WO2006038107A2 - Method for inhibiting fibrogenesis by an rhdecorin-fc fusion protein - Google Patents

Method for inhibiting fibrogenesis by an rhdecorin-fc fusion protein Download PDF

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WO2006038107A2
WO2006038107A2 PCT/IB2005/002999 IB2005002999W WO2006038107A2 WO 2006038107 A2 WO2006038107 A2 WO 2006038107A2 IB 2005002999 W IB2005002999 W IB 2005002999W WO 2006038107 A2 WO2006038107 A2 WO 2006038107A2
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rhdecorin
decorin
fusion protein
tgf
leu
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PCT/IB2005/002999
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WO2006038107A3 (en
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Mengsu Yang
Hsiang-Fu Kung
Yaou Zhang
Celina S.M. Cheng
Pik Yuen Cheung
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Mengsu Yang
Hsiang-Fu Kung
Yaou Zhang
Cheng Celina S M
Pik Yuen Cheung
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Priority to CN2005800371903A priority Critical patent/CN101115839B/en
Publication of WO2006038107A2 publication Critical patent/WO2006038107A2/en
Publication of WO2006038107A3 publication Critical patent/WO2006038107A3/en

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    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K14/00Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4725Proteoglycans, e.g. aggreccan
    • AHUMAN NECESSITIES
    • A61MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61PSPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • CCHEMISTRY; METALLURGY
    • C07ORGANIC CHEMISTRY
    • C07KPEPTIDES
    • C07K2319/00Fusion polypeptide
    • C07K2319/30Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto

Definitions

  • the present invention relates to methods for inhibiting fibrogenesis, especially liver f ⁇ brogenesis, by administering antagonists of hepatic stellate cell activation and fibroblast cell activation.
  • This invention also relates to making a fusion protein construct and to producing the fusion protein.
  • liver fibrosis As to liver fibrosis, fibrosis has an integral role in the final common pathway of structural remodeling that reduces normal organ function following injury. It is one of the most fundamentally destructive and unwanted responses to developmental or inflammatory diseases and is seen in millions of individuals in the advanced stages of many different disease processes including such diseases as cystic fibrosis, interstitial nephritis, hepatic cirrhosis and pulmonary fibrosis following exposure to high oxygen tension. Liver fibrosis is characterized by an excessive deposition of extracellular matrix components in the liver. As described by S.L.
  • liver cell types participate in matrix deposition, the major types being hepatic stellate cells (HSC) and portal fibroblasts as described by B. Tuchweber et al. in an article entitled “Proliferation and Phenotypic Modulation of Portal Fibroblasts in the Early Stages of Cholestatic Fibrosis in the Rat,” Lab Invest 1996; 74:265-278.
  • HSC hepatic stellate cells
  • portal fibroblasts as described by B. Tuchweber et al. in an article entitled “Proliferation and Phenotypic Modulation of Portal Fibroblasts in the Early Stages of Cholestatic Fibrosis in the Rat," Lab Invest 1996; 74:265-278.
  • HSC hepatic stellate cells
  • portal fibroblasts as described by B. Tuchweber et al. in an article entitled “Proliferation and Phenotypic Modulation of Portal Fibroblasts in the Early Stages of Cholestatic Fibrosis in the Rat
  • Fibrogenesis is classically mediated by organ fibroblasts that express abundant amounts of collagen types I and III.
  • the expression of fibrogenesis in liver has been the subject of intense study over the last several years.
  • the cytokine regulation of this process is complex. It is generally believed that collagen types I and III are the principal fibrotic collagens, and that they are well expressed by hepatic stellate cells (HSC) and fibroblast cells.
  • HSC hepatic stellate cells
  • fibroblast cells The expression of these collagens is regulated by a complexity of cytokines.
  • Tumor necrosis factor beta 1 (TGF.beta.l), for example, is an early and pivotal component in the stimulatory process.
  • TGF.beta.l appears to be a regulatory molecule for these collagen genes in the lungs, liver, and kidney.
  • liver fibrosis cirrhosis The most important causes of liver fibrosis cirrhosis are chronic hepatitis B and C infection, and prolonged alcohol abuse. Liver cirrhosis is the clinical end point of liver fibrosis. Until present time, effective methods for reversing liver cirrhosis have not been available, and those with life threatening impairment of liver function can only look to liver transplants for salvage. However, each year the number of new cases of cirrhosis exceeds the number of livers available for transplantation by a factor of 5 to 10. Therefore, prevention of fibrogenesis and early treatment of fibrosis is the best treatment of cirrhosis (Achord JL. 1991, Compr Ther. 17:57-64, Habib et al., 2001, Postgrad Med.
  • the strategies of inhibiting fibrogenesis can be grouped as: (a) Anti-inflammatory agents and antioxidants; (b) Antagonists of cytokines or cytokine receptors; (c) Inhibitors of stellate cell activation; and (d) Anti-collagen agents (D. Montgomery Bissell, 2001, EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 33:179-190). However, most of these agents are either not very effective in treating fibrosis or have severe side effects. There is thus a need to develop new methods for inhibiting liver fibrogenesis and treating liver fibrosis.
  • decorin has been used for limiting TGF- ⁇ activity through the use of gene therapy as discussed in U.S. Patent 5,824,655. Moreover, decorin has been used for treating a pathology as discussed in U.S. Patent 6,436,900. This latter patent does not, however, discuss treatment of liver disease.
  • Decorin is a small leucine-rich multifunctional proteoglycan and it consists of a core protein and a covalently linked glycosaminoglycan chain. The size and molecular weight of decorin core protein in human is 359 amino acids; 39746 Da, in pig is 360 amino acids. The original function of this molecule is involved in fibrils formation.
  • Decorin binds to collagens type I, II and IV in vivo and promotes the formation of fibers with increased stability and changes in solubility.
  • decorin has played a role in cell growth control.
  • the expression of decorin in Chinese hamster ovary (CHO) cells has been demonstrated to lead to decreased growth rate, lowered saturation density, and altered morphology (Yamaguichi and Ruoslahti. Nature 336:244-246 (1988)).
  • These growth-suppressive properties of decorin included (a) that the expression of decorin is markedly elevated during quiescence; (b) that actively proliferating or transformed cells rarely expressed decorin; (c) that decorin expression is abrogated by viral transformation; and (d) that the gene transcription of decorin is suppressed in a variety of tumorigenic cell lines and tumor tissues by methylation of its control regions (Iozzo., et al Crit. Rev. Biochem.
  • TGF-P transforming growth factor-beta
  • ECM extracellular matrix
  • Decorin is involved in the regulation of apoptosis. It was found that decorin decreased apoptosis of endothelial cells cultured in a collagen lattice (Schonherr et al., Eur J Cell Biol. 78:44-55 (1999)). Decorin acts as a signaling molecule in ECs and affects cell survival by Akt- dependent and -independent pathways (Schonherr et al., Ann N Y Acad Sci. 973:149- 52(2002)).
  • Fc fusion proteins have been gaining favor. It is noted that recombinant proteins are an emerging class of therapeutic agents.
  • One such modification is the use of the Fc region of immunoglobulins to make a fusion protein.
  • Antibodies comprise two functionally independent parts, a variable domain known as "Fab", which binds antigen, and a constant domain, known as "Fc" which provides the link to effector functions such as complement or phagocytic cells.
  • the Fc portion of an immunoglobulin has a long plasma half- life, whereas the Fab is short-lived.
  • the Fc fusion protein should keep the bioactivities of parent protein and has longer half-life than its parents, because IgG can circulate for several days (WO 99/25044).
  • the modified decorin antagonist prevented the development of liver fibrosis, especially in course of a viral hepatitis, such as chronic hepatitis B and chronic hepatitis C.
  • the rhDecorin-Fc fusion protein comprises rhDecorin, a peptide linker, and a modified human IgG Fc fragment or domain (denoted by IgGlFc). It was also found that it is preferable to use a flexible peptide linker of about 20 or fewer amino acids in length and that the flexible peptide linker contain of two or more of amino acids selected from the group consisting of glycine, serine, alanine, and threonine.
  • the IgG Fc variant is of non- lytic nature, with the modified Fc fragment formulated by substituting ten new amino acids for the four amino acids normally used. Because of the use of the modified Fc fragment and the peptide linker, the biological half-life of the rhDecorin-Fc fusion protein was found to be 50% longer than that associated with decorin used alone.
  • a method for making or producing the subject recombinant fusion proteins from a mammalian cell line such as a Cos-7 cell line.
  • the rhDecorin-Fc fusion protein is characterized by and exhibits enhanced biological activity relative to that of rhDecorin and has an extended serum half-life without undesirable side effects, leading to improved pharmacokinetics and pharmacodynamics.
  • lower dosages and fewer injections are needed to achieve similar efficacies.
  • the present invention relates to methods for inhibiting liver fibrogenesis in vitro and/or in vivo with directly administered recombinant rhDecorin and rhDecorin-Fc fusion protein.
  • a linker is provided between Decorin and the Fc hinge fragment to form a fusion protein which increases the biologic activity of Decorin and increases the ability to bind to TGF- ⁇ and extends the lifetime of decorin in serum, where the Fc hinge fragment is a modified form of Fc involving the replacement of four N-terminal amino acids with ten different amino acids which extends the lifetime of the Decorin-Fc protein by 50%.
  • Figure IA and IB show the effect of decorin treatment on liver fibrosis after CCl.sub.4 (carbon tetrachloride) treatment for 8 weeks (haematoxylin & eosin (HE) staining), with Figure IA showing the effect on a rat treated with CCl. sub.4 alone and Figure IB showing the effect on a rat treated with CCl.sub.4 and rhDecorin.
  • CCl.sub.4 carbon tetrachloride
  • HE haematoxylin & eosin
  • FIGS. 2A and 2B show the effect of rhDecorin and rhDecorin-Fc on hepatic stellate cell proliferation induced by TGF.beta.l in which LX-2 cells (Human HSC cell line) were exposed to rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 48 hours and in which cell number was evaluated through the measurement of the reduction of the dye 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium (MTT), with Figure 2A showing the effect of rhDecorin on LX-2 cell proliferation induced by TGF.beta.l ((C; control; D: rhDecorin (4 ug/ml); T: TGF.beta.l (2 ng/ml); T+D: TGF.beta.l (2 ng/ml) plus rhDecorin
  • Figures 3 A and 3B show the effect of rhDecorin and rhDecorin-Fc treatment on MMP-2 (matrix metalloproteinases-2) mRNA levels of HSC cells stimulated with TGF.beta.l, in Figure 3A and 3B with LX-2 cells treated with rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 24 hours, with cells harvested for total RNA extraction, with RT-PCR carried out to detect the effect of rhDecorin and rhDecorin-Fc on MMP-2 mRNA levels of LX-2 cells stimulated with TGF.beta.l, and with GADPH used as a control, Figure 3 A showing the effect of rhDecorin treatment on MMP-2 and Figure 3B showing the effect of rhDecorin-Fc treatment on MMP-2 ((C; control; D: rhDecorin; T: TGF.
  • TGF.beta.l (2 ng/ml) plus rhDecorin-Fc (l ⁇ g/ml).
  • Figures 4 A and 4B show the effect of rhDecorin and rhDecorin-Fc treatment on TEvIP-I (tissue inhibitor of matrix metalloproteinases-1) mRNA levels of HSC cells stimulated with TGF.beta.
  • TGF.beta. l (2 ng/ml) plus rhDecorin-Fc (1 ⁇ g/ml).
  • Figures 5A and 5B show the effect of rhDecorin and rhDecorin-Fc treatment on collagen III protein level of HSC cells stimulated with TGF.beta.l, in which LX-2 cells were treated with rhDecorin or rhDecorin-Fc in the presence of 2 ng/ml TGF.beta.l for 24 hours and in which culture mediums were harvested, showing that in Figure 5A, Western Blotting was carried out to evaluate the effect of rhDecorin and rhDecorin-Fc on collagen III protein levels of LX-2 cells stimulated with TGF.beta.l, and showing in Figure 5B the result of quantitation.
  • T TGF.beta.l (2 ng/ml); T+D: TGF.beta.l (2 ng/ml) plus rhDecorin (4 ⁇ g/ml); T+D-Fc: TGF.beta.l (2 ng/ml)
  • T+ Fc TGF.beta.l (2 ng/ml) plus Fc (1 ⁇ g/ml) ).
  • Figure 6 shows the construct of the rhDecorin-Fc fusion protein involving 1 : Decorin; 2: Linker; 3: Immunoglobin Fc fragment; 4: disulfide bonds.
  • Figures 7A and 7B show protein expression of rhDecorin-Fc nucleic acid molecule in which Cos-7 was transfected with rhDecorin-Fc recombinant plasmid, selected with G418 (0.6mg/ml), in which mediums were collected for Western Blotting and in which decorin recombinant plasmid was used as a control, with Figure 7A showing Anti-IgG Fc and Figure 7B showing anti-human decorin antibody conjugated with HRP used to detect rhDecorin-Fc fusion proteins, in which for Figure 7A M: marker; Lane 1-3: Culture media of rhDecorin, rhDecorin- Fc and vector transfected cells.
  • Lane 4 decorin control from Sigma, and in which for Figure 7B M: marker; Lane 1-3: cell lysates of rhDecorin, rhDecorin-Fc and vector transfected cells, Lane 4-6: culture media of rhDecorin, rhDecorin-Fc and vector transfected cells, Lane 7: decorin control from Sigma; and,
  • Figures 8A and 8B show the 50% extension in lifetime of rhDecorin-Fc in which Cos-7 was transfected with rhDecorin-Fc recombinant plasmid and in which the culture medium was collected and incubated at 37 0 C for deferent times, from 0 day to 5 days, with rhDecorin used as the control, and in which Western Blotting was used to assay the stability of rhDecorin, in which for Figure 8A Lane 1, 3, 5, 7, 9 and 1 1 : decorin was incubated at 37 0 C for 0, 1, 2, 3, 4 and 5 days. Lane 2, 4, 6, 8, 10 and 12: Decorin-Fc was incubated at 37 0 C for 0, 1, 2, 3, 4 and 5 days and in which Figure 8B shows the quantitation of Western Blotting.
  • rhDecorin carbon tetrachloride (CCl 4 ) induced liver fibrosis in rats was investigated first.
  • Figure IA compared with controls, eight weeks of CCl 4 treatment induced significant fibrosis with the formation of nodules that lack a central vein with the disappearance of the normal structure of hepatic lobules.
  • Figure 2B two weeks following the initial CCl 4 treatment, rhDecorin was injected together with CCl 4 for the remaining six weeks.
  • addition of rhDecorin significantly decreased the pathologic changes of fibrogenesis.
  • HSC activation is an essential process of liver fibrogenesis. Fibrogenesis presents as proliferation of HSC and the remodeling of extra cellular matrix, including, degradation of collagen IV in the basement membrane of liver cells and deposition of an excess of extracellular matrix components, such as collagen I and collagen III in the liver. In this process, expression of matrix metalloproteinase 2 (MMP-2) and a tissue inhibitor of metalloproteinase 1 (TIMP-I) in mRNA level are up-regulated. Collagen IV is the substrate of MMP-2.
  • MMP-2 matrix metalloproteinase 2
  • TGF-I tissue inhibitor of metalloproteinase 1
  • TMP-I is an inhibitor of MMP-I which can clear up the deposition of collagen I and III. It was found that rhDecorin and rhDecorin-Fc abrogated the effects of TGF- ⁇ on hepatic stellate cells through suppressing cell proliferation, collagen III production, and MMP-2 and TIMP-I expression stimulated by TGF- ⁇ .
  • rhDecorin-Fc may be used as drugs against liver fibrogenesis.
  • Liver fibrogenesis is the active process leading to the deposition of an excess of extracellular matrix components in the liver. It is observed in a number of conditions such as chronic viral hepatitis B and C, alcoholic liver disease, drug-induced liver disease, hemochromatosis, auto-immune hepatitis, Wilson disease, primary biliary cirrhosis, sclerosing cholangitis, liver schistosomiasis and others. Fibrogenesis can occur similarly in other organs, such as lung, kidney, pancreas, heart and skin.
  • liver fibrosis is the established excess deposit of extracellular matrix components in the liver. Its endpoint is liver cirrhosis.
  • rhDecorin-Fc are useful to prevent the development of liver fibrosis that may occur in a patient infected by Hepatitis virus, e.g. hepatitis B virus (HBV), or hepatitis C (HCV) virus.
  • Hepatitis virus e.g. hepatitis B virus (HBV), or hepatitis C (HCV) virus.
  • Chronic viral hepatitis is more particularly aimed at, especially chronic hepatitis B and chronic hepatitis C.
  • patients in need of such treatment is meant any human subject or mammals, including sheep, cattle, dogs, cats, rodents, rabbits or goats, who suffer from an organ disease wherein fibrogenesis is observed or generally results from the development of the disease.
  • treatment and “prevention” include therapy and prophylaxis toward fibrogenesis, at any stage of development of the phenomenon or before it occurs.
  • the invention especially aims at preventing, or reducing or alleviating liver fibrosis in patients suffering from an organ disease.
  • rhDecorin-Fc used in accordance with the invention effectively reduces or prevents the development of liver fibrosis.
  • the invention provides fusion protein which is useful in prevention of fibrosis.
  • the fusion protein of the invention and/or nucleic acid encoding such fusion protein may be administered directly to mammals in need of treatment with an anti-fibrosis protein.
  • the invention thus provides a fusion protein comprising a target protein, referred to herein as decorin, an artificial linker and an immunoglobulin Fc region or fragment.
  • the subject fusion protein is characterized as dimer cross-linked by a pair of disulfide bonds between cysteines in adjacent subunits.
  • the disulfide bond is depicted as linking together the two immunoglobulin heavy chain Fc regions, fragments or domains via a partial of an immunoglobulin hinge region or fragment within each heavy chain, and thus is characteristic of native form of this molecule.
  • the immunoglobulin Fc region or fragment includes at least a portion of the hinge region, a CH2 domain and a CH3 domain (see SEQ. ID. NO. 1 and T).
  • the sequence of nucleic acid in the N-terminus of Fc region was slightly modified, from cctgtctccg ggtaaa to atcactagtg aattcgcggc cgctcgagtc tag (see SEQ. ID. NO.3).
  • SPGK amino acids
  • ITSEF AAARV see SEQ. ID. NO. 4
  • the Fc region is attached via a flexible linker to the C-terminus of Decorin.
  • polypeptide linker is understood to mean a peptide sequence that can link together two proteins that in nature are not naturally linked together.
  • the polypeptide linker preferably comprises a plurality of amino acids such as alanine, glycine and serine or combinations of such amino acids.
  • the polypeptide linker comprises a series of glycine and serine peptides about 19 residues in length (see SEQ. ID. NO. 5 and 6). It is contemplated, however, that the optimal linker length and amino acid composition may be determined by routine experimentation.
  • the recombinant molecule has the configuration X-Fc, where X is a target molecule.
  • the immunoglobulin Fc regions can associate, for example, via interchain disulfide bonds, to produce the type of construct shown in Figure 6.
  • corin is understood to mean full length decorin (see SEQ ID NO: 7 and 8).
  • Group I olive oil alone
  • group II 40% CC14
  • group III 40% CC14 plus rhDecorin.
  • rhDecorin treatment were started at the third week and lasted for six weeks.
  • Routine liver function blood tests including hyaluronan, type IV collagenase, ⁇ GT and transaminases were performed on an automated analyzer. Fibrosis Assessment
  • Fibrosis assessment was done on formalin-fixed, paraffin-embedded sections stained with haematoxylin & eosin (HE). Pictures were taken, using an image analysis system. All samples from a series of experiments were stained simultaneously. Liver fibrosis deposition was judged by senior pathologists and graded with the METAVIR scale, which grades fibrosis from FO (no fibrosis) to F4 (cirrhosis). METAVIR scale is a widely used scale that has excellent inter-observer reliability. Table 1 shows the result.
  • N number of animals used.
  • ALT and AST Serum alanine and aspartate aminotransferase
  • r-GT correlates well with fibrosis.
  • HSC activation is an essential process of liver fibrogenesis. It presents as proliferation of HSC and remodeling of extra cellular matrix, including, degradation of collagen IV in the basement membrane and deposition of an excess of extracellular matrix components, such as collagen I and collagen III, and change of expression of MMPs and TMPs in mRNA level.
  • TGF- ⁇ 1 is one of the most important activation factors in the process.
  • LX-2 Human HSC cell line
  • the full length decorin gene was cloned from human fibroblast cell line, then inserted the gene into a pCDNA3.1 vector (Invitrogen) and the gene was recombined with a poly(histadine) tail in the C-terminal for purification purpose.
  • Cos-7 cells were transfected with the recombinant plasmid. Because most of decorin was secreted into culture medium, the medium was collected and purification of decorin was performed using ProBond Purification system (Invitrogen), in which nickel column was used for His-tag decorin purification.
  • LX-2 cells (2000 /well) were seeded in 96-well microplate for 24 hr.
  • the culture medium was replaced with DMEM supplemented with 0.5 % FBS and the cells were allowed to be starved for 48 hr. After starvation, the medium was replaced with 2% serum supplemented DMEM medium.
  • the reagents were then added to treat cells and LX-2 cells were exposed to rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 48 hours. Cell number was evaluated through the measurement of the reduction of the dye 3-(4,5- dimethylthiazol-2yl)-2,5 diphenyltetrazolium (MTT).
  • MTT 3-(4,5- dimethylthiazol-2yl)-2,5 diphenyltetrazolium
  • MMP-2 Matrix Metalloproteinases-2
  • Tissue Inhibitor of Matrix Metalloproteinases-1 Tissue Inhibitor of Matrix Metalloproteinases-1
  • MMP-2 is the enzyme which can degrade Collagen IV and TMP-I increases fibrosis deposition by inhibiting its degradation by matrix metalloproteinases.
  • the effect of rhDecorin and rhDecorin-Fc on MMP-2 and TMP-I mRNA expression by cultured LX-2 cells was measured. 2x10 5 LX-2 cells were seeded in 6-well cell culture plate for 24hr. Then the culture medium was replaced with DMEM supplemented with 0.5 % FBS. The cells were allowed to be starved for 24 hr. After starvation, the medium was replaced with 2% serum supplemented DMEM medium.
  • reagents were added to treat cells and let LX-2 cells exposed to rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 24 hours. Finally, cells were harvested and lysised using TRIZOL reagent (GIBCOL, MD, USA). The total RNA was then extracted according to manufacturer's protocol. RT-PCR was carried out. The gene expression levels of MMP-2 and TIPM-I were normalized by a housekeeping gene, glyceraldehydes-3 -phosphate dehydrogenase (GADPH).
  • GADPH glyceraldehydes-3 -phosphate dehydrogenase
  • reagents were added to treat cells and LX-2 cells were exposed to rhDecorin or rhDecorin-Fc in the presence of 2 ng/ml TGF.beta.l for 48 hours.
  • the culture medium was harvested and Western Blotting was carried out to evaluate the effect of rhDecorin and rhDecorin-Fc on collagen III levels of LX-2 cells treated with TGF- ⁇ 1.
  • TGF- ⁇ 1 increased LX-2 cell proliferation.
  • the mitogenic effect of TGF- ⁇ 1 (2 ng/ml) on LX-2 was abolished by simultaneous addition of rhDecorin (4ug/ml) or rhDecorin-Fc (lug/ml). Because lug/ml of rhDecorin-Fc can get the same effect of 4ug/ml rhDecorin, rhDecorin-Fc exhibits an enhanced in vitro biological activity of at least 4 fold relative to that of decorin. In the absence of TGF- ⁇ l, rhDecorin or rhDecorin-Fc alone do not have a significant effect on cell proliferation.
  • TGF- ⁇ l (2 ng/ml) increased expression of MMP-2 and TIMP-I in LX-2, whereas rhDecorin and rhDecorin-Fc suppressed the increase.
  • FIG. 5 shows that TGF- ⁇ l (2 ng/ml) increased the Collagen Type III protein level in culture medium of LX-2 cells, whereas rhDecorin and rhDecorin-Fc abolished the effects of TGF- ⁇ 1.
  • Example 3 Construction of the gene encoding the hDecorin-Fc fusion protein
  • a fusion protein is assembled from several DNA segments.
  • total RNA was extracted from human fibroblast, and RT- PCR was carried out to clone decorin.
  • Table 3 shows the sequences of oligonucleotides used for the cloning of the hDecorin-Fc fusion proteins.
  • the resulting DNA fragments of approximately 1077 bp in length are directly inserted into a directional TOPO Cloning vector such as pCDNA 3.1 (Invetrogen) according to instructions of the manufacturer.
  • the sequence of the human decorin gene was confirmed by DNA sequencing.
  • the gene encoding the Fc region or fragment of human IgGl (Fc.sub.. gamma.1) is obtained by reverse transcription and PCR using RNA prepared from human leukocytes and appropriate 5' and 3' primers (Table 3). Resulting DNA fragments of Fc.sub.. gamma.1 containing part of the sequences of the hinge, CH2, and CH3 domains of IgGl was directly inserted into a pGEM-T Easy Vector (Promaga). The sequence of the Fc gene was confirmed by DNA sequencing.
  • the Fc fragment is excised from the Fc plasmid with Not I and is purified by agarose gel electrophoresis. Decorin Plasmid was made a single cut with Not I at the C-terminal of decorin. The purified Fc fragment is then inserted to the cut in the decorin plasmid to form the hDecorin-Fc.sub..gamma.l fusion gene (Fig 6).
  • the fusion gene comprises decorin, a flexible peptide linker and the modified Fc. sub., gamma.1 gene.
  • the Fc fragment was modified using following method: four amino acids were removed from the C-terminal of the Fc fragment during RT-PCR through a 3' primer. After the Fc fragment is inserted to the cut in the decorin plasmid, a sequence that comes from pGEM-T Easy and pCDNA3.1 vector and encodes ten amino acids was introduced to the C-terminal of the Fc fragment. A stop code was also introduced to the C-terminal of Fc fragment (see SEQ ID Nos. 3 and 4). Table 3
  • the sequence between the C-terminal of decorin and the N-terminal of the Fc fragment donated by pGEM-T Easy and pCDNA3.1 vector forms a flexible peptide linker.
  • the presence of a peptide linker between the decorin and Fc moieties increases the flexibility of the decorin domains.
  • the peptide linker has the following favorable characteristics: it consists of 19 amino acids in length and comprises two or more of the following amino acids: glycine, serine, alanine, and threonine (SEQ ID No. 6).
  • the recombinant pCDNA3.1 expression vector plasmid is transfected into a mammalian host cell line to achieve the expression of the hDecrin-Fc fusion protein.
  • a preferred host cell line is Cos-7.
  • a preferred method of transfection is a lipofectin method (Invitrogen). Two days following the transfection, the media are replaced with growth media containing 0.6 mg/ml of G418. Transfectants resistant to the selection drug are tested for the secretion of the fusion protein by anti-human decorin and Fc Western (Fig 7 A and B). The wells producing high levels of the hDecorin-Fc fusion protein were subcloned.
  • hDecorin-Fc Recombinant hDecorin-Fc produced in Cos-7 cells exhibit glycosaminoglycan patterns very similar to that found in the natural decorin.
  • the hDecorin-Fc fusion proteins expressed and produced in accordance with this invention show enhanced biological activities when compared to rHuEPO on a molar basis.
  • the Fc fusion protein is expected to keep the bioactivities of the parent protein and has a longer half-life than its parents.
  • a stability assay of rhDecorin-Fc was carried out by following methods: Culture medium from rhDecorin-Fc transfected Cos-7 cells was collected and incubated at 37 0 C for different times. rhDecorin was used as a control. Western Blotting was used to assay stabilities of rhDecorin-Fc and rhDecorin. Fig. 8 shows that the degradation rate of the Decorin-Fc fusion protein is slower than that of decorin, where 50% of rhDecorin was degraded after five days while only 25% of rhDecorin-Fc was degraded after five days.
  • ORGANISM Homo sapiens SEQUENCE: 1 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 60 ttcccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 120 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 180 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 240 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caa
  • ORGANISM artificial linker SEQUENCE: 3 atcactagtg aattcgcggc cgctcgagtc tag 33

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Abstract

The present invention relates to methods for inhibiting liver fibrogenesis in vitro and/or in vivo with directly administered recombinant rhDecorin and rhDecorin-Fc fusion protein. In one embodiment a linker is provided between Decorin and the Fc hinge fragment to form a fusion protein which increases the biologic activity of Decorin and increases the ability to bind to TGF-ß and extends the lifetime of decorin in serum, where the Fc hinge fragment is a modified form of Fc involving the replacement of four N-terminal amino acids with ten different amino acids which extends the lifetime of the Decorin-Fc protein by 50%.

Description

METHOD FOR INHIBITING FIBROGENESIS BY AN rhDECORIN-Fc FUSION PROTEIN
FIELD OF INVENTION
The present invention relates to methods for inhibiting fibrogenesis, especially liver fϊbrogenesis, by administering antagonists of hepatic stellate cell activation and fibroblast cell activation. This invention also relates to making a fusion protein construct and to producing the fusion protein.
BACKGROUND OF THE INVENTION
As to liver fibrosis, fibrosis has an integral role in the final common pathway of structural remodeling that reduces normal organ function following injury. It is one of the most fundamentally destructive and unwanted responses to developmental or inflammatory diseases and is seen in millions of individuals in the advanced stages of many different disease processes including such diseases as cystic fibrosis, interstitial nephritis, hepatic cirrhosis and pulmonary fibrosis following exposure to high oxygen tension. Liver fibrosis is characterized by an excessive deposition of extracellular matrix components in the liver. As described by S.L. Friedman in an article entitled "Molecular Regulation of Hepatic Fibrosis, an Integrated Cellular Response to Tissue Injury," J Biol Chem 2000; 275:2247-2250, several liver cell types participate in matrix deposition, the major types being hepatic stellate cells (HSC) and portal fibroblasts as described by B. Tuchweber et al. in an article entitled "Proliferation and Phenotypic Modulation of Portal Fibroblasts in the Early Stages of Cholestatic Fibrosis in the Rat," Lab Invest 1996; 74:265-278. During the past decade, a great deal of attention has been given to the stimuli responsible for fibrogenic cell activation in the liver. The major focus has been on growth factors and oxidant stress as described by the above Friedman article. Fibrogenesis is classically mediated by organ fibroblasts that express abundant amounts of collagen types I and III. The expression of fibrogenesis in liver has been the subject of intense study over the last several years. The cytokine regulation of this process is complex. It is generally believed that collagen types I and III are the principal fibrotic collagens, and that they are well expressed by hepatic stellate cells (HSC) and fibroblast cells. The expression of these collagens is regulated by a complexity of cytokines. Tumor necrosis factor beta 1 (TGF.beta.l), for example, is an early and pivotal component in the stimulatory process. It is generally believed that TGF.beta.l appears to be a regulatory molecule for these collagen genes in the lungs, liver, and kidney. (Wahl, S. M., 1992 J. Clin. Immunol. 12:61-74; Sharma, K. and Ziyadeh, F. N., 1993 Seminars in Nephrology 13: 1 16-128; Roberts et al., 1986 Proc. Nat'l Acad. Sci. USA 83:4167-4171.)
The most important causes of liver fibrosis cirrhosis are chronic hepatitis B and C infection, and prolonged alcohol abuse. Liver cirrhosis is the clinical end point of liver fibrosis. Until present time, effective methods for reversing liver cirrhosis have not been available, and those with life threatening impairment of liver function can only look to liver transplants for salvage. However, each year the number of new cases of cirrhosis exceeds the number of livers available for transplantation by a factor of 5 to 10. Therefore, prevention of fibrogenesis and early treatment of fibrosis is the best treatment of cirrhosis (Achord JL. 1991, Compr Ther. 17:57-64, Habib et al., 2001, Postgrad Med. 109:101-13). The strategies of inhibiting fibrogenesis can be grouped as: (a) Anti-inflammatory agents and antioxidants; (b) Antagonists of cytokines or cytokine receptors; (c) Inhibitors of stellate cell activation; and (d) Anti-collagen agents (D. Montgomery Bissell, 2001, EXPERIMENTAL and MOLECULAR MEDICINE, Vol. 33:179-190). However, most of these agents are either not very effective in treating fibrosis or have severe side effects. There is thus a need to develop new methods for inhibiting liver fibrogenesis and treating liver fibrosis.
By way of further background, decorin has been used for limiting TGF-β activity through the use of gene therapy as discussed in U.S. Patent 5,824,655. Moreover, decorin has been used for treating a pathology as discussed in U.S. Patent 6,436,900. This latter patent does not, however, discuss treatment of liver disease. Decorin is a small leucine-rich multifunctional proteoglycan and it consists of a core protein and a covalently linked glycosaminoglycan chain. The size and molecular weight of decorin core protein in human is 359 amino acids; 39746 Da, in pig is 360 amino acids. The original function of this molecule is involved in fibrils formation. Decorin binds to collagens type I, II and IV in vivo and promotes the formation of fibers with increased stability and changes in solubility. (Krusius and Ruoslahti, Proc. Natl. Acad. Sci. USA, 83:7683-7687 (1986); Day et al., Biochem. J., 248:801-805 (1987); Pearson et al., J. Biol. Chem., 258: 15101-15104 (1983); Vogel et al., Biochem. J. 223:587-597 (1984).)
Evidence shows that decorin has played a role in cell growth control. The expression of decorin in Chinese hamster ovary (CHO) cells has been demonstrated to lead to decreased growth rate, lowered saturation density, and altered morphology (Yamaguichi and Ruoslahti. Nature 336:244-246 (1988)). These growth-suppressive properties of decorin included (a) that the expression of decorin is markedly elevated during quiescence; (b) that actively proliferating or transformed cells rarely expressed decorin; (c) that decorin expression is abrogated by viral transformation; and (d) that the gene transcription of decorin is suppressed in a variety of tumorigenic cell lines and tumor tissues by methylation of its control regions (Iozzo., et al Crit. Rev. Biochem. MoI. Biol. 32: 141-174(1997)) Decorin suppresses tumor cell growth through binding to a discrete region of the EGFR, leading to the generation of protein mimetics capable of suppressing EGFR function and activating the EGF receptor/MAP kinase/ p21 axis. (Moscatello et al. J. Clin Invest. 15; 101(2):406-12. (1998); Santra et al., J Biol Chem 20;277:35671-81 (2002)) Decorin also inhibits tumor growth through suppressing tumor cell- mediated angiogenesis (Grant etal., Oncogene 21 :4765-77(2002)).
It was reported that decorin reduces fibrogenesis through interfering with bioactivity of a transforming growth factor-beta (TGF-P), the major stimulator of fibrogenesis. TGF-P regulates not only extracellular matrix (ECM) accumulation and deposition ECM as part of the normal response to tissue injury but also pathological fibrosis. Alterations in TGF-P homeostasis are important in fibrotic diseases of multiple tissues (Wells RG. Am J Physiol Gastrointest Liver Physiol. 279:G845-50. (2000)). As a TGF-P inhibitor, decorin specifically binds and neutralizes TGF-P ligands via its protein moiety to interfere with TGF-P bioactivity in vitro in a dose-dependant manner. In vivo, overexpression of TGF-P resulted in marked lung fibrosis, which was significantly reduced by concomitant overexpression of decorin (Yamaguchi et al., Nature 346: 281-284, (1990 ); KoIb et al., Am J Physiol Lung Cell MoI Physiol. 280:L1327-34(2001)).
Decorin is involved in the regulation of apoptosis. It was found that decorin decreased apoptosis of endothelial cells cultured in a collagen lattice (Schonherr et al., Eur J Cell Biol. 78:44-55 (1999)). Decorin acts as a signaling molecule in ECs and affects cell survival by Akt- dependent and -independent pathways (Schonherr et al., Ann N Y Acad Sci. 973:149- 52(2002)).
In an effort to extend bioactivity, Fc fusion proteins have been gaining favor. It is noted that recombinant proteins are an emerging class of therapeutic agents. One such modification is the use of the Fc region of immunoglobulins to make a fusion protein. Antibodies comprise two functionally independent parts, a variable domain known as "Fab", which binds antigen, and a constant domain, known as "Fc" which provides the link to effector functions such as complement or phagocytic cells. The Fc portion of an immunoglobulin has a long plasma half- life, whereas the Fab is short-lived. (Capon, et al., Nature 337: 525-531 (1989)) The Fc fusion protein should keep the bioactivities of parent protein and has longer half-life than its parents, because IgG can circulate for several days (WO 99/25044).
Note that U.S. Patent 6,277,375 refers to mutant Fc fragments to extend biologic half- lives, as does U.S. Patent 6,660,843. However, neither of these patents applies Fc fragments to decorin.
SUMMARY OF THE INVENTION
By investigating the effects of antagonism on fibrotic conditions related to TGF.beta.l induced hepatic stellate cell activation and liver fibrosis, it has been found that a specially formulated decorin fusion protein increased the attenuation of liver fibrosis by more efficiently inhibiting liver fibrogenesis. hi this protein, a modified Fc fragment was added to rhDecorin, with a peptide linker. The resultant protein was found both to be more effective in treating liver fibrosis and had a biological half-life extended by 50%.
More particularly, it was found that the modified decorin antagonist prevented the development of liver fibrosis, especially in course of a viral hepatitis, such as chronic hepatitis B and chronic hepatitis C.
In one embodiment, the rhDecorin-Fc fusion protein comprises rhDecorin, a peptide linker, and a modified human IgG Fc fragment or domain (denoted by IgGlFc). It was also found that it is preferable to use a flexible peptide linker of about 20 or fewer amino acids in length and that the flexible peptide linker contain of two or more of amino acids selected from the group consisting of glycine, serine, alanine, and threonine. The IgG Fc variant is of non- lytic nature, with the modified Fc fragment formulated by substituting ten new amino acids for the four amino acids normally used. Because of the use of the modified Fc fragment and the peptide linker, the biological half-life of the rhDecorin-Fc fusion protein was found to be 50% longer than that associated with decorin used alone.
In a further embodiment of the present invention, a method is disclosed for making or producing the subject recombinant fusion proteins from a mammalian cell line such as a Cos-7 cell line.
As can be seen, the rhDecorin-Fc fusion protein is characterized by and exhibits enhanced biological activity relative to that of rhDecorin and has an extended serum half-life without undesirable side effects, leading to improved pharmacokinetics and pharmacodynamics. Thus, lower dosages and fewer injections are needed to achieve similar efficacies.
In summary, the present invention relates to methods for inhibiting liver fibrogenesis in vitro and/or in vivo with directly administered recombinant rhDecorin and rhDecorin-Fc fusion protein. In one embodiment a linker is provided between Decorin and the Fc hinge fragment to form a fusion protein which increases the biologic activity of Decorin and increases the ability to bind to TGF-β and extends the lifetime of decorin in serum, where the Fc hinge fragment is a modified form of Fc involving the replacement of four N-terminal amino acids with ten different amino acids which extends the lifetime of the Decorin-Fc protein by 50%.
BRIEF DESCRIPTION OF THE DRAWINGS
These and other features of the subject invention will be better understood in connection with a Detailed Description, in conjunction with the Drawings, of which:
Figure IA and IB show the effect of decorin treatment on liver fibrosis after CCl.sub.4 (carbon tetrachloride) treatment for 8 weeks (haematoxylin & eosin (HE) staining), with Figure IA showing the effect on a rat treated with CCl. sub.4 alone and Figure IB showing the effect on a rat treated with CCl.sub.4 and rhDecorin. (XlOO); Figures 2A and 2B show the effect of rhDecorin and rhDecorin-Fc on hepatic stellate cell proliferation induced by TGF.beta.l in which LX-2 cells (Human HSC cell line) were exposed to rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 48 hours and in which cell number was evaluated through the measurement of the reduction of the dye 3-(4,5-dimethylthiazol-2yl)-2,5 diphenyltetrazolium (MTT), with Figure 2A showing the effect of rhDecorin on LX-2 cell proliferation induced by TGF.beta.l ((C; control; D: rhDecorin (4 ug/ml); T: TGF.beta.l (2 ng/ml); T+D: TGF.beta.l (2 ng/ml) plus rhDecorin (4μg/ml).). (* means control v TGF.beta.l p <0.05; # means TGF.beta.l v TGF.beta.l plus rhDecorin p< 0.05. N= 4.), and with Figure 2B showing the effect of rhDecorin-Fc on LX-2 cell proliferation induced by TGF.beta.l . ((C; control; T: TGF.beta.l (2 ng/ml); D-Fc: rhDecorin-Fc (1 μg/ml);
T+D-Fc : TGF.beta.l (2 ng/ml) plus rhDecorin-Fc (1 μg/ml).). (**means control v TGF.beta.l p<0.01 ; # means TGF.beta.l v TGF.beta.l plus rhDecorin p<0.05. N= 4.);
Figures 3 A and 3B show the effect of rhDecorin and rhDecorin-Fc treatment on MMP-2 (matrix metalloproteinases-2) mRNA levels of HSC cells stimulated with TGF.beta.l, in Figure 3A and 3B with LX-2 cells treated with rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 24 hours, with cells harvested for total RNA extraction, with RT-PCR carried out to detect the effect of rhDecorin and rhDecorin-Fc on MMP-2 mRNA levels of LX-2 cells stimulated with TGF.beta.l, and with GADPH used as a control, Figure 3 A showing the effect of rhDecorin treatment on MMP-2 and Figure 3B showing the effect of rhDecorin-Fc treatment on MMP-2 ((C; control; D: rhDecorin; T: TGF.beta.l (2 ng/ml); D+T: TGF.beta.l (2 ng/ml) plus rhDecorin (4 μg/ml); D-Fc : rhDecorin-Fc (1 μg/ml); T+D-F c:
TGF.beta.l (2 ng/ml) plus rhDecorin-Fc (lμg/ml).).
Figures 4 A and 4B show the effect of rhDecorin and rhDecorin-Fc treatment on TEvIP-I (tissue inhibitor of matrix metalloproteinases-1) mRNA levels of HSC cells stimulated with TGF.beta. l, in which in Figures 4A and 4B respectively LX-2 cells were treated with rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 24 hours, in which rhDecorin and rhDecorin-Fc treated cells were harvested for total RNA abstraction, in which RT-PCR was carried out to detect the effect of rhDecorin and rhDecorin-Fc on TIMP-I mRNA levels of LX-2 cells stimulated with TGF-β, with GADPH as a control, with Figure 4A showing the effect of rhDecorin treatment on TIMP-I and with Figure 4B showing the effect of rhDecorin-Fc treatment on TIMP-I . ((C; control; D: rhDecorin; T: TGF.beta.l (2 ng/ml); D+T: TGF.beta.l (2 ng/ml) plus rhDecorin (4 μg/ml); D-Fc: rhDecorin-Fc (1 μg/ml); T+ D-Fc:
TGF.beta. l (2 ng/ml) plus rhDecorin-Fc (1 μg/ml).);
Figures 5A and 5B show the effect of rhDecorin and rhDecorin-Fc treatment on collagen III protein level of HSC cells stimulated with TGF.beta.l, in which LX-2 cells were treated with rhDecorin or rhDecorin-Fc in the presence of 2 ng/ml TGF.beta.l for 24 hours and in which culture mediums were harvested, showing that in Figure 5A, Western Blotting was carried out to evaluate the effect of rhDecorin and rhDecorin-Fc on collagen III protein levels of LX-2 cells stimulated with TGF.beta.l, and showing in Figure 5B the result of quantitation. T: TGF.beta.l (2 ng/ml); T+D: TGF.beta.l (2 ng/ml) plus rhDecorin (4μg/ml); T+D-Fc: TGF.beta.l (2 ng/ml)
plus rhDecorin-Fc (1 μg/ml). T+ Fc: TGF.beta.l (2 ng/ml) plus Fc (1 μg/ml) ).
Figure 6 shows the construct of the rhDecorin-Fc fusion protein involving 1 : Decorin; 2: Linker; 3: Immunoglobin Fc fragment; 4: disulfide bonds.
Figures 7A and 7B show protein expression of rhDecorin-Fc nucleic acid molecule in which Cos-7 was transfected with rhDecorin-Fc recombinant plasmid, selected with G418 (0.6mg/ml), in which mediums were collected for Western Blotting and in which decorin recombinant plasmid was used as a control, with Figure 7A showing Anti-IgG Fc and Figure 7B showing anti-human decorin antibody conjugated with HRP used to detect rhDecorin-Fc fusion proteins, in which for Figure 7A M: marker; Lane 1-3: Culture media of rhDecorin, rhDecorin- Fc and vector transfected cells. Lane 4: decorin control from Sigma, and in which for Figure 7B M: marker; Lane 1-3: cell lysates of rhDecorin, rhDecorin-Fc and vector transfected cells, Lane 4-6: culture media of rhDecorin, rhDecorin-Fc and vector transfected cells, Lane 7: decorin control from Sigma; and,
Figures 8A and 8B show the 50% extension in lifetime of rhDecorin-Fc in which Cos-7 was transfected with rhDecorin-Fc recombinant plasmid and in which the culture medium was collected and incubated at 370C for deferent times, from 0 day to 5 days, with rhDecorin used as the control, and in which Western Blotting was used to assay the stability of rhDecorin, in which for Figure 8A Lane 1, 3, 5, 7, 9 and 1 1 : decorin was incubated at 370C for 0, 1, 2, 3, 4 and 5 days. Lane 2, 4, 6, 8, 10 and 12: Decorin-Fc was incubated at 370C for 0, 1, 2, 3, 4 and 5 days and in which Figure 8B shows the quantitation of Western Blotting.
DETAILED DESCRIPTION
1. The effects of rhDecorin and rhDecorin-Fc on fibrosis , especially liver fibrosis
The effect of rhDecorin on carbon tetrachloride (CCl4) induced liver fibrosis in rats was investigated first. As shown in Figure IA, compared with controls, eight weeks of CCl4 treatment induced significant fibrosis with the formation of nodules that lack a central vein with the disappearance of the normal structure of hepatic lobules. As shown in Figure 2B, two weeks following the initial CCl4 treatment, rhDecorin was injected together with CCl4 for the remaining six weeks. As can be seen, addition of rhDecorin significantly decreased the pathologic changes of fibrogenesis.
The effect of rhDecorin and rhDecorin-Fc on hepatic stellate cell (HSC) activation induced by TGF-β was also investigated. HSC activation is an essential process of liver fibrogenesis. Fibrogenesis presents as proliferation of HSC and the remodeling of extra cellular matrix, including, degradation of collagen IV in the basement membrane of liver cells and deposition of an excess of extracellular matrix components, such as collagen I and collagen III in the liver. In this process, expression of matrix metalloproteinase 2 (MMP-2) and a tissue inhibitor of metalloproteinase 1 (TIMP-I) in mRNA level are up-regulated. Collagen IV is the substrate of MMP-2. TMP-I is an inhibitor of MMP-I which can clear up the deposition of collagen I and III. It was found that rhDecorin and rhDecorin-Fc abrogated the effects of TGF-β on hepatic stellate cells through suppressing cell proliferation, collagen III production, and MMP-2 and TIMP-I expression stimulated by TGF-β.
In light of these results and taking into account the fact that rhDecorin and rhDecorin-Fc have a very good safety profile, rhDecorin-Fc may be used as drugs against liver fibrogenesis.
Liver fibrogenesis is the active process leading to the deposition of an excess of extracellular matrix components in the liver. It is observed in a number of conditions such as chronic viral hepatitis B and C, alcoholic liver disease, drug-induced liver disease, hemochromatosis, auto-immune hepatitis, Wilson disease, primary biliary cirrhosis, sclerosing cholangitis, liver schistosomiasis and others. Fibrogenesis can occur similarly in other organs, such as lung, kidney, pancreas, heart and skin.
The subject invention is particularly helpful in the treatment of liver fibrosis. "Liver fibrosis" is the established excess deposit of extracellular matrix components in the liver. Its endpoint is liver cirrhosis.
In a preferred embodiment of the invention, rhDecorin-Fc are useful to prevent the development of liver fibrosis that may occur in a patient infected by Hepatitis virus, e.g. hepatitis B virus (HBV), or hepatitis C (HCV) virus.
Chronic viral hepatitis is more particularly aimed at, especially chronic hepatitis B and chronic hepatitis C. By the term "patients in need of such treatment" is meant any human subject or mammals, including sheep, cattle, dogs, cats, rodents, rabbits or goats, who suffer from an organ disease wherein fibrogenesis is observed or generally results from the development of the disease.
The terms "treatment" and "prevention" include therapy and prophylaxis toward fibrogenesis, at any stage of development of the phenomenon or before it occurs.
The invention especially aims at preventing, or reducing or alleviating liver fibrosis in patients suffering from an organ disease.
The use of therapeutically effective amounts of rhDecorin-Fc in accordance with the invention effectively reduces or prevents the development of liver fibrosis.
2. Molecular structure of the rhDecorin-Fc fusion protein
The invention provides fusion protein which is useful in prevention of fibrosis. The fusion protein of the invention and/or nucleic acid encoding such fusion protein may be administered directly to mammals in need of treatment with an anti-fibrosis protein.
The invention thus provides a fusion protein comprising a target protein, referred to herein as decorin, an artificial linker and an immunoglobulin Fc region or fragment.
Because dimeric constructs are preferred, the subject fusion protein is characterized as dimer cross-linked by a pair of disulfide bonds between cysteines in adjacent subunits. In Figure 6, the disulfide bond is depicted as linking together the two immunoglobulin heavy chain Fc regions, fragments or domains via a partial of an immunoglobulin hinge region or fragment within each heavy chain, and thus is characteristic of native form of this molecule.
It should be understood that the immunoglobulin Fc region or fragment includes at least a portion of the hinge region, a CH2 domain and a CH3 domain (see SEQ. ID. NO. 1 and T). In this invention, the sequence of nucleic acid in the N-terminus of Fc region was slightly modified, from cctgtctccg ggtaaa to atcactagtg aattcgcggc cgctcgagtc tag (see SEQ. ID. NO.3). It is noted that in the modification of the Fc region or fragment 4 amino acids, SPGK, were replaced by 10 amino acids, ITSEF AAARV (see SEQ. ID. NO. 4). The Fc region is attached via a flexible linker to the C-terminus of Decorin.
As used herein, the term "polypeptide linker" is understood to mean a peptide sequence that can link together two proteins that in nature are not naturally linked together. The polypeptide linker preferably comprises a plurality of amino acids such as alanine, glycine and serine or combinations of such amino acids. Preferably, the polypeptide linker comprises a series of glycine and serine peptides about 19 residues in length (see SEQ. ID. NO. 5 and 6). It is contemplated, however, that the optimal linker length and amino acid composition may be determined by routine experimentation.
As used herein, the recombinant molecule has the configuration X-Fc, where X is a target molecule. The immunoglobulin Fc regions can associate, for example, via interchain disulfide bonds, to produce the type of construct shown in Figure 6.
As used herein, the term "decorin" is understood to mean full length decorin (see SEQ ID NO: 7 and 8).
EXAMPLES
Example 1
Inhibition of Rat Liver Fibrogenesis through rhDecorin
Material and Methods Animals and Experimental Design
All experiments were carried out using accepted ethical guidelines. Male Wistar rats (Beijing Medical University) weighing 200-250 g were used in this study. The animals had free access to food and drinking water. 40% CC14 (Sigma) was prepared by mixing CC14 with olive oil. Liver damage and fibrosis was induced through giving 40% CC14 at 3ml/kg body weight by subcutaneous injection, two times a week for eight weeks. Control animals for CC14 received only olive oil. rhDecorin was intraperitoneally administered daily at the dose of 20ug/kg/d body weight. rhDecorin was extracted from the culture medium of Cos-7 cells transfected with decorin.
Thus, 3 groups including each 7 animals could be distinguished. Group I: olive oil alone; group II: 40% CC14; group III: 40% CC14 plus rhDecorin. rhDecorin treatment were started at the third week and lasted for six weeks.
At the designated time points, the animals were sacrificed. Liver samples were taken from several lobes and frozen in liquid nitrogen.
Serum samples were also collected.
Liver function tests
Routine liver function blood tests, including hyaluronan, type IV collagenase, γGT and transaminases were performed on an automated analyzer. Fibrosis Assessment
Fibrosis assessment was done on formalin-fixed, paraffin-embedded sections stained with haematoxylin & eosin (HE). Pictures were taken, using an image analysis system. All samples from a series of experiments were stained simultaneously. Liver fibrosis deposition was judged by senior pathologists and graded with the METAVIR scale, which grades fibrosis from FO (no fibrosis) to F4 (cirrhosis). METAVIR scale is a widely used scale that has excellent inter-observer reliability. Table 1 shows the result.
Statistical Analysis
Statistical analysis in Table 1 was performed by WILCOXON in other tables and figures by ANOVA using the SPSSl 1.0 software.
Results
Treatment of rhDecorin on the CC14-treated rats resulted in a reduction in fibrosis. The results were the most significant in the group of rats following 8 weeks of CC14 treatment and 6 weeks of rhDecorin treatment, as compared with CC14 treatment alone (p<0.0 1 by WILCOXON). Biochemical tests of liver function are presented in Table 2.
Table 1. Pathologic changes of rat liver after 8 weeks of CC14 treatment and 6 weeks of rhDecorin treatment
Group No. of rat Pathologic morphology of rat liver slices
Cell damage Fibrosis
+ ++ +++ FO Fl F2 and 3 F4 control 7 7 7 CCL4 7 1 2 4 1 6 decorin 7 2 3 2 2 5
CCL4 v control PO.01 PO.01 CCL4 v decorin P> 0.05 P <0.01
Table 2. Liver function tests after 8 weeks of CCL4 treatment and 6 weeks of rhDecorin treatment
Group N HA cTV ALT AST Y-GT ng/ml ng/ml u/L u/L u/L control 7 35 92tl2 23 46 49± 3.84 32 03± 9 05 100 71± 19 99 15 14± 1 1 71
CCL4 7 130 39+ 52 27 135 98± 28 45 132 97± 32 05 166 57± 28 7 74 43± 35 50 decorin 7 64 41± 13 67 72 60± 30 00 43 63± 18 59 129 86± 17 14 33 00± 1426
CCL4 vcontrol F<0 01 PO Ol POOl PO Ol PO Ol
CCL4 v decorin PO Ol PO 01 POOl PO O5
P<005
The results in Table 2 are expressed as means ± 1 SD.
Difference of the serum biochemical tests of liver function between rats of CC14 treatment alone and CC14 plus rhDecorin is statistically significant.
N= number of animals used.
During liver fibrogenesis, since there is extensive deposition of fibrous tissue, serum levels of the constituents of extracellular matrix such as hyaluronic acid (HA), collagen VI (cIV) and many of their breakdown products will increase as a result of remodeling and recurrent scarring. Serum alanine and aspartate aminotransferase (ALT and AST) levels do not correlate well with fibrosis. However, patients with documented, persistently normal ALT levels usually have mild degrees of hepatitis and either no or mild stages of fibrosis.
The increase of r-GT is found in the condition of liver damage, especially chronic liver damage. Note that r-GT correlates well with fibrosis.
Example 2
Inhibition of Fibrogenesis by rhDecorin and rhDecorin-Fc through suppressing activation of HSC cells
To elucidate the action of rhDecorin and rhDecorin-Fc in fibrogenesis, the effect of rhDecorin and rhDecorin-Fc on activation of HSC cells stimulated by TGF-β 1 was examined. HSC activation is an essential process of liver fibrogenesis. It presents as proliferation of HSC and remodeling of extra cellular matrix, including, degradation of collagen IV in the basement membrane and deposition of an excess of extracellular matrix components, such as collagen I and collagen III, and change of expression of MMPs and TMPs in mRNA level. TGF-β 1 is one of the most important activation factors in the process.
Material and Methods Cells
The inventors used an established Human HSC cell line, named LX-2. These cells have been extensively characterized and exhibit many similarities with primary cultures of HSC. rhDecorin
The full length decorin gene was cloned from human fibroblast cell line, then inserted the gene into a pCDNA3.1 vector (Invitrogen) and the gene was recombined with a poly(histadine) tail in the C-terminal for purification purpose. Cos-7 cells were transfected with the recombinant plasmid. Because most of decorin was secreted into culture medium, the medium was collected and purification of decorin was performed using ProBond Purification system (Invitrogen), in which nickel column was used for His-tag decorin purification.
rhDecorin-Fc
The methods for preparation of rhDecorin-Fc are described in Example 3.
Cell Proliferation Assay
LX-2 cells (2000 /well) were seeded in 96-well microplate for 24 hr. The culture medium was replaced with DMEM supplemented with 0.5 % FBS and the cells were allowed to be starved for 48 hr. After starvation, the medium was replaced with 2% serum supplemented DMEM medium. The reagents were then added to treat cells and LX-2 cells were exposed to rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 48 hours. Cell number was evaluated through the measurement of the reduction of the dye 3-(4,5- dimethylthiazol-2yl)-2,5 diphenyltetrazolium (MTT).
Expression of Matrix Metalloproteinases-2 (MMP-2) and Tissue Inhibitor of Matrix Metalloproteinases-1 (TIMP-I) mRNA levels
MMP-2 is the enzyme which can degrade Collagen IV and TMP-I increases fibrosis deposition by inhibiting its degradation by matrix metalloproteinases. The effect of rhDecorin and rhDecorin-Fc on MMP-2 and TMP-I mRNA expression by cultured LX-2 cells was measured. 2x105 LX-2 cells were seeded in 6-well cell culture plate for 24hr. Then the culture medium was replaced with DMEM supplemented with 0.5 % FBS. The cells were allowed to be starved for 24 hr. After starvation, the medium was replaced with 2% serum supplemented DMEM medium. Then reagents were added to treat cells and let LX-2 cells exposed to rhDecorin or rhDecorin-Fc, either alone, or in the presence of 2 ng/ml TGF.beta.l for 24 hours. Finally, cells were harvested and lysised using TRIZOL reagent (GIBCOL, MD, USA). The total RNA was then extracted according to manufacturer's protocol. RT-PCR was carried out. The gene expression levels of MMP-2 and TIPM-I were normalized by a housekeeping gene, glyceraldehydes-3 -phosphate dehydrogenase (GADPH).
Expression of Collagen Type III Protein Levels
Deposition of an excess of extracellular matrix components, such as Collelagen III is a pathologic process during fibrogenesis. The effect of rhDecorin or rhDecorin-Fc on collagen III expression by cultured LX-2 cells was measured. 2x105 LX-2 cells were seeded in 6-well cell culture plate for 24 hr. Then the culture medium was replaced with DMEM supplemented with 0.5 % FBS and cells were allowed to be starved for 24 hr. After starvation, the medium was replaced with 2% serum supplemented DMEM medium. Then reagents were added to treat cells and LX-2 cells were exposed to rhDecorin or rhDecorin-Fc in the presence of 2 ng/ml TGF.beta.l for 48 hours. The culture medium was harvested and Western Blotting was carried out to evaluate the effect of rhDecorin and rhDecorin-Fc on collagen III levels of LX-2 cells treated with TGF-β 1.
Result Proliferation
As shown on Figure 2, TGF-β 1 increased LX-2 cell proliferation. The mitogenic effect of TGF-β 1 (2 ng/ml) on LX-2 was abolished by simultaneous addition of rhDecorin (4ug/ml) or rhDecorin-Fc (lug/ml). Because lug/ml of rhDecorin-Fc can get the same effect of 4ug/ml rhDecorin, rhDecorin-Fc exhibits an enhanced in vitro biological activity of at least 4 fold relative to that of decorin. In the absence of TGF-βl, rhDecorin or rhDecorin-Fc alone do not have a significant effect on cell proliferation.
MMP-2 and TIMP-I
As shown on Figures 3 and 4, TGF-βl (2 ng/ml) increased expression of MMP-2 and TIMP-I in LX-2, whereas rhDecorin and rhDecorin-Fc suppressed the increase.
Collagen Type III
Figure 5 shows that TGF-βl (2 ng/ml) increased the Collagen Type III protein level in culture medium of LX-2 cells, whereas rhDecorin and rhDecorin-Fc abolished the effects of TGF-β 1. Example 3 1. Construction of the gene encoding the hDecorin-Fc fusion protein
A fusion protein is assembled from several DNA segments. To obtain the gene encoding the leader peptide and mature protein of human decorin, total RNA was extracted from human fibroblast, and RT- PCR was carried out to clone decorin. Table 3 shows the sequences of oligonucleotides used for the cloning of the hDecorin-Fc fusion proteins. The resulting DNA fragments of approximately 1077 bp in length are directly inserted into a directional TOPO Cloning vector such as pCDNA 3.1 (Invetrogen) according to instructions of the manufacturer. The sequence of the human decorin gene was confirmed by DNA sequencing.
The gene encoding the Fc region or fragment of human IgGl (Fc.sub.. gamma.1) is obtained by reverse transcription and PCR using RNA prepared from human leukocytes and appropriate 5' and 3' primers (Table 3). Resulting DNA fragments of Fc.sub.. gamma.1 containing part of the sequences of the hinge, CH2, and CH3 domains of IgGl was directly inserted into a pGEM-T Easy Vector (Promaga). The sequence of the Fc gene was confirmed by DNA sequencing.
To prepare the hDecorin-Fc. sub.. gamma.1 fusion gene, the Fc fragment is excised from the Fc plasmid with Not I and is purified by agarose gel electrophoresis. Decorin Plasmid was made a single cut with Not I at the C-terminal of decorin. The purified Fc fragment is then inserted to the cut in the decorin plasmid to form the hDecorin-Fc.sub..gamma.l fusion gene (Fig 6). The fusion gene comprises decorin, a flexible peptide linker and the modified Fc. sub., gamma.1 gene.
The Fc fragment was modified using following method: four amino acids were removed from the C-terminal of the Fc fragment during RT-PCR through a 3' primer. After the Fc fragment is inserted to the cut in the decorin plasmid, a sequence that comes from pGEM-T Easy and pCDNA3.1 vector and encodes ten amino acids was introduced to the C-terminal of the Fc fragment. A stop code was also introduced to the C-terminal of Fc fragment (see SEQ ID Nos. 3 and 4). Table 3
Name of Primer Sequence
Decorin Forward Primer 5' CAC CAT GAA GGC CAC TAT CAT CCT C 3'
Decorin Reverse Primer 5 ' CTT ATA GTT TCC GAG TTG AAT GGC 3 '
IGG 1 FC Forward Primer 5 ' ACT CAC ACA TGC CCA CCG T 3 ' IGG 1 FC Reverse Primer 5 ' GAG AGG CTC TTC TGC GTG 3 '
The sequence between the C-terminal of decorin and the N-terminal of the Fc fragment donated by pGEM-T Easy and pCDNA3.1 vector forms a flexible peptide linker. The presence of a peptide linker between the decorin and Fc moieties increases the flexibility of the decorin domains. For the present invention, the peptide linker has the following favorable characteristics: it consists of 19 amino acids in length and comprises two or more of the following amino acids: glycine, serine, alanine, and threonine (SEQ ID No. 6).
2. Expression of the fusion protein in transfected cell lines
The recombinant pCDNA3.1 expression vector plasmid is transfected into a mammalian host cell line to achieve the expression of the hDecrin-Fc fusion protein. For stable high levels of expression, a preferred host cell line is Cos-7. A preferred method of transfection is a lipofectin method (Invitrogen). Two days following the transfection, the media are replaced with growth media containing 0.6 mg/ml of G418. Transfectants resistant to the selection drug are tested for the secretion of the fusion protein by anti-human decorin and Fc Western (Fig 7 A and B). The wells producing high levels of the hDecorin-Fc fusion protein were subcloned. Recombinant hDecorin-Fc produced in Cos-7 cells exhibit glycosaminoglycan patterns very similar to that found in the natural decorin. The hDecorin-Fc fusion proteins expressed and produced in accordance with this invention show enhanced biological activities when compared to rHuEPO on a molar basis.
3. In vitro biological assays
In vitro biological assays were carried out in LX-2 cells to test the effects of rhDecorin- Fc on inhibition of fibrogenesis through suppressing activation of HSC cells (see Example 2).
4. In vitro stability studies
Because the Fc portion of an immunoglobulin has a long half-life, the Fc fusion protein is expected to keep the bioactivities of the parent protein and has a longer half-life than its parents. A stability assay of rhDecorin-Fc was carried out by following methods: Culture medium from rhDecorin-Fc transfected Cos-7 cells was collected and incubated at 370C for different times. rhDecorin was used as a control. Western Blotting was used to assay stabilities of rhDecorin-Fc and rhDecorin. Fig. 8 shows that the degradation rate of the Decorin-Fc fusion protein is slower than that of decorin, where 50% of rhDecorin was degraded after five days while only 25% of rhDecorin-Fc was degraded after five days.
SEQ ID No. 1 LENGTH: 660 TYPE: DNA
ORGANISM: Homo sapiens SEQUENCE: 1 actcacacat gcccaccgtg cccagcacct gaactcctgg ggggaccgtc agtcttcctc 60 ttccccccaa aacccaagga caccctcatg atctcccgga cccctgaggt cacatgcgtg 120 gtggtggacg tgagccacga agaccctgag gtcaagttca actggtacgt ggacggcgtg 180 gaggtgcata atgccaagac aaagccgcgg gaggagcagt acaacagcac gtaccgtgtg 240 gtcagcgtcc tcaccgtcct gcaccaggac tggctgaatg gcaaggagta caagtgcaag 300 gtctccaaca aagccctccc agcccccatc gagaaaacca tctccaaagc caaagggcag 360 ccccgagaac cacaggtgta caccctgccc ccatcccggg atgagctgac caagaaccag 420 gtcagcctga cctgcctggt caaaggcttc tatcccagcg acatcgccgt ggagtgggag 480 agcaatgggc agccggagaa caactacaag accacgcctc ccgtgctgga ctccgacggc 540 tccttcttcc tctacagcaa gctcaccgtg gacaagagca ggtggcagca ggggaacgtc 600 ttctcatgct ccgtgatgca tgaggctctg cacaaccact acacgcagaa gagcctctca 660
SEQ ID No. 2 LENGTH: 220 TYPE: PRO
ORGANISM: Homo sapiens SEQUENCE: 2
Thr His Thr Cys Pro Pro Cys Pro Ala Pro GIu Leu Leu GIy GIy Pro
1 5 10 15
Ser VaI Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met He Ser
20 25 30
Arg Thr Pro GIu VaI Thr Cys VaI VaI VaI Asp VaI Ser His GIu Asp
35 40 45
Pro GIu VaI Lys Phe Asn Trp Tyr VaI Asp GIy VaI GIu VaI His Asn
50 55 60
Ala Lys Thr Lys Pro Arg GIu GIu GIn Tyr Asn Ser Thr Tyr Arg VaI 65 70 75 80
VaI Ser VaI Leu Thr VaI Leu His GIn Asp Trp Leu Asn GIy Lys GIu
85 90 95
Tyr Lys Cys Lys VaI Ser Asn Lys Ala Leu Pro Ala Pro He GIu Lys
100 105 110
Thr He Ser Lys Ala Lys GIy GIn Pro Arg GIu Pro GIn VaI Tyr Thr 115 120 125
Leu Pro Pro Ser Arg Asp GIu Leu Thr Lys Asn GIn VaI Ser Leu Thr
130 135 140
Cys Leu VaI Lys GIy Phe Tyr Pro Ser Asp He Ala VaI GIu Trp GIu 145 150 155 160
Ser Asn GIy GIn Pro GIu Asn Asn Tyr Lys Thr Thr Pro Pro VaI Leu
165 170 175
Asp Ser Asp GIy Ser Phe Phe Leu Tyr Ser Lys Leu Thr VaI Asp Lys
180 185 190
Ser Arg Trp GIn GIn GIy Asn VaI Phe Ser Cys Ser VaI Met His GIu
195 200 205
Ala Leu His Asn His Tyr Thr GIn Lys Ser Leu Ser 210 215 220
SEQ ID No. 3
LENGTH: 33 TYPE: DNA
ORGANISM: artificial linker SEQUENCE: 3 atcactagtg aattcgcggc cgctcgagtc tag 33
SEQ ID No. 4 LENGTH: 10 TYPE: PRO ORGANISM: artificial SEQUENCE: 4
He Thr Ser GIu Phe Ala Ala Ala Arg VaI 1 5 10
SEQ ID No. 5 LENGTH: 57 TYPE: DNA ORGANISM: artificial SEQUENCE: 5 aagggtcaag acaattctgc agatatccag cacagtggcg gccgcgggaa ttcgatt 57
SEQ ID No. 6 LENGTH: 19 TYPE: PRO ORGANISM: artificial SEQUENCE: 6
Lys GIy GIn Asp Asn Ser Ala Asp lie GIn His Ser GIy GIy Arg GIy 1 5 10 15
Asn Ser He
SEQ ID No. 7 LENGTH: 1077 TYPE: DNA ORGANISM: Homo sapiens
SEQUENCE: 7 atgaaggcca ctatcatcct ccttctgctt gcacaagttt cctgggctgg accgtttcaa 60 cagagaggct tatttgactt tatgctagaa gatgaggctt ctgggatagg cccagaagtt 120 cctgatgacc gcgacttcga gccctcccta ggcccagtgt gccccttccg ctgtcaatgc 180 catcttcgag tggtccagtg ttctgatttg ggtctggaca aagtgccaaa ggatcttccc 240 cctgacacaa ctctgctaga cctgcaaaac aacaaaataa ccgaaatcaa agatggagac 300 tttaagaacc tgaagaacct tcacgcattg attcttgtca acaataaaat tagcaaagtt 360 agtcctggag catttacacc tttggtgaag ttggaacgac tttatctgtc caagaatcag 420 ctgaaggaat tgccagaaaa aatgcccaaa actcttcagg agctgcgtgc ccatgagaat 480 gagatcacca aagtgcgaaa agttactttc aatggactga accagatgat tgtcatagaa 540 ctgggcacca atccgctgaa gagctcagga attgaaaatg gggctttcca gggaatgaag 600 aagctctcct acatccgcat tgctgatacc aatatcacca gcattcctca aggtcttcct 660 ccttccctta cggaattaca tcttgatggc aacaaaatca gcagagttga tgcagctagc 720 ctgaaaggac tgaataattt ggctaagttg ggattgagtt tcaacagcat ctctgctgtt 780 gacaatggct ctctggccaa cacgcctcat ctgagggagc ttcacttgga caacaacaag 840 cttaccagag tacctggtgg gctggcagag cataagtaca tccaggttgt ctaccttcat 900 aacaacaata tctctgtagt tggatcaagt gacttctgcc cacctggaca caacaccaaa 960 aaggcttctt attcgggtgt gagtcttttc agcaacccgg tccagtactg ggagatacag 1020 ccatccacct tcagatgtgt ctacgtgcgc tctgccattc aactcggaaa ctataag 1077
SEQ ID No. 8 LENGTH: 359 TYPE: PRO ORGANISM: Homo sapiens
SEQUENCE: 8
Met Lys Ala Thr He He Leu Leu Leu Leu Ala GIn VaI Ser Trp Ala
1 5 10 15
GIy Pro Phe GIn GIn Arg GIy Leu Phe Asp Phe Met Leu GIu Asp GIu
20 25 30
Ala Ser GIy He GIy Pro GIu VaI Pro Asp Asp Arg Asp Phe GIu Pro
35 40 45
Ser Leu GIy Pro VaI Cys Pro Phe Arg Cys GIn Cys His Leu Arg VaI
50 55 60
VaI GIn Cys Ser Asp Leu GIy Leu Asp Lys VaI Pro Lys Asp Leu Pro 65 70 75 80
Pro Asp Thr Thr Leu Leu Asp Leu GIn Asn Asn Lys He Thr GIu He
85 90 95
Lys Asp GIy Asp Phe Lys Asn Leu Lys Asn Leu His Ala Leu He Leu
100 105 110
VaI Asn Asn Lys He Ser Lys VaI Ser Pro GIy Ala Phe Thr Pro Leu
1 15 120 125
VaI Lys Leu GIu Arg Leu Tyr Leu Ser Lys Asn GIn Leu Lys GIu Leu
130 135 140
Pro GIu Lys Met Pro Lys Thr Leu GIn GIu Leu Arg Ala His GIu Asn 145 150 155 160
GIu He Thr Lys VaI Arg Lys VaI Thr Phe Asn GIy Leu Asn GIn Met
165 170 175
He VaI He GIu Leu GIy Thr Asn Pro Leu Lys Ser Ser GIy He GIu
180 185 190
Asn GIy Ala Phe GIn GIy Met Lys Lys Leu Ser Tyr He Arg He Ala
195 200 205
Asp Thr Asn He Thr Ser He Pro GIn GIy Leu Pro Pro Ser Leu Thr
210 215 220
GIu Leu His Leu Asp GIy Asn Lys He Ser Arg VaI Asp Ala Ala Ser 225 230 235 240
Leu Lys GIy Leu Asn Asn Leu Ala Lys Leu GIy Leu Ser Phe Asn Ser
245 250 255
He Ser Ala VaI Asp Asn GIy Ser Leu Ala Asn Thr Pro His Leu Arg
260 265 270
GIu Leu His Leu Asp Asn Asn Lys Leu Thr Arg VaI Pro GIy GIy Leu
275 280 285
Ala GIu His Lys Tyr He GIn VaI VaI Tyr Leu His Asn Asn Asn He
290 295 300
Ser VaI VaI GIy Ser Ser Asp Phe Cys Pro Pro GIy His Asn Thr Lys 305 310 315 320
Lys Ala Ser Tyr Ser GIy VaI Ser Leu Phe Ser Asn Pro VaI GIn Tyr 325 330 335 Trp GIu He GIn Pro Ser Thr Phe Arg Cys VaI Tyr VaI Arg Ser Ala
340 345 350
He GIn Leu GIy Asn Tyr Lys
355
While the present invention has been described in connection with the preferred embodiments of the various figures, it is to be understood that other similar embodiments may be used or modifications or additions may be made to the described embodiment for performing the same function of the present invention without deviating therefrom. Therefore, the present invention should not be limited to any single embodiment, but rather construed in breadth and scope in accordance with the recitation of the appended claims.

Claims

WHAT IS CLAIMED IS:
1. A method for inhibiting hepatic fibrogenesis, which method comprises administering an effective amount of an rhDecorin-Fc antagonist of hepatic stellate cell activation to a patient in need of such treatment.
2. The method of Claim 1, wherein the antagonist prevents the development of liver fibrosis in the course of viral hepatitis and/or induced liver damage.
3. The method of Claim 1, wherein the antagonist prevents the development of liver fibrosis in the course of chronic hepatitis and/or induced liver damage.
4. The method of Claim 1, wherein the antagonist is an antagonist of fibrotic conditions related to hepatic stellate cell activation.
5. The method of Claim 1, wherein the antagonist is a decorin fusion protein.
6. The method of Claim 5, wherein the fusion protein is encoded by a nucleic acid molecule which comprises decorin and an IgGI 1 Fc fragment joined by a peptide linker.
7. The method of Claim 6, wherein the peptide linker containing 19 amino acids is present between rhDecorin and wherein the human IgGl Fc; and the peptide linker comprises 8 amino acids selected from the group consisting of glycine, serine, alanine, and threonine.
8. The method of Claim 6, wherein the human IgGl Fc comprises part of hinge, CH2, and CH3 domains of human IgGl with modulation in N-terminal.
9. The method of Claim 6, wherein the rhDecorin -Fc fusion protein exhibits an enhanced in vitro biological activity of at least four-fold relative to that of decorin.
10. The method of Claim 6, wherein rhDecorin-Fc fusion protein exhibits longer life time than its parent protein, decorin.
1 1. A method of producing an rhDecorin-Fc fusion protein, comprising using a Cos-7 cell line producing the rhDecorin -Fc fusion protein in its growth medium in excess of 10 .mu.g per million cells in a 48-hour period.
12. A method for making a recombinant fusion protein comprising rhDecorin, a flexible peptide linker, and a modified human IgGl Fc, comprising the steps of generating a Cos-7 cell line; and, growing the cell line under conditions the recombinant protein is expressed in its growth medium in excess of 10 .mu.g per million cells in a 48 hour period, wherein the recombinant fusion protein exhibits an enhanced in vitro biological activity of at least 4 fold relative to that of decorin without undesirable side effects.
13. The method of Claim 12, wherein the human IgG Fc comprises part of hinge CH2 and CH3 domains of human IgGl with a modified N-terminal, in which 4 amino acids of the IgG 1 Fc N-terminal SPGK are replaced by 10 amino acids, ITSEFAAARV.
PCT/IB2005/002999 2004-08-30 2005-07-18 Method for inhibiting fibrogenesis by an rhdecorin-fc fusion protein WO2006038107A2 (en)

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