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), and seronegative arthropathy. Intestinal diseases include: advanced bowel disease, microscopic or collagenous colitis, eosinophilic gastroenteritis, or pouchitis following proctocolectomy and ileoanal anastomosis, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, multiple sclerosis (MS), asthma (including extrinsic and intrinsic asthma and associated chronic inflammatory states or hyperresponsiveness of the airways), chronic obstructive pulmonary disease (COPD, i.e., chronic bronchitis, emphysema), acute respiratory distress syndrome (ARDS), respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, acute lung injury, allergic bronchopulmonary asthma rugillosis, hypersensitivity pneumonitis, eosinophilic pneumonia, bronchitis, allergic bronchitis bronchiectasis, tuberculosis, hypersensitivity pneumonitis, occupational asthma, asthma-like disorders, sarcoid, reactive airway disease (or dysfunction) syndrome, byssinosis, interstitial lung disease, hypereosinophilic syndrome, rhinitis, sinusitis, and pulmonary parasitosis, airway hyperresponsiveness associated with virally induced conditions (e.g., respiratory syncytial virus (RSV), parainfluenza virus (PIV), rhinovirus (RV), and adenovirus), Guillain-Barré disease, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, GVHD,and the like. TIMP-3 polypeptides, variants, muteins, or derivatives also find use when a decrease in the relative level of TIMP-3 (i.e., a decrease in the ratio of endogenous TIMP-3 to metalloproteases, which may be the result of a decrease in the amount of TIMP-3 or an increase in the amount of metalloproteases) is associated with pathological effects, for example, in myocardial ischemia, reperfusion injury, and in the progression to congestive heart failure.

[0085] Based on the ability of TIMP-3 to inhibit connective tissue degradation, TIMP-3 polypeptides, variants, muteins, or derivatives have applications where inhibiting angiogenesis is useful, for example, in preventing or suppressing tumor growth and preventing parasitic invasion. For example, in the field of tumor invasion and metastasis, the metastatic potential of some specific tumors correlates with an increased ability to synthesize and secrete collagenases and an inability to synthesize and secrete significant amounts of metalloproteinase inhibitors. The TIMP-3 proteins of the present disclosure also have therapeutic applications in inhibiting tumor cell metastasis during removal of primary tumors, chemotherapy and radiation therapy, harvesting of contaminated bone marrow, and shunting of cancerous ascites. Diagnostically, the correlation between the absence of TIMP-3 production in tumor samples and their metastatic potential is useful as a prognostic indicator and as an indicator for possible preventive treatment.

[0086] MMPs also act on the brain's basal lamina and tight junction proteins as part of a pathway for opening the blood-brain barrier (BBB), facilitating the entry of inflammatory cells and soluble mediators into the brain. Therefore, the compositions and methods of the present invention may be useful in treating nervous system disorders characterized by excessive or inappropriate permeabilization of the BBB. Additionally, degradation of matrix proteins around neurons can lead to loss of contact and cell death; therefore, the disclosed TIMP-3 compositions can protect neurons from damage by preserving the basement membrane surrounding them. The TIMP-3 compositions of the present invention are useful for treating or ameliorating the neuroinflammatory response to injury, e.g., cerebral ischemia or traumatic brain injury. The compositions disclosed herein may also be useful in treating neurodegenerative diseases in which inflammation is an underlying cause of the disease, e.g., multiple sclerosis, as well as various forms of neurological disorders and / or myopathies, spinal cord injury, and amyotrophic lateral sclerosis (ALS). Thus, the use of the compositions of the present invention may be in combination with BDNF, NT-3, NGF, CNTF, NDF, SCF, or other regulators of neuronal growth or proliferation. Additionally, the compositions and methods of the present invention may be applicable for cosmetic purposes in that local inhibition of connective tissue breakdown may alter the appearance of the tissue.

[0087] TIMP-3 polypeptides, variants, muteins, or derivatives can be used in ex vivo treatments or administered in vivo to increase endogenous TIMP-3 activity and / or enhance TIMP-3-induced biological activity. The TIMP-3 polypeptides, variants, muteins, or derivatives of the present invention may also be used in vivo under conditions where endogenous TIMP-3 is downregulated or present at low levels. Disorders caused or exacerbated (directly or indirectly) by TIMP-3-inhibitable proteinases can thus be treated, examples of which are provided herein. In one embodiment, the present invention provides a therapeutic method comprising in vivo administration of a TIMP-3 polypeptide, variant, mutein, or derivative to a mammal in need thereof in an amount effective to increase TIMP-3-induced biological activity. In another embodiment, the present invention provides a therapeutic method comprising in vivo administration of a TIMP-3 polypeptide, variant, mutein, or derivative to a mammal in need thereof in an amount effective to increase endogenous levels of TIMP-3.

[0088] In another aspect, the present invention provides TIMP-3 polypeptides, mutants, muteins, or derivatives having improved half-lives in vivo. In one embodiment, the half-life of the TIMP-3 mutein is at least twice that of native TIMP-3; in another embodiment, the half-life is at least 3, 4, 5, 6, 8, or 10 times greater than that of native TIMP-3. In one embodiment, the half-life is determined in a non-human mammal, and in another embodiment, the half-life is determined in a human subject. Further embodiments provide TIMP-3 muteins or variants having a half-life of at least one day in vivo (e.g., when administered to a human subject). In one embodiment, the TIMP-3 polypeptide, mutant, mutein, or derivative has a half-life of at least three days. In another embodiment, the TIMP-3 polypeptide, mutant, mutein, or derivative has a half-life of four days or longer. In another embodiment, the TIMP-3 polypeptide, mutant, mutein, or derivative has a half-life of eight days or longer.

[0089] In another embodiment, a TIMP-3 polypeptide, variant, or mutein is derivatized or modified so that it has a longer half-life compared to underivatized or unmodified TIMP-3 binding protein. A derivatized polypeptide can contain any molecule or substance that confers a desired property to the polypeptide, such as extending its half-life, for a particular use. A derivatized polypeptide can contain, for example, a detectable (or labeling) moiety (e.g., a radioactive, colorimetric, antigenic, or enzymatic molecule; a detectable bead (such as a magnetic bead or an electron-dense (e.g., gold) bead); or a molecule that binds to another molecule (e.g., biotin or streptavidin)), a therapeutic or diagnostic moiety (e.g., a radioactive, cytotoxic, or pharmaceutically active moiety), or a molecule that improves the suitability of the polypeptide for a particular use (e.g., administration to a subject, such as a human subject, or other in vivo or ex vivo use).

[0090] In one such example, a polypeptide is derivatized with a ligand that specifically binds to articular cartilage tissue, as disclosed, for example, in WO2008063291 and / or Rothenfluh et al., Nature Materials 7:248 (2008). Examples of molecules that can be used to derivatize a polypeptide include albumin (e.g., human serum albumin) and polyethylene glycol (PEG). Albumin-binding and pegylated derivatives of polypeptides can be prepared using techniques well known in the art. In one embodiment, the polypeptide is conjugated or otherwise linked to transthyretin (TTR) or a TTR variant. TTR or TTR variants can be chemically modified with, for example, a chemical selected from the group consisting of dextran, poly(n-vinylpyrrolidone), polyethylene glycol, propylene glycol homopolymer, polypropylene oxide / ethylene oxide copolymer, polyoxyethylated polyol, and polyvinyl alcohol (U.S. Patent Application No. 20030195154).

[0091] composition Also included in the present invention are pharmaceutical compositions comprising an effective amount of a polypeptide product of the present invention (i.e., a TIMP-3 polypeptide, variant, mutein, or derivative) together with pharmaceutically acceptable diluents, preservatives, solubilizers, emulsifiers, adjuvants, and / or carriers useful for TIMP-3 therapy (i.e., conditions in which it is useful to increase the endogenous levels of TIMP-3 or to increase the activity of endogenous TIMP-3). Such compositions include diluents of various buffer contents (e.g., Tris-HCl, acetate, phosphate), pH, and ionic strength; additives such as detergents and solubilizers (e.g., Tween 80, Polysorbate 80), antioxidants (e.g., ascorbic acid, sodium metabisulfite), preservatives (e.g., Thimersol, benzyl alcohol), and bulking substances (e.g., lactose, mannitol); covalent attachment of polymers such as polyethylene glycol to proteins (as discussed above; see, e.g., U.S. Pat. No. 4,179,337, incorporated herein by reference); and incorporation of materials into particulate preparations or liposomes of polymeric compounds such as polylactic acid and polyglycolic acid. Such compositions can affect the physical state, stability, in vivo release rate, and in vivo clearance rate of the TIMP-3 binding protein. See, e.g., Remington's Pharmaceutical Sciences, 18th Ed. (1990, Mack Publishing Co., Easton, PA 18042) pages 1435-1712, which is incorporated herein by reference.

[0092] Generally, the effective amount of a polypeptide of the invention will depend on the recipient's age, weight, condition, or severity of disease. See Remington's Pharmaceutical Sciences, supra, pp. 697-773, which is incorporated herein by reference. Typically, a dose of about 0.001 g / kg to about 1 g / kg of body weight may be used, although higher or lower amounts may be used as recognized by a practitioner of ordinary skill. For local (i.e., non-systemic) applications, such as topical or intra-articular applications, the dosage is about 0.001 g / cm. 2 ~Approx. 1g / cm 2 Dosage may be one or more times a day, or less frequently, and may be combined with other compositions described herein. It should be noted that the present invention is not limited to the doses described herein.

[0093] As is understood in the art, pharmaceutical compositions containing the molecules of the present invention are administered to a subject in a manner consistent with the indication. The pharmaceutical composition may be administered by any suitable means, including, but not limited to, parenteral administration, local administration, topical administration, or inhalation. When injected, the pharmaceutical composition may be administered, for example, intravenously, intramuscularly, intralesionally, intraperitoneally, or subcutaneously, by bolus injection or continuous infusion.

[0094] For example, localized administration, such as transdermal delivery and sustained release from implants at the site of disease or injury, is contemplated. Other alternative methods include eye drops; oral administration includes pills, syrups, confectionery tablets, or chewing gum; and topical preparations include lotions, gels, sprays, and ointments. For example, localized administration to the joints or musculoskeletal system includes periarticular, intraarticular, intrabursal, intrachondral, intrasynovial, and intratendinous administration. Administration to the respiratory system includes intrapulmonary, intrapleural, intrapulmonary, intratracheal, intrasinus, and intrabronchial delivery, which can be facilitated, for example, by an inhaler or nebulizer. Intrathecal delivery and other methods useful for introducing compositions into the brain and / or nervous system are also contemplated herein, such as epidural, intrathecal, or epidural administration, as well as perineural, intrasacaudal, intracerebral, intracapsular, and intraspinal administration.

[0095] Further examples of local administration include delivery to tissue in conjunction with surgery or another medical procedure. For example, the pharmaceutical composition may be administered to cardiac tissue during surgery performed to treat or improve cardiac conditions, or during procedures such as cardiac catheterization (e.g., percutaneous coronary intervention). Delivery may be, for example, via intracoronary, intracardiac, intramyocardial, and / or transendocardial routes, and may be guided by the use of endocardial or electromechanical maps of the injection target area of ​​the heart or other techniques, such as magnetic resonance imaging (MRI). The composition may also be delivered by encapsulation in a cardiac patch or as a coating on a stent or other device useful for cardiac conditions.

[0096] In addition to eye drops, the use of ointments, creams, or gels to administer the compositions of the present invention to the eye is also contemplated. Direct administration to the interior of the eye may be achieved by periorbital, conjunctival, intracorneal, subconjunctival, subtenon, retrobulbar, intraocular, and / or intravitreal injection or administration. These and other techniques are discussed, for example, in Gibaldi's Drug Delivery Systems in Pharmaceutical Care (2007, American Society of Health-System Pharmacists, Bethesda, MD).

[0097] Multiple agents work in concert to maintain the dynamic equilibrium between the extracellular matrix and tissue. In treating conditions where this equilibrium is disrupted, one or more other agents may be used in combination with the polypeptides of the present invention. These other agents may be administered simultaneously, sequentially, or in combination. Generally, these other agents may be selected from the list consisting of metalloproteinases, serine proteases, inhibitors of matrix-degrading enzymes, intracellular enzymes, cell adhesion modulators, and factors that regulate the expression of extracellular matrix-degrading proteinases and their inhibitors. Specific examples are listed below, but those skilled in the art will recognize other agents that perform equivalent functions, including additional agents or other forms of the listed agents (e.g., synthetically produced, by recombinant DNA technology, and analogs and derivatives).

[0098] Other degradation inhibitors may also be used if improved or more specific prevention of extracellular matrix degradation is desired. The inhibitor may be selected from the group consisting of alpha-2 macroglobulin, pregnancy zone protein, ovostatin, alpha-1 proteinase inhibitor, alpha-2 antiplasmin, aprotinin, protease nexin-1, plasminogen activator inhibitor (PAI)-1, PAI-2, TIMP-1, and TIMP-2. Others may also be used as recognized by those skilled in the art.

[0099] Intracellular enzymes may also be used in combination with the polypeptides of the present invention. Intracellular enzymes can also affect extracellular matrix degradation, and include, for example, lysozomal enzymes, glycosidases, and cathepsins.

[0100] Cell adhesion modulating agents may also be used in combination with the polypeptides of the present invention. For example, it may be desirable to modulate cell adhesion to the extracellular matrix before, during, or after inhibiting extracellular matrix degradation using the polypeptides of the present invention. Cells that have shown cell adhesion to the extracellular matrix include osteoclasts, macrophages, neutrophils, eosinophils, killer T cells, and mast cells. Cell adhesion modulating agents include peptides containing an "RGD" motif or analog, or mimetic antagonists or agonists.

[0101] Factors that regulate the expression of extracellular matrix-degrading proteinases and their inhibitors include cytokines, such as IL-1 and TNF-α, TGF-β, glucocorticoids, and retinoids. Other growth factors that cause cell proliferation and / or differentiation may also be used if the desired effect is to inhibit extracellular matrix degradation using the polypeptides of the present invention in conjunction with such cellular actions. For example, during inflammation, maintenance of the extracellular matrix (by inhibiting enzyme activity) may be desired, but neutrophil production may also be desired; therefore, G-CSF may be administered. Other factors include erythropoietin, interleukin family members, SCF, M-CSF, IGF-I, IGF-II, EGF, FGF family members, such as KGF, PDGF, and others. Interferon activity, such as interferon α, β, γ, or consensus interferon, may also be desired. Intracellular agents include G proteins, protein kinase C, and inositol phosphatases. The use of the polypeptides of the present invention can provide therapeutic benefit in conjunction with one or more agents involved in inflammation therapy.

[0102] Cell trafficking agents may also be used; for example, inflammation involves the degradation of the extracellular matrix and the movement or trafficking of cells to the site of injury. Preventing the degradation of the extracellular matrix can prevent such cell trafficking. Combining the polypeptides of the present invention with agonists or antagonists of cell trafficking modulators may therefore be desirable in treating inflammation. The cell trafficking modulator is selected from the list consisting of endothelial cell surface receptors (such as E-selectin and integrins); leukocyte cell surface receptors (L-selectin); chemokines, and chemoattractants. For a review of the components involved in inflammation, see Carlos et al., Immunol. Rev. 114:5-28 (1990), which is incorporated herein by reference.

[0103] Additionally, compositions include neu differentiation factor (NDF), and methods of treatment include administering NDF before, during, or after administration of TIMP-3. NDF has been shown to stimulate the production of TIMP-2, and a combination of NDF, TIMP-1, -2, and / or -3 may provide benefit in the treatment of tumors.

[0104] The polypeptide products of the present invention may be coupled to a detectable marker substance (e.g., 125 The nucleic acid products of the invention may also be "labeled" by association with a fluorophore such as 1, 2, 3, 4, 5, 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, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 112, 120, 130, 140, 150, 162, 170, 182, 190, 191, 192, 193, 194, 195, 196, 197, 198, 199,

[0105] As described above, the TIMP-3 polypeptide, mutant mutein, or derivative compositions of the present invention have broad applications in treating a variety of disorders. Thus, another embodiment contemplated herein is a kit comprising a composition of the present invention and, optionally, one or more of the additional compositions described above directed to the treatment of disorders involving extracellular matrix degradation. An additional embodiment is an article of manufacture comprising packaging material and a pharmaceutical product therein, the pharmaceutical product containing a polypeptide, mutant, mutein, or derivative of the present invention, the packaging material comprising labeling indicating therapeutic use for TIMP-3. In one embodiment, the pharmaceutical product may be used for an indication selected from the group consisting of cancer, inflammation, arthritis (including osteoarthritis and the like), dystrophic epidermolysis bullosa, periodontal disease, ulcers, emphysema, bone disorders, scleroderma, wound healing, red blood cell deficiency, cosmetic tissue reconstruction, modulation of fertilization or embryo implantation, and neuronal disorders. The article of manufacture may optionally include labeling instructions for other compositions or other compositions. [Example]

[0106] The following examples are provided for the purpose of illustrating specific embodiments or features of the present invention, but not for the purpose of limiting its scope.

[0107] Example 1 This example describes a method used to determine the effect, if any, of one or more mutations in TIMP-3 on expression in a mammalian expression system. This example describes general vectors and host cell systems; many vectors and host cell systems are known in the art and described herein and are suitable for determining the effect, if any, of specific mutations in the TIMP-3 sequence on expression of recombinant proteins.

[0108] Briefly, DNA encoding TIMP-3 is linked to an expression vector under normal conditions (i.e., DNA encoding TIMP-3 is operably linked to other sequences of the vector so that it can be expressed), and suitable mammalian cells are transformed or transfected with the vector. The transformed or transfected cells are cultured under appropriate conditions to express recombinant protein, and the amount is evaluated qualitatively or semi-quantitatively, for example, by Western blot or SDS-PAGE, or more quantitatively using an assay such as ELISA (R&D Systems, Minneapolis, MN) or ForteBio Octet® (Pall ForteBio Corp, Menlo Park, CA). In this way, the effect of various mutations on the ability of mammalian cells to express TIMP-3 protein, mutant protein, or variant can be determined.

[0109] When one or more mutations are made to introduce an N-linked glycosylation site into a TIMP-3 polypeptide or to enhance a native glycosylation site, it may be desirable to assess the presence and / or extent of glycosylation. Cells can be transformed or transfected as described above, and semiquantitative assays (e.g., Western blots) can be used to determine whether N-linked glycosylation is poorly incorporated, partially incorporated, or fully incorporated.

[0110] Example 2 This example describes a method used to determine whether one or more mutations in TIMP-3 cause an increase in heparin independence. Cells are transformed or transfected as described above and cultured in the presence or absence of heparin. Heparin can be added in various amounts to semi-quantitatively determine the degree of heparin dependency. The amount of TIMP-3 protein, mutant protein, or variant expressed under various conditions is then determined, and a comparison is made to determine whether specific mutations have any effect on whether heparin is required for the release of TIMP-3 protein, mutant protein, or variant from the extracellular matrix, or whether the amount or amount of heparin required is reduced.

[0111] Example 3 This example describes an MMP inhibition assay that uses fluorescence analysis to measure MMP activity, although other methods are known in the art. For example, cleavage of a quenched MMP subtype 5-FAM / QXL520 fluorescence resonance energy transfer (FRET) peptide substrate by an activated MMP subtype or subtype-specific catalytic domain results in an increase in fluorescent signal. FRET peptides for several different MMPs are commercially available, for example, from Anaspec, Fremont, CA. As used herein, TIMP-3 protein may be native TIMP-3 or a TIMP-3 mutant protein, variant, or derivative, and the protein being tested is referred to as a test molecule.

[0112] For the MMP2 activity assay, human pro-MMP2 (Anaspec, Fremont, CA) was activated with 1 mM 4-aminophenylmercuric acetate (APMA, Anaspec, Fremont, CA) for 1 h at 37°C, followed by incubation with various concentrations of the MMP2-sensitive 5-FAM / QXL 520 FRET peptide in assay buffer provided by the supplier in black 384-well Optiplates (PerkinElmer, Waltham, MA) at 37°C. After a 2-h incubation, the fluorescent signal from the reaction plate was measured at excitation (490 nm) and emission (520 nm) in an EnVision multilabel microplate reader (PerkinElmer, Waltham, MA). Relative fluorescence unit (RFU) data are plotted against the test molecule concentration tested in GraphPad Prism 5.0 (GraphPad, San Diego, Calif.) to estimate the half-maximal inhibition constant (IC50).

[0113] For MMP9 activity measurements, the catalytic domain of human MMP9 (Anaspec, Fremont, CA) is incubated with the MMP9-sensitive 5-FAM / QXL 520 FRET peptide and various concentrations of test molecules in black 384-well Optiplates (PerkinElmer, Waltham, MA) at 37°C. After 2 hours of incubation, the fluorescence signal is measured at excitation (490 nm) and emission (520 nm) in an EnVision multilabel microplate reader (PerkinElmer, Waltham, MA). The relative fluorescence unit (RFU) data are plotted against the test molecule concentration tested in GraphPad Prism 5.0 (GraphPad, San Diego, CA) to estimate the half-maximal inhibition constant (IC50).

[0114] For MMP13 activity, test molecules are titrated in assay buffer (20 mM Tris, 10 mM CaCl, 10 μM ZnCl, 0.01% Bridge 35 (Calbiochem / EMD, San Diego, CA), pH 7.5) and added to black polystyrene 96- or 384-well assay plates (Griener Bio-One, Germany). Active MMP13 (Calbiochem / EMD) is diluted in assay buffer and added to the test molecule titration at a final concentration of 50 μL for 10 minutes at room temperature. Alternatively, latent MMP-13 (R&D Systems, Minneapolis, MN) is activated with APMA for 2 hours at 37°C and used in the assay. A fluorogenic substrate, such as Mca-PLGL-Dpa-AR-NH2 fluorogenic MMP substrate or Mca-KPLGL-Dpa-AR-NH2 fluorogenic peptide substrate (R&D Systems), is prepared and added to the MMP-13 enzyme / huTIMP-3 / test molecule solution. MMP-13 activity is measured kinetically, for example, for 20 minutes, using a Molecular Devices fluorescent plate reader (or equivalent).

[0115] The effect of the molecule being tested can be expressed as a percentage of the maximum TIMP-3 inhibition expected for MMP enzymatic activity. Alternatively, quantitative assessment of MMP inhibitory activity may not be necessary; rather, individual test molecules can be evaluated for their ability to inhibit MMPs. Those skilled in the art will recognize that the parameters outlined herein can be varied through the application of routine experimentation. For example, preliminary experiments can be performed using previously tested TIMP-3 and other materials to determine appropriate concentrations of MMPs or latent MMPs. Similarly, the type and appropriate concentration of substrates can be determined. Thus, for example, MMPs can be titrated and compared with previously tested batches of MMPs to optimize assay parameters. Additionally, those skilled in the art can use similar assays to evaluate the effect, if any, of TIMP-3 muteins or variants on the ability to inhibit other MMPs, or various TIMP-3 mutations.

[0116] Example 4 Using standard molecular biology techniques, nucleic acids encoding a number of TIMP-3 mutant proteins were prepared and expressed in mammalian cells essentially as described above. The effect of mutations on the expression of the encoded TIMP-3 mutant proteins was evaluated. The list of mutations made includes the following: G115T、N118D;K45E、K49S;K45E、K49E;K45E、T63E;K45E、Q80E;T63E、H78E;K45E、T63E、H78E;T63E、H78E、Q80E;K45E、T63E、H78E、Q80E;T63E、H78D;T63E、T74E、H78E;T63E、T74E、H78D;L51T、T74E、H78D;T74E、H78E、Q80E;T74E、H78D、Q80E;R43T、T74E、H78D、Q80E;R43E、T74E、H78D、Q80E;R43N、K45T;K45N、V47T;K49N、L51T;K65N、M67T;K75N、P77T;R43N、K45T、K49N、L51T;K45N、V47T、K49N、L51T;R43N、K45T、T63E、T74E、H78E;K45N、V47T、T63E、T74E、H78E;K49N、L51T、T63E、T74E、H78E;K45E、K49N、L51T、T63E;R43T、K49N、L51T、T74E、H78D;R43N、K45T、T74E、H78E;K49N、L51T、T74E、H78E;R43N、K45T、K49N、L51T、T74E、H78E;Q32N、A34T;S38D、D39T;R43N、K45T;V47N、K49T;K49N、L51T;K50N、V52T;L51N、K53T;F57N;P56N、G58T;T63N、K65T;P56N、G58T、T63N、K65T;M67N、M69T;H78N、Q80T;T84N、A86T;K94N、E96T;E96N、N98T;V97N、K99T;K99N、Q101T;T105N、R107T;D110N、K112T;E122N、W124T;R123N、D125T;Q126N;T128N;Q131N、K133T;R132N G134T;R138N、H140T;R138T;H140N、G142T;K142T;K146N、K148T;T158N、K160T;T166N、M168T;M168N;G173T;HS179N、H181T;H181N、A183T;R186N、K188T;R196N、W198T;P200N、D202T;P201N、K203T;D202N;A208Y;A208V;K45S、F57N;K49S、F57N;K68S、F57N;K133S, F57N; K45S, K133S, F57N; and K49S, K68S, F57N. ;

[0117] Further evaluation of the expressed muteins was performed and is described below. Additional muteins are contemplated, including: K49E, K50E, K53E, K99E, R186Q, K188Q; K49S, K50N / V52T, K53E, V97N / K99T, R186N / K188T; K50N / V52T, V97N / K99T, R186N / K188T; K49E, K53E, K 188Q;K50N / V52T, R186N / K188T;K50N / V52T, F57N, R186N / K188T;K45S, K50N / V52T, F5 7N, R186N / K188T; K50N / V52T, F57N, T63N / K65T, R186N / K188T; K45S, K50N / V52T, F57N R186N / K188T; K45S, K49S, K50N / V52T, F57N R186N / K188T; K49S, K50N / V52T, F57N, V97N / K99T, R186N / K188T; K45S, K50N / V52T, F57N, V97N / K99T, R186N / K188T. These mutant proteins can be made and tested as described herein.

[0118] Example 5 This table summarizes the expression and MMP inhibition results obtained for a number of TIMP-3 mutant proteins actually expressed in mammalian cells. For "Mammalian Expression vs. WT," the data are recorded as "+" indicating that expression was substantially the same as that of wild-type (i.e., native) TIMP-3; "++" indicating that expression was increased 2- to 4-fold over that observed with wild-type TIMP-3; or "+++" indicating that expression was increased more than 4-fold over wild-type TIMP-3. The symbol "---" in the enzyme inhibition column indicates that such testing was not performed. Increased expression levels, illustrating fold-increases in expression compared to that observed for wild-type TIMP-3, are determined qualitatively using Western blots or SDS-PAGE Coomassie-stained gels, or through measurement of expression titers measured with a ForteBio Octet® readout, which captures TIMP-3 using anti-TIMP-3 antibodies (such antibodies are commercially available, for example, from EMD Millipore, Billerica, MA; AbCam®, Cambridge, MA; or R&D Systems, Minneapolis, MN). [Table 1] TIFF2026004409000002.tif193164

[0119] Certain of these mutations showed increased expression in mammalian cells compared to wild-type TIMP-3: T63E, T74E, H78E; T63E, T74E, H78D; K65N, M67T; K45N, V47T, T63E, T74E, H78E; K49N, L51T, T63E, T74E, H78E; K49N, L51T, T74E, H78E; K49N, L51T; K50N, V52T; L51N, K 53T; T63N, K65T; K75N, P77T; H78N, Q80T; K94N, E96T; D110N, K112T; Q126N; R138T; G173T; F57N; P56N, G58T; P56N, G58T; T63N, K65T; K45S, F57N; K49S, F57N; K68S, F57N; K133S, F57N; K45S, K133S, F57N; and K49S, K68S, F57N. Of these, a subset (F57N; P56N, G58T; P56N, G58T; T63N, K65T; K45S, F57N; K49S, F57N; K68S, F57N; K133S, F57N; K45S, K133S, F57N; and K49S, K68S, F57N) was expressed at levels more than fourfold higher than those observed with wild-type TIMP-3.

[0120] A detailed comparison of the MMP activity results for some of the mutant proteins and wild-type TIMP-3 (WT) was performed and these results are presented below. [Table 2]

[0121] Example 6 This example describes an assay to assess the ability of TIMP-3 protein to bind to HTB-94™ cells (a chondrocyte cell line commercially available from the American Type Culture Collection, Manassas, VA) by fluorescence-activated cell sorter (FACS) analysis. HTB-94 cells are cultured in HTB-94 medium (high-glucose DMEM containing 10% fetal bovine serum [FBS] and 2 mM L-glutamine) at 37°C under 5% CO2. Cells are grown at 2.5 x 10 in standard cell culture flasks 6-12 weeks prior to staining. 4 HTB-94 cells are seeded at a cell density of 100,000 cells / ml and subcultured every 3-4 days after removal from the flask by trypsinization. Approximately 16 hours before FACS staining, HTB-94 cells are seeded at 100,000 cells per well in 2 ml of HTB94 medium in a standard tissue culture 12-well plate and incubated at 37°C with 5% CO2. Cells are 80-90% confluent before staining.

[0122] After approximately 16 hours, the HTB94 medium was removed from the 12-well plate by aspiration, and 1 ml of 4C staining buffer (phosphate-buffered saline [PBS] 2% FBS 0.15% NaN3) was applied per well. The cell plate was incubated on ice for 1 hour. The staining buffer was aspirated, and 0.9 ml / well of TIMP-3 HIS-Myc-tagged protein (either native TIMP-3 or a TIMP-3 mutant) diluted to 80 micrograms / ml in staining buffer was added, with the same volume of buffer alone added to negative control wells. The cell plate was incubated on ice for 30 minutes, aspirated, and washed twice with 1 ml / well of staining buffer. After the second wash, the buffer was aspirated, and 0.9 ml / well of mouse anti-penta-HIS AlexaFluor 488-conjugated antibody (Qiagen, Valencia, CA) diluted to 20 μg / ml in staining buffer was added. In parallel, an irrelevant mIgG1 AlexaFluor488-conjugated antibody (eBioscience, San Diego, CA) negative control staining reagent diluted to 20 micrograms / ml in staining buffer is added in parallel to replicate wells stained with the known binder TIMP3 HIS-Myc (e.g., K45S, F57N, SEQ ID NO: 23).

[0123] The cell plate is incubated on ice for 30 minutes protected from light, aspirated, and washed twice with 1 ml / well of well staining buffer. After the second wash, the buffer is aspirated and 1 ml of cell dissociation buffer (enzyme-free, PBS, catalog number 13151-014; Life Technologies, Grand Island, NY) is added per well. The cell plate is incubated at 37°C for 5 minutes, and the cells are transferred to a 4 ml FACS tube. The plate wells are rinsed with 1 ml / well of 25C PBS, and the rinse is added to the corresponding FACS tube containing the cells in cell dissociation buffer. The tubes are centrifuged at 1000 RPM for 5 minutes to form a cell pellet, which is aspirated. The cells are resuspended in 300 microliters of 4% paraformaldehyde in PBS (PFA) and may be stored at 4°C protected from light until subjected to FACS.

[0124] Within two days of TIMP3 staining, 8000 fixed HTB94 cell events are acquired using, for example, FL1 on a Becton Dickinson FACS Calibur to detect AlexaFluor 488 fluorescence. The forward scatter (FSC) detector voltage is set at E00, and the side scatter (SSC) detector voltage is set at 316. These detectors are used in combination to measure light reflected from the cells as "forward scatter" and "side scatter," which allows the definition of an HTB-94 cell gate, also known as "gating," and allows separation of cells from non-cellular material in the tube based on their size and granularity. The FL1 detector voltage is set at 370. Analysis is performed using, for example, FlowJo vX.0.6.

[0125] Several TIMP-3 variants were analyzed for binding to HTB-94 cells in this manner, and the results for two separate experiments are shown in Table 3 below (na = not applicable; nd = not performed). Nine TIMP3 HIS-Myc-tagged glycovariants showed no binding to HTB-94 cells, in that no FL1 signal was detected over background in the described method. The results are shown in Table 3 below.

[0126] Example 7 This table summarizes the expression and MMP inhibition results obtained with a number of TIMP-3 mutant proteins that were actually expressed in mammalian cells. [Table 3] TIFF2026004409000005.tif234160TIFF2026004409000006.tif226158TIFF2026004409000007.tif156158

[0127] This table summarizes the glycosylation sites and other properties of a number of TIMP-3 muteins expressed in mammalian cells.

[0128] TIFF2026004409000008.tif230153TIFF2026004409000009.tif225156TIFF2026004409000010.tif213156

[0129] Additional muteins are contemplated, including K45S, F57N, D110N, K112T; K45S, F57N, H78N, Q80T, D110N, K112T; K45S, F57N, H78N, Q80T, D110N, K112T, Q126N; K45S, F57N, H78N, Q80T, K94N, E96T Q126N;K45S, F57N, H78N, Q80T, Q126N, G173T;K45S, F57N, T63N, K65T;K45S, F57N, T63 N, K65T, K94N, E96T; K45S, F57N, T63N, K65T, R138T, G173T; K45N, V47T, F57N, T63N, K65 T, R138T, G173T; K45S, F57N, T63N, K65T, K94N, E96T, R138T; K45N, V47T, F57N, T63N, K6 5T, K94N, E96T, R138T; K45S, F57N, Q126N, R138T, G173T; P56N, G58T, T63N, K65T, K94N, E96T, Q126N, G173T; P56N, G58T, T63N, K65T, D110N, K112T, Q126N, G173T; K49S, K50N, V 52T, K53E, V97N, K99T, R186N, K188T; K50N, V52T, V97N, K99T, R186N, K188T; K49E, K53E , K188Q; K50N, V52T, R186N, K188T; K50N, V52T, F57N, R186N, K188T; K45S, K50N, V52T, F 57N, R186N, K188T; K50N, V52T, F57N, T63N, K65T, R186N, K188T; K45S, K50N, V52T, F57N R186N, K188T; K45S, K49S, K50N, V52T, F57N R186N, K188T; K49S, K50N, V52T, F57N, V97N, K99T, R186N, K188T; K45S, K50N, V52T, F57N, V97N, K99T, R186N, K188T; K45E, K50N, V52T, D110 N, K112T, R138T, G173T, K188E; K45E, F57N, D110N, K112T, R138T, G173T, K188E; K45E, K50N, V52T, K94N, E96T, D110N, K112T, G173T, K188E;K45E、F57N、K94N、E96T、D110N、K112T、G173T、K188E;K45E、K50N、V52T、D110N、K112T、R138T、G173T、R186N、K188T;K45E、F57N、D110N、K112T、R138T、G173T、R186N、K188T;K45E、K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K45E、F57N、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K45E、K50N、V52T、D110N、K112T、R138T、G173T、R186Q、K188Q;K45E、F57N、D110N、K112T、R138T、G173T、R186Q、K188Q;K45E、K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K45E、F57N、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K45E、K50N、V52T、D110N、K112T、R138T、K188E;K45E、F57N、D110N、K112T、R138T、K188E;K45E、K50N、V52T、K94N、E96T、D110N、K112T、K188E;K45E、F57N、K94N、E96T、D110N、K112T、K188E;K45E、K50N、V52T、D110N、K112T、R138T、R186N、K188T;K45E、F57N、D110N、K112T、R138T、R186N、K188T;K45E、K50N、V52T、K94N、E96T、D110N、K112T、R186N、K188T;K45E、F57N、K94N、E96T、D110N、K112T、R186N、K188T;K45E、K50N、V52T、D110N、K112T、R138T、R186Q、K188Q;K45E、F57N、D110N、K112T、R138T、R186Q、K188Q;K45E、K50N、V52T、K94N、E96T、D110N、K112T、R186Q、K188Q;K45E、F57N、K94N、E96T、D110N、K112T、R186Q、K188Q;K50N、V52T、D110N、K112T、R138T、G173T、K188E;K45S、F57N、D110N、K112T、R138T、G173T、K188E;K50N、V52T、K94N、E96T、D110N、K112T、G173T、K188E;K45S、F57N、K94N、E96T、D110N、K112T、G173T、K188E;K50N、V52T、D110N、K112T、R138T、G173T、R186N、K188T;K45S、F57N、D110N、K112T、R138T、G173T、R186N、K188T;K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K45S、F57N、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K50N、V52T、D110N、K112T、R138T、G173T、R186Q、K188Q;K45S、F57N、D110N、K112T、R138T、G173T、R186Q、K188Q;K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K45S、F57N、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K50N、V52T、D110N、K112T、R138T、K188E;K45S、F57N、D110N、K112T、R138T、K188E;K50N、V52T、K94N、E96T、D110N、K112T、K188E;K45S、F57N、K94N、E96T、D110N、K112T、K188E;K50N、V52T、D110N、K112T、R138T、R186N、K188T;K45S、F57N、D110N、K112T、R138T、R186N、K188T;K50N、V52T、K94N、E96T、D110N、K112T、R186N、K188T;K45S、F57N、K94N、E96T、D110N、K112T、R186N、K188T;K50N、V52T、D110N、K112T、R138T、R186Q、K188Q;K45S、F57N、D110N、K112T、R138T、R186Q、K188Q;K50N、V52T、K94N、E96T、D110N、K112T、R186Q、K188Q;K45S、F57N、K94N、E96T、D110N、K112T、R186Q、K188Q;K50N、V52T、K94N、E96T、D110N、K112T、R138T、G173T;K50N、V52T、K94N、E96T、R138T、G173T;K45E、F57N、K94N、E96T、D110N、K112T、R138T、G173T;K45E、F57N、K94N、E96T、R138T、G173T;K45S、F57N、K94N、E96T、D110N、K112T、R138T、G173T;K45S、F57N、K94N、E96T、R138T、G173T;K45N、V47T、H78N、Q80T、Q126N、R186Q、K188Q;K45N、V47T、F57N、H78N、Q80T、Q126N、R186Q、K188Q;K45N、V47T、F57N、H78N、Q80T、K94N、E96T、Q126N、;K45N、V47T、F57N、H78N、Q80T、Q126N、R138T;K45N、V47T、F57N、H78N、Q80T、R138T、R186Q、K188Q;K45N、V47T、F57N、H78N、Q80T、K94N、E96T、D110N、K112T、R186Q、K188Q;K50N、V52T、K94N、E96T、H78N、Q80T、R138T;K50N、V52T、K94N、E96T、H78N、Q80T、R138T、R186Q、K188Q;K45E、F57N、Q126N、R138T、R186Q、K188Q;K45N、V47T、F57N、Q126N、R138T、R186Q、K188Q;K45N、V47T、F57N、H78N、Q80T、R186Q、K188Q;K45S、F57N、H78N、Q80T、Q126N、R138T、R186Q、K188Q;K45S、F57N、H78N、Q80T、K94N、E96T、R138T、R186Q、K188Q;K50N、V52T、K94N、E96T、H78N、Q80T、R138T、;K45N、V47T、F57N、K94N、E96T、D110N、K112T、R186Q、K188Q;K45S、F57N、H78N、Q80T、K94N、E96T、R138T;K45N、V47T、F57N、K94N、E96T、D110N、K112T、R186Q;and K45N, V47T, F57N, K94N, E96T, D110N, K112T, K188Q. Further muteins include K50N, V52T, P56N, G58T, R186N, K188T; K45S, K50N, V52T, P56N, G58T, R186N, K188T; K50N, V52T, P56N, G58T, T63N, K65T, R186N, K188T; K45S, K50N, V52T, P56N, G58T R186N, K188T; K45S, K49S, K50N, V52T, P56N, G58T R186N, K188T; K49S, K50N, V52T, P56N, G58T, V97N, K99T, R186N, K188T; K45S, K50N, V52T, P56N, G58T, V97N, K9 9T, R186N, K188T; K45E, P56N, G58T, D110N, K112T, R138T, G173T, K188E; K45E, P56N, G58T, K94N, E96T, D110N, K 112T, G173T, K188E; K45E, K50N, V52T, D110N, K112T, R138T, G173T, R186N, K188T; K45E, P56N, G58T, D110N, K1 12T, R138T, G173T, R186N, K188T; K45E, P56N, G58T, K94N, E96T, D110N, K112T, G173T, R186N, K188T; K45E, P56N , G58T, D110N, K112T, R138T, G173T, R186Q, K188Q; K45E, P56N, G58T, K94N, E96T, D110N, K112T, G173T, R186Q, K188Q;K45E, P56N, G58T, D110N, K112T, R138T, K188E;K45E, P56N, G58T, K94N, E96T, D110N, K112T, K188E;K45E , P56N, G58T, D110N, K112T, R138T, R186N, K188T; K45E, P56N, G58T, K94N, E96T, D110N, K112T, R186N, K188T; K4 5E, P56N, G58T, D110N, K112T, R138T, R186Q, K188Q; K45E, P56N, G58T, K94N, E96T, D110N, K112T, R186Q, K188Q;K45S、P56N、G58T、D110N、K112T、R138T、G173T、K188E;K45S、P56N、G58T、K94N、E96T、D110N、K112T、G173T、K188E;K45S、P56N、G58T、D110N、K112T、R138T、G173T、R186N、K188T;K45S、P56N、G58T、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K45S、P56N、G58T、D110N、K112; T、R138T、G173T、R186Q、K188Q;K45S、P56N、G58T、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K45S、P56N、G58T、D110N、K112T、R138T、K188E;K45S、P56N、G58T、K94N、E96T、D110N、K112T、K188E;K45S、P56N、G58T、D110N、K112T、R138T、R186N、K188T;K45S、P56N、G58T、K94N、E96T、D110N、K112T、R186N、K188T;K45S、P56N、G58T、D110N、K112T、R138T、R186Q、K188Q;K45S、P56N、G58T、K94N、E96T、D110N、K112T、R186Q、K188Q;K45E、P56N、G58T、K94N、E96T、D110N、K112T、R138T、G173T;K45E、P56N、G58T、K94N、E96T、R138T、G173T;K45S、P56N、G58T、K94N、E96T、D110N、K112T、R138T、G173T;K45S、P56N、G58T、K94N、E96T、R138T、G173T;K45N、V47T、P56N、G58T、H78N、Q80T、Q126N、R186Q、K188Q;K45N、V47T、P56N、G58T、H78N、Q80T、K94N、E96T、Q126N、;K45N、V47T、P56N、G58T、H78N、Q80T、Q126N、R138T;K45N、V47T、P56N、G58T、H78N、Q80T、R138T、R186Q、K188Q;K45N、V47T、P56N、G58T、H78N、Q80T、K94N、E96T、D110N、K112T、R186Q、K188Q;K45E、P56N、G58T、Q126N、R138T、R186Q、K188Q;K45N、V47T、P56N、G58T、Q126N、R138T、R186Q、K188Q;K45N、V47T、P56N、G58T、H78N、Q80T、R186Q、K188Q;K45S、P56N、G58T、H78N、Q80T、Q126N、R138T、R186Q、K188Q;K45S、P56N、G58T、H78N、Q80T、K94N、E96T、R138T、R186Q、K188Q;These mutant proteins include K45N, V47T, P56N, G58T, K94N, E96T, D110N, K112T, R186Q, K188Q; K45S, P56N, G58T, H78N, Q80T, K94N, E96T, R138T; K45N, V47T, P56N, G58T, K94N, E96T, D110N, K112T, R186Q; and K45N, V47T, P56N, G58T, K94N, E96T, D110N, K112T, K188Q. These mutant proteins can be made and tested as described herein.

Claims

1. a mature region that is at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2, and that has at least one mutation, wherein the mutation is K45E; K45N; K45S; V47T; K49N; K49E; K49S; K50N; L51T; L51N; V52T; K53T; P56N; F57N; G58T; T63E; T63N; K65T; K65N; M67T; K68S; T74E; K75N; P77T; H78D; H78E; H78N; Q80E; Q80T; K94 An isolated TIMP-3 mutant protein selected from the group consisting of N; E96T; E96N; V97N; N98T; K99T; D110N; K112T; Q126N; K133S; R138T; R138N; H140T; T158N; K160T; T166N; M168T; G173T; H181N; A183T; R186N; R186Q, R186E, K188T; K188Q, K188E, P201N; K203T; I205F, I205Y, A208G, A208V, and A208Y.

2. An isolated TIMP-3 mutant protein having a mature region whose amino acid sequence is at least 95% identical to the mature region of TIMP-3 set forth in SEQ ID NO: 2, and having the mutation F57N and at least one additional mutation, said mutation being a substitution of one or more K residues of TIMP-3.

3. The TIMP-3 mutein of claim 2, wherein the further mutation introduces an N-linked glycosylation site into the amino acid sequence.

4. An isolated TIMP-3 mutant protein having a mature region whose amino acid sequence is at least 90% identical to the mature region of TIMP-3 set forth in SEQ ID NO: 2, said mutant protein having at least one mutation that introduces at least one N-linked glycosylation site into said amino acid sequence.

5. 5. The TIMP-3 mutein of claim 4, wherein 2, 3, 4, 5, or 6 N-linked glycosylation sites have been introduced.

6. The TIMP-3 mutant protein of claim 3, wherein the N-linked glycosylation site is introduced into a region of the TIMP-3 amino acid sequence selected from the group consisting of: a region including amino acids 48 to 54; a region including amino acids 93 to 100; a region including amino acids 121 to 125; a region including amino acids 143 to 152; a region including amino acids 156 to 164; a region including amino acids 183 to 191; and combinations thereof.

7. 7. The TIMP-3 mutein of claim 6, wherein 2, 3, 4, 5 or 6 (or more) N-linked glycosylation sites are introduced.

8. An isolated TIMP-3 mutein having a mature region that is at least 95% identical in amino acid sequence to the mature region of TIMP-3 set forth in SEQ ID NO:2, said mutein comprising (a) one or more mutations in the TIMP-3 charge patch that cause a change in the properties of the exposed positive charge patch on the TIMP-3 surface, mimicking the charge surface of TIMP-1, TIMP-2, or TIMP-4; (b) one or more mutations that reduce susceptibility to proteolytic cleavage; (c) one or more mutations that result in a decrease in the interaction of the TIMP-3 mutein with the scavenger receptor LRP-1; (d) one or more mutations that result in a reduced interaction of the TIMP-3 mutein with heparin or extracellular matrix components; (e) the addition of one or more cysteinyl residues to the native TIMP-3 sequence; (f) improved pharmacokinetic and / or pharmacodynamic properties; (g) one or more mutations that introduce at least one N-linked glycosylation site; and (h) a combination of the mutations shown in (a) to (g); The isolated TIMP-3 mutein has at least one mutation selected from the group consisting of:

9. i. K45E, K49S; (SEQ ID NO: 5) ii. K45E, K49E; (SEQ ID NO: 6) iii. K45E, T63E; (SEQ ID NO: 7) iv. K45E, Q80E; (SEQ ID NO: 8) v. K45E, T63E, H78E; (SEQ ID NO: 10) vi. T63E, H78E, Q80E; (SEQ ID NO: 11) vii. K45E, T63E, H78E, Q80E; (SEQ ID NO: 12) viii. T63E, T74E, H78E; (SEQ ID NO: 13) ix. T63E, T74E, H78D; (SEQ ID NO: 14) x. L51T, T74E, H78D; (SEQ ID NO: 53) xi. T74E, H78E, Q80E; (SEQ ID NO: 16) xii. T74E, H78D, Q80E; (SEQ ID NO: 17) xiii. K45N, V47T; (SEQ ID NO: 26) xiv. K65N, M67T; (SEQ ID NO: 37) xv. K45N, V47T, T63E, T74E, H78E; (SEQ ID NO: 18) xvi. K49N, L51T, T63E, T74E, H78E; (SEQ ID NO: 19) xvii. K45E, K49N, L51T, T63E; (SEQ ID NO: 20) xviii. K49N, L51T, T74E, H78E; (SEQ ID NO: 21) xix. K49N, L51T; (SEQ ID NO: 27) xx. K50N, V52T; (SEQ ID NO: 30) xxi. L51N, K53T; (SEQ ID NO: 54) xxii. F57N; (SEQ ID NO: 33) xxiii. P56N, G58T; (SEQ ID NO: 31) xxiv. T63N, K65T; (SEQ ID NO: 36) xxv. P56N, G58T, T63N, K65T; (SEQ ID NO: 32) xxvi. K75N, P77T; (SEQ ID NO: 38) xxvii. H78N, Q80T; (SEQ ID NO: 39) xxviii. K94N, E96T; (SEQ ID NO: 40) xxix. E96N, N98T; (SEQ ID NO: 41) xxx.V97N,K99T; (SEQ ID NO: 42) xxxi.D110N,K112T; (SEQ ID NO: 43) xxxii.Q126N; (SEQ ID NO:44) xxxiii. R138N, H140T; (SEQ ID NO: 46) xxxiv.R138T; (SEQ ID NO:45) xxxv.T158N, K160T; (SEQ ID NO: 47) xxxvi. T166N, M168T; (SEQ ID NO: 48) xxxvii. G173T; (SEQ ID NO: 49) xxxviii. H181N, A183T; (SEQ ID NO: 50) xxxix. R186N, K188T; (SEQ ID NO: 51) xl. P201N, K203T; (SEQ ID NO: 52) xli.A208Y; (SEQ ID NO: 55) xlii. A208V; (SEQ ID NO: 56) xxiii. K45S, F57N; (SEQ ID NO: 23) xliv. K49S, F57N; (SEQ ID NO: 28) xlv. K68S, F57N; (SEQ ID NO: 34) xlvi.K133S,F57N; (SEQ ID NO: 35) xlvii. K45S, K133S, F57N; (SEQ ID NO: 24) xlviii. K49S, K68S, F57N (SEQ ID NO: 29) xlix. K45S, F57N, I205F, A208G (SEQ ID NO: 57) 1. K45S, F57N, A208G (SEQ ID NO: 58) li. K45S, F57N, I205Y (SEQ ID NO: 59) lii. K45S, F57N, I205Y, A208G (SEQ ID NO: 60) liii. K45N, V47T, F57N, K75N, P77T, K94N, E96T, R138T, G173T (SEQ ID NO: 61) liv. K45N, V47T, F57N, K94N, E96T, R138T, G173T (SEQ ID NO: 62): lv. K45N, V47T, K50N, V52T, F57N, V97N, K99T (SEQ ID NO: 63) K45S, K50N, V52T, F57N, V97N, K99T, R186N, K188T (SEQ ID NO: 64) lvii. K45S, F57N, K94N, E96T, D110N, K112T, R138T, G173T (SEQ ID NO: 65) lviii. K45S, F57N, T63N, K65T, K94N, E96T, G173T (SEQ ID NO: 66) K45N, V47T, K50N, V52T, F57N, V97N, K99T, R138T, R186N, K188T (SEQ ID NO: 67) lx. K45S, F57N, T63N, K65T, K94N, E96T, Q126N, R138T (SEQ ID NO: 68) lxi. K45N, V47T, K50N, V52T, F57N, V97N, K99T, R186N, K188T (SEQ ID NO: 69) lxii. K45N, V47T, K50N, V52T, V97N, K99T, R138T, R186N, K188T (SEQ ID NO: 70) lxiii. K45S, F57N, H78N, Q80T, K94N, E96T, R138T, G173T (SEQ ID NO: 71) 1xiv. K45S, F57N, K75N, P77T, K94N, E96T, R138T, G173T (SEQ ID NO: 72) K45N, V47T, K50N, V52T, V97N, K99T, G173T, R186N, K188T (SEQ ID NO: 73) lxvi. K45E, F57N, Q126N, R138T, G173T (SEQ ID NO: 74) lxvii. K45S, F57N, T63N, K65T, K94N, E96T, R138T, G173T (SEQ ID NO: 75) lxviii. K45S, K50N, V52T, F57N, V97N, K99T, R138T, R186N, K188T (SEQ ID NO: 76) lxix.K45S.K50N, V52T, F57N, V97N, K99T, G173T, R186N, K188T (SEQ ID NO: 77) lxx. K45N, V47T, F57N, K94N, E96T, G173T, R186N, K188T (SEQ ID NO: 78) K45N, V47T, F57N, K94N, E96T, D110N, K112T, R186N, K188T (SEQ ID NO: 79) lxxii. K45N, V47T, F57N, V97N, K99T, R138T, G173T (SEQ ID NO: 80) lxxiii. K45N, V47T, F57N, K99E G173T, R186N, K188T (SEQ ID NO: 81) lxxiv. K45E, K49E, F57N, K94N, E96T, D110N, K112T, G173T, R186N, K188T (SEQ ID NO: 82) K50N, V52T, K94N, E96T, R138T, G173T (SEQ ID NO: 83) lxxvi. K45E, K50N, V52T, K94N, E96T, D110N, K112T, R138T, G173T (SEQ ID NO: 84) lxxvii. K50N, V52T, K94N, E96T, R138T, G173T, R186N, K188T (SEQ ID NO: 85) lxxviii. K45E, F57N, T63N, K65T, K94N, E96T, G173T, R186N, K188T (SEQ ID NO: 86) K45N, V47T, F57N, K94N, E96T, D110N, K112T, G173T, R186Q, K188Q (SEQ ID NO: 87) lxxx. K45S F57N K94N, E96T R138T G173T (SEQ ID NO: 88) lxxxi. K45E F57N K94N, E96T R138T G173T (SEQ ID NO: 89) lxxxii. K45E F57N K94N, E96T D110N, K112T R138T G173T (SEQ ID NO: 90) lxxxiii. K45E F57N K94N, E96T R138T G173T R186Q, K188Q (SEQ ID NO: 91) lxxxiv. K45E F57N K94N, E96T R138T G173T R186E (SEQ ID NO: 92) lxxxv. K45E F57N K94N, E96T R138T G173T K188E (SEQ ID NO: 93) lxxxvi. K45E F57N K94N, E96T R138T G173T R186N, K188T (SEQ ID NO: 94) lxxxvii. K45E K50N, V52T K94N, E96T D110N, K112T R138T G173T (SEQ ID NO: 95) lxxxviii. K45E K50N, V52T K94N, E96T R138T G173T K188E (SEQ ID NO: 96) lxxxix. K50N, V52T F57N K94N, E96T R138T G173T (SEQ ID NO: 97) xc. K50N, V52T F57N K94N, E96T D110N, K112T R138T (SEQ ID NO: 98), and xci. K45E F57N K94N, E96T D110N, K112T R138T (SEQ ID NO: 99) The TIMP-3 mutant protein of claim 8, selected from the group consisting of:

10. K49E、K50E、K53E、K99E、R186Q、K188Q;K49S、K50N / V52T、K53E、V97N / K99T、R186N / K188T;K50N / V52T、V97N / K99T、R186N / K188T;K49E、K53E、K188Q;K50N / V52T、R186N / K188T;K50N / V52T、F57N、R186N / K188T;K45S、K50N / V52T、F57N、R186N / K188T;K50N / V52T、F57N、T63N / K65T、R186N / K188T;K45S、K50N / V52T、F57N R186N / K188T;K45S、K49S、K50N / V52T、F57N R186N / K188T;K49S、K50N / V52T、F57N、V97N / K99T、R186N / K188T;K45S、K50N / V52T、F57N、V97N / K99T、R186N / K188T;K45S、F57N、D110N、K112T;K45S、F57N、H78N、Q80T、D110N、K112T;K45S、F57N、H78N、Q80T、D110N、K112T、Q126N;K45S、F57N、H78N、Q80T、K94N、E96T Q126N;K45S、F57N、H78N、Q80T、Q126N、G173T;K45S、F57N、T63N、K65T;K45S、F57N、T63N、K65T、K94N、E96T;K45S、F57N、T63N、K65T、R138T、G173T;K45N、V47T、F57N、T63N、K65T、R138T、G173T;K45S、F57N、T63N、K65T、K94N、E96T、R138T;K45N、V47T、F57N、T63N、K65T、K94N、E96T、R138T;K45S、F57N、Q126N、R138T、G173T;P56N、G58T、T63N、K65T、K94N、E96T、Q126N、G173T;P56N、G58T、T63N、K65T、D110N、K112T、Q126N、G173T;K49S、K50N、V52T、K53E、V97N、K99T、R186N、K188T;K50N、V52T、V97N、K99T、R186N、K188T;K49E、K53E、K188Q;K50N、V52T、R186N、K188T;K50N、V52T、F57N、R186N、K188T;K45S、K50N、V52T、F57N、R186N、K188T;K50N、V52T、F57N、T63N、K65T、R186N、K188T;K45S、K50N、V52T、F57N R186N、K188T;K45S、K49S、K50N、V52T、F57N R186N、K188T;K49S、K50N、V52T、F57N、V97N、K99T、R186N、K188T;K45S、K50N、V52T、F57N、V97N、K99T、R186N、K188T;K45E、K50N、V52T、D110N、K112T、R138T、G173T、K188E;K45E、F57N、D110N、K112T、R138T、G173T、K188E;K45E、K50N、V52T、K94N、E96T、D110N、K112T、G173T、K188E;K45E、F57N、K94N、E96T、D110N、K112T、G173T、K188E;K45E、K50N、V52T、D110N、K112T、R138T、G173T、R186N、K188T;K45E、F57N、D110N、K112T、R138T、G173T、R186N、K188T;K45E、K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K45E、F57N、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K45E、K50N、V52T、D110N、K112T、R138T、G173T、R186Q、K188Q;K45E、F57N、D110N、K112T、R138T、G173T、R186Q、K188Q;K45E、K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K45E、F57N、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K45E、K50N、V52T、D110N、K112T、R138T、K188E;K45E、F57N、D110N、K112T、R138T、K188E;K45E、K50N、V52T、K94N、E96T、D110N、K112T、K188E;K45E、F57N、K94N、E96T、D110N、K112T、K188E;K45E、K50N、V52T、D110N、K112T、R138T、R186N、K188T;K45E、F57N、D110N、K112T、R138T、R186N、K188T;K45E、K50N、V52T、K94N、E96T、D110N、K112T、R186N、K188T;K45E、F57N、K94N、E96T、D110N、K112T、R186N、K188T;K45E、K50N、V52T、D110N、K112T、R138T、R186Q、K188Q;K45E、F57N、D110N、K112T、R138T、R186Q、K188Q;K45E、K50N、V52T、K94N、E96T、D110N、K112T、R186Q、K188Q;K45E、F57N、K94N、E96T、D110N、K112T、R186Q、K188Q;K50N、V52T、D110N、K112T、R138T、G173T、K188E;K45S、F57N、D110N、K112T、R138T、G173T、K188E;K50N、V52T、K94N、E96T、D110N、K112T、G173T、K188E;K45S、F57N、K94N、E96T、D110N、K112T、G173T、K188E;K50N、V52T、D110N、K112T、R138T、G173T、R186N、K188T;K45S、F57N、D110N、K112T、R138T、G173T、R186N、K188T;K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K45S、F57N、K94N、E96T、D110N、K112T、G173T、R186N、K188T;K50N、V52T、D110N、K112T、R138T、G173T、R186Q、K188Q;K45S、F57N、D110N、K112T、R138T、G173T、R186Q、K188Q;K50N、V52T、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K45S、F57N、K94N、E96T、D110N、K112T、G173T、R186Q、K188Q;K50N、V52T、D110N、K112T、R138T、K188E;K45S、F57N、D110N、K112T、R138T、K188E;K50N、V52T、K94N、E96T、D110N、K112T、K188E;K45S、F57N、K94N、E96T、D110N、K112T、K188E;K50N、V52T、D110N、K112T、R138T、R186N、K188T;K45S、F57N、D110N、K112T、R138T、R186N、K188T;K50N、V52T、K94N、E96T、D110N、K112T、R186N、K188T;K45S、F57N、K94N、E96T、D110N、K112T、R186N、K188T;K50N、V52T、D110N、K112T、R138T、R186Q、K188Q;K45S、F57N、D110N、K112T、R138T、R186Q、K188Q;K50N、V52T、K94N、E96T、D110N、K112T、R186Q、K188Q;K45S、F57N、K94N、E96T、D110N、K112T、R186Q、K188Q;K50N、V52T、K94N、E96T、D110N、K112T、R138T、G173T;K50N、V52T、K94N、E96T、R138T、G173T;K45E、F57N、K94N、E96T、D110N、K112T、R138T、G173T;K45E、F57N、K94N、E96T、R138T、G173T;K45S、F57N、K94N、E96T、D110N、K112T、R138T、G173T;K45S、F57N、K94N、E96T、R138T、G173T;K45N、V47T、、H78N、Q80T、Q126N、R186Q、K188Q;K45N、V47T、F57N、H78N、Q80T、Q126N、R186Q、K188Q;K45N、V47T、F57N、H78N、Q80T、K94N、E96T、Q126N、;K45N、V47T、F57N、H78N、Q80T、Q126N、R138T;K45N、V47T、F57N、H78N、Q80T、R138T、R186Q、K188Q;K45N、V47T、F57N、H78N、Q80T、K94N、E96T、D110N、K112T、R186Q、K188Q;K50N、V52T、K94N、E96T、H78N、Q80T、R138T;K50N、V52T、K94N、E96T、H78N、Q80T、R138T、R186Q、K188Q;K45E, F57N, Q126N, R138T, R186Q, K188Q; K45N, V47T, F57N, Q126N, R138T, R186Q, K188Q; K45N, V47T, F57N , H78N, Q80T, R186Q, K188Q; K45S, F57N, H78N, Q80T, Q126N, R138T, R186Q, K188Q; K45S, F57N, H78N, Q80T, K 94N, E96T, R138T, R186Q, K188Q; K50N, V52T, K94N, E96T, H78N, Q80T, R138T; K45N, V47T, F57N, K94N, E96T, D110N, K112T, R186Q, K188Q; and K45S, F57N, H78N, Q80T, K94N, E96T, R138T; K45N, V47T, F57N, K94N, E96T; The TIMP-3 mutant protein of claim 8, selected from the group consisting of:

11. An isolated nucleic acid encoding a TIMP-3 mutein according to any one of claims 1 to 10.

12. An expression vector comprising the isolated nucleic acid of claim 11.

13. An isolated host cell transformed or transfected with the expression vector of claim 12.

14. A method for producing a recombinant TIMP-3 mutant protein, comprising culturing the transformed or transfected host cell of claim 13 under conditions that promote expression of the TIMP-3 mutant protein, and recovering the TIMP-3 mutant protein.

15. A composition comprising a TIMP-3 mutant protein according to any one of claims 1 to 10 and a physiologically acceptable diluent, excipient or carrier.

16. A method for treating a condition in which matrix metalloproteinases (MMPs) and / or other proteinases that are inhibited or can be inhibited by TIMP-3 play a causative or exacerbating role, the method comprising administering to an individual suffering from such a condition an amount of the composition of claim 15 sufficient to treat the condition.

17. 17. The method of claim 16, wherein the condition is selected from the group consisting of an inflammatory condition, osteoarthritis, myocardial ischemia, reperfusion injury, and progression to congestive heart failure.

18. 17. The method of claim 16, wherein the condition is selected from the group consisting of asthma, chronic obstructive pulmonary disease (COPD), and idiopathic pulmonary fibrosis (IPF), inflammatory bowel disease (e.g., ulcerative colitis, Crohn's disease, and celiac disease), psoriasis, myocarditis, including viral myocarditis, inflammation associated with atherosclerosis, and arthritic conditions, including rheumatoid arthritis and psoriatic arthritis.

19. wherein the condition is selected from the group consisting of dystrophic epidermolysis bullosa, osteoarthritis, pseudogout, rheumatoid arthritis including juvenile rheumatoid arthritis, ankylosing spondylitis, periodontal disease, ulcers including corneal ulcers, epidermal ulcers, gastric ulcers, post-surgical wound healing, restenosis, emphysema, Paget's disease of bone, osteoporosis, scleroderma, pressure atrophy of bone or tissue such as pressure ulcers, cholesteatoma, wound healing abnormalities, oligoarticular rheumatoid arthritis, polyarticular rheumatoid arthritis, systemic onset rheumatoid arthritis, ankylosing spondylitis, enteropathic arthritis, reactive arthritis, Reiter's syndrome, SEA syndrome (seronegative, enthesopathy, arthropathic syndrome), dermatomyositis, psoriatic arthritis, scleroderma, Systemic lupus erythematosus, vasculitis, myolitis, polymyolitis, dermatomyolitis, osteoarthritis, polyarteritis nodosa, Wegener's granulomatosis, arteritis, polymyalgia rheumatica, sarcoidosis, sclerosis, primary biliary sclerosis, sclerosing cholangitis, Sjogren's syndrome, psoriasis, plaque psoriasis, guttate psoriasis, psoriasis invertis, pustular psoriasis, erythrodermic psoriasis, dermatitis, atopic dermatitis, atherosclerosis, lupus, Still's disease, systemic lupus erythematosus (SLE) ), myasthenia gravis, inflammatory bowel disease, ulcerative colitis, Crohn's disease, celiac disease (non-tropical sprue), enteropathy associated with seronegative arthropathy, microscopic or collagenous colitis, eosinophilic gastroenteritis, or pouchitis occurring after proctocolectomy and ileoanal anastomosis, pancreatitis, insulin-dependent diabetes mellitus, mastitis, cholecystitis, cholangitis, pericholangitis, multiple sclerosis (MS), asthma (including extrinsic and intrinsic asthma and associated chronic inflammatory states or hyperresponsiveness of the airways), chronic obstructive pulmonary disease (COPD, i.e., chronic bronchitis, emphysema), acute respiratory disease Symptoms of respiratory distress syndrome (ARDS), respiratory distress syndrome, cystic fibrosis, pulmonary hypertension, pulmonary vasoconstriction, acute lung injury, allergic bronchopulmonary aspergillosis, hypersensitivity pneumonitis, eosinophilic pneumonia, bronchitis, allergic bronchitis bronchiectasis, tuberculosis, hypersensitivity pneumonitis, occupational asthma, asthma-like disorders, sarcoid, reactive airway disease (or dysfunction) syndrome, assinosis, interstitial lung disease, hypereosinophilic syndrome, rhinitis, sinusitis, and pulmonary parasitosis, airway hyperresponsiveness associated with virus-induced conditions (e.g., respiratory syncytial virus (RSV), parainfluenza virus (PIV), rhinovirus (RV),and adenovirus), Guillain-Barré disease, Graves' disease, Addison's disease, Raynaud's phenomenon, autoimmune hepatitis, graft-versus-host disease (GVHD), cerebral ischemia, traumatic brain injury, multiple sclerosis, neuropathy, myopathy, spinal cord injury, and amyotrophic lateral sclerosis (ALS).

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