HK1191035A - Connective tissue growth factor antibodies - Google Patents

Abstract

The present invention relates to antibodies that bind to CTGF. The antibodies are particularly directed to regions of CTGF involved in biological activities associated with fibrosis. The invention also relates to methods of using the antibodies to treat disorders associated with CTGF including localized and systemic fibrotic disorders including those of the lung, liver, heart, skin, and kidney.

Description

Connective tissue growth factor antibodies

This application is a divisional application of chinese patent application 200480022145.6 entitled "connective tissue growth factor antibody" filed on 6/2/2004.

This application claims priority from U.S. provisional application serial No. 60/475,598, filed on 4/6/2003, which is incorporated herein by reference in its entirety.

Technical Field

The present invention relates to antibodies that bind Connective Tissue Growth Factor (CTGF). The antibodies are specifically directed to the CTGF region involved in biological activities associated with various diseases.

Background

Connective Tissue Growth Factor (connecting Tissue Growth Factor, CTGF)

CTGF is a 36kD cysteine-rich heparin-binding secretory glycoprotein originally isolated from human umbilical vein endothelial cells (see, e.g., Bradham et al (1991) J Cell Biol114:1285-1294; Grotendorst and Bradham, U.S. Pat. No.5,408,040). CTGF belongs to the protein CCN (CTGF,Cyr61,Nov) family (secreted glycoproteins) including the serum-induced immediate early gene product Cyr61, the putative oncogene Nov, the ECM-associated protein FISP-12, the src-induced gene CEF-10, the Wnt-induced secreted protein WISP-3 and the antiproliferative protein HICP/rCOP (Brigstock (1999) Endocr Rev20: 189-. CCN proteins are characterized by conserved 38 cysteine residues, which 38 cysteine residues account for more than 10% of the total amino acid content and form a modular structure with N-terminal and C-terminal domains. The CTGF module structure includes a conserved motif at the N-terminal domain for insulin-like growth factor binding protein (IGF-BP) and von Willebrand factor (VWC), and a thrombospondin (TSP1) and cysteine knot motif at the C-terminal domain.

CTGF expression is induced by members of the transforming growth factor beta (TGF β) superfamily, which includes TGF β -1, -2, and-3, Bone Morphogenic Protein (BMP) -2, and activin, as well as a number of other regulatory modulators including dexamethasone, thrombin, Vascular Endothelial Growth Factor (VEGF), and angiotensin II; and induction by environmental stimuli including hyperglycemia and hypertension (see, e.g., Franklin (1997) IntJ Biochem Cell Biol29:79-89; Wunderlich (2000) Graefes Arch Clin ExpOphthalmol238: 910-. TGF-beta stimulates CTGF expression rapidly and chronically, and without the need for permanent application (Igarashi et al (1993) Mol Biol Cell4: 637-645). Enhanced expression of CTGF by TGF β involves transcriptional activation by DNA regulatory elements present in the CTGF promoter (Grotendorst et al (1996) Cell growth Differ7:469-480; Grotendorst and Bradham, U.S. patent No.6,069,006; Holmes et al (2001) J Biol Chem276: 10594-10601).

CTGF has been shown to enhance the steady state transcription of alpha 1(I) collagen, alpha 5 integrin and fibronectin mRNA, as well as promote cellular processes including proliferation and chemotaxis, in various Cell types in culture (see, e.g., Frazier et al (1996) J Invest Dermatol107: 406. 411; Shi-wen et al (2000) Exp Cell Res259: 213. 224; Klagsburn (1977) Exp CellRes105:99-108; Gupta et al (2000) Kidney Int58: 1389. 1399; Wahab et al (2001) Biochem J359(Pt1):77-87; Uzel et al (2001) J.peroxidol 72: 921. 931; and Riser and cortex (2001) Fan Fail23: 459470). Subcutaneous injection of CTGF in neonatal mice resulted in local deposition of granulation tissue. Similarly, subcutaneous injection of TGF β results in granulation tissue formation and induces high levels of CTGF mRNA in local fibroblasts. In addition, combined or sequential treatment with TGF β and CTGF results in the development of a more persistent granuloma (Mori et al (1999) JCel Physiol181: 153-. Thus, CTGF appears to mediate a subset of the effects elicited by TGF β, particularly the production and deposition of extracellular matrix (ECM). In addition, the ability to respond to CTGF, or the extent of CTGF response, may be dependent on the priming stimulus provided by TGF-beta treatment that results in cellular "compatibility" (International publication WO 96/08140).

Although a series of interacting factors have been identified that modulate tissue composition, CTGF is now of increasing interest for its role in modulating skeletal development, wound healing and extracellular matrix (ECM) remodeling, fibrosis, tumorigenesis and angiogenesis. For example, increased CTGF expression has been observed in liver cirrhosis, lung fibrosis, inflammatory bowel disease, scleroderma and scarring, connective tissue formation, atheromatous plaques. (Absham et al (2000) J Biol Chem275:15220-

CTGF is also upregulated in glomerulonephritis, IgA nephropathy, focal and segmental glomerulosclerosis, and diabetic nephropathy (see, e.g., Riser et al, (2000) J Am Soc Nephrol11: 25-38). An increase in the number of cells expressing CTGF was also observed at the site of chronic tubulointerstitial (tubulointerstitial) injury, and CTGF levels correlated with the extent of injury (Ito et al (1998) Kidney Int53: 853-861). In addition, CTGF expression is also increased in glomeruli and tubulointerstities in various renal diseases associated with scarring and cirrhosis of the renal parenchyma. Elevated levels of CTGF are also associated with liver fibrosis, myocardial infarction, and pulmonary fibrosis. For example, in patients with Idiopathic Pulmonary Fibrosis (IPF), CTGF is strongly upregulated in biopsy and bronchoalveolar lavage fluid cells (Ujike et al (2000) Biochem Biophys Res Commun277: 448-. CTGF therefore represents an effective therapeutic target in diseases such as those described above.

The relevance of CTGF to various aspects of these diseases has been recognized, and methods for treating diseases by modulating CTGF have also been described (see, e.g., grotidinst and Bradham, U.S. patent 5,783,187; international publication WO00/13706; international publication WO 03/049773). Modulation of growth factors, cytokines and cell surface receptors can be performed using monoclonal antibodies, and several therapeutic monoclonal antibodies have been approved or are being developed. (see, e.g., Infliximab (Remicade; Maini et al (1998) Arthritis Rheum41:1552-1563; Targan et al (1997) N Engl J Med337:1029-1035; Basiliximab (Simult) and Daclizumab (Zenapax) (Bumgardner et al (2001) Transplantation72:839-845; Kovarik et al (1999) Transplantation68: 1288-1294; and Trastuzumab (Herceptin; Baselga (2001) Ann col12Suppl1: S49-55.))

Antibodies have been raised against CTGF and have proven effective in vivo, for example, in inhibiting angiogenesis. (see, e.g., Grotendorst and Bradham, U.S. Pat. No.5,408,040; International publication WO 99/07407; Shimo et al (2001) Oncology 61: 315-. In addition, the regulatory nature of CTGF appears to distinguish the domains involved in specific biological activities. For example, the N-terminal half of CTGF has been shown to stimulate cell differentiation and ECM production, while the C-terminal half stimulates cell proliferation. (see, e.g., International publications WO00/35936 and WO 00/35939; Brigstock and Harding, U.S. Pat. No.5,876, 70.) this demonstrates that antibodies directed against different regions of the CTGF molecule exhibit different effects in modulating the biological activity of CTGF. (see, for example, International publications WO00/35936 and WO 00/35939). Currently, there is no clear distinction between anti-CTGF antibodies that produce the desired effect and antibodies that produce multiple effects or are non-neutralizing (see, e.g., international publication WO 99/33878).

There is a clear need in the art for agents that effectively neutralize CTGF activity in diseases. Antibodies, particularly monoclonal antibodies, provide a specific and pharmacokinetic profile suitable for therapeutic agents, and neutralizing antibodies that target the specific activity of CTGF would meet the needs of the art and would be useful in the treatment of CTGF-associated diseases, including pulmonary diseases such as Idiopathic Pulmonary Fibrosis (IPF), and the like; kidney diseases such as diabetic nephropathy, glomerulosclerosis, etc.; and ocular diseases such as retinopathy, macular degeneration, and the like.

Summary of The Invention

The present invention provides antibodies, particularly monoclonal antibodies, and portions thereof, that specifically bind to a region on the N-terminal fragment of a CTGF polypeptide.

In one aspect, the antibodies of the invention specifically bind to a region of human CTGF (SEQ ID NO:2), such as from about amino acid 103 to amino acid 164(SEQ ID NO:21), more specifically from about amino acid 135 to amino acid 157(SEQ ID NO:22), more specifically from about amino acid 142 to amino acid 154(SEQ ID NO:25), or specifically bind to an orthologous region on CTGF derived from another species. In a particular embodiment, the antibody has the same specificity as the antibody produced by the cell line deposited with the ATCC as accession number ______ (deposited with the ATCC at 5/19 of 2004). In particular embodiments, the antibody is substantially identical to mAb1 described below. More preferably, the antibody is substantially similar to CLN1 described below. In another embodiment, an antibody of the invention competes for binding to a CTGF polypeptide with any of the antibodies described above.

In one embodiment, the invention provides a monoclonal antibody or portion thereof comprising at least one member selected from the group consisting of an immunoglobulin heavy chain sequence comprising SEQ ID NO. 14, an immunoglobulin heavy chain sequence comprising the variable region of SEQ ID NO. 14, an immunoglobulin light chain sequence comprising SEQ ID NO. 20, an immunoglobulin light chain sequence comprising the variable region of SEQ ID NO. 20, or a conservative variant thereof. In one embodiment, the antibody comprises an immunoglobulin heavy chain variable region, i.e., amino acid residues 1 through amino acid residue 167 of SEQ ID NO 14. In another embodiment, the antibody comprises an immunoglobulin light chain variable region, i.e., amino acid residue 1 through amino acid residue 136 of SEQ ID NO: 20. In a particular embodiment, the antibody comprises the immunoglobulin heavy chain sequence of SEQ ID NO. 14 and the immunoglobulin light chain sequence of SEQ ID NO. 20. In this embodiment, the invention provides, inter alia, a CLN1 antibody or a portion thereof comprising at least the residues of the antigen binding region of CLN 1.

In certain aspects, the antibodies of the invention are polyclonal antibodies. In other aspects, the antibody is a monoclonal antibody. In certain embodiments, the antibody is a humanized monoclonal antibody; more preferably human monoclonal antibodies. Any of the above antibodies may additionally contain various amounts of glycosylation, which is incorporated by the cell producing the antibody, or is applied and/or modified synthetically; or the antibody has no glycosylation. The antibodies may optionally be pegylated and/or similarly modified to increase plasma half-life, and the like. In various embodiments, the invention provides fragments of the antibodies, particularly wherein the fragments are Fab, F (ab)₂Or an Fv fragment.

In certain aspects, the antibody or portion thereof is produced by a cloned cell line. The cell line may be from any animal model used for monoclonal antibody production, including but not limited to mice, goats, chickens, and the like. In particular, the cell line may be from a mouse. The mouse may be a standard mouse for antibody production, such as BALB/C, or a modified, e.g., transgenic, mouse strain optimized or developed for production of specific isotype, idiotype, or species-specific monoclonal antibodies. In one embodiment, the cell line is a hybridoma cell line that produces and secretes mAb 1. In other embodiments, the cell line produces and secretes an antibody or portion thereof having properties substantially equivalent to mAb 1. In other embodiments, the cell line produces and secretes an antibody or portion thereof having properties substantially equivalent to CLN 1. In a particular embodiment, the invention provides a cell line having ATCC accession number _____ (5/19 of deposit 2004).

In another aspect, the antibody or portion thereof is derived from a non-human transgenic animal, particularly a non-human transgenic mammal, capable of producing human antibodies. The animal may be of any species, including but not limited to mouse, chicken, cow, goat, and the like. In particular, the animal may be a mouse. Such antibodies can be obtained by immunizing a non-human transgenic mammal with a fragment of human CTGF, such as SEQ ID NO:21 or more specifically SEQ ID NO:22, or with orthologous regions on CTGF from a non-human species. In certain embodiments, the antibodies are obtained by immunizing a non-human transgenic mammal with a CTGF fragment selected from the group consisting of SEQ ID NOs 23-26 or with orthologous regions on CTGF from a non-human species. In a specific embodiment, the antibody is obtained by immunizing a transgenic mouse with any one of the CTGF fragments described above. In other embodiments, the antibody is obtained by immunizing a transgenic mouse with a functional equivalent of any of the CTGF fragments described above.

By "specifically binds to a region of CTGF" is meant that the antibody has binding specificity for a particular region of CTGF, which may be defined by a linear amino acid sequence, or by a tertiary, i.e., three-dimensional, conformation of a portion of the CTGF polypeptide. Binding specificity refers to an antibody having substantially higher affinity for a portion of CTGF than for other related polypeptides. By "substantially higher affinity" is meant a measurable increase in affinity for a portion of CTGF, as compared to affinity for other related polypeptides. Preferably, the affinity for a specific part of CTGF is at least 1.5-fold, 2-fold, 5-fold, 10-fold, 100-fold, 103-fold, 104-fold, 105-fold, 106-fold or more greater than the affinity for other proteins. Preferably, binding specificity is determined by affinity chromatography, immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA) or by Fluorescence Activated Cell Sorting (FACS) analysis. More preferably, the binding specificity is obtained by RIA or affinity chromatography as described below.

In a preferred embodiment of the invention, the antibody has an affinity equal to or greater than that of mAb1 as described below as determined by a Scatchard analysis, e.g., of Munson and Pollard (1980, AnalBiochem107: 220). Antibody affinity is defined asThe strength of the overall non-covalent interaction between a single antigen binding site on an antibody and a single epitope on an antigen. Affinity by measuring the association constant (K)_a) To calculate, i.e.

Wherein [ Ab]Is the concentration of the free antigen binding site on the antibody, [ Ag ]]Is the free antigen concentration, [ Ab. Ag ]]Is the concentration of antigen-binding sites on the antibody occupied by the antigen, K_dIs the dissociation constant of the antibody-antigen complex. Preferably, the antibodies of the invention have an affinity for CTGF of greater than Kd =10^-8Preferably higher than 10^-9Preferably higher than 10^-10Particularly for therapeutic purposes. Advantageously, the antibodies of the invention have an affinity similar to or higher than that of mAb1 (i.e., Kd. ltoreq.10)^-9). However, antibodies sharing epitope binding with mAb1 but having lower affinity (i.e., higher Kd) than mAb1 are also encompassed within the invention and potentially useful in various analytical and diagnostic applications as described herein. Such antibodies may additionally be useful for therapeutic applications, particularly if they have a high affinity (avidity) for an antigen, as described below.

The antibodies of the invention may be monovalent, bivalent, or they may be multivalent. In certain embodiments of the invention, it is preferred that the antibodies of the invention are bivalent or multivalent. Any antibody of the invention may be manipulated to improve avidity, for example by combining epitope binding sites in a single antibody construct, for example a tribody or the like. The antibody of the invention may be a single chain antibody.

In certain instances, it is useful that the antibodies of the invention exhibit suitable affinity for CTGF from other species, e.g., for the treatment and prevention of disease in those species. For example, it was shown to have a suitable K for canine CTGF_dThe antibodies of the invention are useful for treating CTGF-associated diseases in dogs. Display Cross-species affinityThe antibodies of the invention, such as mAb1, can also be used as research tools to study CTGF-associated diseases in various animal models. In another aspect, the antibody or portion thereof is encoded by genetic material originally derived from a human. The antibodies can be produced from cultured cells, e.g., using phage display technology, or can be produced in animals, e.g., non-human transgenic animals containing human-derived immunoglobulin genes.

In addition, the invention provides recombinant constructs comprising portions of any of the antibodies of the invention described above and a protein from another source. Specifically included are embodiments that encompass chimeric antibodies comprising a variable region derived from a monoclonal antibody that specifically binds to a region on the N-terminal fragment of CTGF and a constant region derived from another source. The variable region may be derived from any antibody defined herein, particularly comprising an antibody that binds to a region on human CTGF from about amino acid 97 to about amino acid 180 of SEQ ID No. 2, or more particularly from about amino acid 103 to about amino acid 164 of SEQ ID No. 2, or more particularly from about amino acid 134 to about amino acid 158 of SEQ ID No. 2, or more particularly from about amino acid 143 to about amino acid 154 of SEQ ID No. 2, or an antibody that binds to an orthologous region on CTGF from another species. The constant region may be derived from any source. In certain embodiments, the constant region is derived from a constant region of a human immunoglobulin.

The present invention also provides any one of the antibodies described above, wherein the antibody additionally comprises a labeling agent that provides a detectable signal by itself or with other substances. Such labeling agents may be selected from, but are not limited to, the group consisting of enzymes, fluorescent substances, chemiluminescent substances, biotin, avidin, and radioisotopes. The present invention also provides any one of the above antibodies, wherein the antibody additionally comprises a cytotoxic agent or an enzyme.

In other embodiments, an antibody of the invention as described above additionally neutralizes at least one activity associated with CTGF. These CTGF-associated activities include, but are not limited to, stimulating cell migration, production of extracellular matrix by cells in vivo or in vitro, and/or reducing fibrosis in a subject. In particular embodiments, the biological activity is selected from the group consisting of cell growth, differentiation of fibroblasts and/or endothelial cells, and induction of expression of proteins involved in extracellular matrix formation and remodeling, such as collagen (including but not limited to types I, II, III, and IV) and fibronectin.

In certain embodiments, the antibody specifically inhibits cell migration in an ex vivo (ex vivo) assay. Preferably, the antibody inhibits CTGF-stimulated chemotactic migration of smooth muscle cells in a Boyden chamber assay. For example, the antibodies of the invention repeatedly and reproducibly inhibit CTGF-induced migration in the cell migration assay described below. In various embodiments, the antibody specifically reduces fibrosis in an animal model. Preferably, the antibody inhibits the development of fibrosis in an animal model of pulmonary and renal fibrosis. For example, antibodies attenuated bleomycin (bleomycin) -induced pulmonary fibrosis in mice by 60-70%, as determined by histological examination of lung hydroxyproline (collagen) accumulation and/or tissue preparations as described below. In addition, the antibodies reduced collagen accumulation in the rat kidney residual (i.e., 5/6 nephrectomy) model as well as in mice following Unilateral Ureteral Obstruction (UUO), as described below.

In other embodiments, the antibodies of the invention modulate the interaction between the CTGF polypeptide and a cellular receptor, and/or between the CTGF polypeptide and a secreted or membrane associated cofactor, thereby neutralizing the biological activity of CTGF. The cofactor may be any protein, carbohydrate, and/or lipid; in particular embodiments, the cofactor is a member of the TGF- β family of growth factors, e.g., TGF- β, BMP-4, and the like.

In another aspect, the antibody reduces fibrosis in the subject. In various embodiments, the subject is a tissue or organ. In other embodiments, the subject is an animal, preferably a mammal, most preferably a human. When the subject is a tissue, the invention specifically includes endogenous and ex vivo (exvivo) tissues, such as transplanted tissues, cultured growing tissues, and the like. In various embodiments, the tissue is selected from the group consisting of epithelial, endothelial, and connective tissue. When the subject is an organ, the invention specifically includes an organ selected from the group consisting of kidney, lung, liver, eye, heart and skin. In a preferred embodiment, the subject is an animal, in particular an animal of a mammalian species including rat, rabbit, bovine, ovine, porcine, murine, equine and primate species. In a most preferred embodiment, the subject is a human.

In particular embodiments, the antibodies are used to treat or prevent a CTGF-associated disease in a subject having or at risk of having the CTGF-associated disease. These diseases include, but are not limited to, various cancers including acute lymphoblastic leukemia, cutaneous fibromas, breast cancer (Breast cancer), gliomas and glioblastomas, rhabdomyosarcoma and fibrosarcoma, desmoplasia, angiolipoma, angioleiomyoma, desmoplasia cancers, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer and liver cancer, as well as other tumor growth and metastasis. CTGF-associated diseases also include various fibrotic diseases including, but not limited to, idiopathic pulmonary fibrosis, renal fibrosis, glomerulosclerosis, ocular fibrosis (ocular fibrosis), osteoarthritis, scleroderma, cardiac fibrosis, and liver fibrosis. Fibrosis can occur in any organ or tissue, including organs selected from, but not limited to, kidney, lung, liver, heart, and skin; or a tissue selected from, but not limited to, epithelial, endothelial, and connective tissues. In other embodiments, the CTGF-associated disease may be caused by any initiating factor, including but not limited to exposure to a chemical or biological agent, an inflammatory response, an autoimmune response, trauma, surgical procedures, and the like. CTGF-associated diseases also include, but are not limited to, diseases due to hyperglycemia and hypertension. These diseases may occur, for example, as a result of diabetes, obesity, etc., and include diabetic nephropathy, retinopathy and cardiovascular disease.

Accordingly, in various embodiments, the present invention provides antibodies useful for treating or preventing a CTGF-associated disease in a subject. The invention also provides application of the antibodies in preparing medicaments for treating CTGF-related diseases.

In another aspect, the present invention provides a method of neutralizing CTGF-associated activity comprising contacting an antibody of the invention with a CTGF polypeptide, thereby neutralizing the biological activity of CTGF, as described above. The biological activity may be any activity of CTGF, including but not limited to stimulating cell migration and production of extracellular matrix. In various embodiments, neutralization occurs in vitro. In other embodiments, neutralization occurs in vivo in the subject.

In another aspect, the present invention provides a method of treating a CTGF-associated disease in a patient in need thereof using the antibody as described above, the method comprising administering the antibody or a pharmaceutical formulation thereof to the patient, thereby treating the disease. The subject may be a patient diagnosed with or suspected of having a CTGF-associated disease, including, for example, a disease that results in overproduction of extracellular matrix. In a particular aspect, the CTGF-associated disease is selected from cancer or a fibrotic disease. Cancers include, but are not limited to, acute lymphoblastic leukemia, cutaneous fibroids, breast cancer (Breast cancer), breast tumor (Breast carcinoma), gliomas and glioblastomas, rhabdomyosarcoma and fibrosarcoma, desmoplasia, angiolipoma, vascular leiomyoma, desmoplasia cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer and liver cancer, fibrotic diseases include, but are not limited to, idiopathic pulmonary fibrosis, renal fibrosis, glomerulosclerosis, ocular fibrosis, macular degeneration, osteoarthritis, scleroderma, chronic heart failure, cardiac fibrosis and liver fibrosis. In other embodiments, CTGF-associated disorders may be caused by any initiating factor, including, but not limited to, exposure to chemicals and biological agents, inflammatory responses, autoimmune diseases, trauma, surgical procedures, and the like. CTGF-associated diseases also include, but are not limited to, diseases due to hyperglycemia and hypertension. These diseases may occur, for example, as a result of diabetes, obesity, etc., and include diabetic nephropathy, retinopathy and cardiovascular disease.

In another aspect, the invention provides a composition comprising an antibody as described above and at least one additional component. The component may include any compound, molecule, or active agent, including, for example, proteins, nucleic acids, carbohydrates, lipids, and the like. In addition, the components may include various solvents, salts, and other carriers and/or excipients. In some embodiments, the composition is a pharmaceutical composition comprising an antibody as described above and at least one additional component selected from the group consisting of solvents, stabilizers, and excipients. In a particular embodiment, the pharmaceutical composition comprises the antibody in admixture with a pharmaceutically acceptable carrier. The pharmaceutical composition may additionally contain a second therapeutic agent, such as an Angiotensin Converting Enzyme (ACE) inhibitor, an advanced glycation end product cleavage or inhibitor, or the like. The present invention also provides a medicament comprising an antibody as described above for treating a subject having a CTGF-associated disease. Such diseases include, but are not limited to, various cancers and fibrotic diseases; diseases resulting from pathologies such as myocardial infarction, arthritis and inflammation; and diseases caused by diabetes, obesity, and the like, which may include diabetic nephropathy, retinopathy, cardiovascular disease, and the like.

In another embodiment, the present invention provides a polypeptide sequence selected from the group consisting of SEQ ID NO. 14, amino acid 1 to amino acid 167 of SEQ ID NO. 14, SEQ ID NO. 20, and amino acid 1 to amino acid 136 of SEQ ID NO. 20. Conservative variants of these polypeptides are also encompassed by the invention. In another embodiment, the present invention provides specific fragments of human CTGF selected from the group consisting of SEQ ID NOs 21-26, as well as orthologous CTGF fragments obtained from non-human species.

The above polypeptides may be "altered" polypeptides, as defined below.

In another embodiment, the invention provides a polynucleotide sequence encoding an antibody or portion thereof of the invention. In particular embodiments, the polynucleotide sequence is selected from the group consisting of: the polynucleotide sequence encoding SEQ ID NO. 14, the polynucleotide sequence encoding amino acids 1 to 167 of SEQ ID NO. 14, the polynucleotide sequence of SEQ ID NO. 13, and the polynucleotide comprising nucleotides 1 to 501 of SEQ ID NO. 13. In other embodiments, the polynucleotide sequence is selected from the group consisting of: the polynucleotide sequence encoding SEQ ID NO. 20, the polynucleotide sequence encoding amino acid 1 to amino acid 136 of SEQ ID NO. 20, the polynucleotide sequence of SEQ ID NO. 19, and the polynucleotide comprising nucleotide 1 to nucleotide 408 of SEQ ID NO. 19.

The polynucleotide may be an "altered" polynucleotide, as defined below.

The present invention also provides recombinant polynucleotides comprising any of the polynucleotide sequences described above operably linked to a vector sequence comprising replication and transcription control sequences. In one aspect, the recombinant polynucleotide encodes the amino acid sequence of SEQ ID NO. 14 or a variable region thereof. In another aspect, the recombinant polynucleotide comprises SEQ ID NO 13. In another aspect, the recombinant polynucleotide encodes the amino acid sequence of SEQ ID NO. 20 or a variable region thereof. In another aspect, the recombinant polynucleotide comprises SEQ ID NO 19.

The invention also provides a host cell transfected with at least one recombinant polynucleotide as described above. Host cells include any prokaryotic and eukaryotic host cells, including, for example, clonal cell lines maintained by culture methods known to those skilled in the art. Host cells also include transgenic plants and animals derived from transformed cells such as stem cells. In one embodiment, the host cell comprises a cell co-transfected with a polynucleotide encoding SEQ ID No. 14 and a polynucleotide encoding SEQ ID No. 20 that produces a functional antibody having substantially the same characteristics as mAb 1. In a particular embodiment, the antibody is CLN 1. In another specific embodiment, the host cell is ATCC accession number ______ (Collection: 5/19/2004).

These and other embodiments of the present invention will be readily understood by those skilled in the art with reference to the description herein, all of which are specifically contemplated.

Brief Description of Drawings

FIGS. 1A and 1B show the structure and sequence conservation of connective tissue growth factor. FIG. 1A shows the modular domain structure of CTGF including conserved motifs for insulin-like growth factor binding protein (IGF-BP) and Von Willebrand's factor (VWC) in the N-terminal fragment and for thrombospondin (TSP1) and cysteine knot motif (CT) in the C-terminal fragment. FIG. 1B shows multiple sequence alignments between human CTGF (hCGGF), bovine CTGF (bCGTGF), porcine CTGF (pCTGF), rat CTGF (rCTGF), and murine CTGF (FISP12) orthologs. This comparison was generated using the CLUSTAL W program (version 1.74; Thompson et al, (1994) Nucleic acids sRs 22:4673- - "4680) using default parameters. The asterisks in the figure indicate that the amino acid residues are completely conserved in the species shown.

Fig. 2A and 2B show Scatchard (Scatchard) plots of competitive binding of labeled and unlabeled human CTGF to anti-CTGF antibodies mAb2 and mAb1, respectively. mAb1 is an exemplary antibody of the invention.

FIG. 3A shows the Fab antibody fragment (Mr45kD) from papain digestion of the corresponding IgG antibody mAb1 followed by protein A-agarose affinity chromatography, as confirmed by SDS-PAGE (lane 2). Figure 3B shows binding of Fab fragments and corresponding IgG to CTGF with increasing chaotropic agent (thiocyanate) concentration.

Fig. 4A and 4B show Scatchard (Scatchard) plots of competitive binding of labeled recombinant human CTGF and unlabeled rat CTGF to anti-CTGF antibodies mAb2 and mAb1, respectively.

Fig. 5A, 5B and 5C show the therapeutic benefit of the antibodies of the invention in a model of pulmonary interstitial fibrosis. Figure 5A shows the effect of antibody treatment on bleomycin-induced increases in hydroxyproline content in mouse lungs. The number of animals per group is indicated in parentheses below each bar and the treatment groups are shown along the x-axis. And SA: brine; BL: bleomycin; AbsJ: a collection of 3 monoclonal antibodies of the invention; mAb1 exemplary antibodies of the invention. Values are expressed as mean ± SE. Fig. 5B and 5C show hematoxylin and eosin stained paraffin sections from the proximal lung lobule (pulmony proximal acini) of mice exposed to bleomycin by intratracheal injection and subsequently treated with saline or an antibody of the invention, respectively. In fig. 5B, the thin alveolar septa minutus (alveolular septa acinus) has an abnormal appearance, and there are inflammatory cells and fibrosis. In fig. 5C, the majority of parenchyma is normal, with only moderate degrees of alveolar septa thickening.

Fig. 6A, 6B and 6C show therapeutic benefit of the antibodies of the invention in a model of tubulointerstitial fibrosis in the kidney. Fig. 6A shows reduced fibrosis due to Unilateral Ureteral Obstruction (UUO) following treatment with antibody mAb1 of the present invention or antibody mAb3 directed against the C-terminus of CTGF. The degree of fibrosis is expressed as the ratio of hydroxyproline to proline in the obstructed kidney compared to the contralateral unobstructed kidney (mean ± SE). Figures 6B and 6C show trichrome-stained paraffin sections of obstructed kidneys receiving saline or antibody treatment, respectively.

Fig. 7A and 7B show therapeutic benefit of the antibodies of the invention in a glomerular fibrosis model in the kidney. Fig. 7A and 7B show light micrographs of trichrome-stained, disabled kidney tissue after receiving saline or antibody treatment, respectively.

Fig. 8A, 8B, 8C, 8D, 8E, 8F and 8G show induction of localized subcutaneous granulomas in neonatal mice. On the left, fig. 8A and 8B show the formation of granulomas at the sites of subcutaneous injection of TGF β alone or TGF β and CTGF, respectively. On the right, fig. 8C to 8G show histological groups (panels) representing scoring systems (from 0 (normal) to 4 (fibrosis)) for assessing the therapeutic benefit of antibodies.

Fig. 9 shows the degree of fibrosis in the localized subcutaneous granuloma model with and without treatment with anti-CTGF antibody. mAb1 is an exemplary antibody of the invention, while mAb3 is an anti-CTGF antibody that specifically binds to a C-terminal CTGF epitope.

Fig. 10A, 10B, 10C, and 10D show therapeutic benefit of the antibodies of the invention on organ fibrosis in a model of systemic sclerosis. Each group showed changes in collagen accumulation in various organs following treatment with saline (control), TGF β and CTGF, or TGF β and CTGF concurrent with antibody treatment.

FIGS. 11A and 11B show schematic flowsheets for cloning the immunoglobulin heavy chain and immunoglobulin light chain of exemplary antibody mAb1 of the invention. Fig. 11A shows a comparison of heavy chain PCR fragments used to determine the mAb1 heavy chain coding sequence (CDS). Fig. 11B shows a comparison of light chain PCR fragments used to determine the light chain coding sequence (CDS) of mAb 1.

Fig. 12A and 12B show binding studies between CTGF and TGF β. Fig. 12A shows the degree of binding between TGF β and CTGF, a CTGF fragment encoded by exon 3 (exon 3), or a CTGF fragment encoded by exon 5 (exon 5) in the presence or absence of anti-CTGF antibody. Fig. 12B shows the extent to which anti-CTGF antibodies inhibit the interaction of TGF β and CTGF. In the figure, the antibodies include exemplary antibodies mAb1 and mAb4 of the present invention and antibody mAb3 that specifically binds to the C-terminal CTGF epitope.

Detailed Description

Before the present compositions and methods are described, it is to be understood that this invention is not limited to the particular methodologies, protocols, cell lines, assays, and reagents described, as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments of the invention, and is not intended to limit the scope of the invention which will be expressed in the claims.

It must be noted that, as used herein and in the claims, the singular forms "a," "an," and "the" include the plural forms unless the context clearly dictates otherwise. Thus, for example, reference to "a fragment" includes a plurality of such fragments, reference to "an antibody" is a reference to one or more antibodies and equivalents thereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described. All publications cited herein are incorporated herein by reference in their entirety for the purpose of describing and disclosing the methodologies, reagents, and tools reported in the publications that might be used in connection with the invention. Nothing herein is to be construed as an admission that the invention is not entitled to antedate such disclosure by virtue of prior invention.

The practice of the present invention will employ, unless otherwise indicated, conventional methods of chemistry, biochemistry, molecular biology, cell biology, genetics, immunology and medicine. These techniques are explained fully in the literature. See, for example, Gennaro, A.R., ed (1990) Remington's pharmaceutical Sciences,18th ed., Mack Publishing Co., Colowick, S.et al, eds., Methods In Enzymology, Academic Press, Inc., Handbook of Experimental immunology, Vols.I-IV (D.M.Weir and C.C.Blackwell, eds.,1986, Blackwell scientific Publications), Maniatis, T.T., eds. (1989) Molecular Cloning: ALORAL, 2nd edition, Vols.I-III, Cold Spring borg Laboratory, Experimental Press, Molecular Cloning, sample, Molecular analysis, Ready, Molecular testing, Press, Molecular testing, Ready, Molecular testing, PCR, Molecular testing, Ready, Molecular testing.

Definition of

By "connective tissue growth factor" or "CTGF" is meant an amino acid sequence of substantially purified CTGF derived from any species, particularly mammalian species, including rat, rabbit, bovine, ovine, porcine, murine, equine, and primate, preferably human species, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.

The term "N-terminal fragment" of CTGF refers to any polypeptide comprising a sequence derived from the amino-terminal portion of CTGF polypeptide, or to any variant or fragment thereof. The N-terminal fragment may include all or part of CTGF from the initial methionine residue to the "hinge" region without cysteine as shown in fig. 1A and 1B, or a sequence not including any of these segments. In addition, the N-terminal fragment may include all or part of the insulin-like growth factor binding protein motif and/or the von Villebrand type C domain (SEQ ID NO:21) as shown in FIG. 1B, or none of this sequence. The N-terminal fragment of CTGF may also include sequences that do not contain all or part of the cysteine domain, or any such segment. In addition, the N-terminal fragment of CTGF may be any 15 or more contiguous amino acids contained in any of the above N-terminal fragments.

In one aspect, an "N-terminal fragment" of CTGF refers to a polypeptide sequence derived from the amino-terminal portion of human CTGF. Such fragments may comprise the entire region from amino acid residue 1 to about amino acid residue 198 of SEQ ID NO. 2, or from about amino acid 23 to about amino acid 198 of SEQ ID NO. 2. The boundary of the N-terminal fragment within the hinge region may optionally be defined by one of several protease cleavage sites defined in SEQ ID NO 2, such as a chymotrypsin cleavage site between residues 179 and 180, between residues 182 and 183, and between residues 188 and 189; a plasmin cleavage site between residues 183 and 184 and between residues 196 and 197; a bone morphogenetic protein-1 cleavage site between residues 169 and 170. In addition, the N-terminal fragment of human CTGF may include all or part of the region from amino acid 27 to amino acid 97 of SEQ ID NO. 2, amino acid 103 to amino acid 166 of SEQ ID NO. 2, or amino acid 167 to amino acid 198 of SEQ ID NO. 2, or none of these sequences. In addition, the N-terminal fragment of human CTGF may be any 15 or more contiguous amino acids contained in any of the above N-terminal fragments.

In a particular embodiment, the CTGF N-terminal fragment of the present invention comprises a sequence selected from the group consisting of the following regions of human CTGF (SEQ ID NO:2) and orthologous fragments thereof derived from a different species, in particular mammalian species including rat, rabbit, bovine, ovine, porcine, murine and equine: amino acid residue 23 to amino acid residue 96 (encoded by exon 2); amino acid residue 27 to amino acid residue 97(IGF-BP motif); amino acid residue 97 to amino acid residue 180 (encoded by exon 3); amino acid residue 103 to amino acid residue 166(VWC domain); amino acid residue 167 to amino acid residue 198 (hinge region without cysteine); amino acid residue 23 through amino acid residue 180 (encoded by exons 2 and 3); amino acid residues 27 to 166(IGF-BP and VWC); and amino acid residue 23 through amino acid residue 198. (see FIG. 1B)

The term C-terminal fragment of CTGF refers to any polypeptide comprising a sequence derived from the carboxy-terminal portion of the CTGF amino acid polypeptide sequence, or to any variant or fragment thereof. The C-terminal fragment of CTGF may include all or part of the cysteine-free region of the CTGF polypeptide (amino acids 167 to 198 of SEQ ID NO:2), or none of this sequence.

The C-terminal fragment may include all or part of the CTGF from the cysteine-free hinge to the terminus of the protein, or none of this sequence. In addition, the C-terminal fragment may include all or part of the thrombospondin motif and/or the cysteine knot motif, or none of these sequences. In addition, the C-terminal fragment of CTGF may be any 15 or more contiguous amino acids contained within any of the aforementioned C-terminal fragments.

In some aspects, the C-terminal fragment can comprise the entire region of SEQ ID NO 2 from amino acid residue 181 to about amino acid residue 349. The boundary of the C-terminal fragment within the hinge region may optionally be defined by one of several protease cleavage sites defined in SEQ ID NO 2, such as the chymotrypsin, plasmin and bone morphogenetic protein-1 cleavage sites described above. In addition, the C-terminal fragment comprises the following region selected from human CTGF (SEQ ID NO:2) and sequences of orthologous fragments thereof derived from a different species, particularly mammalian species including rat, rabbit, bovine, ovine, porcine, murine and equine: amino acid 201 to amino acid 242 of SEQ ID NO. 2, amino acid 247 to amino acid 349 of SEQ ID NO. 2, amino acid 248 to amino acid 349 of SEQ ID NO. 2, or amino acid 249 to amino acid 346 of SEQ ID NO. 2. In addition, the C-terminal fragment of human CTGF may be any 15 or more contiguous amino acids contained within any of the C-terminal fragments described above.

The term "cysteine-free region" or "hinge region" of CTGF refers to any polypeptide derived from about amino acid residue 167 to about amino acid residue 198 of human CTGF (SEQ ID NO:2) and orthologous fragments thereof derived from a different species, particularly mammalian species including rat, rabbit, bovine, ovine, porcine, murine and equine.

The term "amino acid sequence" or "polypeptide" as used herein refers to an oligopeptide, peptide, polypeptide or protein sequence, as well as fragments thereof, to naturally occurring or synthetic molecules. A polypeptide or amino acid fragment is any portion of a polypeptide that retains at least one structural and/or functional characteristic of the polypeptide. CTGF fragments include any portion of the CTGF polypeptide sequence that retains at least one structural or functional characteristic of CTGF. When referring to an "amino acid sequence" to refer to a polypeptide sequence of a naturally occurring protein molecule, the term "amino acid sequence" or the like is not meant to limit the amino acid sequence to the entire native sequence associated with the referenced protein molecule.

The term "immunogenicity" relates to the ability of a substance, when introduced into the body, to stimulate an immune response and produce antibodies. Substances that exhibit immunogenic properties are said to be immunogenic. Immunogenic substances may include, but are not limited to, a variety of macromolecules such as proteins, lipoproteins, polysaccharides, nucleic acids, bacterial and bacterial components, viruses and viral components. The immunogenic agent typically has a molecular weight of greater than 10 kDa. An antigenic fragment refers to a fragment of CTGF polypeptide that retains at least one biological or immunological representation of CTGF polypeptide activity, preferably a fragment of about 5 to 15 amino acids in length.

The term "antibody" refers to intact molecules and fragments thereof, such as Fab, F (ab')2 and Fv fragments, including polyclonal and monoclonal antibodies, which are capable of binding epitope determinants. Antibodies that bind CTGF or fragments of CTGF may be prepared using the intact polypeptide or using fragments containing small peptides of interest as immunizing antigens. Polypeptides or oligopeptides used to immunize animals (e.g., mice, rats, rabbits, chickens, turkeys, goats, etc.) can be obtained from translation of RNA or chemical synthesis, and can be conjugated to a carrier protein if desired. Commonly used carriers chemically coupled to the peptides include, for example, bovine serum albumin, thyroglobulin, and Keyhole Limpet Hemocyanin (KLH).

The term "monoclonal antibody" as used herein refers to a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical in specificity and affinity, except that minor amounts of naturally occurring mutations may be present. Note that a monoclonal antibody composition may contain more than one monoclonal antibody.

Monoclonal antibodies included within the scope of the invention include hybrid antibodies and recombinant antibodies (e.g., "humanized" antibodies), regardless of the species of origin or immunoglobulin class or subclass, as well as antibody fragments (e.g., Fab, F (ab')2, and Fv) having at least one of the unique characteristics of the antibodies described herein. Preferred embodiments include antibodies that bind to substantially the same epitope as recognized by monoclonal antibody mAb1 and/or have an affinity for that epitope that is greater than or equal to the affinity of mAb 1.

The term "monoclonal" refers to the antibody characteristics as a population of substantially homogeneous antibodies, and should not be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies of the invention can be prepared by the hybridoma method first described by Kohler and Milstein (1975, Nature256:495-497), or can be prepared by recombinant DNA methods. See, for example, Celltech Therapeutics Ltd, European patent EP-0120694, Cabilly et al, U.S. Pat. No.4,816,567, or Mage and Lamoyi (1987; In:Monoclonal Antibody Production Techniques and Applications,Marcel Dekker,Inc.New York,pp.79-97)。

the term "neutralizing antibody" as used herein refers to an antibody, preferably a monoclonal antibody, that substantially inhibits or abrogates the biological activity of CTGF. Typically, the neutralizing antibody will inhibit the binding of CTGF to a cofactor, such as TGF β, inhibit the binding of CTGF to a CTGF-specific receptor associated with the target cell, inhibit the binding of CTGF to another biological target. In a particular embodiment, the neutralizing antibody inhibits CTGF biological activity to an extent about equal to or greater than that inhibited by mAb 1. Preferably, the neutralizing antibody inhibits CTGF biological activity to a degree about equal to or greater than that inhibited by CLN 1.

The phrase "CTGF-associated disease" as used herein refers to pathologies and diseases associated with abnormal or altered expression or activity of CTGF. Aberrant expression of CTGF has been associated with cell proliferative disorders such as those caused by endothelial cell proliferation, cell migration, tumor-like growth, systemic (general) tissue scarring, and a variety of disorders characterized by inappropriate deposition of extracellular matrix.

CTGF-associated diseases include, but are not limited to, diseases that include angiogenesis and other processes that play an important role in such pathologies as proliferative vitreoretinopathy; cancers, including acute lymphoblastic leukemia, cutaneous fibroma, breast cancer (breast cancer), glioma and glioblastoma, rhabdomyosarcoma and fibrosarcoma, connective tissue hyperplasia, angiolipoma, vascular leiomyoma, connective tissue proliferative cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer and liver cancer; other tumor growth and metastasis, etc.

CTGF-associated diseases also include fibrotic diseases and associated pathologies, such as excessive scarring resulting from local or systemic fibrosis, chronic or acute fibrosis of organs such as kidney, lung, liver, eye, heart, skin, and the like; or a tissue selected from, but not limited to, epithelial, endothelial, and connective tissues. Fibrosis can also occur in the eye and joints. Such CTGF-associated diseases include, for example, cardiac fibrosis, myocardial remodeling including reactive cardiac fibrosis or myocardial infarction or congestive heart failure; lung diseases including interstitial pulmonary fibrosis, and the like; fibrosis associated with dialysis, including peritoneal dialysis, such as Continuous Ambulatory Peritoneal Dialysis (CAPD); fibrosis around the dura mater; renal fibrosis; pulmonary fibrosis; fibrosis of the stroma; fibrosis of the skin; and fibrosis resulting from acute or repetitive trauma including surgery, chemotherapy, radiation therapy, allograft rejection, chronic and acute transplant rejection (e.g. kidney, liver or other organs); bronchiolitis obliterans, e.g. after lung transplantation; inflammation and infection, for example due to disease or injury.

Additionally, CTGF-associated diseases include, but are not limited to, sclerosing lesions, including systemic sclerosis, scleroderma, keloids, excessive scarring, and other skin diseases and lesions; atherosclerosis, such as lesions including atherosclerotic plaques, and atherosclerosis associated with diabetes, peritoneal dialysis, and the like; arthritis, including rheumatoid arthritis, osteoarthritis, and other joint inflammations, and the like; interstitial diseases, including interstitial fibrosis; crohn's disease; inflammatory bowel disease; retinopathies including, for example, proliferative vitreoretinopathy, nonproliferative diabetic retinopathy, proliferative diabetic retinopathy and macular degeneration (including age-related and juvenile (Stargardt's) diseases, and pigment epithelial detachment); nephropathy, including diabetic nephropathy, IgA-related nephropathy, nephropathy due to toxicity, lupus nephropathy, etc.; and lesions associated with chemotoxic tubular destruction (tubular destruction).

CTGF-associated diseases also include, but are not limited to, diseases caused by hyperglycemia, hypertension, formation of advanced glycation end products (AGEs), and the like. Such diseases may be caused by diabetes, obesity, etc., and include diabetic nephropathy, retinopathy and cardiovascular disease. Additionally, CTGF-associated diseases may be caused by any initiating factor, including but not limited to exposure to chemicals or biological agents, inflammatory responses, autoimmune reactions, trauma, surgical procedures, and the like. In some embodiments, the methods are used to treat patients predisposed to CTGF-associated diseases due to pathologies including, but not limited to, myocardial infarction, arthritis, and local or systemic inflammation.

As referred to herein, "proliferative" processes and diseases include pathological conditions characterized by continued multiplication of cells resulting in overgrowth of cell populations within tissues. The cell population need not be transformed, tumorigenic, or malignant cells, but may also include normal cells. For example, CTGF may be pathologically involved by inducing a proliferative lesion in the intimal layer of the arterial wall leading to atherosclerosis or by stimulating neovascularization.

"cancer" refers to any spontaneous growth of tissue, including uncontrolled abnormal growth of cells, or to any malignancy of potentially unrestricted growth, which spreads locally through invasion and systemically through metastasis. Cancer also refers to any abnormal state characterized by cancer.

The term "fibrosis" refers to abnormal processing of fibrous tissue or fibrosis. Fibrosis can result from various injuries or diseases, and can often result from chronic graft rejection associated with various organ transplants. Fibrosis typically involves the abnormal production, accumulation or deposition of extracellular matrix components, including, for example, increased overproduction and deposition of collagen and fibronectin. "fibrosis" is used herein in its broadest sense to refer to any overproduction or deposition of extracellular matrix proteins. There are many examples of fibrosis, including scar tissue formation after a heart attack, which impairs the blood supply to the heart. Diabetes often leads to kidney damage/scarring, resulting in progressive loss of kidney function; and results in vision loss in the eye. After surgery, scar tissue may form between internal organs, causing contractures, pain, and in some cases infertility. Major organs such as the heart, kidney, liver, eye and skin are prone to chronic scarring commonly associated with other diseases. Hypertrophic scars (non-malignant tissue masses) are the most common form of fibrosis caused by burns and other wounds. In addition, there are many other fibroproliferative diseases including scleroderma, keloids and atherosclerosis, which are associated with systemic tissue scarring, tumor-like growth in the skin or persistent vascular scarring that impairs blood-carrying capacity, respectively.

The term "nucleic acid" or "polynucleotide" refers to an oligonucleotide, a nucleotide sequence or a polynucleotide, or any fragment thereof, and also to DNA or RNA of natural or synthetic origin, which may be single-or double-stranded and may represent a sense or antisense strand, and also to Peptide Nucleic Acid (PNA), or to any DNA-like or RNA-like material of natural or synthetic origin. A polynucleotide fragment is any portion of a polynucleotide sequence that retains at least one structural or functional characteristic of the polynucleotide. A polynucleotide fragment may be of variable length, for example greater than 60 nucleotides in length, at least 100 nucleotides in length, at least 1000 nucleotides in length, or at least 10000 nucleotides in length.

"altered" polynucleotides include those having deletions, insertions, or substitutions of different nucleotides that result in the production of a polynucleotide encoding the same or a functionally equivalent polypeptide. Also included within this definition are sequences that display polymorphisms that are readily or poorly detectable with specific oligonucleotide probes or by deletion of inappropriate or unexpected hybridization to alleles, which polymorphisms have positions that differ from the normal chromosomal position of the target polynucleotide sequence.

An "altered" polypeptide may contain deletions, insertions, or substitutions of amino acid residues that produce a silent change and produce a functionally equivalent polypeptide. The desired amino acid substitutions may be made based on similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or amphiphilicity of the residues, so long as the biological or immunological activity of the encoded polypeptide is retained. For example, negatively charged amino acids may include aspartic acid and glutamic acid; positively charged amino acids may include lysine and arginine; amino acids with uncharged polar head groups having similar hydrophilicity values can include leucine, isoleucine and valine, glycine and alanine, asparagine and glutamine, serine and threonine, phenylalanine and tyrosine.

A polypeptide or amino acid "variant" refers to an amino acid sequence in which one or more amino acids are changed from a particular amino acid sequence. Polypeptide variants may have conservative changes, where the substituted amino acid has similar structural or chemical properties as the amino acid being substituted, e.g., replacement of leucine with isoleucine. Variants may also have non-conservative changes in which the substituted amino acid has different physical properties than the amino acid being substituted, e.g., replacement of glycine with tryptophan. Similar minor variations may also include amino acid deletions or insertions, or both. Preferably, the amino acid variants retain certain structural or functional characteristics of the particular polypeptide. Guidance in determining which amino acid residues may be substituted, inserted or deleted can be found, for example, using computer programs well known in the art, such as LASERGENE software (DNASTAR inc., Madison, WI).

A polynucleotide variant is a variant of a particular polynucleotide sequence that preferably has at least about 80%, more preferably at least about 90%, and most preferably at least about 95% polynucleotide sequence similarity to the particular polynucleotide sequence. One skilled in the art will appreciate that due to the degeneracy of the genetic code, a large number of variant polynucleotide sequences encoding particular proteins can be produced, some of which have very low homology to any known or naturally occurring gene. Thus, the invention includes every possible variation of a polynucleotide sequence that can be made by selecting combinations based on possible codon usage. These combinations were generated according to the standard codon triplet genetic code, and all of these variations were considered to be specifically disclosed.

A "deletion" is a change in an amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.

The term "insertion" or "addition" refers to a change in a polypeptide or polynucleotide that results in the addition of one or more amino acid residues or nucleotides, respectively, as compared to a naturally occurring molecule.

The term "functional equivalent" as used herein refers to a polypeptide or polynucleotide having at least one functional and/or structural feature of a particular polypeptide or polynucleotide. Functional equivalents may contain modifications that allow for the performance of specific functions. The term "functional equivalent" is intended to include a fragment, mutant, hybrid, variant, analog or chemical derivative of a molecule.

The term "microarray" refers to any arrangement of nucleic acids, amino acids, antibodies, etc. on a substrate. The substrate may be any suitable support, such as beads, glass, paper, nitrocellulose, nylon, or any suitable membrane, and the like. The substrate can be any rigid or semi-rigid support, including but not limited to membranes, filters, wafers, chips, slides, fibers, beads including magnetic and non-magnetic beads, gels, tubes, plates, polymers, microparticles, capillaries, and the like. The substrate may provide a surface for coating and/or may have various surface forms such as wells, needles, grooves, channels, wells, to which nucleic acids, amino acids, etc. may be bound.

The term "sample" is used herein in its broadest sense. The sample may be from any source, for example, from cells in bodily fluids, secretions, tissues, cells, or cultures including, but not limited to, saliva, blood, urine, serum, plasma, vitreous humor, synovial fluid, cerebrospinal fluid, amniotic fluid, and organ tissue (e.g., biopsy tissue); from chromosomes, organelles, or any other membrane isolated from a cell; from genomic DNA, cDNA, RNA, mRNA, etc.; and blots or traces from clarified cells or tissues, or from such cells or tissues. The sample may be from any source, e.g., a human or non-human mammalian subject, etc. The invention also includes samples from animal models of any disease. The sample may be in solution or may be immobilized or bound to a substrate. A sample may refer to any material suitable for testing for the presence of CTGF or CTGF fragments, or for screening for molecules that bind to CTGF or fragments thereof. Methods for obtaining these samples are within the level of skill in the art.

The term "hybridization" refers to the process by which a nucleic acid sequence binds to a complementary sequence through base pairing. Hybridization conditions can be defined, for example, by the salt or formamide concentration in the prehybridization and hybridization solutions, or by the hybridization temperature, as is well known in the art. Hybridization can occur under various stringency conditions.

In particular, stringency can be increased by decreasing salt concentration, increasing formamide concentration, or raising hybridization temperature. For example, in the present invention, hybridization under high stringency conditions can occur in about 50% formamide at about 37 ℃ to 42 ℃, and hybridization under reduced stringency conditions can occur in about 35% to 25% formamide at about 30 ℃ to 35 ℃. In particular, hybridization generally occurs under the highest stringency conditions, i.e., hybridization occurs at 42 ℃ in 50% formamide, 5 XSSPE, 0.3% SDS, and 200. mu.g/ml sheared and denatured salmon sperm DNA.

The temperature range corresponding to a particular level of stringency can be further narrowed by methods known in the art, for example, by calculating the purine to pyrimidine ratio of the nucleic acid of interest and adjusting the temperature accordingly. To remove non-specific signals, the blot can then be washed at, for example, room temperature or up to and including 60 ℃ under conditions of increased stringency up to 0.1 XSSC and 0.5% SDS. Variations of the above ranges and conditions are well known in the art.

Invention of the invention

The present invention provides antibodies that specifically bind Connective Tissue Growth Factor (CTGF). The antibody is a polyclonal or monoclonal antibody, preferably a monoclonal antibody, more preferably a human monoclonal antibody. The antibody is an antibody to an N-terminal fragment of CTGF, as shown in fig. 1. More particularly, the antibody is an antibody to a CTGF fragment extending from residue 97 to residue 180 of SEQ ID NO. 2. In particular embodiments, the antibody is an antibody to a fragment of CTGF extending from about residue 103 to about residue 164, particularly from about residue 134 to about residue 158 of SEQ ID No. 2. More particularly, the antibody is an antibody to a CTGF fragment from about residue 143 to about residue 154 of SEQ ID NO. 2.

In particular embodiments, the antibodies neutralize the biological activity of CTGF. Biological activities of CTGF include cell proliferation, differentiation, gene expression, and the like. In particular embodiments, the biological activity is selected from the group consisting of cell differentiation, e.g., differentiation or transdifferentiation of fibroblasts, myofibroblasts, endothelial cells, etc., from various precursor cells; induction of expression of proteins involved in extracellular matrix formation and remodeling, including, for example, type I collagen, fibronectin, and the like; synergistic induction of signaling cascades associated with various factors including, but not limited to, TGF- β, IGF, VEGF, angiotensin II, endothelin, and the like; cellular responses to various environmental stimuli, including but not limited to a group consisting of increased glucose levels (hyperglycemia), increased mechanical stress (hypertension), and the like.

Although the present invention is not limited by the mechanism by which antibodies neutralize CTGF activity, antibodies can bind to CTGF and prevent its interaction with specific cellular receptors. The receptor may have a high affinity for CTGF, and stimulates the production of intracellular signals resulting in proliferation, differentiation, induction of gene expression, and/or alteration of cell morphology or function by binding to CTGF. The specific biological response of cells to CTGF depends on the current state of the cell and the surrounding environment. Alternatively, the receptor may have a low CTGF binding affinity and by binding to CTGF may, for example, localize CTGF to a high affinity receptor to facilitate recognition and response to CTGF. Alternatively, the antibody may bind to CTGF in a tissue or organ and facilitate titration or elimination of CTGF from the body.

Alternatively or in combination with the above mechanisms, the antibodies may bind to CTGF and prevent interaction of CTGF with secreted or membrane-bound cofactors. Such cofactors include, inter alia, members of the TGF superfamily, e.g., TGF-1, -2, and-3; activin-A, -B, -C, and-E; BMP-2, -3, -4, -5, -6, -7, -8a, -8b, -9, -10, -11, and-15; and GDF-3, -5, -6, -7, -9, and-10. For example, CTGF has been shown to bind to and modulate the activity of TGF-1 and BMP-4 (Abreu et al (2002) Nat Cell Biol4: 599-604). The present invention provides evidence that the region of CTGF that binds to TGF- β is encoded by exon 3 (FIG. 1B, nucleotide 418-669 of SEQ ID NO: 1) and that antibodies that bind to this region prevent interaction between CTGF and TGF β (example 12, described below). In addition, antibodies binding in this region of CTGF were shown to neutralize specific CTGF-associated processes in animal models. For example, antibodies binding to this region of CTGF have been shown to specifically inhibit cell migration in ex vivo assays and reduce fibrosis in animal models. Exemplary antibodies of the invention are mAbl and-CLN 1; antibody CLN1 was produced by the cell line ATCC accession No. deposited with the american type culture collection (Manassus VA) on day 5, month 19, 2004.

Regardless of the mechanism of action, the present invention provides methods of using antibodies to treat various diseases and disorders associated with CTGF. Diseases and disorders associated with CTGF include, but are not limited to, nephropathy, pulmonary fibrosis, retinopathy, scleroderma, liver fibrosis, heart failure, arthritis, and atherosclerosis. In addition, CTGF-associated pathologies are due to a variety of factors including, but not limited to, hyperglycemia, hypertension, diabetes, obesity, and the like, as well as diabetic nephropathy, retinopathy, cardiovascular disease, and the like. Since CTGF is overexpressed in many diseases, including the above-described diseases, it is expected that the present invention will treat patients having CTGF-associated diseases with CTGF antibodies to improve or stabilize the pathology, maintain or restore organ function, improve quality of life, and prolong survival time.

For example, the antibodies are specifically directed to regions of CTGF involved in biological activities associated with both fibrotic and non-fibrotic aspects of various diseases, such as interstitial pulmonary fibrosis, diabetic nephropathy and retinopathy, macular degeneration, and the like. The present invention also relates to methods of using the antibodies to treat disorders associated with CTGF, including localized and systemic fibrotic disorders, such as those of the lung, liver, heart, skin, and kidney; and local scarring due to, for example, trauma, surgery, and the like.

The antibodies of the invention may also be used in any method that involves binding to CTGF. Such methods include, for example, purification of CTGF or CTGF fragments by affinity chromatography, e.g., detection of CTGF or CTGF fragments in a sample using ELISA or immunohistochemical techniques; the CTGF-associated disease is diagnosed by using a method for detecting CTGF to determine the level of CTGF in the patient sample and to compare with the level of CTGF in the standard sample.

Antibodies against CTGF

Modulation of the amount and/or activity of secreted cytokines using, for example, monoclonal antibodies has been demonstrated, and some therapeutic antibodies have been licensed or in the development phase (see, for example, Abciximab (Reopro; Centocor, Inc., Malvern PA), Infliximab (Remicade; Maiini et al (1998) Arthritis Rheum41: 1552-10353; Targan et al (1997) N Engl J Med337: 1029-Bas 1035); iliximab (Simmulct) and Daclizumab (Zenapax) (Bumgardner et al (2001) Transplantation72:839-845; Kovak et al (1999) Transplantation68:1288-1294); and Trastuzumab (Herceptin; Baselin Onla S55: S12-S55-11)). Numerous methods for producing antibodies, including production in animals, plants, fungi and bacteria, synthetic construction and ex vivo culture are known to those skilled in the art and can be utilized.

Antibodies of the invention can be prepared using any technique directed to the production of antibody molecules. Techniques for the production of monoclonal or polyclonal antibodies in vivo and in vitro are well known in the art (see, e.g., Pound (1998)Immunochemical Protocols,Humana Press,Totowa NJ;Harlow and Lane(1988)Antibodies:A Laboratory Manual,Cold Spring Harbor Laboratory,New York;Goding(1986)Monoclonal Antibodies:Principles and Practice,2"d Edition,Academic Press;Schook(1987)Monoclonal Antibody Production Techniques and ApplicationsMarcel Dekker, Inc.). Techniques for generating chimeric antibodies are also well known in the art, such as the generation of single chain antibodies (see, e.g., Morrison et al (1984) Proc Natl Acad Sci USA81: 6851-. Antibodies with related specificity but unique idiotypic components can be generated by a variety of suitable methods, such as by chain shuffling (chain shuffling) from a randomly combined immunoglobulin library (see, e.g., Burton (1991) Proc)Natl Acad Sci USA88:11120-11123)。

Antibodies can also be generated in vivo by induction in lymphocyte populations or by screening immunoglobulin libraries or panels of highly specific binding reagents (see, e.g., Orlandi et al (1989) Proc NatlAcad Sci USA86: 3833-. Antibody fragments containing specific binding sites for the target polypeptide can also be generated. Such antibody fragments include, but are not limited to, F (ab ')2 fragments, which can be produced by pepsin digestion of the antibody molecule, and Fab fragments, which can be produced by reducing F (ab')₂Disulfide bonds of the fragments. Alternatively, Fab expression libraries can be constructed to quickly and easily identify monoclonal Fab fragments of the desired specificity (see, e.g., Huse et al (1989) Science254: 1275-.

Monoclonal antibodies of the invention may also be prepared using hybridoma methods (see, e.g., Kohler and Milstein (1975) Nature256:495-497), or by recombinant DNA methods (see, e.g., Celltech Therapeutics Ltd., European Patent No. EP0120694; Cabilly et al, U.S. Pat. No.4,816,567; and Mage and Lamoyi (1987) In:Monoclonal Antibody Production Techniques and Applications,Marcel Dekker,Inc.,NewYork,pp.79-97)。

in the hybridoma method, a mouse or other suitable host animal is immunized with CTGF or fragments thereof by subcutaneous, intraperitoneal, or intramuscular routes to elicit lymphocytes that produce or are capable of producing antibodies that specifically bind to the polypeptide used for immunization. Alternatively, the host animal may be a transgenic mammal,

it has a transgene encoding a human immunoglobulin gene and has an inactivated endogenous immunoglobulin locus. The transgenic mammal responds to the immunogen by producing human antibodies (see, e.g., Lonberg et al, WO93/12227(1993), U.S. Pat. No.5,877,397, and Nature148: 1547. sub.1553 (1994); and Kucherlapati et al (1991) WO 91/10741). Alternatively, lymphocytes can be immunized in vitro and then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycolFusing to form hybridoma cells (see, e.g., Goding (1986)Monoclonal Antibodies:Principles and Practice2nd Edition, Academic Press, pp.59-103). Alternatively, antibody-producing human cells, particularly B lymphocytes, are suitable for fusion with a myeloma cell line. Peripheral blood B lymphocytes, which are more readily available, are preferred when spleen, tonsils or lymph nodes from an individual biopsy can be used. Alternatively, human B cells can be directly immortalized by Epstein-Barr virus (see, e.g., Cole et al (1995)Monoclonal Antibodies and Cancer Therapy,Alan R.Liss,Inc.,pp.77-96)。

Preferred myeloma cell lines for use in hybridoma-producing fusion procedures are those that fuse efficiently, support stable high-level expression of antibodies by selected antibody-producing cells, are enzyme deficient such that they cannot grow in certain selected media that support the desired hybridoma growth, and cannot produce antibodies themselves. Myeloma cell lines that can be used for producing hybridomas in the present invention include, for example, P3X63Ag8, P3X63Ag8-653, NS1/l.Ag4.1, Sp210-Agl4, FO, NSO/U, MPC-11, MPC11-X45-GTG1.7, S194/5XXO Bul; rat-derived R210.RCY3, Y3-Ag1.2.3, IR983F and 4B 210; and human-derived U-266, GM1500-GRG2, LICR-LON-HMy2, UC729-6 (see, e.g., Goding (1986) Monoclonal Antibodies: Principles and Practice,2nd Edition, Academic Press, pp.65-66; and Campbell (1984) In:Monoclonal Antibody Technology: Laboratory Techniques in Biochemistry and Molecular Biology,Vol.13(Burden and Von Knippenberg,eds.)Amsterdam,Elseview,pp.75-83)。

the hybridoma cells are seeded and grown in a suitable medium that preferably contains one or more agents that inhibit the growth or survival of the unfused parent myeloma cells. For example, if the parental myeloma cells lack hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium of the hybridoma typically includes an agent that prevents the growth of HGPRT-deficient cells, such as hypoxanthine, aminopterin, and thymidine (HAT medium).

Determining production of monoclonal antibodies directed against CTGF or CTGF fragments by the medium in which the hybridoma cells are grown. Preferably, the binding specificity is determined by affinity chromatography, immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA), or by a Fluorescence Activated Cell Sorting (FACS) assay. The monoclonal antibodies of the invention are those that bind to CTGF, and are additionally those that neutralize the biological activity of CTGF, as exemplified below.

Antibodies generated as described above are optionally screened to detect antibodies that bind well to the N-terminal fragment of CTGF. In one embodiment, the antibody is directed against a fragment of CTGF extending from about residue 24 to about residue 180 of SEQ ID No. 1. In another embodiment, the antibody is directed against a fragment of CTGF extending from about residue 96 to about residue 180 of SEQ ID No. 1. In a particular embodiment, the screen detects antibodies that bind sufficiently to the same epitope recognized by the antibody mAbl, as determined, for example, by the competition assay described below. In another particular embodiment, the screen detects antibodies that bind sufficiently to the same epitope recognized by antibody CLN1, as determined, for example, by the competition assay described below. It should be noted that "identical epitope" does not mean the exact amino acid or carbohydrate bound by the reference antibody, which can be determined by epitope mapping (epitop mapping), for example, using the CTGF variants of alanine scanning. By "epitope-identical" is meant the complete form of the CTGF domain blocked by binding to CTGF by a natural reference antibody. Of course, "identical epitope" includes CTGF domain residues or carbohydrates that structurally interact with or bind to the reference Complementarity Determining Regions (CDRs) of mAbl or CLN 1.

In a preferred embodiment of the invention, the monoclonal antibody has an affinity equal to or greater than that of mABl, as determined by, for example, the scatchard analysis by Munson and Pollard (1980, AnalBiochem107: 220).

In the identification of hybridoma cellsAfter production of neutralizing antibodies of desired specificity and affinity, the clones are typically subcloned by limiting dilution methods and grown by standard methods (Goding (1986)Monoclonal Antibodies:Principles and Practice2nd Edition, academic Press, pp.59-104). Suitable media for this purpose include, for example, Dulbecco's Modified Eagle's Medium or RPMI-1640 medium. In addition, the hybridoma cells can also be grown in vivo in animals as ascites tumors.

Monoclonal antibodies secreted by the subclones are isolated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification methods, such as by protein A-Sepharose (Sepharose), hydroxyapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

The DNA encoding the monoclonal antibodies of the invention is readily isolated and sequenced using conventional methods, e.g., by using oligonucleotide probes that specifically bind to the heavy and light chains encoding the antibodies. Once isolated, the DNA may be ligated into an expression or cloning vector, which is then transfected into host cells that do not otherwise produce immunoglobulin, such as simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells. The cells thus transformed are cultured under conditions suitable for the synthesis of monoclonal antibodies in recombinant host cell culture. An exemplary cell line is deposited at 19.5.2004 with ATCC accession No. ____.

The DNA is optionally modified to alter the properties of the immunoglobulin it encodes. Variants of immunoglobulins are well known. For example, chimeric antibodies are produced by substituting the coding sequences for the heavy and light chain constant domains of one species, such as mouse, with homologous sequences from another species, such as human (see, e.g., Boss et al, International publication No. WO84/03712; Cabilly et al, U.S. Pat. No.4,816,567; or Morrison et al (1984) Proc Nat Acad Sci81: 6851). In a particular embodiment, a humanized form of a mouse antibody may be produced by substituting the framework domains, i.e., constant regions, of a human antibody for the Complementarity Determining Regions (CDRs), i.e., variable domains, of a mouse antibody (see, e.g., international publication No. wo 92/22653). In some embodiments, selected murine framework residues are also replaced by human acceptor immunoglobulins. In addition, the selected Fc domain may be any of IgA, IgD, IgE, IgG-1, IgG-2, IgG-3, IgG-4, or IgM. The Fc domain optionally has effector functions such as ability to complementarily bind.

The anti-CTGF antibodies of the invention may also be fused to a component that provides additional capabilities such as detection or cytotoxic effects. The fusion of the immunoglobulin of the invention with a cytotoxic component is, for example, produced by linking the immunoglobulin coding sequence to all or part of the coding sequence of a cytotoxic non-immunoglobulin polypeptide. Such non-immunoglobulin polypeptides include polypeptide toxins such as ricin, diphtheria toxin, or pseudomonas exotoxin. The conjugates can also be prepared by in vitro methods. For example, immunotoxins may be constructed using disulfide substitution reactions or by forming a thioether bond between an immunoglobulin and a toxin polypeptide. Suitable reagents for this purpose include, for example, iminothiolate and methyl-4-mercaptobutylspermidine (methyl-4-mercaptobiuridate). Typically, such non-immunoglobulin fusion polypeptides replace the constant regions of the antibodies of the invention. Alternatively, they replace the variable region of one antigen binding site of an antibody of the invention.

Substitution of the Fv or CDR of an antibody with antigen specificity other than CTGF results in a chimeric antibody comprising one antigen binding site specific for CTGF and another antigen binding site specific for a different antigen. In this embodiment, the light chain is deleted and the Fv of the heavy chain is replaced by the desired polypeptide. These antibodies are called bivalent or multivalent antibodies depending on the number of immunoglobulin "arms" the Fc domain has, e.g., IgG is bivalent and IgM is multivalent. In addition to the non-immunoglobulins described above, the antibodies are also made multivalent by recombination of antibodies with more than one specificity. For example, antibodies in some embodiments bind CTGF (as described elsewhere herein), but also bind another growth factor, e.g., TGF β, VEGF, FGF, other CCN family members such as CYR61, etc., or a cytokine. Examples of antibodies against these factors are well known. Multispecific, multivalent antibodies are produced by co-transforming cells with DNA encoding the heavy and light chains of both antibodies, and the expressed antibody portion having the desired structure is recovered by techniques such as immunoaffinity chromatography. Alternatively, such antibodies are produced from monovalent antibodies by in vitro recombination in a conventional manner.

The monovalent antibody itself is also produced by conventional techniques. Recombinant expression of the light chain and the modified heavy chain is suitable. The heavy chains are typically truncated at any point within the Fc region to prevent heavy chain cross-linking. Or the relevant cysteine is substituted or deleted with another residue to prevent cross-linking. In vitro methods are also used to generate monovalent antibodies, e.g., Fab fragments are prepared by cleaving intact antibodies with an enzyme.

Diagnosis of

The antibodies of the invention are useful for qualitative and quantitative detection of CTGF in a sample. The sample may be from any source, including conditioned medium that allows cells to grow in culture, tissue samples such as biopsies and organ transplants, bodily fluids including blood, urine, vesicular fluid, cerebrospinal fluid, vitreous fluid, synovial fluid, and the like. In one embodiment, the detection of CTGF is used to diagnose the state of cells growing in culture, e.g., with respect to differentiation, matrix production, and the like. CTGF has various autocrine and paracrine effects on cultured cells, and the level of CTGF present in the cell layer or in the conditioned medium may be indicative of the current state of the cell or predictive of a future state of the cell (see, e.g., international publication No. wo 96/38168). In other embodiments, the detection of CTGF is used to determine the status of a tissue or organ. For example, a given transplanted organ may be evaluated by determining the level of CTGF expressed by cells in the organ, which indicates the relative health of the organ and the suitability of the transplant. CTGF levels may also be determined in biopsied tissue to determine the status of the organ or the stage of the cancer and potential metastatic predisposition.

In a preferred embodiment, the antibody is used to diagnose a disease or disorder associated with CTGF (see, e.g., international publication No. wo 03/024308). In one aspect, the invention provides antibodies for diagnosing a CTGF-associated disorder by obtaining a sample, detecting and quantifying the level of CTGF in the sample, and comparing the level of CTGF in the sample to a standard amount of CTGF, wherein an increase or decrease in the level of CTGF in the sample indicates the presence of a CTGF-associated disorder. Diseases associated with abnormal (e.g., increased or decreased) levels of CTGF include, but are not limited to, diseases associated with altered expression and deposition of proteins associated with the extracellular matrix. Such diseases include, for example, cancers such as breast, pancreatic and gastrointestinal cancers, atherosclerosis, arthritis, retinopathies such as diabetic retinopathy, renal pathologies such as diabetic nephropathy, heart, lung, liver and kidney fibrosis, and diseases associated with chronic inflammation and/or infection. CTGF-associated diseases are also associated with pathologies such as myocardial infarction, diabetes, peritoneal dialysis, chronic and acute transplant rejection, chemotherapy, radiation therapy and surgery.

In another aspect, the present invention provides antibodies useful for identifying whether an individual has a predisposition to develop a CTGF-associated disease. The predisposition may initially manifest itself as hyperglycemia, hypertension, or obesity in the subject. In addition, the predisposition may be suspected to arise as a result of an event experienced by the subject, such as myocardial infarction, surgery, orthopedic surgery or paralytic immobilization (paralytic immobilization), congestive heart failure, pregnancy or varicose veins in the subject.

In another aspect, the present invention provides antibodies that monitor the progression of, or monitor the efficacy of a treatment for, a CTGF-associated disease. For example, a method of using the antibody can include obtaining a sample from a subject over time; detecting and quantifying the level of CTGF in each sample; the CTGF levels in subsequent samples are compared to the CTGF levels in earlier or previous samples. Changes in CTGF levels between samples over time indicate progression of the CTGF-associated disease or efficacy of treatment of the CTGF-associated disease.

For diagnostic applications, the antibodies of the invention are typically labeled with a detectable moiety. The detectable label may be any component capable of directly or indirectly producing a detectable signal. For example, the detectable component may be a radioisotope, such as 3H, 14C, 32p, 35S, or 125I; fluorescent or chemiluminescent compounds, such as fluorescein isothiocyanate, rhodamine or luciferin; or an enzyme such as alkaline phosphatase, beta-galactosidase or horseradish peroxidase.

Any method known in the art for separately conjugating the antibody to a detectable component can be used (see, e.g., Hunter et al (1962) Nature144:945; David et al (1974) Biochemistry13:1014; Pain et al (1981) J Immunol Meth40:219; and Nygren (1982) JHistochem Cytomem 30: 407). The antibodies of the invention can be used In any assay method, such as competitive binding assays, direct and indirect sandwich assays, and immunoprecipitation assays (Zola (1987) In:Monoclonal Antibodies:A Manual of Techniques,CRC Press,Inc.,pp.147-158.)。

competitive binding assays rely on the ability of a labeled standard (which may be CTGF or an immunoreactive portion thereof) to compete with the sample analyte (CTGF) for binding to a limited amount of antibody. The amount of CTGF in the sample is inversely proportional to the amount of standard bound to the antibody. To facilitate determination of the amount of standard bound to the antibody, the antibody is typically insoluble before or after competition, so that the standard and analyte bound to the antibody can be routinely separated from unbound standard and analyte.

Sandwich assays involve the use of two antibodies, each capable of binding a different immunogenic portion or epitope of the protein to be tested. In a sandwich assay, a sample analyte binds to a first antibody immobilized on a solid support, after which a second antibody binds to the analyte, thereby forming an insoluble three-part complex (David and Greene, U.S. Pat. No.4,376,110). The second antibody may itself be labeled with a detectable component (direct sandwich assay) or an anti-immunoglobulin antibody assay (indirect sandwich assay) labeled with a detectable component may be used. For example, one sandwich assay is an ELISA assay, in which case the detectable component is an enzyme. One exemplary assay that can use the antibodies of the invention is described in international publication No. wo 03/024308.

The antibodies of the invention are also useful for in vivo imaging, wherein a detectable component, such as a radiopaque agent, a radioisotope, or a fluorescent component, such as a Green Fluorescent Protein (GFP) -labeled antibody, is administered to a host, preferably into the bloodstream, and the presence and location of the labeled antibody in the host is determined. This imaging technique is used for staging and treating CTGF-associated diseases such as fibrotic diseases. The antibody may be labeled with any moiety detectable in the host, whether by nuclear magnetic resonance, radiology, or other detection means known in the art.

Treatment of

The present invention provides antibodies for the treatment of various diseases and disorders associated with CTGF. The antibodies of the invention have been found to reduce the deleterious effects caused by CTGF production or activity in certain diseases, as exemplified below. In addition, the antibodies show advantageous pharmacokinetic properties, making them useful as superior therapeutic agents for the treatment of CTGF-associated diseases.

The anti-CTGF antibodies of the invention inhibit the development of fibrosis in animal models, for example, inhibit the development of lung and kidney fibrosis. In particular, the antibodies reduced bleomycin-induced pulmonary fibrosis in mice by 60-70% as determined by inhibition of lung hydroxyproline (collagen) accumulation and histological examination of tissue preparations. In addition, the antibodies reduced collagen accumulation in the rat residual kidney (i.e., 5/6 nephrectomy) model and in mice following Unilateral Ureteral Obstruction (UUO). The antibodies also reduced fibrosis induced by a combination of subcutaneous or intraperitoneal perfusion of CTGF and TGF in neonatal mice. In addition, the antibodies reduce complications associated with organ failure, such as improving kidney function in various models of chronic and acute renal failure. No toxicity was observed in animals using these antibodies. Because CTGF is overexpressed in a variety of fibrotic diseases, including diffuse and limited scleroderma, osteoarthritis, diabetic nephropathy, retinopathy, and the like, the present invention includes the use of CTGF antibodies to treat patients having CTGF-associated diseases to ameliorate or stabilize the pathology, restore organ function, improve quality of life, and prolong survival.

Accordingly, the antibodies of the invention are particularly useful for therapeutic applications to prevent or treat CTGF-associated diseases in a subject. Such diseases include, but are not limited to, angiogenesis and other processes that play a key role in certain pathologies and cancers, such as atherosclerosis, glaucoma, and the like; such cancers include acute lymphoblastic leukemia, cutaneous fibromas, breast cancer, breast tumors (breast carcinomas), gliomas and glioblastomas, rhabdomyosarcomas and fibrosarcomas, desmoplasia, angiolipomas, angioleiomyomas, desmoplasia, and prostate, ovarian, colorectal, pancreatic, gastrointestinal and liver cancers and other tumor growth and metastasis.

In addition, the antibodies of the invention are useful for therapeutic applications to prevent or treat CTGF-associated diseases, including fibrosis. In one aspect, the antibodies of the invention are administered to a subject to prevent or treat CTGF-associated disorders, including but not limited to disorders that exhibit altered expression or deposition of extracellular matrix-associated proteins, such as fibrotic disorders. In various aspects, fibrosis can be localized to a particular tissue, such as epithelial, endothelial, or connective tissue; or to an organ such as the kidney, lung or liver. Fibrosis can also occur in the eye and joints. In other aspects, the fibrosis can be systemic, including multiple organ and tissue systems. CTGF-associated diseases include, for example, atherosclerosis, arthritis, retinopathies such as diabetic retinopathy; renal disorders such as diabetic nephropathy; heart, lung, liver and kidney fibrosis, and diseases associated with chronic inflammation and/or infection.

In another aspect, the present invention provides an antibody for preventing a CTGF-associated disease in a subject having a predisposition to develop the CTGF-associated disease. The predisposition may include, for example, hyperglycemia, hypertension, or obesity of the subject. Such diseases may occur, for example, as a result of diabetes, obesity, and the like, including diabetic nephropathy, retinopathy, and cardiovascular disease. In addition, the predisposition may be suspected to arise as a result of an event experienced by the subject, such as myocardial infarction, surgery, peritoneal dialysis, chronic and acute transplant rejection, chemotherapy, radiation therapy, trauma, plastic surgery or paralytic immobilization, congestive heart failure, pregnancy or varicose veins in the subject.

In particular embodiments, as exemplified herein, the antibodies of the invention are administered to a subject to treat fibrosis of an organ, such as the lung or kidney. The antibodies are shown herein to provide benefits in various models of lung and kidney fibrosis (see, e.g., examples 7-9). In another particular embodiment, the antibodies of the invention are administered to a subject to reduce local or systemic sclerosis (see, e.g., examples 11 and 12). In additional embodiments, the antibodies are administered to a subject to treat or prevent ocular diseases such as proliferative vitreoretinopathy, diabetic retinopathy, macular degeneration, and the like. Since CTGF is involved in many diseases, the invention further includes treating a patient having a CTGF-associated disease with an antibody of the invention to improve or stabilize pathology and organ function, improve quality of life, and prolong survival.

For therapeutic use, the antibodies of the invention are administered to a mammal, preferably a human, in a pharmaceutically acceptable dosage form. The antibody may be administered intravenously as a bolus and/or by continuous infusion over a period of time, and/or by intramuscular, subcutaneous, intraarterial, intrasynovial, intrathecal, intravitreal, intracranial, oral, topical, or inhalation routes. The antibodies, when suitably active, may also be administered by intratumoral, peritumoral, intralesional or perilesional (perilesional) routes to exhibit local as well as systemic therapeutic effects.

Such dosage forms contain pharmaceutically acceptable carriers that are not inherently toxic and therapeutically effective. Such carriers include, for example, ion exchangers, aluminum stearate, lecithin; serum proteins such as human serum albumin; buffers such as phosphate or glycine; sorbic acid, potassium sorbate, partial glyceride mixtures of saturated vegetable fatty acids, water, salt; or electrolytes such as protamine sulfate, sodium chloride, metal salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, cellulose polymers, and polyethylene glycol. Carriers for topical or gel-based formulations of antibodies include polysaccharides such as carboxymethylcellulose or sodium methylcellulose, polyvinylpyrrolidone, polyacrylates, polyoxyethylene polyoxypropylene block polymers, polyethylene glycol and wood wax alcohols (wood wax alcohols). Conventional storage forms (depot forms) include, for example, microcapsules, nanocapsules (nano-capsules), liposomes, plasters, sublingual tablets, and polymeric matrices such as polylactic acid-polyglycolic acid copolymers. When present in an aqueous solution rather than a lyophilized formulation, the antibody is typically formulated at a concentration of about 0.1mg/ml to 100mg/ml, although variations outside of these ranges are possible.

For the prevention or treatment of disease, the appropriate dosage of the antibody will depend on the type of disease to be treated (as described above), the severity and course of the disease, whether the antibody is administered prophylactically or therapeutically, the course of prior treatment, the patient's clinical history and response to the antibody, and the judgment of the attending physician. The antibodies are suitable for administration to a patient at one time or over a series of treatments.

Depending on the type and severity of the disease, approximately 0.015-15 mg antibody/kg patient body weight is the initial candidate dose to be administered to the patient, whether, for example, by one or more separate administrations or continuous infusion. For repeated administrations over several days or longer, the treatment is repeated depending on the disease condition until the desired suppression of disease symptoms occurs. However, other dosage regimens may be used and are not excluded from the scope of the invention.

According to another embodiment of the invention, the efficacy of an antibody in the prevention or treatment of a disease may be improved by continuous administration of the antibody or in combination with another therapeutic agent that is effective for the same clinical goal, such as another antibody directed against a different epitope than the primary antibody, or one or more conventional therapeutic agents known to be useful for a desired therapeutic indication, e.g., the prevention or treatment of a pathology associated with excessive extracellular matrix production, such as fibrosis or cirrhosis, the inhibition of tumor growth or metastasis, the inhibition of neovascularization, or the reduction of inflammation. Such formulations may ameliorate symptoms or improve outcome by similar mechanisms of action, such as anti-TGF antibodies, or by a different mechanism, such as interferon-gamma. Such formulations may additionally ameliorate symptoms associated directly or indirectly with or contributing to CTGF-associated disorders such as Angiotensin Converting Enzyme (ACE) inhibitors and angiotensin receptor blockers (Arb).

For example, scleroderma patients receiving perfusion with the stable prostacyclin agonist Iloprost (Iloprost) often exhibit an improvement in skin tightness, consistent with inhibition of scar tissue formation by dermal fibroblasts. Prostaglandins have been shown to exert inhibitory effects on collagen synthesis, and some evidence suggests that iloprost blocks CTGF induction in scleroderma (Korn et al (1980) J Clin Invest65:543-554; Goldstein and Polger (1982) J Biol Chem257:8630-8633; and Stratton et al (2001) J Clin Invest108: 241-250). CTGF increased 7-fold in the vesicular fluid in scleroderma patients compared to healthy controls, whereas patients given intravenous administration of iloprost showed a significant reduction in CTGF in the vesicular fluid (Stratton et al (2001) J Clin Invest108: 241-250). Taken together, these results suggest that some of the benefits of iloprost treatment in scleroderma may result from anti-fibrotic effects mediated by reduced CTGF levels. Given the use of chronic systemic administration of potent vasodilators with anti-platelet prostacyclin analogs in scleroderma patients, treatment with anti-CTGF antibodies alone or in combination with reduced levels of iloprost may provide a safe and effective scleroderma treatment.

Additional applications

The antibodies of the invention are also useful as affinity purifiers. In this method, the antibody to CTGF is immobilized on a suitable support, such as Sephadex (Sephadex) resin or filter paper, using methods well known in the art. The immobilized antibody is then contacted with a sample containing CTGF to be purified, after which the support is washed with a suitable solvent, thus removing substantially all of the material in the sample except for CTGF bound to the immobilized antibody. Finally, the support is washed with another suitable solvent, such as glycine buffer (ph5.0), which will release CTGF from the antibody.

Examples

The invention will be further understood by reference to the following examples, which are intended to be illustrative of the invention. The scope of the invention is not limited to the exemplary embodiments, which are intended merely to illustrate certain aspects of the invention. Any functionally equivalent method is within the scope of the invention. Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description and accompanying drawings. Such modifications are intended to fall within the scope of the appended claims.

Example 1: production of recombinant human CTGF

Recombinant human CTGF baculovirus (baculovir) constructs were generated as described by Segarini et al ((2001) J Biol Chem276: 40659-40667). Briefly, CTGF cDNA containing only open reading frames was generated by PCR using DB60R32(Bradham et al (1991) J Cell Biol114:1285-94) as a template using primers 5'-gctccgcccgcagtgggatccATGaccgccgcc-3' and 5 ' -ggatccggatccTCAtgccatgtctccgta, which add BamHI restriction enzyme sites at the ends of the amplified product. Native start and stop codons are indicated in capital letters.

The resulting amplified DNA fragment was digested with BamHI, electrophoretically purified on an agarose gel, and subcloned directly into the BamHI site of the baculovirus PFASTBAC1 expression plasmid (Invitrogen corp., CarlsbadCA). The sequence and orientation of the expression cassette (expression cassette) was verified by DNA sequencing. The resulting CTGF expression cassette is then transferred into bacmid DNA by site-specific recombination in bacteria. This bacmid was subsequently used TO generate fully recombinant CTGF baculovirus in spodoptera frugiperda (spodoptera frugiperda) SF9 insect cells according TO the protocol provided by the manufacturer (BAC-TO-BAC expression system manual, Invitrogen). The titer of the recombinant baculovirus in Sf9 insect cells was expanded using standard procedures known in the art.

Hi5 insect cells were adapted to suspension growth by serial passage of shake flask cultured cells and enrichment of isolated cells at each passage. The suspended Hi5 cells were cultured in 1L SF900II SFM medium (Invitrogen) supplemented with 20 μ g/ml gentamicin (Mediatech, inc., Herndon VA) and 1x lipids (Invitrogen) at 110rpm at 27 ℃ in disposable 2.8LFernbach flasks (Corning inc., Acton MA) placed on a shaker. When the cell density reaches 1.0-1.5x10⁶Cell/ml, viability of>At 95%, cells were infected with recombinant baculovirus at a multiplicity of infection (MOI) of 10. The culture was then incubated at 27 ℃ for an additional 40-44 hours. Conditioned medium containing rhCTGF was collected, cooled on ice, and centrifuged at 5000x g. The supernatant was then passed through a 0.45mm filter.

Alternatively, recombinant rat CTGF was generated by inserting clone 2-4-7(Schmidt et al, U.S. Pat. No.6,348,329) encoding rat CTGF into the pMK33 expression vector (constructed by Michael Koelle, Stanford university doctor's paper, 1992). The rat CTGF expression construct was transfected into Schneider2 cells (American type culture Collection, Manassas VA; Schneider (1972) J Embryol Exp Morphol27: 353-. Cells were grown for 6 weeks in medium containing 300. mu.g/ml hygromycin B, followed by 3 days of antibiotic-free selection. By adding 500. mu.M CuSO₄And 100. mu.M ZnSO₄CTGF expression was induced and after 4 days the medium was harvested and clarified by centrifugation and filtration as described above.

CTGF produced by the above method was purified as follows. 4 liters of conditioned medium were loaded onto a 5ml HI-TRAP heparin column (Amersham biosciences Corp., Piscataway NJ) pre-equilibrated with 50mM Tris (pH7.5), 150mM NaCl. CTGF was eluted from the column with an increasing NaCl salt gradient. The eluted fractions were screened by SDS-PAGE and fractions containing CTGF were pooled.

Heparin-purified CTGF was diluted with pyrogen-free double distilled water to a final conductivity of 5.7mS and pH was adjusted to 8.0. A Q-Sepharose strong anion exchange column (Amersham Biosciences) containing about 23ml of resin was used in series with a Carboxymethyl (CM) POROS polystyrene column (Applied Biosystems) containing about 7ml of resin to remove endotoxin and capture and elute purified rhCTGF. Before loading, the tandem column was washed with 0.5M NaOH, then 0.1M NaOH, and finally with equilibration buffer. The sample was loaded onto the tandem column, the Q-Sepharose column was removed, and CTGF was eluted from a CM POROS column (Applied Biosystems) with a gradient of 350mM to 1200mM NaCl. The purity of the eluted fractions containing CTGF was assessed by SDS-PAGE analysis before a final sample pool was formed.

Example 2 Generation of N-terminal and C-terminal fragments of CTGF

N-terminal and C-terminal fragments of CTGF were prepared as follows. Recombinant human CTGF prepared and purified as described above was digested at room temperature for 6 hours by treatment with chymotrypsin beads (Sigma Chemical co., st.louis, MO) at 1.5mg CTGF per unit chymotrypsin. The mixture was centrifuged, the chymotrypsin beads discarded, and the supernatant containing the enzymatically cleaved rhCTGF was diluted 1:5 with 50mM Tris, pH 7.5. The diluted supernatant was applied to a Hi-Trap heparin column. The flow-through (flow-through) containing the N-terminal fragment of CTGF was collected. The heparin column was washed with 350mM NaCl and bound CTGF C-terminal fragments were eluted as described above using a linear gradient of 350mM to 1200mM NaCl. Fractions were analyzed by SDS-PAGE and fractions containing the C-terminal fragment of CTGF were pooled.

The heparin column flow through containing the N-terminal fragment of CTGF was adjusted to 0.5M ammonium sulfate/50 mM Tris, pH7.5, and then loaded on a 15ml phenyl sepharose HP column (Amersham-Pharmacia) that had been pre-equilibrated with 0.5M ammonium sulfate/50 mM Tris, pH 7.5. The column was washed with 15 column volumes of 0.5M ammonium sulfate/50 mM Tris, pH7.5, and the bound N-terminal fragment of CTGF was eluted with a linear gradient of 0.5M to 0M ammonium sulfate/50 mM Tris, pH7.5, about 15 column volumes. Fractions were analyzed by SDS-PAGE and fractions containing the N-terminal fragment of CTGF were pooled. The combined solutions were concentrated with ultracellamicon YM10 ultrafiltration membrane (Millipore corp., Bedford MA) and the buffer was exchanged with 50mM Tris,400mM NaCl (ph 7.2).

Example 3 production of human anti-CTGF monoclonal antibodies

Fully human monoclonal antibodies against human CTGF were prepared using HUMAB mouse strains HCo7, HCo12, and HCo7 + HCo12(Medarex, inc., Princeton NJ). Mice were immunized with 25-50mg of recombinant human CTGF in complete freund's adjuvant with up to 10 Intraperitoneal (IP) or subcutaneous (Sc) injections over a 2-4 week period. Immune responses were monitored by retroorbital bleeding. Plasma was screened by ELISA (as described below) and mice with sufficient titers of anti-CTGF immunoglobulin were used for fusion. Mice were boosted intravenously with antigen 3 and 2 days prior to sacrifice and spleens removed.

A single cell suspension of splenic lymphocytes from immunized mice was fused with one-fourth the number of P3X63-ag8.653 non-secreting mouse myeloma cells (american type culture collection (ATCC), Manassas VA) with 50% PEG (Sigma, st. The cells were cultured at about 1X10⁵Cells/well were plated in flat-bottomed microtiter plates and incubated for about 2 weeks in high glucose DMEM (Mediatech, Herndon VA) containing L-glutamine and sodium pyruvate, 10% fetal bovine serum, 10% P388D1(ATCC) conditioned medium, 3-5% origen (Igen International, Gaithersburg MD), 5mM HEPES, 0.055mM 2-mercaptoethanol, 50mg/ml gentamicin, and 1XHAT (Sigma). After 1-2 weeks, the cells were cultured in medium in which HAT was replaced by HT. Each well was then screened by ELISA (as described below). Antibody secreting hybridomas were replated, rescreened, and if positive for anti-CTGF antibodies, subcloned at least 2 times by limiting dilution. The stable subclones were then cultured in vitro to produce small amounts of antibody in tissue culture medium for characterization. One clone from each hybridoma that retains the reactivity of the parental cells was used to generate a 5-10 flask cell bank, stored in liquid nitrogen.

ELISA assays were performed as described by Fishwild et al (1996, Nature Biotech14: 845-. Briefly, microtiter plates were coated with 1-2 μ g/ml purified recombinant CTGF in PBS at 50 μ l/well, incubated overnight at 4 ℃ and then blocked with 200 μ l/well of 5% chicken serum in PBS/Tween (0.05%). Dilutions of plasma or hybridoma culture supernatants from CTGF-immunized mice were added to each well and incubated for 1-2 hours at ambient temperature. The plates were washed with PBS/Tween and then incubated with goat anti-human IgG Fc polyclonal antibody conjugated with horseradish peroxidase (HRP) for 1 hour at room temperature. After washing, the plates were developed with 0.22mg/ml ABTS substrate (Sigma) and analyzed with a spectrophotometer at 415-495 nm.

Example 4 antibody identification

Hybridomas producing anti-human CTGF antibodies were prepared as described in example 3. Cloned hybridoma cells were grown in Dulbecco's modified Eagle's Medium-high glucose/RPMI 1640(50:50) containing 8mM L-glutamine, 1/2X non-essential amino acids and 10% fetal bovine serum. Cells expanded for antibody production were grown in the same medium containing 1.5% low IgG fetal bovine serum at 37 ℃ under 6% CO2 for 4-9 days. The resulting conditioned media is cell depleted and concentrated by elution with a tangential flow filtration/concentration system. The concentrate was passed through a protein A column and bound monoclonal antibody was eluted with 100mM glycine, pH 3. The eluate was neutralized with 1M Tris, pH8.0 and dialyzed against PBS.

4.1 epitope mapping (epitope mapping)

Epitope mapping of antibodies by competitive binding experiments is well known to those skilled in the art of Immunology (see, e.g., Van Der gel et al (1999) Clinical and Experimental Immunology118: 487-96). Each antibody population isolated from cells propagated from a unique clonal hybridoma cell is mapped using standard binding and blocking experiments and assigned to a specific binding domain on human CTGF (see, e.g.,Antibodies:A Laboratory Manual(1988)Harlow and Lane(eds),Cold Spring Harbor Laboratory Press;Tietz Textbook of Clinical Chemistry,2^nd ed.,(1994)Chapter10(Immunochemical Techniques),Saunders;and Clinical Chemistry:Theory,Analysis,Correlation(1984) chapter 10 (electrochemical technologies) and Chapter11 (comparative Biding Assays), C.V.Mosby, St.Louis). Independent binding domains were initially defined by antibody competition experiments in which two different antibodies were incubated sequentially on CTGF-coated plates. Two antibodies are assigned to the same binding domain if steric hindrance of the first antibody prevents the second antibody from binding to CTGF. It is understood, however, that two antibodies may have different epitopes that are sufficiently close to each other to be considered members of the same binding domain.

Binding domains spanning all 4 exons of human CTGF were identified. All binding domains are conformationally constrained such that the antibody binds to CTGF under non-reducing conditions in a western blot assay. Some antibodies also bind to CTGF under reducing conditions in western blot assays, suggesting that each of these antibodies binds to a linear epitope on the CTGF protein. In addition, antibodies representing a subset of the binding domains showed cross-reactivity with mouse CTGF in western blot analysis. Antibodies from each panel with the highest affinity for intact CTGF were used for further identification and analysis.

More elaborate epitope mapping was performed by ELISA analysis using specific recombinantly expressed CTGF fragments. For example, antibodies recognizing an epitope on the N-terminal domain of CTGF were identified by ELISA analysis of immobilized fragments derived from recombinant expression of exon 2 and/or exon 3 of the CTGF gene. In this way, antibodies that specifically recognize the N-terminal domain or N-terminal fragment of CTGF were selected and further identified. Antibodies that specifically recognize the C-terminal domain or C-terminal fragment of CTGF were also selected and further identified.

The epitope defined by mAb1 binds to a linear epitope on the N-terminal fragment of CTGF encoded by exon 3. Generating a series of regions covering the region encoded by the polynucleotide of exon 3With these peptides, ELISA tests were performed to further define the epitope of mAb 1. The results are summarized in Table 1, where "+ means binding between the peptide and mAb1 and" - "means mAb1 does not bind to the peptide. Bold italics "C" indicates the cysteine residue in the peptide that is critical for mAb1 binding. Underline "C"denotes a cysteine residue added to the terminus that is not part of the native CTGF sequence.

TABLE 1 binding of mAb1 to the truncated peptide series encoded by exon 3

Peptides	Sequence of	mAb1 binding	SEQ ID NO:
				N-CTGF		＋
Exon 3		＋
				Pep135	CPLCSMDVRLPSPDCPFPRRVKLP	＋	22
PC5444	PLSSMDVRLPSPDS	－
				PC5445	RLPSPDSPFPRRVKLPGK	＋	23
PEP5	RLPSPDCPFPRRVKL	＋	24
				P40340	RLPSPDCPFPRRV	＋	25
P40341	RLPSPDSPFPRRV	－
				P40342	LPSPDCPFPRRVKL	＋	26
10MER	SPDSPFPRRV	－
				10MER2	SPDCPFPRRV	－
9MER	PDSPFPRRV	－
				9MER2	CPFPRRVKL	－
8MER	DSPFPRRV	－
				8MER2	CFPRRVKL	－
7MER	CPRRVKL	－
				6MER	CRRVKL	－
5MER	CRVKL	－

Thus, mAb1 is a member of a panel of antibodies that bind to the N-terminal region of CTGF. The linear epitope on CTGF that is sufficiently essential for binding to mAb1 is defined by amino acid residues L143 to V154 of human CTGF (SEQ ID NO: 2). Further confirmation of the binding specificity of MAb1 for this peptide was obtained by RIA and affinity chromatography. Antibodies sharing this epitope in part or in whole are specifically included in the invention. In addition, antibodies that compete with mAb1 for binding to CTGF or fragments thereof are also specifically included within the invention. 4.2 antibody affinity to CTGF

Antibody affinity is defined as the strength of the overall non-covalent interaction between a single antigen binding site on an antibody and a single epitope on an antigen. The affinity was calculated by measuring the association constant (Ka) as follows:

wherein [ Ab]Is the concentration of the free antigen binding site on the antibody, [ Ag ]]Is the free antigen concentration, [ Ab. Ag ]]Is the concentration of antigen-binding sites on the antibody occupied by the antigen, K_dIs the dissociation constant of the antibody-antigen complex.

The affinity of each antibody population identified by epitope mapping was measured using RIA, in which intact rhCTGF was labeled with radioiodine as described below, and added to wells containing immobilized monoclonal antibodies. Using chloramine-T method¹²⁵I radiolabelling of recombinant human CTGF (see Greenwood et al (1963) Biochem J89: 114-123). Typically, at least 60% of¹²⁵I is incorporated and the specific activity of the labeled CTGF is at least 1x10⁵cpm/ng, of course lower specific activity labeled CTGF may also be used in radioimmunoassays. Will be Ca-free²⁺And Mg²⁺Goat anti-human IgG, gamma Fc specific capture antibody (Jackson ImmunoResearch) in DPBS (Mediatech, HerndonVA) of (a) was added to wells of a MAXISORP break part microtiter plate (Nalge Nunc International, RochesterNY) to allow binding overnight at 4 ℃. Then used in the presence of Ca²⁺And Mg²⁺1% BSA in DPBS blocked the pores at 4 ℃ for at least 4 hours. The blocking solution was removed and 100. mu.l of Ca-free solution was added²⁺And Mg²⁺The DPBS of (1) at a concentration of 2-50ng/ml, binds overnight at 4 ℃. Will be in a constant amount¹²⁵I]Serial dilutions of unlabeled CTGF in rhCTGF were added to the wells and incubated for 4-8 hours at room temperature. Then used in the presence of Ca²⁺And Mg²⁺The wells were washed 4 times with 0.1% Tween20(Mediatech) in DPBS, and the individual wells of the microtiter plate were separated and counted in a gamma counter.

The affinity was estimated graphically by the scatchard method (1948, Ann NY Acad Sci51: 660-72). The total concentration of labeled CTGF applied to the microtiter plate was calculated as follows:

wherein cpm _ applied is the count from a control vial loaded in parallel with the CTGF mixture in wells of a microtiter plate; cpm/fmol is [ 2 ]¹²⁵I]Specific Activity of CTGF, [ CTGF]_{cold_stock}Is the concentration of unlabeled CTGF added to each well and dilution is the dilution factor of the unlabeled CTGF.

The concentration of CTGF bound to the antibody was calculated from the ratio of the count bound to the wells to the total concentration of CTGF applied to the wells:

the concentration of free (unbound) CTGF is the difference between the total concentration of CTGF applied and the concentration of bound CTGF.

[CTGF]_free＝[CTGF]_total－[CTGF]_bound

The scatchard plot for determining the affinity of the antibodies of the invention is shown in figure 2. FIG. 2A is a graph showing that the antibody mAb2 of the invention is conjugated to [ alpha ]¹²⁵I]Binding of rhCTGF. FIG. 2B is a graph showing that the exemplary antibody mAb1 of the invention is directed to [ alpha ], [ alpha¹²⁵I]Binding of rhCTGF. For CTGF with similar ratios of bound and unboundDots are given more attention (weight) because they have a bound count that is well above the blank (and therefore a well-defined bound count), but still much lower than the total applied count (and therefore a well-defined free count). Maximal binding (B)_max) And K_dExpressed as x-intercept and y-intercept, respectively.

Affinity of mAb1 for CTGF (K)_d) Less than 10^-9M, which is a typical affinity found in commercially successful antibody therapeutics (see, e.g., Maini et al (1998) Arthritis Rheum41:1552-1563; Targan et al (1997) N Engl J Med337:1029-1035; Bumgardneret al (2001) Transplantation72:839-45; and Kovarik et al (1999) Transplantation68: 1288-94). Thus, mAb1 is a suitable candidate for therapeutic use and shares epitope binding with mAb1 as described above and has affinity for CTGF (i.e., K) similar to or greater than mAb1_d≤10^-9) Are also suitable candidates for similar therapeutic uses. Sharing epitope binding with mAb1 but having lower affinity (i.e., higher K) than mAb1_d) Are also encompassed within the invention and may potentially be used in the various assay and diagnostic applications described. These antibodies may additionally be used in therapeutic applications, particularly if they have high affinity for antigens, as described below.

4.3 Antibody Avidity (Antibody affinity)

For antibodies with more than one antigen binding site (multivalent), affinity at one binding site does not always reflect the true strength of the antibody-antigen interaction. When a multivalent antibody binds to an antigen with multiple repeating epitopes, one antigen interaction at one binding site of the antibody increases the chance of the antigen interacting with other binding sites. Avidity measures the strength of a functional combination of an antibody with its antigen, which correlates both with the affinity of the reaction between the epitope and paratope and with the valency of the antibody and antigen. Thus, avidity provides a more accurate measure of the tendency of an antibody to dissociate.

High affinity may compensate for low affinity. For example, the IgM antigen binding site is generally less avidity than IgG, but the multivalency of IgM renders it highly avidity, thereby rendering it effective for binding antigen.

To determine the avidity of the antibodies of the invention, Fab fragments were first prepared by conventional papain digestion of the corresponding immunoglobulins. The Fab fragments were then separated from the Fc and undigested antibody using immobilized protein a.

Approximately 1ml of immobilized papain slurry containing 0.5ml of the deposited (settled) gel, 250. mu.g of papain and 3.5BAEE units was washed with digestion buffer (DB;20mM sodium phosphate, 10mM EDTA, 20mM cysteine, pH7.0), 3 times with 1ml digestion buffer each and 1 time with 10ml digestion buffer each. The papain slurry was then resuspended in 0.3ml DB, mixed with 1.1ml antibody (ca. 5mg, pH7), and shaken overnight at 37 ℃. The antibody digest was then separated from the resin and the Fab fragments were separated from the Fc fragment and undigested antibody by affinity chromatography with protein a. The purity of the Fab fragments was monitored by SDS-PAGE (FIG. 3A).

Monovalent and bivalent binding were distinguished by eluting antigen-bound antibodies with varying concentrations of thiocyanate. By increasing the chaotropic ion (thiocyanate) concentration in solution, low affinity associations (e.g., monovalent binding of Fab to antigen) are disrupted first, while higher affinity associations (e.g., divalent binding of IgG to ligand) are not disturbed. Thus, by increasing the thiocyanate concentration, two different combinations can be distinguished.

The plates were coated with 10. mu.g/ml CTGF or CTGF peptide in 50mM bicarbonate buffer (pH8.5) overnight at 4 ℃, blocked with blocking agent casein/TBS overnight at 4 ℃ and then incubated with 100. mu.g/ml antibody or the corresponding Fab in blocking agent casein/TBS overnight at room temperature with shaking. The plates were then incubated with a dilution (1:1) of thiocyanate (0-7.6M) in 100mM phosphate buffer (pH6.0) at room temperature for 15 minutes with shaking, followed by alkaline phosphatase-mouse anti-human (Fab')₂The conjugate (1:1000 dilution) was incubated at room temperature for 45 minutes. Add 1M diethanolamine, 0.5mM MgCl₂Alkaline phosphatase substrate (1 mg/ml; Sigma) in (pH9.8), plates were incubated at room temperature and the absorbance at 405nm was measured after 2, 10, 20 and 60 minutes.

The affinity index (affinity index) is the concentration of chaotropic agent (thiocyanate) that reduces the initial absorbance by 50%. For the exemplary antibody mAb1 of the invention, the affinity index for dissociating Fab from CTGF was 0.46M, while the affinity index for dissociating intact IgG from CTGF was 1.8M (fig. 3B). Thus, mAb1 binds (avidity) to antigen primarily at double valency and dissociates from antigen much more slowly than monovalent bound antibody. Other antibodies of the invention that share epitope binding parameters with mAb1 may be similarly bivalent, or they may be monovalent or multivalent. Any antibody of the invention can be manipulated to improve avidity, for example by combining epitope binding sites in a single antibody construct, such as a tribody et al (see, e.g., Schoonjans et al (2000) JImmunol165: 7050-.

4.5 Cross-reactivity

The cross-reactivity of the antibodies was determined using the radioimmunoassay described above (example 4.2), but the unlabeled rhCTGF was replaced by another unlabeled competitor, rat CTGF derived from Normal Rat Kidney (NRK) cells. NRK cells were cultured until confluent, and then the medium was changed to serum-free medium containing 2ng/ml TGF-. beta.2, 50. mu.g/ml heparin and 250. mu.g/ml BSA. After 2 days of culture, the conditioned medium was collected, centrifuged to remove debris, and incubated with heparin-agarose beads (1/100v/v bead suspension: medium) at 4 ℃ for 2 hours with shaking. The mixture was then centrifuged, the beads were collected and washed with PBS, and then lysed in SDS buffer.

The mAb2 and [ 2 ] in the presence of an increasing concentration of unlabeled rat CTGF¹²⁵I]The scatchard plot of binding of rhCTGF is shown in fig. 4A; the mAb1 and [ 2 ] in the presence of an increasing concentration of unlabeled rat CTGF¹²⁵I]The scatchard plot of binding of rhCTGF is shown in fig. 4B. As shown, mAb1 bound to both human and rat CTGF, while mAb2 bound to human CTGF but not rat CTGF.

For mAb1, the scatchard plot competing with rat CTGF (fig. 4B) had a smaller slope, lower apparent affinity and higher apparent B than the scatchard plot competing with rhCTGF (fig. 2B)_max. Thus, although rat CTGF competed with human CTGF for binding mAb1, the antibody had a higher affinity for recombinant human CTGF than for recombinant rat CTGF. mAb1 also cross-reacted with mouse and monkey CTGF (data not shown). Antibodies that exhibit suitable affinity for CTGF from other species may be used to treat and prevent disease in those species. For example, it was shown to have a suitable K for canine CTGF_dThe antibodies of the invention are useful for treating CTGF-associated diseases in dogs. Antibodies of the invention such as mAb1, which exhibit cross-species affinity, can also be used as a research tool to study CTGF-associated diseases in various animal models.

4.5 glycosylation

The above radioimmunoassay (example 4.2) was used to determine the effect of antibody glycosylation on antigen binding affinity. Antibody mAb was treated with the peptide N-glycosidase F (PNGase F) cleaving oligosaccharides from N-linked glycoproteins in PBS, 0.5M EDTA, pH8.0 for 18 days at 37 ℃. After incubation, the reaction solution was used directly or fractionated on a protein a-agarose FASTFLOW column (Amersham Bioscience, Piscataway NJ) and eluted with 0.1M glycine-HCl, ph 2.5. The recovery of antibody after fractionation was approximately 87% and the endotoxin level was 0.30 EU/mg. Deglycosylation was confirmed by SDS-PAGE. The binding activity of deglycosylated antibodies to human recombinant CTGF was the same as the binding activity of the glycosylated form of the antibody within experimental error.

Production of recombinant proteins such as antibodies in cultured cells or non-homologous species can result in non-native glycosylation due to different glycosylation patterns produced by different cells. Specific glycosylation is required for the activity of some proteins, and altered glycosylation reduces activity; for example, in the case of an antibody, the affinity for an antigen decreases. In certain systems, such as plants and eggs, protein production may also produce glycosylation patterns that are immunogenic, thereby reducing the ability to use these proteins in certain applications. The ability of the antibodies of the invention to exhibit the same activity in both glycosylated and non-glycosylated forms demonstrates that the invention is not limited by the presence of glycosylation, particularly species-specific glycosylation.

Example 5: cell migration assay

Cell migration is a normal and important cellular event, for example, during development and wound healing. Cell migration is also a factor in disease pathology such as the formation of fibrotic lesions, from which isolated cells respond more to chemotactic stimuli than cells from corresponding normal tissues.

The ability of the antibodies of the invention to inhibit CTGF-stimulated smooth muscle cell chemotactic migration was analyzed using a Boyden chamber assay (Boyden chamber assay) as follows. Rat Arterial Smooth Muscle Cells (ASMC) in medium containing 0.1% Fetal Calf Serum (FCS) were added to the upper chamber of the Boyden chamber and medium containing 300ng/ml rhCTGF, 10% FCS or 0.1% FCS alone was added to the lower chamber. A collagen-coated filter with a pore size of 8 μm separates the upper and lower chambers. Cells were allowed to adhere and migrate through the filter for 2-3 hours. The filters are then removed, the cells on the filters are fixed and stained, and the cells that have migrated through the filters are counted. Incubation with 300ng/ml rhctgf increased the number of cells migrating through the filter by about 5-fold relative to the 0.1% FCS control. The increase in migration stimulated by CTGF was approximately 27% of the chemotactic effect seen with 10% FCS stimulation with various chemokines.

The ability of the antibodies of the invention to inhibit CTGF-mediated cell migration was tested using the assay described above, except that anti-CTGF antibodies (at 30 and 300mg/ml) or pooled human IgG were also added to the lower chamber. Cells were counted for each sample in each assay for 4 fields of each of 3 independent filters. The results are shown in Table 2.

TABLE 2 inhibition of CTGF-mediated cell migration

As shown in table 2, antibodies that bind CTGF within the epitope defined by mAb1 inhibited CTGF-mediated cell migration in a dose-dependent manner. The antibodies of this epitope group were the only anti-CTGF antibodies tested that repeatedly and reproducibly inhibited CTGF-induced migration.

Various processes such as angiogenesis, chondrogenesis, and carcinogenesis require changes in cell adhesion and migration. CTGF is associated with both cell adhesion and migration, and the ability of anti-CTGF antibodies to affect one activity differently relative to another provides various compositions of different therapeutic agents for treating CTGF-associated pathologies. The antibodies provided by the present invention clearly demonstrate differential activity with respect to neutralization of CTGF activity. As shown below, these capabilities provide unique therapeutic potential in this class of anti-CTGF antibodies.

Example 6 pulmonary diseases

Intratracheal (IT) instillation of bleomycin in mice is a widely used model system for the study of pulmonary fibrosis and the screening of potentially desirable anti-fibrotic drugs. The ability of the antibodies of the invention to reduce bleomycin-induced pulmonary fibrosis in vivo was tested using the method described by Wang et al (2000) Biochem Pharmacol60:1949-1958 as follows.

Male C57BL/6 mice were randomly divided into 2 groups. Mice were anesthetized with isoflurane and then separately injected intratracheally with a single dose of bleomycin dissolved in 0.9% saline at 0.1 units/50 μ l/mouse or 0.9% saline alone. Each group was treated immediately after isolation with Intraperitoneal (IP) administration of saline or antibody, followed by treatment every 1 day for a total of 7 doses. 14 days after IT instillation, mice were euthanized by exsanguination of the abdominal aorta under anesthesia and lung tissue was collected.

Lung collagen content was analyzed by measuring hydroxyproline and proline levels using the method of Palmerini et al (1985; J Chromatogr339:285-292) except that L-lilacin (Aldrich) was used as an internal standard instead of 3, 4-dehydroproline. Briefly, tissue samples were hydrolyzed in 6N HCl at 105 ℃ for 22 hours. The samples were pre-column derivatized with o-phthalaldehyde followed by 4-chloro-7-nitrobenzofuran (Aldrich) to form fluorescent adducts of proline and hydroxyproline. The fluorescence adduct was separated by reverse phase HPLC and then subjected to fluorescence detection.

Fig. 5 shows the results of therapeutic administration of Saline (SA), an exemplary antibody mAb1 of the present invention, and compares the ability of the CTGF-specific antibody repertoire (AbsJ) to inhibit pulmonary fibrosis following bleomycin treatment. As shown in FIG. 5A, bleomycin treatment (BL + SA) significantly increased lung hydroxyproline content, which is 168% of control (SA + SA; 220. + -. 15. mu.g/lung). Subsequent treatment with pooled antibodies of the invention (BL + AbsJ) however showed a 60% reduction in lung hydroxyproline compared to bleomycin alone treatment. Similarly, subsequent treatment with mAb1 (BL + mAb1) showed a 70% reduction in lung hydroxyproline compared to bleomycin alone.

Histological examination of the mouse lungs revealed normal lung parenchyma tissue in the control group (not shown). However, in bleomycin-treated lungs, an increase in fibrotic area was clearly visible (fig. 5B at the arrows). Therapeutic administration of the antibodies of the invention after bleomycin treatment showed a significant reduction in fibrosis (fig. 5C), although some lung lobes still had a slight degree of interstitial fibrosis. Accordingly, the antibodies of the invention provide therapeutic benefits when administered to a patient suffering from or at risk of a pulmonary disease, such as Idiopathic Pulmonary Fibrosis (IPF).

Example 7 renal diseases

7.1 renal failure

Tubulointerstitial fibrosis is a major factor in renal disease associated with several progression to end-stage renal failure (Sharma et al (1993) Kidney Int44: 774-. Unilateral Ureteral Obstruction (UUO), characterized by decreased renal function and increased interstitial fibrosis, was used as an experimental model to induce tubulointerstitial injury and fibrosis (Fern et al (1999) J Clin Invest103: 39-46).

Mice were anesthetized with isoflurane and left ureteral ligation was performed according to the method described by Moriyama et al (1998; Kidney Int54: 110-. Mice were treated 1 time with Intraperitoneal (IP) administered saline or antibody for a total of 7 doses immediately after surgery and every 1 day thereafter. 14 days after UUO, animals were anesthetized and sacrificed by exsanguination of the descending aorta. The right and left kidneys were separately decapsulated and weighed. Half of each kidney was histologically fixed in 10% formalin (trichrome staining), and the other half weighed and stored at-70 ℃ for hydroxyproline assay. Hydroxyproline and proline were determined as described above.

As shown in figure 6A, UUO increased renal collagen content by approximately 4-fold as determined by measuring the hydroxyproline to proline ratio of the obstructed left kidney relative to the unobstructed right kidney in each mouse. Treatment with antibody mAb1 of the invention resulted in a statistically significant reduction in dose-dependent fibrosis in the obstructed kidney (fig. 6A). However, antibody mAb3 which binds to the C-terminal epitope of CTGF showed no significant effect. Trichrome staining of UUO kidneys identified areas of increased collagen accumulation (fig. 6B, arrows), whereas treatment with the antibodies of the invention showed a significant decrease in collagen staining in obstructed kidneys (fig. 6C).

Alternatively, renal fibrosis can be studied in a rat kidney residual (remnants kidney) model of progressive renal failure. This model involves 2/3 unilateral nephrectomy combined with contralateral total nephrectomy (5/6 total nephrectomy), which induces degenerative parenchymal changes associated with chronic renal failure in the residual kidney, and the animals become uremic and exhibit marked proteinuria, glomerulosclerosis, interstitial fibrosis and tubular atrophy. (see, e.g., Frazier et al (2000) Vet Pathol37:328-335; and Gandhi et al (1998) Kidney Int54: 1157-1165).

A5/6 nephrectomy was performed as described by Frazier et al (2000, Vet Pathol37: 328-335). Male 5-week-old Sprague-Dawley rats (Harlan, Indianapolis IN) with an average weight of 120g were anesthetized with ketamine and xylazine and the left kidney head 1/3 and tail 1/3 were excised. The animals were stopped bleeding briefly by a gauze cotton swab, the abdominal cavity was washed with saline, 0.2ml butrophenol and sutured. One week after the initial surgery, the contralateral kidney was completely removed.

Rats were divided into saline-treated and antibody-treated groups, and treatment was initiated 2 weeks after 5/6 nephrectomy. Saline or antibody was given every 3 days at a dose of 5mg/kg by IP injection (0.5 mL each) for 15 days (5 injections total). Blood and urine samples were taken weekly from randomized nephrectomized rats to follow renal disease progression and correlate renal dysfunction with histological changes. Results from renal fibrosis analysis, urinalysis and serum chemistry assays were compared between groups at 18 and 28 days after treatment initiation.

Renal fibrosis was assessed independently blindly by two pathologists; three different morphological stains were used to examine 3 histological sections of each kidney: hematoxylin/eosin, Masson's trichrome and picric acid-sirius red (sirius red). In addition, immunohistochemistry was performed on frozen sections to assess the type of collagen deposition at each location in the kidney. Quantitative collagen assessment (hydroxyproline/proline ratio) was performed and renal function was assessed using urinalysis and serum chemistry of samples collected at euthanasia.

Histologically intermediate differences in fibrosis were observed between untreated and antibody treated kidney residues (figure 7). At 3 days post-treatment, blinded subjective evaluation gave a mean fibrosis score of 12.6 for the saline-treated group, while the corresponding score for the antibody-treated group was 10.7(p < 0.05). Statistically significant differences in tissue fibrosis scores were maintained between antibody and saline treated rats at 14 days post-treatment, with a mean fibrosis score of 16.9 in the saline treated group and 14.4 in the antibody treated group (p < 0.05). Quantitative hydroxyproline content analysis of collagen also demonstrated a trend towards reduced fibrosis in the antibody treated group relative to the saline treated group, but the differences were not statistically significant.

Qualitative differences were also noted between treatment groups. Although most collagen deposition in the antibody-treated group was limited to the corticocancellous and medullary mesenchyme, fibrosis in saline-treated rats was multifocal, with diffuse distribution in cortex and medulla. The most significant histopathological difference is the amount of glomerular fibrosis. Many rats in the saline treated group had moderate to severe glomerulosclerosis with periarticular cystic fibrosis, thickened Bowman's membrane, adhesions and glomerular degeneration. These changes were low to mild in the kidneys of other groups of rats including antibody treatment. Collagen accumulation was observed with Masson's trichrome and picric acid-sirius red stain.

In a progressive renal failure model, the antibodies of the invention reduce tissue degradation and improve kidney function. Accordingly, the antibodies of the invention provide therapeutic benefits when administered to a patient suffering from or at risk of suffering from renal disease, such as glomerulonephritis, IgA nephropathy, glomerulosclerosis and renal failure and tubular destruction due to toxins, among others.

7.2 diabetic nephropathy

Diabetes results in multiple organ failure, including but not limited to kidney, heart and eye. One major component of pathological progression in diabetic organ failure is fibrosis. An established model of diabetic nephropathy is a mouse carrying a loss-of-function mutation in the leptin receptor (Ob-R; encoded by the db gene). Key features common between db/db mice and human diabetic nephropathy include renal hypertrophy, glomerular enlargement, proteinuria and mesangial matrix broadening.

Antibodies of the invention were tested using a db/db mouse model of diabetic nephropathy as follows. 8-week-old db/db mice (Harlan, Indianapolis IN) and littermate heterozygous db/+ mice were treated by intraperitoneal injection of an antibody of the invention (CLN 1; see below) or a control human IgG (cIgG). In all animals, an initial injection of 300 μ g of antibody was followed by a 100 μ g dose administered 3 times per week for 60 days. Blood samples were collected and body weights were measured periodically at the start of and during treatment. Food consumption was also recorded.

By 11 weeks, there was a clear difference in body weight, blood glucose levels and food consumption between diabetic (db/db) and non-diabetic (db/+) animals. Treatment with the antibodies of the invention or control antibodies did not significantly affect any of these parameters. However, various measurements of kidney function confirmed a clear difference between diabetic and non-diabetic mice. As shown in table 3, diabetic mice showed increased kidney weight, creatinine clearance, and Albumin Excretion Rate (AER) relative to non-diabetic mice. However, diabetic animals treated with the antibodies of the invention showed normalized values for all parameters. All data are expressed as mean ± SEM. The number of mice per group (n) was from 9 to 15.

TABLE 3 renal function in db/db and db/+ mice

Relative to db/+ mouse P <0.01, relative to db/+ mouse P <0.01 and relative to cggg treated db/db mouse P <0.05

^△Db/db mouse P relative to cIgG treatment<0.01,^□Db/db mouse P relative to db/+ mouse and cIgG treatment<0.01

Since CTGF is induced by high glucose and mediates various activities, including ECM production in tissues as a result of injury, for example, due to formation and accumulation of advanced glycation end products (AGEs), etc., various pathologies associated with diabetes, such as diabetic nephropathy, can be prevented using the antibodies of the present invention.

Example 8: eye diseases

Increased expression of CTGF is known to be associated with a variety of ocular diseases including Proliferative Vitreoretinopathy (PVR), macular degeneration and diabetic retinopathy (see, e.g., Hinton et al (2002) Eye16: 422-866; He et al (2003) Arch Ophthalmol121:1283-1288; and Tikellis et al (2004) Endocrinology145: 860-866). The role of CTGF and the use of anti-CTGF therapeutic agents has been described (see International publication WO 03/049773). The antibodies of the present invention represent a unique therapeutically effective class of anti-CTGF therapeutic agents for such ocular diseases. The ability of the antibodies of the invention to eliminate ocular complications is tested in the following ocular model.

8.1 diabetic retinopathy

Animal models of diabetes, such as db/db mice, are described in example 7.2 above. Any of these models can be used to demonstrate the efficacy of the antibodies of the invention in treating diabetic retinopathy. One particular model of diabetic retinopathy is provided below, in which animals are injected with Streptozotocin (STZ), a toxin known to be the insulin-secreting islet beta cell.

Diabetes is induced in rats (e.g., Long-Evans, Sprague-Dawley, etc.) by, for example, injecting Streptozotocin (STZ), e.g., intraperitoneally, at about 60-85 mg/kg body weight. To improve survival, rats may be given 10% sugar water for 24 hours and/or 2-4 units of insulin per day after STZ injection. Various factors are measured, e.g., after 4,8 and 12 weeks, including, e.g., body weight, urinary protein excretion rate, blood glucose, glycated hemoglobin, blood pressure, etc. Control animals injected with buffer alone were operated simultaneously. Half of the STZ-treated and control rats are additionally treated with an antibody of the invention, e.g., by intravenous, intraperitoneal, or intraocular injection. Animals were exposed to food and water ad libitum throughout the study. Animals were sacrificed at 12 weeks, eyes were harvested and examined for histological changes.

A decrease in pathological changes in the antibody-treated animals relative to untreated controls is indicative of the efficacy of the treatment for diabetic retinopathy. Since CTGF is induced by high glucose and mediates various activities including ECM production in tissues as a result of damage due to, for example, formation and accumulation of advanced glycation end products (AGEs), etc., pathologies associated with diabetes, such as diabetic retinopathy, can be prevented with anti-CTGF therapeutic agents (see, for example, international publication WO 03/049773). The antibodies of the present invention represent a unique therapeutically effective class of anti-CTGF therapeutic agents for ocular diseases such as diabetic retinopathy.

8.2PVR

Rabbit Retinal Pigment Epithelium (RPE) cells were isolated from adult rabbit eyes and cultured in DMEM supplemented with 10% fetal bovine serum. Sub-confluent cultures (typically at passage 2-3) were used in all subsequent injections. At the time of injection, cultured RPE cells were collected and resuspended to approximately 2.5 × 10 in PBS⁶Cells/ml. Approximately 0.2ml of aqueous humor was removed from each recipient rabbit eye with a 25 gauge (gauge) needle, and RPE cells were injected transsclerally with a 27 gauge needle to a site 3mm posterior to the limbus, just above the optic disc. After RPE cell injection, 0.1ml of PDGF BB (50-150ng), CTGF (200-400 ng) or PDGF and CTGF in PBS were injected through the same entry site. The non-injected eye of each animal was used as a control. Optionally, CTGF may be re-injected at day 7 and/or day 14 after the 1 st injection. Half of the animals are additionally treated with the antibodies of the invention, for example, by intravenous, intraperitoneal or intraocular injection. Depending on the injection site, the antibody may be provided daily or not administered so frequently, e.g., on days 7, 10, 14, etc.

Animals were examined using an indirect ophthalmoscopic procedure to monitor PVR development and extent, which was classified according to the parameters described by Fastenberg (Fastenberg et al (1982) Am J Ophthalmol 93: 565-. The animals were then sacrificed and analyzed for the extent of membrane formation and the extent of fibrosis in the eye by histological examination. In addition, the retina and fibrotic membrane can be collected to measure collagen content.

Alternatively, PVR was induced in rabbit eyes by subretinal injection of dispase using a model and procedure adapted from Frenzel et al (1998, Invest Ophthamol Vis Sci 39: 2157-. Subretinal blebs (subclinical blebs) were formed with 50ml (0.05U) of dispase (Sigma Chemical Co.) in PBS. Half of the animals are additionally treated with the antibodies of the invention, for example, by intravenous, intraperitoneal or intraocular injection. Retinal detachment was induced in about 75% of rabbits not receiving injection of the antibody of the present invention one week after surgery, and in about 100% of these animals two weeks after surgery. The degree of fibrosis of the epiretinal membrane (epiretinal membrane) was examined.

A decrease in pathological changes in antibody-treated animals relative to untreated controls is indicative of the effect of PVR treatment. Since CTGF has been shown to be associated with tissue damage in PVR models, anti-CTGF agents have been proposed as therapeutic agents for such diseases (see, e.g., international publication WO 03/049773). The antibodies of the present invention represent a unique therapeutically effective class of anti-CTGF therapeutic agents for ocular diseases such as PVR.

Example 9: hardening of

Sclerosis is generally characterized by diffuse fibrosis, degenerative changes and vascular abnormalities in the skin (scleroderma), joints and internal organs, in particular the oesophagus, the GI tract, the lungs, the heart and the kidneys.

9.1 local granuloma induction

Neonatal mice develop persistent localized fibrosis when a combination of human-derived TGF- β 2 and CTGF is administered by continuous 7 days of subcutaneous injection (Mori et al (1999) J Cell Physiol181: 153-.

The day after birth, mice were divided into 3 treatment groups and administered by subcutaneous injection for 7 consecutive days into 40 μ l1% Mouse Serum Albumin (MSA), PBS containing 800ng TGF- β 2, 400ng CTGF or both TGF- β 2 and CTGF in the subscapular region. The TGF- β 2 and CTGF combination group was further divided into 2 groups, with group 1 additionally receiving 40 μ g of antibody mAb1 of the present invention. On day 11, animals were sacrificed and injection site sections were processed and stained with Mason's trichrome for histological evaluation. Slides were randomized and qualitatively assessed blindly by 3 scientists, scoring from 0 (no change) to 4 (fibrous tissue) based on the degree of fibrosis or connective tissue broadening (see figure 8). Cumulative scores from all observers for each slide were then calculated and the mean values between groups were compared using the ANOVA test.

The group mean scores for vector control, TGF-. beta.2, and the combination of TGF-. beta.2 and CTGF were 0.75, 6.83, and 9.00, respectively (Table 4).

Table 4 histological scoring of granulomas in neonatal mice

Treatment of	Mean score	Standard error of	Group size
				Carrier	0.75	0.48	4
TGF-β2	6.83	0.65	6
				TGF-β2+rhCTGF	9.00	0.72	7
TGF-β2+CTGF+mAb1	6.17	1.40	6
				TGF-β2+CTGF+FG-3025	7.50	1.50	4

Group score of 1 slide from 3 different observers

The group of antibody treatments scored a mean of 6.17, which was a statistically significant decrease (p <0.05) when compared to the corresponding TGF- β 2 and CTGF combination, while treatment with the non-neutralizing anti-CTGF antibody mAb3 against the C-terminus did not reduce fibrosis. Thus, the antibodies of the invention are particularly effective in reducing localized sclerosing damage to tissue.

9.2 Neonatal Systemic Fibrosis (Neoneatal Systemic Fibrosis)

Newborn mice were divided into several groups and injected intraperitoneally daily for 21 consecutive days with the following drugs: 300. mu.g/kg/day TGF β, 300. mu.g/kg/day CTGF, 300. mu.g/kg/day TGF β and CTGF each in combination, or 5mg/kg of antibody mAb1 of the present invention injected IP 30 minutes prior to growth factor treatment. The pup stays with its mother during the treatment. On day 21, animals were sacrificed and major organs removed and total proline and hydroxyproline were measured as described above.

Daily injections of TGF β induced mild systemic fibrosis, while CTGF alone did not produce any response. TGF β and CTGF in combination induced systemic fibrosis with extensive collagen deposition in several organs including liver, lung, heart, GI tract, diaphragm and kidney (fig. 10); a large amount of intestinal adhesions; and an increase in mortality of 25%. Administration of the antibodies of the invention in combination with growth factor treatment reduced or prevented organ fibrosis (fig. 10) and intestinal adhesions and prevented death. Thus, the antibodies of the invention are also effective in reducing sclerosing damage to various tissues and organs when administered systemically. The results in examples 10.1 and 10.2 clearly show that the antibodies of the invention are therapeutically effective for treating sclerosing conditions when administered locally or systemically.

9.3 scleroderma

The antibodies of the invention are useful for ameliorating fibrosis associated with scleroderma. Methods for measuring the extent and severity of skin diseases in scleroderma are known in the art (see, e.g., Rodnan et al (1979) Arthritis Rheum22:130-40; Aghassi et al (1995) Arch Dermatol131:1160 1161; Brennan et al (1982) Br J Rheumatotol 31:457-460; Kahale et al (1986) Clin Exp Rheumatotol 4: 369; Falanga and Bucala (1993) J Am AcadDerm29:47-51; Seyger et al (1997) J Am Acourm 37:793 Derma 796; Seyger et al (1990) J Am Acrm Derm39:220 ack 225; Bl et al J1990: 67790: 1990) J Amoto J67793 Derma 7978; Rheumato J67102; 1990: 1990) J67790: 19835; 1990: 794J 67666-1996).

For example, the modified Rodnan skin score measures skin firmness in standardized firmness units having a resolution of 0.1 units using a Type OO Rex DD-3 digital sclerometer (RexGauge Company, Buffalo Grove IL). The durometer measurements were made at all identical skin sites measured by the Rodnan skin score. Skin scores and sclerometer readings were taken at baseline scan, before administration of the antibodies of the invention, every 3 months during administration and subsequent follow-up. Each measurement was repeated 4 times, using analysis of variance structures and homogeneous correlation coefficient calculations to determine site-related repeat variability and patient variability (Fleiss (1971) Psychol Bull76: 378-. The consistency between skin scores and sclerometer scores was also assessed using correlation techniques, both providing total and subset scores at a given time point. Post-extension correlation analysis was also performed (e.g., the on-entry sclerometer score was correlated with the skin score at t + 3 months or t + 6 months of treatment with antibody). Disease activity and functional status information can also be collected, including collagen synthesis data (PIIINP measurements). A reduction in the symptoms and/or complications of scleroderma, as measured by any one of the methods described above, confirms the therapeutic efficacy of the antibodies of the invention.

Example 10: osteoarthritis

The antibodies of the invention were tested in one of the following models to confirm their therapeutic efficacy in osteoarthritis. In the examples described below, the concentration of the antibody used is in the range of about 0.015-15 mg antibody/kg body weight of the subject, i.e., a dose of about 5mg antibody/kg body weight is considered suitable.

Animals such as 12 week old male C57BL/6 mice are housed in standard cages and fed a standard diet with tap water ad libitum.

10.1 Intra-articular AdCTGF injection in the mouse Knee joints

An adenovirus expression vector construct (AdCTGF) containing CTGF was prepared using the adesy system (Qbiogene, Carlsbad CA) according to the procedure provided by the manufacturer. Briefly, polynucleotides encoding full-length human CTGF were inserted into the PSHUTTLE-CMV plasmid (Qbiogene) using standard molecular cloning techniques. The pShuttle-CMV-CTGF construct was then linearized and co-transfected with the PADEASY-1 plasmid (Qbiogene) by electroporation into competent E.coli (E.coli) BJ-5183 cells. AdCTGF was amplified and purified using the procedure described by Kim et al (2001, J Biol Chem276:38781-38786) and an empty adenovirus vector was used as a control. AdCTGF and control viruses were found to form plaques in the range of 1.0-2.1X10¹⁰Per mol) and viral particles (range 0.9-1.5X10¹²In mol) are similar.

AdCTGF or control adenovirus (1X 10)⁷Plaque forming unit) by intra-articular injection; the antibodies of the invention are administered by intra-articular, intravenous, intraperitoneal or subcutaneous injection. The antibody may be injected simultaneously with the administration of the adenovirus, or treatment may be initiated before and after injection of AdCTGF. Animals receiving control adenovirus were similarly injected with anti-CTGF antibody or control antibody. The non-injected knee joints served as controls for antibody effects.

The knee joints were separated at various days, e.g., 1, 3, 7, 14 and/or 28 days after injection of AdCTGF, decalcified in EDTA/polyvinylpyrrolidone for 14 days, and stored at-20 ℃ using the procedure described in Stoop et al (2001, Osteoarthritis Cartilage9: 308-315). Analyzing the histology of the joints to measure synovial thickness and proteoglycan reduction (depletion); in situ hybridization and immunohistochemistry were performed to identify CTGF expression and expression of additional factors including collagen (type I and/or type III) and the like. Synovial fluid was collected to determine the levels of CTGF, metalloproteases, etc. Efficacy of treatment with an anti-CTGF antibody was demonstrated by a reduction in osteoarthritis-associated parameters relative to animals injected with AdCTGF and treated with a control antibody.

10.2 Intra-articular injection of AdTGF β in murine Knee joints

Alternatively, antibodies can be tested in an animal model of Osteoarthritis as described by Bakker et al (2001, Osteoarthritis Cartilige 9: 128-136). For example, an antibody of the invention or a control antibody can be injected intra-articularly at 1X10⁷Concurrent, subsequent or prior injection of an adenovirus construct (AdTGF β) expressing TGF β in pfu. The non-injected knee joints served as controls for antibody effect. On each day, e.g., days 3, 7, 14, etc., animals from each group are sacrificed and tissues are isolated and processed. Analyzing the histology of the joints to measure synovial thickness, proteoglycan reduction, osteophyte formation, and the like; in situ hybridization and immunohistochemistry were performed to identify CTGF expression and expression of additional factors including collagen (type I and/or type III) and the like. Synovial fluid was collected to determine the levels of TGF β, CTGF, metalloproteases, etc. The efficacy of treatment with the antibodies of the invention is demonstrated by a reduction in osteoarthritis-related parameters relative to animals injected with AdTGF β and treated with control antibodies.

10.3 Intra-articular injection of papain in mouse Knee joints

Alternatively, antibodies can be tested using the procedure described by van der Krann et al (1989, Am J Pathol 135: 1001-1014). Intra-articular injection of papain induces osteophyte formation, fibrosis and reduction of articular cartilage proteoglycans. The papain model was initiated by injecting 1 unit papain solution (Sigma, st. The left knee of each animal was used as an internal control. The antibodies of the invention may be administered simultaneously, subsequently or previously with the intra-articular injection of papain (0.5%/knee) by intra-articular, intravenous, intraperitoneal, or subcutaneous injection. On each day, e.g., days 3, 7, 14, etc., animals from each group are sacrificed and tissues are isolated and processed. Analyzing the histology of the joints to measure synovial thickness, proteoglycan reduction, osteophyte formation, and the like; in situ hybridization and immunohistochemistry were performed to identify CTGF expression and expression of additional factors including collagen (type I and/or type III) and the like. Synovial fluid was collected to determine the levels of TGF β, CTGF, metalloproteases, etc. The efficacy of treatment with the anti-CTGF antibody was demonstrated by a reduction in osteoarthritis-related parameters relative to animals injected with papain and treated with the control antibody.

Example 11 cloning and expression

Although the following examples describe the cloning and expression of one particular antibody of the invention, the methods are applicable to all antibodies described and claimed herein.

An exemplary antibody mAb1 of the invention was first identified as part of a composite human antibody secreted by a hybridoma cell line (8C 12-F10; prepared as described in example 3).

11.1 cloning and sequencing of mAb1 heavy chain

Messenger RNA was isolated from 8C12-F10 cell cultures using the MICRO-FAST TRACK kit (Invitrogen) according to the protocol provided by the manufacturer. Two pools of cDNA (pool) were then generated via second strand synthesis using a cDNA cell cycle kit (cDNA cell cycle kit, Invitrogen) according to the protocol provided by the manufacturer and one of the following heavy chain antisense primers:

AB90(TGCCAGGGGGAAGACCGATGG;SEQ ID NO:3)

m19H1504R(GCTGGGCGCCCGGGAAGTATGTA;SEQ ID NO:4)

the heavy chain variable region sequences were cloned by PCR amplification of an AB 90-primed cDNA pool using an AB90 primer and one of a series of V region primers, including primers corresponding to conserved secretory signal sequences encoding the 5' ends of the respective coding regions and the framework region 1 sequence encoding the mature immunoglobulin initiation region. Pfu DNA polymerase (Stratagene) was used with the following changes according to the recommended manufacturer's protocol: the reaction is typically carried out in a total volume of 50. mu.l, containing 1. mu.l of cDNA, 0.75. mu.M of each forward and reverse primer, 200. mu.M of each dNTP and 1. mu.l of Pfu polymerase (2.5 units/. mu.l). Initial incubation at 94 ℃ for 2 minutes before addition of enzyme was performed using a countdown (countdown) thermocycler program. The following cycle parameters were then used: 45 seconds at 94 ℃, 45 seconds at 65 ℃ and 1 minute at 72 ℃ for 10 cycles; 30 cycles at 94 ℃ for 45 seconds, 55 ℃ for 45 seconds and 72 ℃ for 1 minute; then 10 minutes at 72 ℃ for 1 cycle.

Only one heavy chain signal sequence primer AB87(ATGGAGTTTGGRCTGAGCTG; SEQ ID NO:5) that binds to the VH3 family heavy chain V region produced a significant product. The 453 nucleotide long PCR product was cloned into the PCR BLANT II-TOPO vector (Invitrogen), each clone was screened for the correct insert size, and 3 clones corresponding to the PCR product were sequenced. All three clones obtained the same sequence.

Heavy chain constant and UTR region sequences were cloned by PCR amplification of the m19H 1504R-directed cDNA pool. A601 nucleotide long PCR fragment was amplified using sense primer VH3-3329-51F (CGGCGGTGTTTCCATTCGGTGAT; SEQ ID NO:6) and heavy chain constant region antisense primer m19H553R (GGGCGCCTGAGTTCCACGACAC; SEQ ID NO:7), which corresponds to the 5' end of the heavy chain segment. The PCR products were cloned into the PCR-BLUNT II vector (Invitrogen) using topoisomerase mediated cloning according to the manufacturer's instructions, followed by sequencing of the inserts. Similarly, a 505 nucleotide long PCR fragment was amplified with sense primer m19H439F (GTCTTCCCCCTGGCACCCTCCTC; SEQ ID NO:8) and antisense primer m19H943R (CCCGCGGCTTTGTCTTGGCATTAT; SEQ ID NO:9), and a 503 nucleotide long PCR fragment was amplified with sense primer m19H1002F (CTGGCTGAATGGCAAGGAGTA; SEQ ID NO:10) and antisense primer m19H 1504R. Both fragments were cloned into the PCR-BLUNT II vector (Invitrogen) and sequenced as described above. The 4th heavy chain PCR fragment, 586 nucleotides long, was amplified using sense primer m19H645F (GGGCACCCAGACCTACATC; SEQ ID NO:11) and antisense primer m19H1230R (CTCCGGCTGCCCATTGCTCTCC; SEQ ID NO:12) and was directly sequenced.

FIG. 11A shows an alignment of the PCR fragments of the clones, which provides the full-length nucleotide sequence (SEQ ID NO:13) encoding the heavy chain (SEQ ID NO:14) of mAb 1. The amino acid sequence of the heavy chain variable region most closely resembles that of the VH3 germline gene DP-44. Although it is not known which D segment was used, the sequence of mAb1 most closely resembles the DH4 family. The JH region most closely matches the germline JH4 and JH 5. The heavy chain constant region of mAb1 matched GenBank accession No. BC016381, indicating a G1m (3) allotype (allotype).

11.2 cloning and sequencing of the light chain of mAb1

Messenger RNA was isolated from 8C12-F10 cell cultures using the MICRO-FAST TRACK kit (Invitrogen) according to the protocol provided by the manufacturer. Two cDNA pools were then generated via second strand synthesis using a cDNA cell cycle kit (cDNA cell cycle kit, Invitrogen) according to the protocol provided by the manufacturer and one of the following light chain antisense primers:

AB16(CGGGAAGATGAAGACAGATG;SEQ ID NO:15)

Ck-760R(AAGGATGGGAGGGGGTCAGG;SEQ ID NO:16)

the light chain variable region sequences were cloned by PCR amplification of an AB 16-primed cDNA pool using an AB16 primer and one of a series of V region primers, including primers corresponding to conserved secretory signal sequences encoding the 5' ends of the respective coding regions and the framework region 1 sequence encoding the mature immunoglobulin initiation region. Pfu DNA polymerase (Stratagene) was used according to the recommended manufacturer's protocol with the changes and cycling parameters described above.

Only one light chain signal sequence primer AB123(CCCGCTCAGCTCCTGGGGCTCCTG; SEQ ID NO:17) that binds to the VK1 family heavy chain V region produced a significant product. The 408 nucleotide long PCR product was cloned into the PCRBUNT II-TOPO vector (Invitrogen), individual clones were screened for the correct insert size, and 3 clones corresponding to the PCR product were sequenced. All three clones obtained the same sequence.

The light chain constant region sequence was cloned by PCR amplification of a Ck-760R-directed cDNA pool. The entire coding region and the 5' UTR region of the light chain were amplified with light chain antisense primers L1522m (TCAGWCYCAGTCAGGACACAGC; SEQ ID NO:18) and Ck-760R. The 788 nucleotide long fragment was cloned into the PCR BLUNT II vector (Invitrogen) and sequenced. The resulting plasmid was designated 41m 6.

FIG. 11B shows an alignment of the PCR fragments of the clones, which provided the full-length nucleotide sequence (SEQ ID NO:19) encoding the light chain (SEQ ID NO:20) of mAb 1. The amino acid sequence of the light chain variable region most closely matched the region encoded by germline Vk L15 and Jk2 nucleotide sequences. The light chain constant region of mAb1 was identical to the reported human germline kappa light chain immunoglobulin gene sequence (Whitehurst et al (1992) Nucleic Acids Res20: 4929-4930).

11.3 Generation of mAb1 heavy and light chain expression constructs

Full length mAb1 heavy chain cDNA was generated in two steps from the heavy chain PCR product described above and shown in fig. 11A by overlap extension PCR. Two 5' PCR products were combined together in a PCR overlap extension reaction using remote primers (digital primers) VH3-3329-51F and m19H943R to generate a single fragment 991 nucleotides long. Similarly, two 3' PCR products were combined together in a PCR overlap extension reaction using the remote primers VH3-3329-51F and m19H943R to generate a single fragment of 860 nucleotides in length. The two PCR extension reaction products were then gel purified and amplified together with the remote primers VH3-3329-51F and m19H1504R to yield the 1407 nucleotide long cDNA (residues 441 to 1847 of SEQ ID NO:13) coding sequence for the full length mAb1 heavy chain.

The heavy chain cDNA was then cloned into the PCR-BLANT II TOPO vector (Invitrogen) to generate plasmid 43a 4. The mAb1 heavy chain coding region was then subcloned by digesting plasmid 43a4 with BamHI and XbaI restriction enzymes, followed by ligation of the cleaved insert into the PCDNA5-FRI expression vector (Invitrogen) that had been previously digested with BamHI and Nhe restriction enzymes. The resulting insert of expression vector 44a1 was sequence verified, and was then similarly reverse subcloned into the PBK-CMV vector (Clontech) to produce plasmid 47a4, and into the pCEP-Pu vector (E.Kohfeldt, Max-platform-institute fur Biochemie) derived from the pCEP4 vector (Invitrogen) to produce plasmid 49a 1.

The 708 nucleotide long cDNA encoding full length mAb1 light chain (residues 415 to 1122 of SEQ ID NO:19) was excised from plasmid 41m6 as described above using HindIII and XhoI restriction enzymes and ligated into the PCDNA5-FRT vector (Invitrogen) that had previously been digested with HindIII and XhoI restriction enzymes to produce mammalian expression plasmid 42b 2. The insert of plasmid 42b2 was sequence verified, and was then similarly reverse subcloned into the PBK-CMV vector (Clontech) to produce plasmid 47b3, and into the pCEP-Pu vector (E.Kohfeldt, Max-Planck-institute fur Biochemie) to produce plasmid 49b 1.

11.4 transfection and expression of antibody chain constructs

COS7 cells were transfected with plasmid 44a1(mAb1 heavy chain) and 42b2(mAb1 light chain) alone and COS7 cells were co-transfected with both plasmids using standard procedures. Conditioned media was assayed as described in example 4 (see above) to detect the presence of antibody. Only the media from cells co-transfected with 44a1 and 42b2 expressed human antibodies with CTGF binding activity as measured by ELISA using the procedure described above. The antibody produced by co-transfected COS7 cells, referred to herein as CLN1, bound the N-terminal half (N-terminal half) of CTGF with an affinity of 0.8 nM.

CLN1 was also expressed in genetically modified Chinese Hamster Ovary (CHO) cells. A CHO cell line expressing exemplary antibody CLN1 was deposited at the american type culture collection (Manassas VA) on year 2004, 5/19 with ATCC accession No. ______. Cell lines can be optimized and antibody expression enhanced using various techniques known in the art, such as the gene amplification described by Wigler et al (1980; Proc Natl Acad Sci USA77:3567-3570) and modified by Ringold et al (1981; J Mol ApplGenet1:165-175), Gasser et al (1982; Proc Natl Acad Sci USA79:6522-6526) and Kaufman et al (1985; Mol Cell Biol5: 1750-1759).

Example 12 interaction of CTGF with TGF beta

The antibodies of the invention specifically bind to the region of CTGF defined by the residues encoded by exon 3 (FIG. 1B; nucleotide 418 to nucleotide 669 of SEQ ID NO: 1). This region encompasses amino acids 97 to 180 of SEQ ID NO. 2 and includes the von Willebrand type C domain (amino acids 103 to 164 of SEQ ID NO. 2) and the epitope of mAb1 (amino acids 134 to 158 of SEQ ID NO. 2). Abreu et al (2002, Nat Cell Biol4:599-604) report that the domain corresponding to the VWC domain of CTGF is important for the interaction between CTGF and TGF β or BMP-4 and that the interaction modulates the activity of TGF β and BMP-4. The following experiment confirmed that the region encoded by exon 3 is sufficiently necessary for binding of CTGF to TGF β, and that the antibody of the present invention can block the interaction between CTGF and TGF β.

The interaction between CTGF and TGF β was determined using the following procedure. Wells of 96-well maxisorp elisa plates (Nalge Nunc) were coated with 10 μ g/ml CTGF, a CTGF fragment encoded by exon 3 or a CTGF fragment encoded by exon 5 (all in PBS) or with PBS overnight at 4 ℃. All wells were then blocked with 1% BSA in PBS followed by incubation for 1 hour at room temperature in 50. mu.l of a solution containing TGF β at a concentration of 0, 1, 3.3, 10, 33, 100, 333, or 1000ng/ml and MAB612 or MAB1835 mouse anti-TGF β monoclonal antibody (R & D Systems, Minneapolis MN) at 100, 300, or 1000ng/ml in PBS,0.05% Tween-20. MAB1835 recognizes bovine, mouse and human TGF-beta 1 and TGF-beta 2 and blocks TGF-beta binding to mouse thymocytes. MAB612 recognizes TGF-. beta.2 but does not inhibit TGF-. beta.activity. The wells were rinsed with PBS,0.05% Tween-20, and then incubated for 1 hour at room temperature in a solution containing alkaline phosphatase conjugated goat anti-mouse IgG antibody diluted in PBS,0.05% Tween-20. The plates were washed again, p-nitrophenyl phosphate (PNPP) in 1M ethanolamine, 1mM MgSO4, pH9.8 was added, the wells were incubated for a suitable period of time to develop color, and the reaction was stopped by the addition of NaOH. The absorbance at λ 405nm was measured with a spectrophotometer.

Fig. 12 shows that CTGF and CTGF fragments encoded by exon 3 interact with TGF to an equal extent, whereas CTGF fragments encoded by exon 5 do not show any binding activity to TGF. Interestingly, the anti-TGF β antibody MAB612 was able to detect CTGF-bound TGF β in a dose-dependent manner, but the neutralizing antibody MAB1835 was unable to detect CTGF-bound TGF β at any concentration tested (data not shown). This suggests that CTGF competes with MAB1835 for binding to TGF β.

anti-CTGF antibodies were tested for their ability to block binding between CTGF and TGF β. As shown in fig. 12, the antibodies of the invention exemplified by mAb4 and mAb1 blocked the binding of CTGF and the CTGF fragment encoded by exon 3 to TGF β, while anti-CTGF antibodies directed against the C-terminus of CTGF did not block binding. These results support the mechanism of action of the antibodies of the invention to specifically block the interaction between CTGF and TGF β and potentially the interaction between CTGF and other members of the TGF β superfamily.

Various modifications of the invention in addition to those shown and described above will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to be included within the scope of the appended claims.

All references cited herein are incorporated by reference in their entirety.

Instructions for the preservation of microorganisms (second of PCT rule 13)

PCT/RO/134 Table (7 months in 1998; 1 month in 2004 reprinted)

Claims (45)

1. An isolated antibody that specifically binds to the sequence set forth in SEQ ID NO. 25 and is capable of neutralizing the biological activity of CTGF.

2. The antibody of claim 1, wherein the antibody modulates the interaction between the CTGF polypeptide and the secreted or membrane cofactor, thereby neutralizing the biological activity.

3. The antibody of claim 1, wherein the biological activity is cell migration.

4. The antibody of any one of the preceding claims, wherein the antibody reduces fibrosis in a subject.

5. The antibody of claim 4, wherein the fibrosis occurs within a tissue selected from the group consisting of: epithelial tissue, endothelial tissue, and connective tissue.

6. The antibody of claim 4, wherein the fibrosis occurs within an organ selected from the group consisting of: kidney, lung, liver, heart and skin.

7. The antibody of any one of the preceding claims, wherein the antibody has an affinity for CTGF of about 10^-9M。

8. The antibody of any one of the preceding claims, wherein the antibody is a single chain antibody.

9. The antibody of any one of the preceding claims, wherein the antibody is a humanized antibody.

10. The antibody of any one of the preceding claims, wherein the antibody is a chimeric antibody.

11. The antibody of any one of the preceding claims, wherein the antibody is a multivalent antibody.

12. The antibody of any one of the preceding claims, wherein the antibody is glycosylated.

13. The antibody of any one of the preceding claims, wherein the antibody is non-glycosylated.

14. The antibody of any one of the preceding claims, wherein the antibody is conjugated to a cytotoxic agent or enzyme.

15. The antibody of any one of the preceding claims, wherein the antibody is detectably labeled.

16. The antibody of claim 15, wherein the detectable label is an enzyme, a fluorescent moiety, a chemiluminescent moiety, biotin, avidin, or a radioisotope.

17. The antibody of any one of the preceding claims, wherein the antibody is a monoclonal antibody.

18. Use of an isolated antibody that binds to CTGF, wherein the variable region of an immunoglobulin heavy chain is the variable region of amino acids 1-167 of SEQ ID No. 14 and the variable region of an immunoglobulin light chain is the variable region of amino acids 1-136 of SEQ ID No. 20.

19. The use of claim 18, wherein the binding to CTGF is for affinity chromatography, enzyme-linked immunosorbent assay (ELISA), Radioimmunoassay (RIA), immunoprecipitation, immunohistochemistry, fluorescence-activated cell sorting (FACS), diagnosing CTGF-associated disease, monitoring progression of CTGF-associated disease, monitoring efficacy of treatment for CTGF-associated disease, neutralizing biological activity of CTGF.

20. The use according to claim 18, wherein the binding to CTGF is used for qualitative or quantitative detection of CTGF in a sample.

21. The use of claim 20, wherein the sample is conditioned cell culture medium, biopsy tissue, organ transplant, blood, urine, vesicular fluid, cerebrospinal fluid, vitreous humor, or synovial fluid.

22. The use according to claim 19, wherein diagnosing the CTGF-associated disorder comprises obtaining a sample, detecting the level of CTGF in the sample, and comparing the level of CTGF in the sample to a standard amount of CTGF, wherein an increase or decrease in the level of CTGF in the sample indicates the presence of the CTGF-associated disorder.

23. The use according to claim 19, wherein the CTGF-associated disease is selected from the group consisting of: breast cancer, pancreatic cancer, gastrointestinal cancer, atherosclerosis, arthritis, diabetic retinopathy, diabetic nephropathy, heart, lung, liver fibrosis, chronic inflammation related diseases and chronic infection related diseases.

24. The use according to claim 19, wherein the CTGF-associated disease is associated with: myocardial infarction, diabetes, peritoneal dialysis, chronic transplant rejection, chemotherapy, radiation therapy and surgery.

25. The use of claim 19, wherein monitoring CTGF-associated disease progression comprises: obtaining samples from the subject over time, detecting and quantifying the level of CTGF in each sample, and comparing the level of CTGF in subsequent samples to the level of CTGF in earlier or prior samples, wherein a change in the level of CTGF between samples over time indicates the progression of or the efficacy of treatment for the CTGF-associated condition.

26. The use of any one of claims 18 to 25, wherein the antibody is detectably labeled.

27. The use of claim 26, wherein the detectable label is an enzyme, a fluorescent moiety, a chemiluminescent moiety, biotin, avidin, or a radioisotope.

28. A pharmaceutical composition comprising an effective amount of the antibody of claim 1, wherein the pharmaceutical composition comprises a pharmaceutically acceptable carrier.

29. The pharmaceutical composition of claim 28, further comprising: an Angiotensin Converting Enzyme (ACE) inhibitor, an advanced glycation end product cleaving agent or an advanced glycation end product inhibitor.

30. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is for treating a subject having a disease selected from the group consisting of idiopathic pulmonary fibrosis, diabetic nephropathy, chronic heart failure, and cirrhosis of the liver.

31. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is for treating a subject predisposed to a disease caused by a condition selected from the group consisting of: hypertension, diabetes, myocardial infarction and arthritis.

32. The pharmaceutical composition of claim 28, wherein the pharmaceutical composition is for treating a subject predisposed to a disease caused by local or systemic inflammation.

33. Use of the antibody of claim 1 for the manufacture of a medicament for treating or preventing a CTGF-associated disease in a subject having or at risk of having the CTGF-associated disease.

34. The use of claim 33, wherein the subject is predisposed to or diagnosed with hypertension, diabetes, myocardial infarction, or arthritis.

35. The use of claim 33, wherein the subject is predisposed to or diagnosed with a local or systemic inflammation.

36. The use of claim 33, wherein the disease is a cell proliferative disease.

37. The use of claim 36, wherein the cell proliferative disorder is angiogenesis, atherosclerosis, glaucoma or cancer.

38. The use of claim 37, wherein the cancer is acute lymphoblastic leukemia, dermatofibroma, breast cancer desmogenesis, angiolipoma, vascular leiomyoma, desmogenesis cancer, prostate cancer, ovarian cancer, colorectal cancer, pancreatic cancer, gastrointestinal cancer, or liver cancer.

39. The use of claim 33, wherein the subject is predisposed to or diagnosed with a fibrotic disease.

40. The use of claim 39, wherein the fibrotic disease is idiopathic pulmonary fibrosis, diabetic nephropathy, diabetic retinopathy, osteoarthritis, scleroderma, chronic heart failure, or cirrhosis of the liver.

41. A polynucleotide sequence comprising a sequence selected from the group consisting of:

(a) a polynucleotide sequence encoding SEQ ID NO. 14;

(b) a polynucleotide sequence encoding amino acids 1-167 of SEQ ID NO. 14;

(c)SEQ ID NO:13；

(d) nucleotides 1-501 of SEQ ID NO 13.

42. A polynucleotide sequence comprising a sequence selected from the group consisting of:

(a) a polynucleotide sequence encoding SEQ ID NO. 20;

(b) a polynucleotide sequence encoding amino acids 1-136 of SEQ ID NO. 20;

(c)SEQ ID NO:19；

(d) nucleotides 1-408 of SEQ ID NO 19.

43. A recombinant polynucleotide comprising the polynucleotide sequence of claim 41 or 42 operably linked to a vector sequence comprising replication and transcription control sequences.

44. The recombinant polynucleotide of claim 43, wherein said polynucleotide encodes the amino acid sequence of SEQ ID NO. 20 or SEQ ID NO. 14.

45. The recombinant polynucleotide of claim 42, wherein said polynucleotide comprises the amino acid sequence of SEQ ID NO 19 or SEQ ID NO 13.