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Title:  Human TIMP-1 antibodies
United States Patent: 
7,091,323
Issued: 
August 15, 2006

Inventors: 
Pan; Clark (Castro Valley, CA), Knorr; Andreas M. (Erkrath, DE), Schauer; Michael (Wuppertal, DE), Hirth-Dietrich; Claudia (Wuppertal, DE), Kraft; Sabine (Planegg, DE), Krebs; Barbara (Bergisch Gladbach, DE)
Assignee: 
Bayer Corporation (Pittsburgh, PA), Bayer Aktiengesellschaft (Leverkusen, DE)
Appl. No.: 
10/128,520
Filed: 
April 24, 2002


 

Pharm Bus Intell & Healthcare Studies


Abstract

Human antibodies that bind to TIMP-1 can be used as reagents to diagnose and treat disorders in which TIMP-1 is elevated, such as liver fibrosis, alcoholic liver disease, cardiac fibrosis, acute coronary syndrome, lupus nephritis, glomerulosclerotic renal disease, benign prostate hypertrophy, colon cancer, lung cancer, and idiopathic pulmonary fibrosis.

DETAILED DESCRIPTION OF THE INVENTION

The invention provides human antibodies that bind to TIMP-1. These antibodies are useful for a variety of therapeutic and diagnostic purposes.

Characteristics of Human TIMP-1 Antibodies

"Antibody" as used herein includes intact immunoglobulin molecules (e.g., IgG.sub.1, IgG.sub.2a, IgG.sub.2b, IgG.sub.3, IgM, IgD, IgE, IgA), as well as fragments thereof, such as Fab, F(ab')2, scFv, and Fv, which are capable of specific binding to an epitope of a human and/or rat TIMP-1 protein. Antibodies that specifically bind to TIMP-1 provide a detection signal at least 5-, 10-, or 20-fold higher than a detection signal provided with other proteins when used in an immunochemical assay. Preferably, antibodies that specifically bind to human and/or rat TIMP-1 do not detect other proteins in immunochemical assays and can immunoprecipitate the TIMP-1 from solution.

The K.sub.d of human antibody binding to TIMP-1 can be assayed using any method known in the art, including technologies such as real-time Bimolecular Interaction Analysis (BIA) (Sjolander & Urbaniczky, Anal. Chem. 63, 2338 45, 1991, and Szabo et al., Curr. Opin. Struct. Biol. 5, 699 705, 1995). BIA is a technology for studying biospecific interactions in real time, without labeling any of the interactants (e.g., BIAcore.TM.). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.

In a BIAcore.TM. assay, some human antibodies of the invention specifically bind to human TIMP-1 with a K.sub.d of about 0.1 nM to about 10 .mu.M, about 2 nM to about 1 .mu.M, about 2 nM to about 200 nM, about 2 nM to about 150 nM, about 50 nM to about 100 nM, about 0.2 nM to about 13 nM, about 0.2 nM to about 0.5 nM, about 2 nM to about 13 nM, and about 0.5 nM to about 2 nM. More preferred human antibodies specifically bind to human TIMP-1 with a K.sub.d selected from the group consisting of about 0.2 nM, about 0.3 nM, about 0.5 M, about 0.6 nM, about 2 nM, about 7 nM, about 10 nM, about 11 nM, and about 13 nM.

Other human antibodies of the invention specifically bind to rat TIMP-1 with a K.sub.d of about 0.1 nM to about 10 .mu.M, about 2 nM to about 1 .mu.M, about 2 nM to about 200 nM, about 2 nM to about 150 nM, about 50 nM to about 100 nM, about 1.3 nM to about 13 nM, about 1.8 nM to about 10 nM, about 2 nM to about 9 nM, about 1.3 nM to about 9 nM, and about 2 nM to about 10 nM. Preferred K.sub.d s range from about 0.8 nM, about 1 nM, about 1.3 nM, about 1.9 nM, about 2 nM, about 3 nM, about 9 nM, about 10 nM, about 13 nM, about 14 nM, and about 15 nM.

Preferably, antibodies of the invention neutralize an MMP-inhibiting activity of the TIMP-1. The MMP can be, for example, MMP-1, MMP-2, MMP-3, MMP-7, MMP-8, MMP-9, MMP-10, MMP-11, MMP-12, MMP-13, MMP-19, MMP-20 or MMP-23.

IC.sub.50 for neutralizing MMP-inhibiting activity of TIMP-1 can be measured by any means known in the art. Preferably, IC.sub.50 is determined using the high throughput fluorogenic assay described in Bickett et al., Anal. Biochem. 212, 58 64, 1993. In a typical fluorogenic assay, the IC.sub.50 of a human antibody for neutralizing human TIMP-1 MMP-inhibiting activity ranges from about 0.1 nM to about 200 nM, about 1 nM to about 100 nM, about 2 nM to about 50 nM, about 5 nM to about 25 nM, about 10 nM to about 15 nM, about 0.2 nM to about 11 nM, about 0.2 nM to about 4 nM, and about 4 nM to about 11 nM. The IC.sub.50 for neutralizing human TIMP-1 MMP-inhibiting activity of some human antibodies is about 0.2 nM, about 0.3 nM, about 0.4 nM, about 4 nM, about 7 nM, about 9 nM, and about 11 nM.

A typical IC.sub.50 for neutralizing rat TIMP-1 MMP-inhibiting activity ranges from about 0.1 nM to about 300 nM, about 1 nM to about 100 nM, about 2 nM to about 50 nM, about 5 nM to about 25 nM, about 10 nM to about 15 nM, about 1.1 nM to about 14 nM, about 1.6 nM to about 11 nM, about 3 nM to about 7 nM, about 1.1 nM to about 7 nM, about 1.1 nM to about 11 nM, about 3 nM to about 11 nM, and about 3 nM to about 14 nM. The IC.sub.50 for neutralizing rat TIMP-1 MMP-inhibiting activity of some human antibodies is about 1.1 nM, about 1.6 nM, about 3 nM, about 7 nM, about 11 nM, about 14 nM, about 19 nM, about 20 nM, about 30 nM, and about 100 nM.

Preferred human antibodies of the invention are those for which the K.sub.d for binding to TIMP-1 and the IC.sub.50 for neutralizing the MMP-inhibiting activity of the TIMP-1 are approximately equal.

A number of human antibodies having the TIMP-1 binding and MMP-inhibiting activity neutralizing characteristics described above have been identified by screening the MorphoSys HuCAL.RTM. Fab 1 library. The CDR cassettes assembled for the HuCAL.RTM. library were designed to achieve a length distribution ranging from 5 to 28 amino acid residues, covering the stretch from position 95 to 102. Knappik et al., J Mol. Biol. 296, 57 86, 2000. Some clones, however, had shorter VHCDR3 regions. In fact, it is a striking feature of anti-human TIMP-1 human antibodies identified from this library that they all exhibit the combination VH312 and a relatively short VHCDR3 region, typically four amino acids.

In some embodiments of the invention, the VHCDR3 region of a human antibody has an amino acid sequence shown in SEQ ID NOS:1 43. In other embodiments of the invention, the VLCDR3 region of a human antibody has an amino acid sequence shown in SEQ ID NOS:44 86. See Tables 2, 3, and 7. Human antibodies which have TIMP-1 binding and MMP-inhibiting activity neutralizing characteristics of antibodies such as those described above and in Tables 2, 3, and 7 also are human antibodies of the invention.

Obtaining Human Antibodies

Human antibodies with the TIMP-1 binding and MMP-activity neutralizing characteristics described above can be identified from the MorphoSys HuCAL.RTM. library as follows. Human or rat TIMP-1, for example, is coated on a microtiter plate and incubated with the MorphoSys HuCAL.RTM. Fab phage library (see Example 1, below). Those phage-linked Fabs not binding to TIMP-1 can be washed away from the plate, leaving only phage which tightly bind to TIMP-1. The bound phage can be eluted, for example, by a change in pH or by elution with E. coli and amplified by infection of E. coli hosts. This panning process can be repeated once or twice to enrich for a population of antibodies that tightly bind to TIMP-1. The Fabs from the enriched pool are then expressed, purified, and screened in an ELISA assay. The identified hits are then screened in the enzymatic assay described in Bickett et al., 1993, and Bodden et al., 1994. Those Fabs that lead to the degradation of the peptide are likely the ones which bind to TIMP-1, thereby blocking its interaction to MMP-1.

The initial panning of the HuCAL.RTM. Fab 1 library also can be performed with TIMP-1 as the antigen in round one, followed in round 2 by TIMP-1 peptides fused to carrier proteins, such as BSA or transferrin, and in round 3 by TIMP-1 again. Human TIMP-1 peptides which can be used for panning include human TIMP-1 residues 2 12 (TCVPPHPQTAF, SEQ ID NO:87; CTSVPPHPQTAF, SEQ ID NO:88; STCVPPHPQTAF, SEQ ID NO:89; STSVPPHPQTAFC, SEQ ID NO:90), 28 36 (CEVNQTTLYQ, SEQ ID NO:91), 64 75 (PAMESVCGYFHR, SEQ ID NO:92), 64 79 (PAMESVCGYFHRSHNR, SEQ ID NO:93; CPAMESVSGYFHRSHNR, SEQ ID NO:94; PAMESVSGYFHRSHNRC, SEQ ID NO:95), and 145 157 (CLWTDQLLQGSE, SEQ ID NO:96). These peptide sequences are selected from regions of human TIMP-1 that are predicted to interact with MMPs. See Gomis-Ruth et al., Nature 389, 77 81, 1997. Directing Fabs toward the MMP-interacting region of human TIMP-1 in round 2 should increase the chance of identifying Fabs that can block the ability of human TIMP-1 to inhibit human MMP-1 activity.

Another method that can be used to improve the likelihood of isolating neutralizing Fabs is the panning on human TIMP-1 and eluting the binding Fabs with human MMP-1. This strategy should yield higher affinity antibodies than would otherwise be obtained.

Details of the screening process are described in the specific examples, below. Other selection methods for highly active specific antibodies or antibody fragments can be envisioned by those skilled in the art and used to identify human TIMP-1 antibodies.

Human antibodies with the characteristics described above also can be purified from any cell that expresses the antibodies, including host cells that have been transfected with antibody-encoding expression constructs. The host cells are cultured under conditions whereby the human antibodies are expressed. A purified human antibody is separated from other compounds that normally associate with the antibody in the cell, such as certain proteins, carbohydrates, or lipids, using methods well known in the art. Such methods include, but are not limited to, size exclusion chromatography, ammonium sulfate fractionation, ion exchange chromatography, affinity chromatography, and preparative gel electrophoresis. A preparation of purified human antibodies is at least 80% pure; preferably, the preparations are 90%, 95%, or 99% pure. Purity of the preparations can be assessed by any means known in the art, such as SDS-polyacrylamide gel electrophoresis. A preparation of purified human antibodies of the invention can contain more than one type of human antibody with the TIMP-1 binding and neutralizing characteristics described above.

Alternatively, human antibodies can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques (Merrifield, J. Am. Chem. Soc. 85, 2149 54, 1963; Roberge et al., Science 269, 202 04, 1995). Protein synthesis can be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 431A Peptide Synthesizer (Perkin Elmer). Optionally, fragments of human antibodies can be separately synthesized and combined using chemical methods to produce a full-length molecule.

The newly synthesized molecules can be substantially purified by preparative high performance liquid chromatography (e.g., Creighton, PROTEINS: STRUCTURES AND MOLECULAR PRINCIPLES, WH Freeman and Co., New York, N.Y., 1983). The composition of a synthetic polypeptide can be confirmed by amino acid analysis or sequencing (e.g., using Edman degradation).

Assessment of Therapeutic Utility of Human Antibodies

To assess the ability of a particular antibody to be therapeutically useful to treat, liver fibrosis, for example, the antibody can be tested in vivo in a rat liver fibrosis model. Thus, preferred human antibodies of the invention are able to block both human and rat TIMP-1 activity. If desired, human Fab TIMP-1 antibodies can be converted into full immunoglobulins, for example IgG.sub.1 antibodies, before therapeutic assessment. This conversion is described in Example 5, below.

To identify antibodies that cross-react with human and rat TIMP-1, an ELISA can be carried out using rat TIMP-1. Functional cross-reactivity can be confirmed in an enzymatic assay, as described in Bickett et al., Anal. Biochem. 212, 58 64, 1993. The assay uses human or rat TIMP-1, human MMP-1 or rat MMP-13 (the rat counterpart of human MMP-1), and a synthetic fluorogenic peptide substrate. Enzyme activity of uncomplexed MMP-1 (or MMP-13) is assessed by observing an increase in a fluorescence signal.

Antibodies that block human and/or rat TIMP-1 activity can be screened in an ELISA assay that detects the decrease of TIMP-1/MMP-1 complex formation in cultures of HepG2 cells. Antibodies that meet this criteria can then be tested in a rat liver fibrosis model to assess therapeutic efficacy and correlate this efficacy with the ability of the antibodies to block TIMP-1 inhibition of MMP-1 in vitro.

Antibodies that demonstrate therapeutic efficacy in the rat liver fibrosis model can then be tested for binding to and blockade of TIMP-2,-3, and -4 in an in vitro enzymatic assay. Blocking the minimum number of TIMPs necessary for efficacy in liver fibrosis or other TIMP-associated pathology is preferable to minimize potential side effects.

Polynucleotides Encoding Human TIMP-1 Antibodies

The invention also provides polynucleotides encoding human TIMP-1 antibodies. These polynucleotides can be used, for example, to produce quantities of the antibodies for therapeutic or diagnostic use.

Polynucleotides that can be used to encode the VHCDR3 regions shown in SEQ ID NOS: 1 43 are shown in SEQ ID NOS:226 268, respectively. Polynucleotides that can be used to encode the VLCDR3 region shown in SEQ ID NOS:44 86 are shown in SEQ ID NOS: 183 225, respectively. Polynucleotides that encode heavy chains (SEQ ID NOS:140 182) and light chains (SEQ ID NOS:97 139) of human antibodies of the invention that have been isolated from the MorphoSys HuCAL.RTM. library are shown in SEQ ID NOS:269 311 and SEQ ID NOS:312 354, respectively.

Polynucleotides of the invention present in a host cell can be isolated free of other cellular components such as membrane components, proteins, and lipids. Polynucleotides can be made by a cell and isolated using standard nucleic acid purification techniques, or synthesized using an amplification technique, such as the polymerase chain reaction (PCR), or by using an automatic synthesizer. Methods for isolating polynucleotides are routine and are known in the art. Any such technique for obtaining a polynucleotide can be used to obtain isolated polynucleotides encoding antibodies of the invention. For example, restriction enzymes and probes can be used to isolate polynucleotides which encode the antibodies. Isolated polynucleotides are in preparations that are free or at least 70, 80, or 90% free of other molecules.

Human antibody-encoding DNA molecules of the invention can be made with standard molecular biology techniques, using mRNA as a template. Thereafter, DNA molecules can be replicated using molecular biology techniques known in the art and disclosed in manuals such as Sambrook et al. (1989). An amplification technique, such as PCR, can be used to obtain additional copies of the polynucleotides.

Alternatively, synthetic chemistry techniques can be used to synthesize polynucleotides encoding antibodies of the invention. The degeneracy of the genetic code allows alternate nucleotide sequences to be synthesized that will encode an antibody having, for example, one of the VHCDR3, VLCDR3, light chain, or heavy chain amino acid sequences shown in SEQ ID NOS:1 43, 44 86, 97 139, or 140 182, respectively.

Expression of Polynucleotides

To express a polynucleotide encoding a human antibody of the invention, the polynucleotide can be inserted into an expression vector that contains the necessary elements for the transcription and translation of the inserted coding sequence. Methods that are well known to those skilled in the art can be used to construct expression vectors containing sequences encoding human antibodies and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Such techniques are described, for example, in Sambrook et al. (1989) and in Ausubel et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, John Wiley & Sons, New York, N.Y., 1995. See also Examples 1 3, below.

A variety of expression vector/host systems can be utilized to contain and express sequences encoding a human antibody of the invention. These include, but are not limited to, microorganisms, such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors, insect cell systems infected with virus expression vectors (e.g., baculovirus), plant cell systems transformed with virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids), or animal cell systems.

The control elements or regulatory sequences are those non-translated regions of the vector--enhancers, promoters, 5' and 3' untranslated regions--which interact with host cellular proteins to carry out transcription and translation. Such elements can vary in their strength and specificity. Depending on the vector system and host utilized, any number of suitable transcription and translation elements, including constitutive and inducible promoters, can be used. For example, when cloning in bacterial systems, inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or pSPORT1 plasmid (Life Technologies) and the like can be used. The baculovirus polyhedrin promoter can be used in insect cells. Promoters or enhancers derived from the genomes of plant cells (e.g., heat shock, RUBISCO, and storage protein genes) or from plant viruses (e.g., viral promoters or leader sequences) can be cloned into the vector. In mammalian cell systems, promoters from mammalian genes or from mammalian viruses are preferable. If it is necessary to generate a cell line that contains multiple copies of a nucleotide sequence encoding a human antibody, vectors based on SV40 or EBV can be used with an appropriate selectable marker.

Large scale production of human TIMP-1 antibodies can be carried out using methods such as those described in Wurm et al., Ann. N.Y. Acad. Sci. 782, 70 78, 1996, and Kim et al., Biotechnol. Bioengineer. 58, 73 84, 1998.

Pharmaceutical Compositions

Any of the human TIMP-1 antibodies described above can be provided in a pharmaceutical composition comprising a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier preferably is non-pyrogenic. The compositions can be administered alone or in combination with at least one other agent, such as stabilizing compound, which can be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. A variety of aqueous carriers may be employed, e.g., 0.4% saline, 0.3% glycine, and the like. These solutions are sterile and generally free of particulate matter. These solutions may be sterilized by conventional, well known sterilization techniques (e.g., filtration). The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions such as pH adjusting and buffering agents, etc. The concentration of the antibody of the invention in such pharmaceutical formulation can vary widely, i.e., from less than about 0.5%, usually at or at least about 1% to as much as 15 or 20% by weight and will be selected primarily based on fluid volumes, viscosities, etc., according to the particular mode of administration selected. See U.S. Pat. No. 5,851,525. If desired, more than one type of human antibody, for example with different K.sub.d for TIMP-1 binding or with different IC.sub.50s for MMP-inhibiting activity neutralization, can be included in a pharmaceutical composition.

The compositions can be administered to a patient alone, or in combination with other agents, drugs or hormones. In addition to the active ingredients, these pharmaceutical compositions can contain suitable pharmaceutically-acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations which can be used pharmaceutically. Pharmaceutical compositions of the invention can be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, intraventricular, transdermal, subcutaneous, intraperitoneal, intranasal, parenteral, topical, sublingual, or rectal means.

After pharmaceutical compositions have been prepared, they can be placed in an appropriate container and labeled for treatment of an indicated condition. Such labeling would include amount, frequency, and method of administration.

Methods of Decreasing MMP-Inhibiting Activity of Human TIMP-1

The invention provides methods of decreasing an MMP-inhibiting activity of human or rat TIMP-1. Such methods can be used therapeutically, as described below, or in a research setting. Thus, the methods can be carried out in a cell-free system, in a cell culture system, or in vivo. In vivo methods of decreasing MMP-inhibiting activity of human or rat TIMP-1 are described below.

Human TIMP-1 is contacted with a human antibody that binds to the human TIMP-1, thereby decreasing the MMP-inhibiting activity of the human TIMP-1 relative to human TIMP-1 activity in the absence of the antibody. The antibody can be added directly to the cell-free system, cell culture system, or to an animal subject or patient, or can be provided by means of an expression vector encoding the antibody.

Diagnostic Methods

The invention also provides diagnostic methods, with which human or rat TIMP-1 can be detected in a test preparation, including without limitation a sample of serum, lung, liver, heart, kidney, colon, a cell culture system, or a cell-free system (e.g., a tissue homogenate). Such diagnostic methods can be used, for example, to diagnose disorders in which TIMP-1 is elevated. Such disorders include, but are not limited to, liver fibrosis, alcoholic liver disease, cardiac fibrosis, acute cardiac syndrome, lupus nephritis, glomerulosclerotic renal disease, benign prostate hypertrophy, lung cancer, colon cancer, and idiopathic pulmonary fibrosis. When used for diagnosis, detection of an amount of the antibody-TIMP-1 complex in a test sample from a patient which is greater than an amount of the complex in a normal sample identifies the patient as likely to have the disorder.

The test preparation is contacted with a human antibody of the invention, and the test preparation is then assayed for the presence of an antibody-TIMP-1 complex. If desired, the human antibody can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or luciferase.

Optionally, the antibody can be bound to a solid support, which can accommodate automation of the assay. Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads). Any method known in the art can be used to attach the antibody to the solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached to the antibody and the solid support. Binding of TIMP-1 and the antibody can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.

Therapeutic Methods

The invention also provides methods of ameliorating symptoms of a disorder in which TIMP-1 is elevated. These disorders include, without limitation, liver fibrosis alcoholic liver disease, cardiac fibrosis, acute coronary syndrome, lupus nephritis, glomerulosclerotic renal disease, idiopathic pulmonary fibrosis, benign prostate hypertrophy, lung cancer, colon cancer, and scarring. See, e.g., Inokubo et al., Am. Heart J. 141, 211 17, 2001; Ylisirnio et al., Anticancer Res. 20, 1311 16, 2000; Holten-Andersen et al., Clin. Cancer Res. 6, 4292 99, 2000; Holten-Andersen et al., Br. J. Cancer 80, 495 503, 1999; Peterson et al., Cardiovascular Res. 46, 307 15, 2000; Arthur et al., Alcoholism: Clinical and Experimental Res. 23, 840 43, 1999; Iredale et al., Hepatol. 24, 176 84, 1996.

Human antibodies of the invention are particularly useful for treating liver fibrosis. All chronic liver diseases cause the development of fibrosis in the liver. Fibrosis is a programmed uniform wound healing response. Toxic damage or injury caused by foreign proteins cause the deposition of extracellular matrix such as collagen, fibronectin, and laminin. Liver fibrosis and cirrhosis can be caused by chronic degenerative diseases of the liver such as viral hepatitis, alcohol hepatitis, autoimmune hepatitis, primary biliary cirrhosis, cystic fibrosis, hemochromatosis, Wilson's disease, and non-alcoholic steato-hepatitis, as well as chemical damage.

Altered degradation and synthesis of extracellular matrix (particularly collagens) play central roles in pathogenesis of liver fibrosis. In the early phases, hepatic stellate cells (HSC) are initially activated and release matrix metalloproteases with the ability to degrade the normal liver matrix. When HSC are fully activated, there is a net down-regulation of matrix degradation mediated by increased synthesis and extracellular release of tissue inhibitors of metalloprotease (TIMP)-1 and -2. The dynamic regulation of activity of metalloproteases during liver fibrosis makes them and their inhibitors targets for therapeutic intervention.

Human antibodies of the invention are also particularly useful for treating lung fibrosis. Lung airway fibrosis is a hallmark of airway remodeling in patients with chronic asthma, so human antibodies of the invention are also particularly useful for chronic asthma. Airway remodeling is a well-recognized feature in patients with chronic asthma. TIMP-1 but not TIMP-2 levels were significantly higher in untreated asthmatic subjects than in glucocorticoid-treated subjects or controls (p<0.0001), and were far greater than those of MMP-1, MMP-2, MMP-3, and MMP-9 combined (Mautino et al., Am J Respir Crit Care Med 1999 160:324 330). TIMP-1 mRNA and protein expression are selectively and markedly increased in a murine model of bleomycin-induced pulmonary fibrosis (Am. J. Respir. Cell Mol. Biol. 24:599 607, 2001). This specific elevation of TIMP-1 without increase in MMPs in asthma patients suggests that inhibition of TIMP-1 by an antibody can restore normal collagen degradation in the lung.

Human antibodies of the invention are also particularly useful for treating cancer. TIMP-1 protein has been found to be elevated in plasma of colon (Holten-Andersen et al., Br J Cancer 1999, 80:495 503) and prostate (Jung et al., Int J Cancer, 1997, 74:220 223) cancer patients, and high TIMP-1 plasma level correlates with poor clinical outcome of colon cancer (Holten-Andersen et al., Clin Cancer Res 2000 6:4292 4299). TIMP-1 induces dose-dependent proliferation of breast tumorigenic clonal cell line and tyrosine phosphorylation (Luparello et al, Breast Cancer Res Treat, 1999, 54:235 244). Therefore, the use of antibody against TIMP-1 may block its ability to induce cancer.

Human TIMP-1 antibodies can be used to prevent or diminish scar formation, such as scar formation after surgery (particularly ophthalmic surgery) or injury (such as a burn, scrape, crush, cut or tear injury).

In one embodiment of the invention, a therapeutically effective dose of a human antibody of the invention is administered to a patient having a disorder in which TIMP-1 is elevated, such as those disorders described above. Symptoms of the disorder, including deposition of extracellular matrix, as well as loss of tissue or organ function, are thereby ameliorated.

Determination of a Therapeutically Effective Dose

The determination of a therapeutically effective dose is well within the capability of those skilled in the art. A therapeutically effective dose refers to that amount of human antibody that reduces MMP-inhibiting activity of the TIMP-1 relative to the activity which occurs in the absence of the therapeutically effective dose.

The therapeutically effective dose can be estimated initially either in cell culture assays or in animal models, usually rats, mice, rabbits, dogs, or pigs. The animal model also can be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans. A rat liver fibrosis model is described in Example 6.

Therapeutic efficacy and toxicity, e.g., ED.sub.50 (the dose therapeutically effective in 50% of the population) and LD.sub.50 (the dose lethal to 50% of the population) of a human antibody, can be determined by standard pharmaceutical procedures in cell cultures or experimental animals. The dose ratio of toxic to therapeutic effects is the therapeutic index, and it can be expressed as the ratio, LD.sub.50/ED.sub.50.

Pharmaceutical compositions that exhibit large therapeutic indices are preferred. The data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use. The dosage contained in such compositions is preferably within a range of circulating concentrations that include the ED.sub.50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.

The exact dosage will be determined by the practitioner, in light of factors related to the patient who requires treatment. Dosage and administration are adjusted to provide sufficient levels of the human antibody or to maintain the desired effect. Factors that can be taken into account include the severity of the disease state, general health of the subject, age, weight, and gender of the subject, diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long-acting pharmaceutical compositions can be administered every 3 to 4 days, every week, or once every two weeks depending on the half-life and clearance rate of the particular formulation.

Polynucleotides encoding human antibodies of the invention can be constructed and introduced into a cell either ex vivo or in vivo using well-established techniques including, but not limited to, transferrin-polycation-mediated DNA transfer, transfection with naked or encapsulated nucleic acids, liposome-mediated cellular fusion, intracellular transportation of DNA-coated latex beads, protoplast fusion, viral infection, electroporation, "gene gun," and DEAE- or calcium phosphate-mediated transfection.

Effective in vivo dosages of an antibody are in the range of about 5 mg to about 50 mg/kg, about 50 mg to about 5 mg/kg, about 100 mg to about 500 mg/kg of patient body weight, and about 200 to about 250 mg/kg of patient body weight. For administration of polynucleotides encoding the antibodies, effective in vivo dosages are in the range of about 100 ng to about 200 ng, 500 ng to about 50 mg, about 1 mg to about 2 mg, about 5 mg to about 500 mg, and about 20 mg to about 100 mg of DNA.

The mode of administration of human antibody-containing pharmaceutical compositions of the invention can be any suitable route which delivers the antibody to the host. Pharmaceutical compositions of the invention are particularly useful for parenteral administration, i.e., subcutaneous, intramuscular, intravenous, or intranasal administration.
 


Claim 1 of 12 Claims

1. A purified human antibody, wherein the antibody: binds to a tissue inhibitor of metalloprotease-1 (TIMP-I); neutralizes a matrix metalloprotease (MMP)-inhibiting activity of the TIMP-1; and comprises: a VHCDR1 region comprising an amino acid sequence as set forth in SEQ ID NO:355; a VHCDR2 region comprising an amino acid sequence as set forth in SEQ ID NO:359; a VHCDR3 region comprising an amino acid sequence as set forth in SEQ ID NO:360; a VLCDR1 region comprising an amino acid sequence as set forth in SEQ ID NO:363; a VLCDR2 region comprising an amino acid sequence as set forth in SEQ ID NO:364; and a VLCDR3 region comprising an amino acid sequence as set forth in SEQ ID NO:48.

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