Patent Application
20090162375
The present invention relates to resistin antagonists such as antibodies and their use in treating osteoarthritis and diseases with fibrotic pathologies. The invention also relates to antigens useful for generating anti-resistin antibodies.
Resistin is a secreted factor that belongs to the FIZZ (Found in Inflammatory Zone, also known as RELM) protein family containing a conserved C-terminal Cys-rich domain (Steppan et al., Proc. Natl. Acad. Sci. U.S.A. 98:502-506 (2001)). It was originally described in mice as an adipocyte-derived polypeptide that provided the link between obesity and insulin resistance (Steppan et al., Nature 409:307-312 (2001)). Human resistin, on the other hand, is a macrophage/monocyte-derived factor recognized as a potent proinflammatory factor. Human resistin induces cytokine release from various cells of immune origin, up-regulates the expression of adhesion molecules and promotes angiogenesis in endothelial cells (Burnett et al., Atherosclerosis 182:241-8 (2005); Mu et al., Cardiovasc. Res. 70:146-57 (2006); Verma et al., Circulation 108:736-40 (2003); Bokarewa et al., J. Immunol. 174:5789-95 (2005)). The effect of resistin is likely mediated through the NF-κb pathway (Bokarewa et al., supra).
A resistin receptor has not been identified, which limits the understanding of resistin biology. Recently, the three-dimensional structure of mouse resistin has been solved (Patel et al., Science 304:1154-8 (2004). Mouse resistin forms non-covalent trimeric oligomers that may aggregate into disulphide-linked hexameric structures. There is no structural information on human resistin.
Interstitial lung disease (ILD) is a collective term for more than 100 different diseases with similar clinical, radiological and physiological characteristics. The underlying causes can be due to systemic illness or occupational exposure. However, some ILD have unknown underlying etiology. These fibrotic lung diseases are difficult to treat, with biopsies being required for diagnosis.
The most common fibrotic lung disease is UIP (usual interstitial pneumonia) with an incidence of between 3-29 per 100,000 (Coultas et al., Am. J. Respir. Crit. Care Med. 150:967-72 (1994)). UIP develops insidiously over a few months to several years and often radiological changes appear before clinical symptoms. Characteristic histological findings of UIP include the presence of patchy, heterogenous lung fibrosis, which, as the disease progresses can result in alveolar collapse, bronchiolectasis and honeycombing. Nonspecific interstitial pneumonia (NSIP) presents with similar clinical symptoms to UIP. However, although certain histological findings are similar, this patient group tends to have less fibrosis (MacDonald et al., Radiology 221:600-5 (2001)). Furthermore, NSIP patients are more responsive to corticosteroids and in one study, NSIP patients had considerably improved prognosis (Daniil et al., Am. J. Respir. Crit. Care Med. 160:899-905 (1999)).
There have been reports in the literature that there are differences in fibroblasts obtained from fibrotic lung disease compared to cells obtained from non-fibrosis related lung disease, such as samples isolated from the normal margins of lung tumor resections. Tissue sections from UIP patients express increased levels of CXCL8 (also known as Interleukin 8 or IL8), an angiogenic chemokine, and decreased levels of an angiostatic chemokine CXCL10 (IFN-inducible Protein of 10 kDa, IP10), thereby suggesting an imbalance in net angiogenesis in the lungs of UIP patients (Keane et al., J. Immunol. 159:1437-43 (1997)). Moreover, fibroblasts from these patients also expressed increased CXCL8 suggesting that the fibroblast is the main effector cell causing the angiogenic imbalance (Keane et al., supra). More recently it has been shown that fibroblasts from UIP patients express and secrete increased levels of CCL7 (Monocyte Chemotactic Protein 3, MCP3) (Choi et al., Am. J. Respir. Crit. Care Med. 170:508-15 (2004)). Studies comparing TGFβ1-induced gene expression in fibroblasts from non-fibrotic tissue, UIP and scleroderma-induced lung fibrosis, have also demonstrated distinct phenotypes and responses of fibroblasts depending on the environment the cells have been isolated from (Renzoni et al., Respir. Res. 5:24 (2004)).
Currently four members of the FIZZ family have been identified in rodents and two in humans. Among them FIZZ1 (RELMα) is implicated in the development of lung fibrosis in mice. FIZZ1 was discovered in lavage fluid in a murine allergic pulmonary model (Holcomb et al., EMBO J. 19:4046-55 (2000)). In addition, up-regulation of the FIZZ1 gene was found in lung from bleomycin-induced fibrotic mice (Liu et al., Am. J. Pathol. 164):1315-26 (2004)). Intravenous injection of FIZZ1 into mice showed marked increase of CD68-positive macrophages in the lungs (Yamaji-Kegan et al., J. Physiol. Lung Cell. Mol. Physiol. Aug. 4, 2006 e-print). In vitro studies showed that FIZZ1 has mitogenic properties and may mediate fibrosis through the stimulation of smooth muscle cell proliferation and induction of actin and collagen deposition in lung fibroblasts (Teng et al., Circ. Res. 92:1065-7 (2003)). Intratracheal instillation of FIZZ1 resulted in significant increase of VEGF production suggesting the role of FIZZ1 in pulmonary angiogenesis (Tong et al., Respir. Res. 7:37 (2006)). A FIZZ1 orthologue has not been identified in the human genome. Interestingly, human resistin (FIZZ3) shows a greater similarity in expression pattern to murine FIZZ1 then murine resistin suggesting a potential functional similarity. However, the relationship between human resistin and lung fibrosis remains largely unexplored.
Thus, in view of the above, a need exists for structural information on human resistin and for therapeutics, such as resistin antagonists, to treat lung fibrosis and its associated symptoms.
Osteoarthritis (OA) is a chronic and progressive joint disease, primarily affecting the knees, hips, spine and hands. It is the most common cause of musculoskeletal disability in the elderly, with a prevalence of 10-30% in persons over age 65 (Wang et al., Altern. Med. Rev. 9:275-96, (2004)). OA can cause a substantial burden of disability and economic cost, particularly with an aging population (Lawrence et al., Arthritis Rheum. 41:778-799, (1998)). Despite its frequency in the population, OA remains a poorly understood condition for which few therapeutic options are available (McAlindon and Dieppe, Br. J. Rheumatol. 29:471-473, (1990)).
The risk factors for OA include family history, age, obesity, and joint trauma. The major pathologic feature of OA is the loss of articular cartilage, the tissue that provides a low-friction, wear-resistant bearing surface in joints and distributes stresses to underlying bone. It also involves other components of the joint including osteophyte formation and concomitant alteration of synovium and subchondral bone metabolism. These pathologic changes are associated with pain, stiffness, and loss of function. At the molecular level, OA is characterized by an imbalance between chondrocyte anabolism and catabolism that results in destruction of cartilage extracellular matrix (ECM). Accordingly, novel therapeutics that block cartilage degeneration and inhibit ECM degradation would be useful.
Recent evidence suggests that inflammation may affect disease progression and pain in OA. Varying degrees of inflammation are observed on arthroscopy or in synovial biopsy specimens in OA. There is increasing evidence indicating that alterations in synovial tissue metabolism are present in a significant proportion of OA patients (Pelletier et al., Arthritis Rheum. 44:1237-1247, (2001)). In addition, correlation was found between the severity of synovitis and the progression of joint destruction (Ayral et al., Osteoarthritis Cartilage 13:361-367, (2005)). Several markers have been used to assess synovitis, including serum hyaluronan (HA), N-propeptide of type III procollagen, YKL40, and glucosyl-galactosyl-pyridinoline (Glc-Gal-PYD) (Gineyts et al., Rheumatology (Oxford) 40:315-323, (2001)). Systemic level of C-Reactive Protein (CRP) has provided added value in predicting the outcomes of knee OA and rapidly progressive hip OA (Chevalier, Rev. Rheum. Engl. Ed. 64:562-577, (1997)).
It has been shown that resistin accumulates locally in the inflamed joints of rheumatoid arthritis (RA) patients and that its levels correlate with the intensity of inflammation as defined by the intra-articular white blood cell count and IL-6 levels. Injection of resistin into healthy mouse joints can induce arthritis. The effect of resistin is likely mediated through the NF-κb pathway (Bokarewa et al., J. Immunol. 174:5789-5795, (2005)).
Recently, resistin has been found to be present in both OA and RA synovial fluid samples. Concentrations in RA have been observed to be 10-fold higher than in OA. Positive correlations have been found between resistin levels and systemic inflammatory markers such as erythrocyte sedimentation rate and the levels of C-reactive protein in both OA and RA patients (Schaffler et al., JAMA 290:1709-1710, (2003)). However, the relationship between resistin and OA remains largely unexplored. Thus, there remains a need to determine if resistin serves as a biological marker for OA and to employ resistin antagonists to diagnose or treat OA and its associated symptoms.
One aspect of the invention is an antagonist of resistin. In one embodiment, the resistin antagonist is an isolated antibody reactive with resistin and neutralizes at least one of its biological activities.
Another aspect of the invention is an isolated antibody reactive with an epitope located at residues 50 to 65 (SEQ ID NO: 3) of human resistin.
Another aspect of the invention is an isolated antibody reactive with an epitope located at residues 78 to 93 (SEQ ID NO: 4) of human resistin.
Another aspect of the invention is an isolated polypeptide comprising the peptide ESQSVTSRGDLATSPR (SEQ ID NO: 5).
Another aspect of the invention is an isolated polypeptide comprising the peptide SGSWDVRAETTSHSQS (SEQ ID NO: 6).
Another aspect of the invention is an isolated nucleic acid encoding the amino acid sequence of SEQ ID NO: 5.
Another aspect of the invention is an isolated nucleic acid encoding the amino acid sequence of SEQ ID NO: 6.
Another aspect of the invention is a method of treating fibrosis or alleviating the symptoms of fibrosis in a mammalian subject by reducing the level or effect of resistin.
Another aspect of the invention is a diagnostic method for determining the risk or progression of OA in a mammalian subject by employing resistin as a novel biomarker for such disorder. In one embodiment, the diagnostic method comprises determining the risk or progression of OA by measuring the level of resistin protein in a biological sample of a mammalian subject and comparing the measured resistin level to a standard of resistin levels in the biological sample in a healthy population. An elevated resistin level compared to the standard is predictive of increased risk of OA.
Another aspect of the invention is an antagonist of resistin. In one embodiment, the resistin antagonist is an isolated antibody reactive with resistin and neutralizes at least one of its biological activities.
Another aspect of the invention is a method of treating OA or alleviating the symptoms of OA in a mammalian subject by reducing the level or effect of resistin. In one embodiment, the method comprises administering to the mammalian subject a therapeutically effective amount of a resistin antagonist of the invention. In another embodiment, the method comprises administering to the mammalian subject a therapeutically effective amount of a resistin antibody of the invention.
All publications, including but not limited to patents and patent applications, cited in this specification are herein incorporated by reference as though fully set forth.
This invention relates to the role of resistin, a cytokine, as a novel therapeutic target for resistin antagonists to treat diseases associated with fibrotic pathologies such as ILD, scleroderma, hypertrophic scarring, as well as diseases associated with significant remodeling in the lung such as asthma and chronic obstructive pulmonary disorder (COPD).
In the present invention, it has been shown that resistin protein is detected in fibrotic lungs. Immunohistochemistry with anti-resistin monoclonal antibodies indicate that resistin is expressed in monocyte/macrophage cells that are involved in fibrotic and remodeling processes. Resistin secretion is induced by pro-fibrotic modulators suggesting that elevated local levels of resistin might be present in fibrotic lung. Resistin directly affects primary lung fibroblasts by inducing genes associated with extracellular matrix (ECM) accumulation and myofibroblast differentiation, two processes that contribute to pathogenesis of lung fibrosis. Resistin also induces the expression of pro-fibrotic growth factors and their receptors in lung fibroblasts. Furthermore, resistin stimulates inflammatory response in lung fibroblasts by increasing the release of pro-inflammatory cytokines. Resistin affects fibrotic cells more then non-fibrotic fibroblasts suggesting that resistin signaling is up-regulated in diseased fibroblasts. Finally, in the present inventions it is shown that resistin expression and secretion are induced by pro-fibrotic modulators. Taken together, these data suggest that resistin is able to exacerbate fibrosis by contributing to fibroblast activation and lung inflammation. Accordingly, inhibition of resistin will be beneficial for the treatment of diseases associated with aberrant fibroblast function.
Further, the present invention describes a model of the three-dimensional structure of human resistin that was based on the crystal structure of the mouse protein. This model is used to predict a potential receptor-binding site based on structural similarity to the C1q/TNF superfamily. Two polyclonal antibodies have been raised against peptides spanning the predicted receptor-binding site and the antibodies' neutralizing activity was confirmed in in vitro functional assay. The data suggest that the identified epitopes are important for resistin function and can be used for generation of therapeutics agents neutralizing resistin.
In an embodiment of the invention, the epitope peptides are conjugated to a carrier protein or fused to a larger protein scaffold for use as an antigen for immunization and/or for phage display panning for generation of neutralizing anti-resistin antibody. The disclosed functional epitopes can be used for screening a panel of antibodies to identify those with neutralizing activity.
This invention also relates to the role of resistin, an adipokine, as a novel therapeutic target for resistin antagonists to treat OA and as a biomarker for OA. In the present invention, it has been shown that resistin protein is detected in synovial fluid and serum of post-traumatic injury OA patients. The observation originated from protein detection and data analysis of various protein levels in synovial fluid and serum samples from post-traumatic injury OA patients. Upon principal component analysis of the data, resistin as a variable was found to change in concert with other proinflammatory cytokines such as TNF-α and IL-6 in both serum and synovial fluid samples. This finding suggests that resistin likely plays a proinflammatory role in the pathological process of OA and relates to the release of matrix molecule fragments, as well as a variety of other disorders with an inflammatory component. Further, it is expected that systemic levels of various cytokines including IL-1, TNF-α, and IL-6 could be useful for predicting the outcomes of knee OA based on an association or correlation between systemic levels of proinflammatory cytokines and progression of joint destruction.
Further, in the present invention it is shown that resistin protein was present in paired synovial fluid and serum samples of traumatic injury and OA patients. Resistin levels are higher in patients immediately after the injury and in the early stage of OA and declined with time. Also, resistin protein was expressed in macrophage-like cells in synovium and in a subset of degenerative chondrocytes. Thus, intra-articular resistin could be produced from joint resident cells and local resistin concentrations may be quite different from the circulating level of resistin. In addition, recombinant resistin caused a pro-inflammatory response in immune cells that in turn induced degenerative processes in cartilage. Moreover, direct treatment of cartilage explants with recombinant human resistin resulted in increased ECM degradation, induction of prostaglandin E(2) levels and stimulation of pro-inflammatory cytokine/chemokine secretion. Taken together, these data suggest that resistin is a potent pro-inflammatory factor in joints that contributes to cartilage degradation in OA.
The present invention provides novel methods for evaluating the risk or progression of OA by evaluating levels of resistin in a mammalian subject, preferably a human. One embodiment of the present invention is a method for diagnosing the risk of OA, comprising measuring the resistin levels in a biological sample from a mammalian subject.
As used herein, the term “biological sample” includes any sample from a human patient, e.g., a body fluid such as synovial fluid. Other examples of a biological sample include blood, serum, plasma, and other fluid or tissue. The measurement of the concentration of resistin protein may employ any suitable resistin antibody to detect the protein. Such antibody may be presently extant in the art or presently used commercially, or may be developed by techniques now common in the field of immunology. For example, the LINCO human adipokine kit (Linco Research, St Charles, Mo.) can be used to determine resistin levels in serum and synovial fluid.
The measurement of resistin serves as a novel biomarker for OA. According to the method of this invention, to determine the risk or progression of OA, the level of resistin protein in a biological sample of a mammalian subject is measured and compared to a reference standard of resistin levels in a population. An elevated resistin level compared to the standard is predictive of increased risk of OA. The reference standard is established by measuring resistin levels of the biological samples from a normal population. Preferably, the standard is provided by using the same assay technique as is used for measurement of the subject's resistin levels, to avoid any error in standardization.
Another embodiment of the present invention is a method for diagnosing the risk of OA, comprising measuring the resistin levels in a biological sample from a mammalian subject in combination with measuring one or more other OA biomarkers. Such additional biomarkers include, but are not limited to, high-sensitivity C-reactive protein, markers of inflammation. Correlation between the resistin level and a level indicative of OA risk for a known OA biomarker further confirms the risk or progression of OA. Thus the measurement of resistin may serve to confirm indications of OA provided by assays for known biomarkers or may serve to additional stratification of OA patient population. Alternatively, the measurement of resistin may serve to more accurately diagnose the OA risk than the known biomarkers or may serve for additional stratification of the OA patient population.
Another embodiment of the present invention is a method for monitoring the progress of OA, comprising repeatedly measuring resistin levels over a given time period. The method may be useful to determine the degree of success of a particular therapeutic regimen for OA and may indicate circumstances in which a change of therapy is necessary.
The present invention provides an antagonist against resistin. In one embodiment, the resistin antagonist is an antibody having the properties of binding resistin and neutralizing at least one resistin biological activity.
The term “antagonist” is used in the broadest sense and includes a molecule that is capable of, directly or indirectly, partially or fully counteracting, reducing or inhibiting one or more biological activities of resistin.
The term “antibody” is used in the broadest sense and includes immunoglobulin or antibody molecules including polyclonal antibodies, monoclonal antibodies including murine, human, humanized and chimeric monoclonal antibodies and antibody fragments.
In general, antibodies are proteins or polypeptides that exhibit binding specificity to a specific antigen. Intact antibodies are heterotetrameric glycoproteins, composed of two identical light chains and two identical heavy chains. Typically, each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide linkages varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has at one end a variable domain (VH) followed by a number of constant domains. Each light chain has a variable domain at one end (VL) and a constant domain at its other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain and the light chain variable domain is aligned with the variable domain of the heavy chain. Antibody light chains of any vertebrate species can be assigned to one of two clearly distinct types, namely kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains. Immunoglobulins can be assigned to five major classes, namely IgA, IgD, IgE, IgG and IgM, depending on the heavy chain constant domain amino acid sequence. IgA and IgG are further sub-classified as the isotypes IgA1, IgA2, IgG1, IgG2, IgG3 and IgG4.
The term “antibody fragments” means a portion of an intact antibody, generally the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab′, F(ab′)2 and Fv fragments, and single chain antibody molecules.
“CDRs” are defined as the complementarity determining region amino acid sequences of an antibody which are the hypervariable regions of immunoglobulin heavy and light chains. See, e.g., Kabat et al., Sequences of Proteins of Immunological Interest, 4th ed., U.S. Department of Health and Human Services, National Institutes of Health (1987). There are three heavy chain and three light chain CDRs or CDR regions in the variable portion of an immunoglobulin. Thus, “CDRs” as used herein refers to all three heavy chain CDRs, or all three light chain CDRs or both all heavy and all light chain CDRs, if appropriate.
CDRs provide the majority of contact residues for the binding of the antibody to the antigen or epitope. CDRs of interest in this invention are derived from donor antibody variable heavy and light chain sequences, and include analogs of the naturally occurring CDRs, which analogs also share or retain the same antigen binding specificity and/or neutralizing ability as the donor antibody from which they were derived.
The term “monoclonal antibody” (mAb) as used herein means an antibody (or antibody fragment) obtained from a population of substantially homogeneous antibodies. Monoclonal antibodies are highly specific, typically being directed against a single antigenic determinant. The modifier “monoclonal” indicates the substantially homogeneous character of the antibody and does not require production of the antibody by any particular method. For example, murine mAbs can be made by the hybridoma method of Kohler et al., Nature 256:495-497 (1975). Chimeric mAbs containing a light chain and heavy chain variable region derived from a donor antibody (typically murine) in association with light and heavy chain constant regions derived from an acceptor antibody (typically another mammalian species such as human) can be prepared by the method disclosed in U.S. Pat. No. 4,816,567. Humanized mabs having CDRs derived from a non-human donor immunoglobulin (typically murine) and the remaining immunoglobulin-derived parts of the molecule being derived from one or more human immunoglobulins, optionally having altered framework support residues to preserve binding affinity, can be obtained by the techniques disclosed in Queen et al., Proc. Natl. Acad. Sci. (USA), 86:10029-10032 (1989) and Hodgson et al., Bio/Technology, 9:421 (1991). Exemplary human framework sequences useful for humanization are disclosed at, e.g., www.ncbi.nlm.nih.gov/entrez/query.fcgi; www.ncbi.nih.gov/igblast; www.atcc.org/phage/hdb.html; www.mrc-cpe.cam.ac.uk/ALIGNMENTS.php; www.kabatdatabase.com/top.html; ftp.ncbi.nih.gov/repository/kabat; www.sciquest.com; www.abcam.com; www.antibodyresource.com/onlinecomp.html; www.public.iastate.edu/˜pedro/research_tools.html; www.whfreeman.com/immunology/CH05/kuby05.htm; www.hhmi.org/grants/lectures/1996/vlab; www.path.cam.ac.uk/˜mrc7/mikeimages.html; mcb.harvard.edu/BioLinks/Immunology.html; www.immunologylink.com; pathbox.wustl.edu/˜hcenter/index.html; www.appliedbiosystems.com; www.nal.usda.gov/awic/pubs/antibody; www.m.ehime-u.ac.jp/˜yasuhito/Elisa.html; www.biodesign.com; www.cancerresearchuk.org; www.biotech.ufl.edu; www.isac-net.org; baserv.uci.kun.nl/˜jraats/links1.html; www.recab.uni-hd.de/immuno.bme.nwu.edu; www.mrc-cpe.cam.ac.uk; www.ibt.unam.mx/vir/V_mice.html; http://www.bioinf.org.uk/abs; antibody.bath.ac.uk; www.unizh.ch; www.cryst.bbk.ac.uk/−ubcg07s; www.nimr.mrc.ac.uk/CC/ccaewg/ccaewg.html; www.path.cam.ac.uk/˜mrc7/humanisation/TAHHP.html; www.ibt.unam.mx/vir/structure/stat_aim.html; www.biosci.missouri.edu/smithgp/index.html; www.jerini.de; imgt.cines.fr; and Kabat et al., Sequences of Proteins of Immunological Interest, U.S. Dept. Health (1987), each entirely incorporated herein by reference.
Fully human mabs lacking any non-human sequences can be prepared from human immunoglobulin transgenic mice by techniques referenced in, e.g., Lonberg et al., Nature 368:856-859 (1994); Fishwild et al., Nature Biotechnology 14:845-851 (1996) and Mendez et al., Nature Genetics 15:146-156 (1997). Human mAbs can also be prepared and optimized from phage display libraries by techniques referenced in, e.g., Knappik et al., J. Mol. Biol. 296:57-86 (2000) and Krebs et al., J. Immunol. Meth. 254:67-84 (2001).
The present invention relates to resistin antagonists capable of binding human resistin and modulating NF-Kb mediated signaling. The present invention also relates to resistin antagonists capable of binding human resistin and modulating resistin-mediated activities including, but not limited to, osteoarthritis, aberrant activation of fibroblasts, induction of pro-fibrotic gene expression and stimulation of inflammatory responses. Such antagonists include anti-resistin antibodies having the properties of binding resistin and modulating resistin signaling. One aspect of the invention is an antibody reactive with human resistin that inhibits resistin-mediated release of other inflammatory cytokines including, but not limited to MCP-1, IL-8 and IL-6. These antibodies are useful as research reagents, diagnostic reagents and therapeutic agents. In particular, the antibodies of the invention are useful as therapeutic agents for treating osteoarthritis, fibrosis or alleviating symptoms of fibrosis.
Exemplary antibodies may be antibodies of the IgG, IgD, IgGA or IgM isotypes. Additionally, such antibodies can be post-translationally modified by processes such as glycosylation, isomerization, aglycosylation or non-naturally occurring covalent modification such as the addition of polyethylene glycol moieties (pegylation) and lipidation. Such modifications may occur in vivo or in vitro. Fully human, humanized and affinity-matured antibody molecules or antibody fragments are within the scope of the invention as are mimetibodies, fusion proteins and chimeric proteins.
The antibodies of the invention may bind to one or both of the human resistin epitopes 50ECQSVTSRGDLATCPR65 (SEQ ID NO: 3) or 78CGSWDVRAETTCHCQC93 (SEQ ID NO: 4). These resistin epitopes can be isolated and modified for use as antigen to produce and screen monoclonal antibodies against human resistin. For example, the epitope sequences can be modified to remove the cysteine residues to prevent disulfide bonding. These residues can be replaced with serine. Exemplary modified resistin epitopes comprise an isolated polypeptide having the amino acid sequence ESQSVTSRGDLATSPR (SEQ ID NO: 5) and SGSWDVRAETTCHSQS (SEQ ID NO: 6). Polynucleotides encoding these polypeptides are also included in the present invention.
The antibodies of the invention may bind human resistin with a Kd less than or equal to about 10−7, 10−8, 10−9, 10−10, 10−11 or 10−12 M. The affinity of a given antibody against human resistin can be determined experimentally using any suitable method. Such methods may utilize Biacore or KinExA instrumentation, ELISA or competitive binding assays known to those skilled in the art. Antibody molecules binding human resistin with a desired affinity can be selected from libraries of variants or fragments by techniques including antibody affinity maturation and other suitable art-recognized techniques.
Another aspect of the present invention is pharmaceutical compositions comprising at least one resistin antibody and a pharmaceutically acceptable carrier or diluent known in the art. The carrier or diluent can be a solution, suspension, emulsion, colloid or powder.
A resistin antibody of the invention is formulated as a pharmaceutical composition in a therapeutically effective amount. The term “effective amount” generally refers to the quantities of antibody necessary for effective therapy, i.e., the partial or complete alleviation of the symptom or disorder for which treatment was sought. Included within the definition of effective therapy are prophylactic treatments intended to reduce the likelihood of onset of the above-described symptoms or disorders.
Another aspect of the present invention is isolated nucleic acid molecules comprising, complementary to or having significant identity with a polynucleotide encoding an antibody heavy or light chain having CDR amino acid sequences. Other polynucleotides which, given the degeneracy of the genetic code or codon preferences in a given expression system, encode the heavy or light chain variable region CDRs are also within the scope of the invention.
Typically, the nucleic acids of the present invention are used in expression vectors for the preparation of the resistin antibody polypeptides of the invention. Vectors within the scope of the invention provide necessary elements for eukaryotic expression, including viral promoter driven vectors, such as CMV promoter driven vectors, e.g., pcDNA3.1, pCEP4 and their derivatives, Baculovirus expression vectors, Drosophila expression vectors and expression vectors that are driven by mammalian gene promoters, such as human Ig gene promoters. Other examples include prokaryotic expression vectors, such as T7 promoter driven vectors, e.g., pET41, lactose promoter driven vectors and arabinose gene promoter driven vectors.
The present invention also relates to cell lines expressing resistin antibodies. The host cells can be prokaryotic or eukaryotic cells. Exemplary eukaryotic cells are mammalian cells, such as but not limited to, COS-1, COS-7, HEK293, BHK21, CHO, BSC-1, HepG2, 653, SP2/0, NS0, 293, HeLa, myeloma, lymphoma cells, or any derivative thereof. Most preferably, the host cells are HEK293, NS0, SP2/0 or CHO cells. The cell lines may be generated by stable or transient transfection procedures that are well known in the art.
The present invention further provides methods for expressing a resistin antibody comprising culturing the cell lines under conditions wherein the resistin antibody is expressed in detectable or recoverable amounts. The present invention also provides methods for generating resistin antibody comprising translating the resistin antibody encoding nucleic acids under conditions in vitro or in situ, such that the resistin antibody is expressed in detectable or recoverable amounts. The present invention also encompasses a resistin antibody produced by the above methods.
A resistin antibody can be recovered and purified by well-known methods including, but not limited to, protein A purification, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylatpatite chromatography and lectin chromatography. High performance liquid chroatography (HPLC) can also be employed for purification.
The resistin antibodies are useful as, inter alia, research reagents and therapeutic agents. In one aspect, the present invention relates to a method of modifying the biological activities of resistin comprising providing resistin antibody of the present invention to a mammal in need thereof. Examples of such biological activities of resistin include aberrant activation of fibroblasts, stimulation of myoblast differentiation, induction of pro-fibrotic genes, release of pro-inflammatory cytokines, induction of collagen I accumulation as well as other activities known to those skilled in the art. Specifically, the resistin antibody may decrease or inhibit resistin-mediated cell signaling cascades, such as but not limited to the NF-Kb pathway.
The present invention further provides methods for alleviating the symptoms of, or treating fibrosis comprising administering a therapeutically effective amount of a resistin antibody pharmaceutical composition to a patient in need thereof. As described above, such composition comprises an effective amount of resistin antibody and a pharmaceutically acceptable carrier or diluent. The effective amount for a given therapy, whether curative or preventative, will generally depend upon may different factors, including means of administration, target site and other medicants administered. Thus, treatment doses will need to be titrated to optimize safety and efficacy. In addition, the resistin antibody may be administered singly or in combination with at least one further compound, protein or composition useful for treating fibrosis.
The mode of administration can be any suitable route to deliver the pharmaceutically effective amount of resistin antibody of the present invention to a host. For example, the resistin antibody can be delivered via parenteral administration, such as subcutaneous, intraarcheal, intramuscular, intradermal, intravenous or intranasal administration, or any other means known in the art.
In another aspect, the present invention relates to a method of modifying the biological activities of resistin comprising providing resistin antibody of the present invention to a mammal in need thereof where such biological activities of resistin include cartilage degradation, release of pro-inflammatory markers (PGE2 and cytokines), disruption of extra-cellular matrix biosynthesis as well as other activities known to those skilled in the art. Specifically, the resistin antibody may decrease or inhibit resistin-mediated cell signaling cascades, such as but not limited to the NF-Kb pathway.
The present invention further provides methods for alleviating the symptoms of, or treating OA comprising administering a therapeutically effective amount of a resistin antibody pharmaceutical composition to a patient in need thereof. As described above, such composition comprises an effective amount of resistin antibody and a pharmaceutically acceptable carrier or diluent. The effective amount for a given therapy, whether curative or preventative, will generally depend upon may different factors, including means of administration, target site and other medicants administered. Thus, treatment doses will need to be titrated to optimize safety and efficacy. In addition, the resistin antibody may be administered singly or in combination with at least one further compound, protein or composition useful for treating OA.
The mode of administration can be any suitable route to deliver the pharmaceutically effective amount of resistin antibody of the present invention to a host. For example, the resistin antibody can be delivered via parenteral administration, such as intra-articular, subcutaneous, intramuscular, intradermal, intravenous or intranasal administration, or any other means known in the art.
The present invention is further described with reference to the following examples. These examples are merely to illustrate aspects of the present invention and are not intended as limitations of this invention.
Lung biopsy samples from UIP, NSIP and the normal margins of lung tumors were homogenized in PBS containing Complete Protease Inhibitor (Roche). Resistin levels were measured by ELISA (Linco Research Inc., St. Charles, Mo.) following the manufacturer's instructions. Total protein was measured in each lung biopsy sample using the BCA protein assay (Pierce Biotechnology, Inc., Rockford, Ill.). Resistin levels were then normalized to the amount of protein in each patient sample and are shown in
Pulmonary fibroblasts were isolated from lung biopsies taken from UIP patients (n=4); now referred to herein as “fibrotic fibroblasts” or ‘UIP fibroblasts’. Fibroblasts were also isolated from lung tissue taken during lung tumor resection (n=5) and these samples were confirmed to be non-fibrotic by histological analysis. These non-fibrotic tissue derived fibroblasts are referred to herein as “non-fibrotic fibroblasts” (NF).
Human lung fibroblasts were plated into 24 well plates (Costar, Corning, N.Y.) at 100,000 cells/well and allowed to adhere for 8 hours. The cells were then washed with PBS and cultured overnight in serum free media (DMEM with 1-Glutamine, Pen/Strep). Cells were then stimulated for 24 hrs in the presence or absence of TGFβ-1 (1 or 10 ng/mL) or resistin (1 or 10 ug/mL). Human recombinant resistin was purchased from Peprotech, Inc (Rocky Hill, N.J.). The purity of the protein was confirmed using SDS-PAGE and mass spectroscopy and endotoxin levels were found to be low (0.9 EU/mg). Supernatants were removed and analyzed for resistin protein using commercially available ELISA or Luminex. RNA was subsequently isolated using RNeasy Plus Mini-Kits (QIAGEN, Valencia, Calif.) as per manufacturer's instructions and reverse transcribed into cDNA using TaqMan® Reverse Transcription Reagents (Applied Biosystems, Foster City, Calif.). Profibrotic gene expression was determined by real time PCR using the Taqman® Universal PCR Master Mix (Applied Biosystems) and pre-developed Taqman® Gene Expression Assays (Applied Biosystems) as per manufacturer's instructions. Quantitative gene expression was calculated using the comparative CT method, where CT values are determined as the threshold cycle number for which gene expression is first detected. Fold changes in gene expression for the genes of interest were first normalized to the housekeeping gene 18S, giving ΔCT values. Fold changes in gene expression due to in vitro stimulation were calculated by ΔΔCT=ΔCT(unstimulated)−ΔCT(stimulated), where the unstimulated sample served as calibrator. To investigate potential pro-fibrotic activities of resistin the expression level of procollagen I, alpha smooth muscle actin (αSMA), CTGF, TGFβ1, TGFbR, PDGFR, hyaluronan receptor CD44, fibrillin (fbn1), fibronectin (fn1) and IL-6 were evaluated in untreated and resistin-treated lung fibroblasts.
Accumulation of collagen deposition and the presence of myofibroblasts are two key hallmarks of fibrosis. TGFβ1, a known pro-fibrotic modulator used as a positive control in the study, induced an up-regulation of both procollagen I and αSMA in lung fibroblasts (
As shown in
Alterations in extracellular matrix (ECM) components as well as aberrant matrix turnover are hallmarks of various fibrotic diseases or diseases associated with tissue remodeling. The effect of resistin on several of the ECM genes including the hyaluronan receptor CD44, fibrillin (fbn1) and fibronectin (fn1) was evaluated. Resistin enhanced the expression of CD44, fibrillin and fibronectin specifically in fibrotic fibroblasts (
Cell-free supernatants from the fibroblasts isolated from the lung biopsies that were stimulated in vitro with human recombinant resistin were analyzed for cytokine levels by the LINCO human cytokine kit (Linco Research). Resistin stimulated the release of IL-6 (
Human CD14+ monocytes purified from freshly isolated primary blood mononuclear cells were purchased from AllCells, LLC (Emeryville, Calif.). The cells were plated in 96 U-bottom well plate at a density of 75,000 cells per well in RPMI containing 10% fetal bovine serum (FBS). Cells were treated with 0 or 1 ng/ml TGFβ (R&D Systems, Minneapolis, Minn.) and PDGF (R&D Systems). After 48 h of culture, resistin levels in the supernatants were measured by ELISA kit. To induce macrophage differentiation monocytes were plated in RPMI containing 10% FBS in 96 well flat-bottom plates and allowed to adhere, then the cells were treated similarly to described above. Data in
As shown in
Lung biopsy samples from either the normal margins of lung tumors, UIP or NSIP patients (total n=9; n=3 of each patient cohort) were stained with an anti-resistin antibody (R&D Systems) at concentration 2 μg/ml. In lung specimens, resistin protein was detected in macrophages, histiocytes (tissue-restricted macrophages) and mast cells. Co-staining of non-fibrotic lung specimen with high degree of inflammation with CD68 antibody showed that resistin co-localized with CD68-positive macrophage cells (data not shown). Expression of human resistin in monocytes and macrophage cells has been previously reported (Jung et al., Cardiovasc. Res., 69:76-85 (2006); Lehrke et al., PLOS Med., 1:e45 (2004)) as well as confirmed by our finding (
Amino acid sequences of human (SEQ ID NO: 1) and mouse resistin (SEQ ID NO: 2) were obtained from Swiss-Prot under the accession codes Q9HD89 and Q99P87, respectively and aligned.
The crystal structure of mouse resistin (Patel et al., supra) is available from the Protein Data Bank under the accession code 1RFX. Based on this structure, a 3D model of human resistin was constructed by replacing amino acids according to the sequence alignment. The computer program COOT (Emsley, Acta Crystallogr. D. Biol. Crystallogr. 60:2126-32 (2004)) was used for this purpose. The side chain conformation was adjusted to avoid close contacts with other residues. The most energetically favorable rotamers were chosen whenever possible. No changes in the main-chain conformation were necessary. The resulting model contained no residues in the forbidden conformation, no steric clashes, and no residues in an unfavorable environment, such as uncompensated charges in the interior of the molecule, or extensive hydrophobic patches on the surface of the molecule. Given that the sequence similarity to the mouse protein is relatively high and the stereochemical criteria are met, the resulting three-dimensional model provides a good approximation of the real structure.
The polypeptide chain of resistin is folded into an N-terminal α-helix (residues 22-47) followed by a globular domain (residues 48-108), which has a β-structure of a jelly-roll type. Three protein monomers form a trimer in which the α-helices form a coiled-coil, and the globular domains form a compact head with an extensive interface that covers about 30% of the surface of each monomer. An earlier theoretical model of resistin is available from the Protein Data Bank under accession code 1LV6. This earlier model is not in agreement with the crystal structure data now available for mouse resistin.
Several lines of evidence suggest that the side surface of the globular domains of the resistin trimer is a potential receptor-binding site. For example, resistin is active in a trimeric form (Patel et al., surpa), therefore only the surface accessible to solvent in the trimer is considered as a potential receptor-binding site. Further, as part of C1q/TNF structural superfamily of proteins, resistin may bind to its receptor in a way common to other family members that is at the side surface of the globular domain. The C1q/TNF superfamily is registered in the Pfam database under the accession code CL0100. It includes a number of cytokines, such as adiponectin, TNFα and β, and CD40L. Members of the superfamily have a characteristic helical/jelly-roll fold and a coiled-coil homotrimeric structure. Crystal structures are available for many members of the superfamily as well as for their complexes with the corresponding receptors. These structures indicate that receptor binding occurs at the side surface of the globular domains, usually at the domain interface, as observed for instance in APO2L+DR5 and TNFβ+TNFR55. Receptor was never observed being bound to the top of the globular domain or to the helical domain.
Also, the sequence similarity in the resistin family of proteins is much higher within the globular domain than in the helical domain suggesting its functional importance. Amino acid homology between human and mouse resistin is ˜36% in the helical domain versus ˜66% in the globular domain. Similarly, the homology between human and bovine or porcine resistin is ˜28% in the helical domain versus ˜85% in globular domain.
Lastly, the three-fold symmetry of the globular head of the resistin trimer implies that the receptor, if bound to the top of the head, should have an internal three-fold symmetry of its recognition site, which is very unlikely. This practically eliminates the possibility of binding to the top of the head.
There are only two fragments of the human resistin polypeptide chain that are on the side surface of the globular domain and that are solvent accessible in the trimeric form. These include residues 50-65 (SEQ ID NO: 3) and 78-93 (SEQ ID NO: 4). Both peptides contain two types of residues, those that are accessible and therefore may interact with the receptor, and those that are buried and therefore may not be involved in the interaction. Amino acids that are not on the surface might be replaced in order to achieve better biophysical properties of peptide. The following substitutions were made in the peptides to avoid non-specific disulfide cross-linking: C51S, C63S (peptide 1) and C78S, C89S, C91S, C93S (peptide 2) resulting in the following peptide epitope sequences:
Together, these two peptides cover 64% of the surface of the globular domain. An advantage of peptide 2815 is that all residues except the two cysteines are exposed in resistin, so that regardless of the folding of the peptide in the free form, the potential epitope residues will be available for antibody generation. Peptide 2816 forms a β-hairpin in the protein. Only one face of the β-hairpin may be involved in receptor recognition. The key residues here are R84-A85-E86. Another advantage of the peptide 2816 is that this region is 100% conserved in higher mammals and 75% conserved with respect to murine resistin. Given the high sequence similarity in this fragment, it is possible to raise an antibody that will cross-react with resistin protein from multiple species.
Generation of polyclonal antibodies: The epitope peptides 2815 and 2816 were chemically synthesized on an ABI433A Peptide Synthesizer using RINK Resin SS (Advanced ChemTech, SA5030, Lot#17046). The peptide was cleaved from the resin, precipitated, filtered, washed and dried. Peptides were sent to Invitrogen Corporation (Carlsburg, Calif.) where they were fused to KLH protein for immunization. Two rabbits per peptide were immunized using the standard procedures (Cat. number M0300). ELISA assays on the peptide indicated antibody titers and the rabbits were terminally bled after the four-week immunization procedure. The antibody was affinity purified using peptide conjugated to serum albumin. The polyclonal antibody raised against the 2815 and 2816 peptides are referred to as C2815 and C2816, respectively.
Binding to full length human resistin: The ability of polyclonal antibodies C2815 and C2816 to bind full length human resistin was tested using a standard sandwich ELISA format. A plate was coated with 2 μg/ml C2815 or C2816 in 0.2M carbonate-bicarbonate buffer, pH 8.5 overnight at 4° C. To eliminate non-specific binding plates were blocked with 280 μl of 0.5% bovine serum albumin in PBS for 2 hours at 37° C. Human resistin at different concentrations was added to the plate and incubated overnight at 4° C. For detection biotin labeled anti-resistin antibody BAM1359 (R&D System, Minneapolis, Minn.) with europium was added followed by enhancement solution. Fluorescence signal was read on the Victor plate reader and data was plotted in GraphPad Prism.
The results in
Neutralizing activity: The neutralizing activity of C2816 and C2815 was evaluated in a cytokine release assay. Briefly, 2 ug/ml of human or mouse resistin was incubated with increasing concentrations of each antibody at room temperature for twenty minutes. The resistin-antibody mixture was added to isolated human primary blood mononuclear cells (PBMC) and incubated for twenty-four hours. Cell-free supernatants were collected and MCP-1 levels were detected by a commercially available ELISA kit (R&D System, Minneapolis, Minn.).
The results in
Synovial fluid and serum samples were obtained at various times following traumatic injury (including meniscus, ligament and OC fracture injury) or from patients diagnosed with osteoarthritis (over a 10-year timeframe). The samples were collected from 263 patients. Adipokines including adiponectin, resistin, and PAI-1 were detected using the LINCO human adipokine kit (Linco Research, St Charles, Mo.).
Resistin was detected in both synovial fluid and serum samples in post-injury and OA patients. Its level was higher immediately after the injury and in the early stage of OA and declined with time. According to the piecewise linear model, resistin level in synovial fluid changed from approximately 3000 pg/mL immediately after injury or diagnosis to approximately 900 pg/mL after 6.3 weeks (an approximately 71% decrease), and remained steady at that level (
Synovium and cartilage tissues from 3 OA and 3 RA patients were stained with an anti-resistin antibody at the concentration of 4 mg/ml (R&D System, Minneapolis, Minn.). Resistin protein was detected in macrophage-like cells in synovium from both OA and RA patients (data not shown). Cartilage tissue was negative for resistin staining except for one RA cartilage specimen where the anti-resistin antibody stained a subset of degenerated chondrocytes (data not shown). The data suggest that joint resident cells may be responsible for the secretion of resistin and accumulation of intra-articular resistin, and thus, the intra-articular resistin levels may vary from serum resistin concentrations. Therefore, intra-articular resistin levels would be expected to increase in situations where macrophage-like cell numbers are increasing due to injury-induced inflammation or cartilage degradation.
Human peripheral blood mononuclear cells (PBMCs) and monocytes from two normal healthy volunteers were isolated using the Lymphoprep™ gradient (Greiner Bio-one, Inc, Longwood, Fla.). The cells were treated with different concentrations of human recombinant resistin (Peprotech, Inc, Rocky Hill, N.J.). Following 2 days of culture in complete DMEM media containing 10% fetal bovine serum, conditioned media was collected and cytokines/chemokines including IL-1a, IL-1b, IL-1ra, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12 p70, IL-12p40, IL-13, IL-15, IL-17, EGF, eotaxin, francline, MIP-1b, IP-10, G-CSF, sCD40L, TGFα, VEGF, GM-CSF, IFN-G, IP-10, MCP-1, MIP-1a, TNF-α, and RANTES were assayed using the LINCO human cytokine kit (Linco Research).
Recombinant resistin induced the release of multiple cytokines and chemokines from PBMCs and monocytes. IL-6, IL-8 and MCP-1 secretion from human PBMC and monocytes is shown in
Human PBMCs or monocytes were treated with human recombinant resistin and conditioned media was collected as described in Example 11. Femoral head articular cartilage isolated from 3-week old mice was cultured in 1:3 diluted conditioned media for 3 days. Media was collected and glycosaminoglycans (GAG) was measured by the Dimethylmethylene blue (DMMB) assay. The DMMB assay is based on the metachromatic shift in absorption maximum that occurs when the dye is complexed with sulfated glycosaminoglycans. Briefly, culture medium is mixed with the dye and the absorbance is read on a plate reader at 520 nm. Samples are compared to a standard curve to determine the concentration of GAG.
Resistin caused the release of molecules from PBMCs and monocytes that drive cartilage degradation (
Femoral heads from the hip joints of 3-week old mice were isolated and cultured in vitro for 2 days in complete DMEM containing 10% FBS to allow equilibration. Femoral head articular cartilage was then treated with different concentrations of human recombinant resistin for 3 days. Conditioned media was collected and glycosaminoglycan content was measured as described in Example 12. Cartilage tissue was fixed and prepared for histology. Briefly, 5 mm sections were deparaffinized, hydrated, and stained for 10 minutes in 0.1% Toluidine Blue. Glycosaminoglycans (GAG) stain dark blue in Toluidine Blue. Therefore, a decrease in the staining intensity indicates loss of GAG containing proteoglycans from the cartilage that in turn would imply pathological destruction of cartilage tissue.
As shown in
Mouse cartilage explants were treated with different concentrations of human recombinant resistin for 3 days. Cytokines including IL-1α, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-7, IL-10, IL-12 p70, IL-13, IL-15, IL-17, IP-10, G-CSF, GM-CSF, IFN-γ, IP-10, MCP-1, MIP-1α, TNF-α, KC and RANTES were detected in conditioned media using LINCO mouse cytokines kit (MCYTO-70K-PMX22, Luminex, St Charles, Mo.). Prostaglandin E2 (PGE2) levels were measured in conditioned media by an ELISA kit (KGE004, R&D Systems).
Resistin stimulated the release of MCP-1, KC, and IL-6 from mouse cartilage explants (
The present invention now being fully described, it will be apparent to one of ordinary skill in the art that many changes and modifications can be made thereto without departing from the spirit or scope of the appended claims.
This application claims the benefit of U.S. Provisional Application Nos. 60/862,846, filed 25 Oct. 2006, 60/863,464, filed 30 Oct. 2006, and 60/974,607, filed 24 Sep. 2007.
| Number | Date | Country | |
|---|---|---|---|
| 60862846 | Oct 2006 | US | |
| 60863464 | Oct 2006 | US | |
| 60974607 | Sep 2007 | US |
