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WO2009097262A2 - Chitotriosidase et procédés d'utilisation de celle-ci - Google Patents

Chitotriosidase et procédés d'utilisation de celle-ci Download PDF

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Publication number
WO2009097262A2
WO2009097262A2 PCT/US2009/032102 US2009032102W WO2009097262A2 WO 2009097262 A2 WO2009097262 A2 WO 2009097262A2 US 2009032102 W US2009032102 W US 2009032102W WO 2009097262 A2 WO2009097262 A2 WO 2009097262A2
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Prior art keywords
chtrase
antibody
activity
copd
subject
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WO2009097262A3 (fr
Inventor
Anthony Coyle
Peter Kiener
Alexander Kozhich
Jennifer Reed
Marina Pretolani
Michel Aubier
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
MedImmune LLC
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Assistance Publique Hopitaux de Paris APHP
Institut National de la Sante et de la Recherche Medicale INSERM
MedImmune LLC
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Publication of WO2009097262A2 publication Critical patent/WO2009097262A2/fr
Publication of WO2009097262A3 publication Critical patent/WO2009097262A3/fr
Anticipated expiration legal-status Critical
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • C12Q1/6883—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for diseases caused by alterations of genetic material
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2600/00—Oligonucleotides characterized by their use
    • C12Q2600/158—Expression markers

Definitions

  • COPD chronic obstructive pulmonary disease
  • Chitinases can be subdivided into true chitinase protein family members, as represented by the enzymatically active proteins, acidic mammalian chitinase (AMCase) and chitotriosidase (ChTRase) and the pseudo chitinases, which lack enzymatic activity and include YKL40 (also called cartilage glycoprotein-39).
  • AMCase acidic mammalian chitinase
  • ChTRase chitotriosidase
  • pseudo chitinases which lack enzymatic activity and include YKL40 (also called cartilage glycoprotein-39).
  • enzymatically active chitinases are endo- ⁇ -l,4-N-acetylglucosamidases that degrade chitin, an abundant wall- and exoskeleton-derived polysaccharide that protects nematodes, fungal cells and insects from the animal and plant hosts that they invade (Gooday GW EXS. 87:158-169 (1999); Herrera-Estrella A et al. EXS. 87:171-184 (1999)).
  • chitin does not have a mammalian counterpart, elevated chitinolytic activity and prominent expression of ChTRase and AMCase have been associated with pathologic conditions that are characterized by tissue inflammation and remodeling, including atherosclerosis and asthma (Boot GR et al. Arterioscler Thromb Vase Biol. 19(3):687-694 (1999); Artieda M et al. Arterioscler Thromb Vase Biol. 23(9):1645-1652 (2003); Zhu Z et al. Science. 304(5677):1678-1682 (2004)).
  • All publications and patent applications herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference.
  • the present invention provides an isolated monoclonal antibody that binds to human chitotriosidase (ChTRase) or an antigenic fragment thereof, wherein said antibody binds said human ChTRase or antigenic fragment thereof with a dissociation constant (Kd) of equal to or less than 100 nM.
  • an antibody of the invention is a humanized or human antibody.
  • an antibody of the invention is an antagonist of ChTRase.
  • the present invention relates to biomarkers that are indicative of COPD.
  • the present invention provides methods for identifying a subject who is at risk of having COPD or an analagous disease.
  • a method of the invention comprises assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject.
  • the present invention also provides methods of monitoring the progression of COPD in a subject.
  • a method of the invention comprises assaying the level of ChTRase or ChTRase activity in a test sample of the subject.
  • the present invention additionally provides methods for evaluating the effect of one or more COPD treatment regimens on a subject with COPD.
  • a method of the invention comprises assaying the level of ChTRase or ChTRase activity in a test sample of the subject following treatment.
  • the present invention also provides methods of differentially diagnosing asthma and COPD who is at risk of having COPD, asthma or an analagous disease.
  • a method of the invention comprises assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject.
  • the present invention encompasses a diagnostic kit for diagnosing COPD.
  • a kit of the invention comprises one or more reagents that substantially specifically bind to ChTRase.
  • the present invention also provides compostions comprising one or more antibodies of the invention and at least one excipient.
  • the present invention provides methods for treating COPD.
  • a method of the invention comprises administering to a subject in need thereof a therapeutically effective amount of a ChTRase antagonist.
  • a method of the invention comprises administering an anti-ChTRase antibody of the invention.
  • the present invention relates to methods for reducing the percent of ChTRase- positive macrophages in a subject having COPD.
  • a method of the invention comprises administering a ChTRase antagonist.
  • a method of the invention comprises administering an anti-ChTRase antibody of the invention.
  • the present invention provides methods for reducing the ChTRase levels and/or ChTRase activity in the BAL of a subject having COPD.
  • a method of the invention comprises administering a ChTRase antagonist.
  • a method of the invention comprises administering an anti-ChTRase antibody of the invention.
  • the present invention includes methods for reducing macrophage activation associated with COPD in a subject.
  • a method of the invention comprises administering a ChTRase antagonist.
  • a method of the invention comprises administering an anti-ChTRase antibody of the invention.
  • FIG. 1 BAL chitino lytic activity and levels of ChTRase in subjects with COPD and asthma.
  • A BAL chitino lytic activity and
  • B levels of ChTRase in never- smokers, in smokers without and with COPD [defined as COPD (-) and COPD (+), respectively] and in mild, moderate and severe asthmatics.
  • C Percent inhibition of chitino lytic activity induced by 25 ng/ml of recombinant human ChTRase by a neutralizing anti-ChTRase, but not anti-AMCase, antibody.
  • ChTRase red chromogen
  • C faint ChTRase-positive cells were identified in the bronchial epithelium and the submucosa (arrow) of smokers without COPD.
  • D In smokers with COPD, substantially more ChTRase- positive cells infiltrate the bronchial epithelium and the subepithelial areas (arrows).
  • Original objective x 40 In smokers with COPD, substantially more ChTRase- positive cells infiltrate the bronchial epithelium and the subepithelial areas (arrows).
  • FIG. 3 Immunolocalization of ChTRase in lung tissue sections.
  • A Scant ChTRase-positive cells (red chromogen) in the alveolar septa and
  • B absence of immunostaining in the bronchiolar epithelium of a never-smoker.
  • C Presence of ChTRase- expressing cells in the alveoli and
  • D at the vicinity of the bronchiolar epithelium of a smoker without COPD.
  • D Absence of ChTRase immunostaining in the bronchiolar epithelium.
  • FIG. 1 ChTRase expression and release by alveolar macrophages. Expression of ChTRase (red chromogen) in alveolar macrophages collected by BAL from never-smokers (A), smokers without COPD (B) and smokers with COPD (C). Original magnifications x 40.
  • D Mean proportions of alveolar macrophages expressing ChTRase. * P ⁇ 0.05, as compared to never-smokers, f P ⁇ 0.05, as compared to smokers without COPD.
  • E Mean levels of ChTRase mRNA, normalized according to the amount of ubiquitin C (defined as UBC) mRNA, in alveolar macrophages.
  • FIG. 6 In vitro effects of the chitinase inhibitor, allosamidin.
  • A Chitinolytic activity (in fluorescence units, FU) induced by the addition of 4- methylumbelliferyl- ⁇ -D-N,N',N"-triacetylchitotriose to 0.005 to 10 ng/ml of purified human ChTRase to.
  • B Inhibition of ChTRase (5 ng/ml)-mediated chitinolytic activity by 0.5 to 1000 ng/ml allosamidin.
  • C,D,E Effect of 5 ng/ml of allosamidin on TNF- ⁇ (10 ng/ml)- induced IL-8 (C), MCP-I (D) and MMP-9 (E) release in the supernatant of alveolar macrophages from smokers with COPD.
  • Columns represent mean values of 5 independent experiments and error bars indicate SEM. * P ⁇ 0.05, as compared to medium-stimulated alveolar macrophages; f P ⁇ 0.05, as compared to TNF- ⁇ -stimulated alveolar macrophages.
  • the present inventors have unexpectly found that smokers with COPD, but not subjects with mild, moderate or severe asthma, had greater BAL chitinolytic activity, increased levels of ChTRase, more ChTRase-positive cells in bronchial biopsies and an elevated proportion of alveolar macrophages expressing ChTRase than smokers without COPD, or never-smokers.
  • ChTRase accounted for approximately 80% of BAL chitinolytic activity in smokers with COPD, while AMCase was undetectable.
  • the present invention in based in part on the discovery that BAL chitinolytic activity and ChTRase in BAL and biopsies of smokers with and without COPD were associated with airflow obstruction and with emphysema. Furthermore, tumor-necrosis factors- ⁇ stimulated ChTRase release only from alveolar macrophages from smokers with COPD, and exposure of these cells to ChTRase promoted the release of interleukin-8, monocyte-chemoattractant protein- 1 and MMP-9. Additionally, treatment of alveolar macrophages with the chitinase inhibitor, allosamidin, significantly reduced tumor-necrosis factors- ⁇ -induced chemokine release.
  • blocking antibodies to selectively inhibit ChTRase activity the inventors found that the presence of this enzyme accounted for approximately 80% of the BAL chitino lytic activity found in smokers with COPD.
  • the residual 20% activity that is not inhibited by the anti-ChTRase antibody is likely a result of incomplete inhibition of ChTRase activity by the anti-ChTRase neutralizing antibody, as shown by dose-response enzymatic assays.
  • BAL chitinase residual activity might be attributable to the presence of small quantities of AMCase.
  • the present invention provides antibodies that bind to human chitotriosidase (ChTRase) or an antigenic fragment thereof.
  • Chitotriosidase also referred to as ChT, ChTRase
  • GenBank Ace No. NM 003465.
  • an anti-ChTRase antibody of the invention binds to human ChTRase or an antigenic fragment thereof with a dissociation constant (Kd) of equal to or less than about 1000 nM, about 100 nM, about 10 nM, about 1 nM, about 100 pM, about 10 pM, or about 1 pM.
  • Kd dissociation constant
  • an anti-ChTRase antibody of the invention is a chimeric antibody; a single-chain Fv (scFv); an Fab fragment; an F(ab') fragment; an intrabody; or a synthetic antibody.
  • an anti-ChTRase antibody o fht einvention is a humanized or human antibody.
  • an anti-ChTRase antibody of the invention binds to
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the binding of human ChTRase to a ligand (e.g., chitin).
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the binding of human ChTRase to a ligand (e.g., chitin) by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • the baseline chitin binding activity of ChTRase may be measured in the presence of a control antibody.
  • an anti-ChTRase antibody of the invention binds to
  • ChTRase and may inhibit or reduce ChTRase mediated signaling (e.g., ChTRase mediated signaling in alveolar macrophages).
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce ChTRase mediated signaling by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • the baseline ChTRase mediated signaling may be measured in the presence of a control antibody.
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the enzymatic activity of ChTRase. In one embodiment, an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the enzymatic activity of ChTRase by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • the baseline enzymatic activity of ChTRase may be measured in the presence of a control antibody
  • an anti-ChTRase antibody of the invention may bind to the catalytic domain of ChTRase. In another embodiment, an anti-ChTRase antibody of the invention may bind to the chitin binding domain of ChTRase.
  • ChTRase epitopes contemplated herein include, but are not limited to, the epitopes listed in Table 4.
  • a ChTRase antibody of the invention binds to at least one epitope listed in table 4. Table 4. Antigenic Epitopes of ChTRase
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated in vitro activation of alveolar macrophages.
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated in vitro activation of alveolar macrophages by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • the baseline ChTRase mediated in vitro activation of alveolar macrophages may be measured in the presence of a control antibody.
  • ChTRase mediated macrophage activation may be monitored by measuring the level of IL-8, MCP-I or MMP-9 secretion.
  • IL-8, MCP-I or MMP-9 secretion may be monitored by any method known to one of skill in the art, for example, by not limited to, ELISA assay and biological assay.
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated activation of IL-8 secretion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated activation of IL-8 secretion by between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • the baseline ChTRase mediated activation of IL-8 secretion may be measured in the presence of a control antibody.
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated activation of MCP-I secretion by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated activation of MCP-I secretion by between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • the baseline ChTRase mediated activation of MCP-I secretion may be measured in the presence of a control antibody.
  • an anti-ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated activation of MMP-9 expression by about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • an anti- ChTRase antibody of the invention binds to ChTRase and may inhibit or reduce the ChTRase mediated activation of MMP-9 expression by between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • the baseline ChTRase mediated activation of MMP-9 expression may be measured in the presence of a control antibody.
  • an anti-ChTRase antibody of the invention does not inhibit or reduce ChTRase mediated signaling. In another embodiment, an anti-ChTRase antibody of the invention does not inhibit or reduce the enzymatic activity of ChTRase. In a further embodiment, an anti-ChTRase antibody of the invention does not inhibit or reduce the ChTRase mediated in vitro activation of alveolar macrophages.
  • a method of the invention for identifying a subject at risk or suspected of having COPD or a fibrotic lung disease comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL), bronchial biopsy, sputum, blood, serum, plasma, or urine; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD or a fibrotic lung disease.
  • BAL bronchoalveolar lavage
  • the subject has one or more symptoms associated with COPD, one or more physiologic parameters associated with COPD, or both.
  • the severity of the subject's COPD correlates with the extent of the elevation of the ChTRase or ChTRase activity level in the test sample.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • the control value is the level of ChTRase or ChTRase activity in subjects lacking COPD.
  • the level of ChTRase activity is measured using a fluorescent substrate.
  • the level of ChTRase activity is measured using a Chitin Azure substrate.
  • the level of ChTRase is assayed by determining mRNA levels or protein levels. In one embodiment, the level of ChTRase is assayed by determining the percent of ChTRase-positive cells in the sample. In another embodiment, the level of ChTRase is assayed by determining the percent of ChTRase-positive cells in the sample, wherein the cells are alveolar macrophages. In one embodiment, the test sample is bronchoalveolar lavage (BAL).
  • BAL bronchoalveolar lavage
  • Chitinolyitic activity and ChTRase activity measurement assays are well known by one of skill in the art.
  • the teachings of the present invention may be practiced by employing any one of the known chitinolyitic activity and ChTRase activity measurement assays.
  • Particularly applicable are the fluorometric assays utilizing a labeled substrate, for example, but not limited to, 4-methylumbelliferyl- ⁇ -d-NlS[N-triacetylchitotrioside (see, e.g., Bargagli et al. Resp. Medicine 101(10):2176-81 (2007)).
  • Additional ChTRase activity assays are described in U.S. Patent Publication No. 2004/0166554 and Schoonhoven et al. Clin Chim Acta. 381(2):136-9 (2007).
  • ChTRase protein concentration in a sample may be determined using any one of the measurement techniques known to one of skill in the art.
  • ChTRase protein concentration in a sample may be determined by ELISA, quantitative Western, protein CHIP.
  • the use of any other assay described in the art for measuring protein concentration is contemplated herein.
  • ChTRase mRNA concentration in a sample may be determined using any one of the measurement techniques known to one of skill in the art.
  • ChTRase mRNA concentration in a sample may be determined by quantitative PCR, microarray hybridization, quantitative Northern blot. The use of any other assay described in the art for measuring mRNA concentration is contemplated herein.
  • ChTRase mRNA concentration in a sample may be expressed either as absolute concentration (e.g., copy/ unit volume or mg/unit volume) or as a ratio between the copy numbers of ChTRase mRNA and an internal standard mRNA (e.g., GAPDH, ubiquitin C).
  • the prevalence of ChTRase expressing cells in a sample may be determined using any one of the techniques known to one of skill in the art. For example, ChTRase expressing cells in a sample may be detected by immunohistochemistry, intracellular immuno-staining followed by flow cytometry, or by in situ hybridization. The use of any other assay described in the art for measuring protein concentration is contemplated herein.
  • Example 1 of the instant application provides non- limiting examples of the assyas that may be used to determine ChTRase protein levels, ChTRase mRNA levels, ChTRase activity levels, chitinolytic activity levels, or the prevalence of ChTRase expressing cells in a sample.
  • a method of the invention for identifying a subject at risk or suspected of having COPD comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • the subject has one or more symptoms associated with COPD, one or more physiologic parameters associated with COPD, or both.
  • the severity of the subject's COPD correlates with the extent of the elevation of the ChTRase or ChTRase activity level in the test sample.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • the control value is the level of ChTRase or ChTRase activity in corresponding samples from subjects lacking COPD.
  • the level of ChTRase activity is measured using a fluorescent substrate.
  • the level of ChTRase activity is measured using a Chitin Azure substrate.
  • the level of ChTRase is assayed by determining mRNA levels or protein levels.
  • the level of ChTRase is assayed by determining the percent of ChTRase-positive cells in the sample. In another embodiment, the level of ChTRase is assayed by determining the percent of ChTRase- positive cells in the sample, wherein the cells are alveolar macrophages. In one embodiment, the test sample is bronchoalveolar lavage (BAL).
  • BAL bronchoalveolar lavage
  • a method of the invention for identifying a subject at risk or suspected of having COPD or a fibrotic lung disease comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL); and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is at risk of having COPD or a fibrotic lung disease.
  • the level of ChTRase protein in the test sample is measured.
  • the level of ChTRase protein in the test sample is between about 2 and about 100 ng/ml, between about 3 and about 100 ng/ml, between about 4 and about 100 ng/ml, between about 5 and about 100 ng/ml, between about 6 and about 100 ng/ml, between about 7 and about 100 ng/ml, between about 8 and about 100 ng/ml, between about 9 and about 100 ng/ml, between about 10 and about 100 ng/ml, between about 15 and about 100 ng/ml, or between about 2 and about 100 ng/ml.
  • the level of ChTRase protein in the test sample is at least about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, or about 20 ng/ml.
  • the control sample is a corresponding sample from subjects lacking COPD. In a further embodiment, the control sample is a corresponding sample from subjects lacking COPD, wherein the level of
  • ChTRase protein in the control sample is between about 0 and about 2 ng/ml, or about 0 and about 3 ng/ml.
  • the control value is between about 0 to about 2 ng/ml, about 0 to about 3 ng/ml, about 0 and about 4 ng/ml, about 0 ChTRase protein.
  • a method of the invention for identifying a subject at risk or suspected of having COPD comprises: assaying for elevated level of ChTRase or
  • ChTRase activity in a test sample of the subject wherein the test sample is BAL; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • the level of ChTRase protein in the test sample is measured.
  • the level of ChTRase protein in the test sample is between about 2 and about 100 ng/ml, between about 3 and about 100 ng/ml, between about 4 and about 100 ng/ml, between about 5 and about 100 ng/ml, between about 6 and about 100 ng/ml, between about 7 and about 100 ng/ml, between about 8 and about 100 ng/ml, between about 9 and about 100 ng/ml, between about 10 and about 100 ng/ml, between about 15 and about 100 ng/ml, or between about 2 and about 100 ng/ml.
  • the level of ChTRase protein in the test sample is at least about 2 ng/ml, about 3 ng/ml, about 4 ng/ml, about 5 ng/ml, about 6 ng/ml, about 7 ng/ml, about 8 ng/ml, about 9 ng/ml, about 10 ng/ml, or about 20 ng/ml.
  • the control sample is a corresponding sample from subjects lacking COPD. In a further embodiment, the control sample is a corresponding sample from subjects lacking COPD, wherein the level of
  • ChTRase protein in the control sample is between about 0 and about 2 ng/ml, about 0 and about 3 ng/ml, or about 0 and about 4 ng/ml.
  • the control value is between about 0 to about 2 ng/ml, or about 0 and about 3 ng/ml ChTRase protein.
  • a method of the invention for identifying a subject at risk or suspected of having COPD or a f ⁇ brotic lung disease comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL); and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is at risk of having COPD or a f ⁇ brotic lung disease.
  • the level of ChTRase activity in the test sample is measured.
  • the level of ChTRase activity in the test sample is between about 2x10 3 and about 100 xl O 3 FU/ml, between about 3x10 3 and about 100 x 10 3 FU/ml, between about 4x 10 3 and about 100 x 10 3 FU/ml, between about 5x 10 3 and about 100 xl O 3 FU/ml, between about 6x10 3 and about 100 xl O 3 FU/ml, between about 7x10 3 and about 100 xl O 3 FU/ml, between about 8x10 3 and about 100 xl O 3 FU/ml, between about 9x10 3 and about 100 xl O 3 FU/ml, between about 10x10 3 and about 100 xl O 3 FU/ml, between about 15x10 and about 100 xlO FU/ml, or between about 20x10 and about 100 xlO 3 FU/ml.
  • the level of ChTRase activity in the test sample is at least about 2 xlO 3 FU/ml, about 3 xlO 3 FU/ml, about 4 xlO 3 FU/ml, about 5 xlO 3 FU/ml, about 6 xlO 3 FU/ml, about 7 xlO 3 FU/ml, about 8 xlO 3 FU/ml, about 9 xlO 3 FU/ml, about 10 xlO 3 FU/ml, about 15 xlO 3 FU/ml, or about 2O xIO 3 FU/ml.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the level of ChTRase activity in the control sample is between about 0 and about 2.5 xlO 3 , or about 0 and about 3 xlO 3 FU/ml. In a further embodiment, the control value is between about 0 and about 2.5 xlO 3 , or about 0 and about 3 xlO 3 FU/ml ChTRase activity.
  • a method of the invention for identifying a subject at risk or suspected of having COPD comprises: assaying for elevated level of ChTRase or
  • ChTRase activity in a test sample of the subject wherein the test sample is BAL; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • the level of ChTRase activity in the test sample is measured.
  • the level of ChTRase activity in the test sample is between about 2x10 3 and about 100 xl O 3 FU/ml, between about 3x10 3 and about 100 x 10 3 FU/ml, between about 4x10 3 and about 100 xl O 3 FU/ml, between about 5x10 3 and about 100 x 10 3 FU/ml, between about 6x10 3 and about 100 xl O 3 FU/ml, between about 7x10 3 and about 100 xl O 3 FU/ml, between about 8x10 3 and about 100 xl O 3 FU/ml, between about 9x10 3 and about 100 xl O 3 FU/ml, between about 10x10 3 and about 100 xl O 3 FU/ml, between about 15x10 3 and about 100 xl O 3 FU/ml, or between about 2OxIO 3 and about 100 xl O 3 FU/ml.
  • the level of ChTRase activity in the test sample is at least about 2 x 10 3 FU/ml, about 3 x 10 3 FU/ml, about 4 x 10 3 FU/ml, about 5 x 10 3 FU/ml, about 6 xlO 3 FU/ml, about 7 xlO 3 FU/ml, about 8 xlO 3 FU/ml, about 9 xlO 3 FU/ml, about 10 xlO 3 FU/ml, about 15 xlO 3 FU/ml, or about 2O xIO 3 FU/ml.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the level of ChTRase activity in the control sample is between about 0 and about 2.5 xlO 3 , or about 0 and about 3 xlO 3 FU/ml. In a further embodiment, the control value is between about 0 and about 2.5 xlO 3 , or about 0 and about 3 xlO 3 FU/ml ChTRase activity.
  • a method of the invention for identifying a subject at risk or suspected of having COPD or a fibrotic lung disease comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL); and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is at risk of having COPD or a fibrotic lung disease.
  • the level of ChTRase activity is assessed by measuring total chitinolytic activity in the test sample.
  • the total chitinolytic activity in the test sample is between about 2x10 3 and aboutto about 100 xlO 3 FU/ml, between about 3x10 3 and about 100 xl O 3 FU/ml, between about 4x10 3 and about 100 x 10 3 FU/ml, between about 5x10 3 and about 100 xl O 3 FU/ml, between about 6x10 3 and about 100 x 10 3 FU/ml, between about 7x10 3 and about 100 xl O 3 FU/ml, between about 8x10 3 and about 100 xl O 3 FU/ml, between about 9x10 3 and about 100 xl O 3 FU/ml, between about 10x10 3 and about 100 xl O 3 FU/ml, between about 15x10 3 and about 100 xl O 3 FU/ml, or between about 2OxIO 3 and about 100 xl O 3 FU/ml.
  • the total chitinolytic activity in the test sample is at least about 2 xlO 3 FU/ml, about 3 xlO 3 FU/ml, about 4 xlO 3 FU/ml, about 5 xlO 3 FU/ml, about 6 xlO 3 FU/ml, about 7 xlO 3 FU/ml, about 8 xlO 3 FU/ml, about 9 xlO 3 FU/ml, about 10 xlO 3 FU/ml, about 15 xlO 3 FU/ml, or about 20 xlO 3 FU/ml.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the total chitinolytic activity in the control sample is between about 0 and about 2.5 xlO 3 , or about 0 and about 3 xlO 3 FU/ml.
  • control value is between about 0 and about 2.5 xl O 3 , or about 0 and about 3 xlO 3 FU/ml total chitino lytic activity.
  • a method of the invention for identifying a subject at risk or suspected of having COPD comprises: assaying for elevated level of ChTRase or
  • ChTRase activity in a test sample of the subject wherein the test sample is BAL; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • the level of ChTRase activity is assessed by measuring total chitino lytic activity in the test sample.
  • the total chitino lytic activity in the test sample is between about 2x10 3 and about 100 xl O 3 FU/ml, between about 3x10 and about 100 xlO FU/ml, between about 4x10 and about 100 xlO 3 FU/ml, between about 5x10 3 and about 100 xl O 3 FU/ml, between about 6x10 3 and about 100 x 10 3 FU/ml, between about 7x10 3 and about 100 xl O 3 FU/ml, between about 8x10 3 and about 100 x 10 3 FU/ml, between about 9x10 3 and about 100 xl O 3 FU/ml, between about 10x10 3 and about 100 xl O 3 FU/ml, between about 15x10 3 and about 100 xl O 3 FU/ml, or between about 2OxIO 3 and about 100 xl O 3 FU/ml.
  • the total chitino lytic activity in the test sample is at least about 2 xlO 3 FU/ml, about 3 xlO 3 FU/ml, about 4 xlO 3 FU/ml, about 5 xlO 3 FU/ml, about 6 xlO 3 FU/ml, about 7 xlO 3 FU/ml, about 8 x 10 3 FU/ml, about 9 x 10 3 FU/ml, about 10 x 10 3 FU/ml, about 15 x 10 3 FU/ml, or about 20 xlO 3 FU/ml.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the total chitinolytic activity in the control sample is between about 0 and about 2.5 xlO 3 , or about 0 and about 3 xlO 3 FU/ml.
  • control value is between about 0 and about 2.5 xl O 3 , or about 0 and about 3 xlO 3 FU/ml total chitinolytic activity.
  • a method of the invention for identifying a subject at risk or suspected of having COPD or a fibrotic lung disease comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL); and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is at risk of having COPD or a fibrotic lung disease.
  • BAL bronchoalveolar lavage
  • the level of ChTRase is assessed by determining the percentage of ChTRase positive alveolar macrophages in the test sample.
  • the percentage of ChTRase positive alveolar macrophages in the test sample is between about 50 and about 100%, between about 60 and about 100%, between about 70 and about 100%, between about 80 and about 100%, between about 90 and about 100%,.
  • the percentage of ChTRase positive alveolar macrophages in the test sample is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the percentage of ChTRase positive alveolar macrophages in the control sample is between about 0 and about 50%. In a further embodiment, the control value is between about 0 and about 50% ChTRase positive alveolar macrophages.
  • a method of the invention for identifying a subject at risk or suspected of having COPD comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is BAL; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • the level of ChTRase is assessed by determining the percentage of ChTRase positive alveolar macrophages in the test sample.
  • the percentage of ChTRase positive alveolar macrophages in the test sample is between about 50 and about 100%, between about 60 and about 100%, between about 70 and about 100%, between about 80 and about 100%, between about 90 and about 100%,. In another embodiment, the percentage of ChTRase positive alveolar macrophages in the test sample is at least about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 100%. In another embodiment, the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the percentage of ChTRase positive alveolar macrophages in the control sample is between about 0 and about 50%. In a further embodiment, the control value is between about 0 and about 50% ChTRase positive alveolar macrophages.
  • a method of the invention for identifying a subject at risk or suspected of having COPD or a f ⁇ brotic lung disease comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL); and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is at risk of having COPD or a fibrotic lung disease.
  • BAL bronchoalveolar lavage
  • the level of ChTRase is assessed by measuring the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample.
  • the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample is between about 0.4 and about 5, between about 0.5 and about 5, between about 0.6 and about 5, between about 0.7 and about 5, between about 0.8 and about 5, between about 0.9 and about 5, between about 1 and about 5, or between about 2 and about 5.
  • the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample is at least about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1, about 2, about 3, about 4, or about 5.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • the control sample is a corresponding sample from subjects lacking COPD, wherein the ratio of ChTRase mRNA and ubiquitin C mRNA in the control sample is between about 0 and about 0.4, about 0 and about 0.45, or about 0 and about 0.5.
  • a method of the invention for identifying a subject at risk or suspected of having COPD comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is BAL; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • the level of ChTRase is assessed by measuring the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample is measured.
  • the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample is between about 0.4 and about 5, between about 0.5 and about 5, between about 0.6 and about 5, between about 0.7 and about 5, between about 0.8 and about 5, between about 0.9 and about 5, between about 1 and about 5, or between about 2 and about 5.
  • the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample is at least about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.65, about 0.7, about 0.75, about 0.8, about 0.85, about 0.9, about 0.95, about 1, about 2, about 3, about 4, or about 5.
  • the control sample is a corresponding sample from subjects lacking COPD.
  • the control sample is a corresponding sample from subjects lacking COPD, wherein the ratio of ChTRase mRNA and ubiquitin C mRNA in the control sample is between about 0 and about 0.4, or about 0 and about 0.45, or about 0 and about 0.5.
  • the control value is between about 0 and about 0.4, or about 0 and about 0.45, or about 0 and about 0.5 ChTRase mRNA to ubiquitin C mRNA ratio.
  • a method of the invention for identifying a subject at risk or suspected of having COPD or a f ⁇ brotic lung disease comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is a bronchial biopsy sample; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is at risk of having COPD or a f ⁇ brotic lung disease.
  • the level the percentage of ChTRase positive cells in the test sample is measured.
  • the percentage of ChTRase positive cells in the test sample is between about 10 and about 100 cells/mm 2 , between about 15 and about 100 cells/mm 2 , between about 20 and about 100 cells/mm 2 , between about 25 and about 100 cells/mm 2 , between about 30 and about 100 cells/mm 2 , between about 40 and about 100 cells/mm 2 , between about 50 and about 100 cells/mm 2 , between about 60 and about 100 cells/mm 2 , between about 70 and about 100 cells/mm 2 , or between about 80 and about 100 cells/mm 2 .
  • the percentage of ChTRase positive cells in the test sample is at least about 10 cells/mm 2 , about 15 cells/mm 2 , about 20 cells/mm 2 , about 25 cells/mm 2 , about 30 cells/mm 2 , about 40 cells/mm 2 , about 50 cells/mm 2 , about 60 cells/mm 2 , about 70 cells/mm 2 , about 80 cells/mm 2 , about 90 cells/mm 2 , or about 100 cells/mm 2 .
  • the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the percentage of ChTRase positive cells in the control sample is between about 0 and about 10 cells/mm 2 , or about 0 and about 15 cells/mm 2 . In a further embodiment, the control value is between about 0 and about 10, or about 0 and about 15 ChTRase positive cells/mm 2 .
  • a method of the invention for identifying a subject at risk or suspected of having COPD comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is a bronchial biopsy sample; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • the level the percentage of ChTRase positive cells in the test sample is measured.
  • the percentage of ChTRase positive cells in the test sample is between about 10 and about 100 cells/mm 2 , between about 15 and about 100 cells/mm 2 , between about 20 and about 100 cells/mm 2 , between about 25 and about 100 cells/mm 2 , between about 30 and about 100 cells/mm 2 , between about 40 and about 100 cells/mm 2 , between about 50 and about 100 cells/mm 2 , between about 60 and about 100 cells/mm 2 , between about 70 and about 100 cells/mm 2 , or between about 80 and about 100 cells/mm 2 .
  • the percentage of ChTRase positive cells in the test sample is at least about 10 cells/mm 2 , about 15 cells/mm 2 , about 20 cells/mm 2 , about 25 cells/mm 2 , about 30 cells/mm 2 , about 40 cells/mm 2 , about 50 cells/mm 2 , about 60 cells/mm 2 , about 70 cells/mm 2 , about 80 cells/mm 2 , about 90 cells/mm 2 , or about 100 cells/mm 2 .
  • the control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD, wherein the percentage of ChTRase positive cells in the control sample is between about 0 and about 10 cells/mm 2 , or about 0 and about 15 cells/mm 2 . In a further embodiment, the control value is between about 0 and about 10, or about 0 and about 15 ChTRase positive cells/mm 2 .
  • the invention also provides methods of monitoring the progression of COPD in a subject, comprising determining the level of ChTRase or ChTRase activity in a plurality of test samples over time, wherein the test samples are BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine.
  • the subject has one or more symptoms associated with COPD, one or more physiologic parameters associated with COPD, or both.
  • the severity of the subject's COPD correlates with the ChTRase or ChTRase activity level in the test sample.
  • the ChTRase or ChTRase activity level is compared between one or more test samples.
  • ChTRase or ChTRase activity level of one or more test samples is compared to a control value.
  • a reduced ChTRase or ChTRase activity level in the test samples over time correlates with a reduction in the severity of a subject's COPD.
  • an increased ChTRase or ChTRase activity level in the test samples over time correlates with an increase in the severity of a subject's COPD.
  • no change in the ChTRase or ChTRase activity level in the test samples over time correlates with no change in the severity of a subject's COPD.
  • an increased ChTRase or ChTRase activity level in the test samples over time correlates with an increase in the severity of a subject's COPD.
  • the level of ChTRase activity is measured using a fluorescent substrate.
  • the level of ChTRase activity is measured using a Chitin Azure substrate.
  • the level of ChTRase is assayed by determining mRNA levels or protein levels. In a further embodiment, the level of ChTRase is assayed by determining the percent of ChTRase-positive cells in the sample. In a specific embodiment, the level of ChTRase is assayed by determining the percent of ChTRase-positive alveolar macrophages in the sample.
  • the invention further encompasses methods of evaluating the effect of one or more COPD treatment regimens on a subject with COPD, comprising: comparing levels of ChTRase or ChTRase activity in BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine of the subject following treatment of the subject to levels of ChTRase or ChTRase activity in the BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine of the subject prior to treatment, and/or comparing levels of ChTRase or ChTRase activity in the BAL, sputum, blood, serum, plasma, or urine of the subject following treatment with a control value or with the levels of ChTRase or ChTRase activity in the BAL, sputum, blood, serum, plasma, or urine, respectively, of a control subject.
  • the subject has one or more symptoms associated with COPD, one or more physiologic parameters associated with COPD, or both.
  • the severity of the subject's COPD correlates with the ChTRase or ChTRase activity level in the test sample.
  • a reduced ChTRase or ChTRase activity level in the test sample following treatment correlates with a reduction in the severity of a subject's COPD.
  • an increased ChTRase or ChTRase activity level in the test sample following treatment correlates with an increase in the severity of a subject's COPD.
  • no change in the ChTRase or ChTRase activity level in the test sample following treatment correlates with no change in the severity of a subject's COPD.
  • the level of ChTRase activity is measured using a fluorescent substrate.
  • the level of ChTRase activity is measured using a Chitin Azure substrate.
  • the level of ChTRase is assayed by determining mRNA levels or protein levels.
  • the level of ChTRase is assayed by determining the percent of ChTRase-positive cells in the sample.
  • the level of ChTRase is assayed by determining the percent of ChTRase-positive alveolar macrophages in the sample.
  • a method of the invention for identifying a subject that an active smoker comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL), bronchial biopsy, sputum, blood, serum, plasma, or urine; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is an active smoker.
  • BAL bronchoalveolar lavage
  • control sample is a corresponding sample from at least one subject that has never smoked.
  • control value is the level of ChTRase or ChTRase activity in subjects that have never smoked.
  • level of ChTRase activity is measured using a fluorescent substrate.
  • the level of ChTRase activity is measured using a Chitin Azure substrate.
  • the level of ChTRase is assayed by determining mRNA levels or protein levels.
  • the level of ChTRase is assayed by determining the percent of ChTRase-positive cells in the sample.
  • the level of ChTRase is assayed by determining the percent of ChTRase-positive cells in the sample, wherein the cells are alveolar macrophages.
  • the test sample is bronchoalveolar lavage (BAL).
  • a method of the invention for identifying a subject that is an active smoker comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL); and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is an active smoker.
  • the level of ChTRase is assessed by determining the percentage of ChTRase positive alveolar macrophages in the test sample.
  • the percentage of ChTRase positive alveolar macrophages in the test sample is between about 30 and about 100%, between about 30 and about 90%, between about 35 and about 80%, between about 40 and about 80%, between about 45 and about 80%,. In another embodiment, the percentage of ChTRase positive alveolar macrophages in the test sample is at least about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 55%, or about 60%. In another embodiment, the control sample is a corresponding sample from at least one subject that has never smoked.
  • control sample is a corresponding sample from subjects that have never smoked, wherein the percentage of ChTRase positive alveolar macrophages in the control sample is between about 0 and about 30%. In a further embodiment, the control value is between about 0 and about 30% ChTRase positive alveolar macrophages.
  • a method of the invention for identifying a subject that is an active smoker comprises: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL); and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a corresponding control sample indicates that the subject is an active smoker.
  • the level of ChTRase is assessed by measuring the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample.
  • the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample is between about 0.1 and about 3, between about 0.15 and about 3, between about 0.2 and about 3, between about 0.25 and about 3, between about 0.3 and about 3, or between about 0.35 and about 3.
  • the ratio of ChTRase mRNA and ubiquitin C mRNA in the test sample is at least about 0.1, about 0.15, about 0.2, about 0.25, about 0.3, about 0.35, about 0.4, about 0.45, about 0.5, about 0.55, about 0.6, about 0.7, about 1, about 2, or about 3.
  • the control sample is a corresponding sample from subjects that have never smoked.
  • control sample is a corresponding sample from subjects that have never smoked, wherein the ratio of ChTRase mRNA and ubiquitin C mRNA in the control sample is between about 0 and about 0.05, about 0 and about 0.1, or about 0 and about 0.15. In a further embodiment, the control value is between about 0 and about 0.05, about 0 and about 0.1, or about 0 and about 0.15 ChTRase mRNA to ubiquitin C mRNA ratio.
  • the invention also provides diagnostic kits for diagnosing COPD, said kit comprising one or more reagents that substantially specifically bind to ChTRase.
  • a diagnostic kit of the invention comprises one or more reagents for the detection of ChTRase activity.
  • a diagnostic kit of the invention comprises one or more reagents for the detection of polynucleotide encoding the ChTRase protein or a complementary polynucleotide thereof.
  • a diagnostic kit of the invention comprises one or more reagents for the detection of the ChTRase protein or a fragment thereof.
  • a diagnostic kit of the invention further comprises instructions for using the reagent to diagnose COPD, or for using the reagent to assess the severity of COPD in the test subject, or both.
  • the invention further provides methods for treating COPD comprising administering to a subject in need thereof a therapeutically effective amount of a ChTRase antagonist.
  • a method of the invention for treating COPD comprises administering to a subject in need thereof a therapeutically effective amount of a ChTRase antagonist, wherein the ChTRase antagonist is an anti-ChTRase antibody of the invention.
  • the invention also relates to methods for reducing the percent of ChTRase- positive macrophages in a subject having COPD, comprising administering to said subject an amount of a ChTRase antagonist sufficient to reduce the percent of ChTRase-postive macrophages.
  • the ChTRase-positive macrophages are present in BAL fluid.
  • a method of the invention for reducing the percent of ChTRase-positive macrophages in a subject having COPD comprises administering to said subject an amount of a ChTRase antagonist sufficient to reduce the percent of ChTRase- postive macrophages, wherein the ChTRase antagonist is an anti-ChTRase antibody of the invention.
  • the percent of ChTRase-positive macrophages is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • the invention also provides methods for reducing the ChTRase levels and/or ChTRase activity in the BAL of a subject having COPD, comprising administering to said subject an amount of a ChTRase antagonist sufficient to reduce the ChTRase levels and/or ChTRase activity.
  • a method for reducing the ChTRase levels and/or ChTRase activity in the BAL of a subject having COPD comprises administering to said subject an amount of a ChTRase antagonist sufficient to reduce the ChTRase levels and/or ChTRase activity, wherein the ChTRase antagonist is an anti-ChTRase antibody of the invention.
  • the ChTRase or ChTRase activity level is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • the level of ChTRase is assayed by determining mRNA levels or protein levels.
  • the level of ChTRase is assayed by determining the percent of ChTRase-positive alveolar macrophages.
  • the level of ChTRase activity is measured using a fluorescent substrate.
  • the level of ChTRase activity is measured using a Chitin Azure substrate.
  • the present invention also encompasses methods for reducing macrophage activation associated with COPD in a subject, comprising administering to said subject an amount of a ChTRase antagonist sufficient to reduce macrophage activation.
  • a method for reducing macrophage activation associated with COPD in a subject comprises administering to said subject an amount of a ChTRase antagonist sufficient to reduce macrophage activation, wherein the ChTRase antagonist is an anti-ChTRase antibody of the invention.
  • macrophage activation is monitored by measuring the level of IL-8, MCP-I or MMP-9 secretion.
  • macrophage activation is monitored by measuring the level of IL-8, MCP-I or MMP-9 secretion, wherein the level of IL-8 or MCP-I secretion is measured by ELISA assay and wherein the level of MMP-9 secretion is measured by ELISA assay or a biological activity assay.
  • a method for reducing macrophage activation associated with COPD in a subject comprises administering to said subject an amount of a ChTRase antagonist sufficient to reduce macrophage activation, wherein macrophage activation is monitored by measuring the level of IL-8.
  • the level of IL-8 is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • a method for reducing macrophage activation associated with COPD in a subject comprises administering to said subject an amount of an anti-ChTRase antibody sufficient to reduce macrophage activation, wherein macrophage activation is monitored by measuring the level of IL-8.
  • the level of IL-8 is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100% as compared to a control antibody.
  • a method for reducing macrophage activation associated with COPD in a subject comprises administering to said subject an amount of a ChTRase antagonist sufficient to reduce macrophage activation, wherein macrophage activation is monitored by measuring the level of MCP-I .
  • the level of MCP-I is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • a method for reducing macrophage activation associated with COPD in a subject comprises administering to said subject an amount of an anti-ChTRase antibody sufficient to reduce macrophage activation, wherein macrophage activation is monitored by measuring the level of MCP-I .
  • the level of MCP-I is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100% as compared to a control antibody.
  • a method for reducing macrophage activation associated with COPD in a subject comprises administering to said subject an amount of a ChTRase antagonist sufficient to reduce macrophage activation, wherein macrophage activation is monitored by measuring the level of MMP-9 expression and/or activity.
  • the level of MMP-9 expression and/or activity is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100%.
  • a method for reducing macrophage activation associated with COPD in a subject comprises administering to said subject an amount of an anti-ChTRase antibody sufficient to reduce macrophage activation, wherein macrophage activation is monitored by measuring the level of MMP-9 expression and/or activity.
  • the level of MMP-9 expression is decreased by between about 5% and about 90%, between about 20% and about 75%, between about 25% and about 50%, between about 20% and about 100%, between about 20% and about 80%, between about 20% and about 60%, between about 20% and about 40%, between about 40% and about 100%, between about 40% and about 80%, between about 40% and about 60%, between about 60% and about 100%, between about 60% and about 80%, or between about 80% and about 100% as compared to a control antibody.
  • a "ChTRase antagonist" according to the present invention is preferably one that modulates, decreases or inhibits at least one activity of a ChTRase. In one embodiment, a ChTRase antagonist decreases at least one activity of a ChTRase. In another embodiment, a ChTRase antagonist inhibits at least one activity of a ChTRase.
  • Activities of ChTRase include, but are not limited to, chitinase activity, macrophage induction, induction of IL-8 secretion by alveolar macrophages, induction of MCP-I secretion by alveolar macrophages, and induction of MMP-9 expression by alveolar macrophages.
  • Additional activities of ChTRase include, but are not limited to, saccaride (e.g., GIcNAc) binding activity, simulation of inflammatory responses, and those mediated directly by a ChTRase, such as production of IL-8, MCP-I and MMP-9), cell surface receptor binding, signal transduction activity.
  • ChTRase activities of a ChTRase include those which mediate, directly and/or indirectly, any of the variety of changes associated with inflammatory disease including, but not limited to, apoptosis, tissue inflammation, increased lung volume, increased eosinophils in bronchioalveolar ravage (BAL) fluid, increased lymphocytes in BAL fluid, increased total cells in BAL fluid, increased alveolus size, increased deposition of crystals comprising chitinase-like molecules in lung tissue, increased airway resistance, increased mucus metaplasia, increased mucin expression, increased parenchymal fibrosis, increased airway remodeling (e.g., airway thickening, mucus metaplasia, epithelial hypertrophy and airway fibrosis), increased subepithelial fibrosis, increased collagen deposition in airway tissue, epithelial hypertrophy in the lung tissue, focal organization of crystalline material into Masson body-like fibrotic foci, airway hyperresponsiveness
  • a ChTRase antagonist of the invention is a specific antagonist of ChTRase.
  • a ChTRase antagonist of the invention antagonizes multiple chitinases.
  • a ChTRase antagonist of the invention antagonizes a single activity of ChTRase.
  • a ChTRase antagonist of the invention antagonizes more then one activity of ChTRase.
  • a ChTRase antagonist of the invention antagonizes at least one activity of ChTRase by at least about 10% or at least about 15 %, or at least about 20 %, or at least about 30 %, or at least about 40%, or at least about 50 %, or at least about 60%, or at least about 70 %, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%.
  • a ChTRase antagonist of the invention antagonizes at least one activity of ChTRase by at least 10% or at least 15 %, or at least 20 %, or at least 30 %, or at least 40%, or at least 50 %, or at least 60%, or at least 70 %, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500%.
  • a ChTRase antagonist of the invention reduces at least one activity of ChTRase by at least about 2 fold, or at least about 5 fold, or at least about 10 fold, or at least about 20 fold, or at least about 30 fold, or at least about 40 fold, or at least about 50 fold, or at least about 60 fold, or at least about 70 fold, or at least about 80 fold, or at least about 90 fold, or at least about 100 fold, or at least about 200 fold, or at least about 500 fold, or at least about 1000 fold.
  • a ChTRase antagonist of the invention reduces at least one activity of ChTRase by at least 2 fold, or at least 5 fold, or at least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or at least 500 fold, or at least 1000 fold.
  • a ChTRase antagonist of the invention may bind to one or more specific ChTRase protein domain or fragment thereof.
  • a ChTRase antagonist binds the signal sequence of ChTRase.
  • a ChTRase antagonist binds the catalytic domain (functional or nonfunctional) of ChTRase.
  • a ChTRase antagonist binds the chitin binding domain of ChTRase.
  • the ChTRase antagonists are, in one embodiment, those that decrease or inhibit the ability of ChTRase to macrophage induction, induction of IL-8 secretion by alveolar macrophages, induction of MCP-I secretion by alveolar macrophages, and induction of MMP-9 expression by alveolar macrophages.
  • a ChTRase antagonist of the invention decreases ChTRase mediated inflammatory responses including, but not limited to, those mediated directly by ChTRase, such as production of IL-8, MCP-I and MMP-9.
  • the ChTRase antagonists employed in the methods of the present invention are those that modulate one or more activity of ChTRase including, but not limited to, chitinolytic activity, saccharide (e.g., GIcNAc) binding activity, chemotatic activity, receptor binding activity, signal transduction activity, receptor binding activity.
  • chitinolytic activity e.g., GIcNAc
  • saccharide e.g., GIcNAc
  • a ChTRase antagonist when administered to a subject in need thereof, improves (e.g., reduces the severity and/or symptoms of) one or more clinical indicator, including but not limited to, cellular infiltration of the lung (e.g., increased eosinophils in bronchioalveolar ravage (BAL) fluid, increased lymphocytes in BAL fluid, increased total cells in BAL fluid), airway function, airway inflammation, airway remodeling (e.g., airway thickening, mucus metaplasia, epithelial hypertrophy and airway fibrosis), airway hyperresponsiveness (AHR), joint inflammation, connective tissue damage, bone damage, joint mobility.
  • BAL bronchioalveolar ravage
  • AHR airway hyperresponsiveness
  • a ChTRase antagonist of the invention when administered to a subject in need thereof, improves (e.g., reduces the severity and/or symptoms of) one or more clinical indicator by at least about 10% or at least about 15 %, or at least about 20 %, or at least about 30 %, or at least about 40%, or at least about 50 %, or at least about 60%, or at least about 70 %, or at least about 80%, or at least about 90%, or at least about 100%, or at least about 200%, or at least about 300%, or at least about 400%, or at least about 500%.
  • a ChTRase antagonist of the invention antagonizes at least one activity of ChTRase by at least 10% or at least 15 %, or at least 20 %, or at least 30 %, or at least 40%, or at least 50 %, or at least 60%, or at least 70 %, or at least 80%, or at least 90%, or at least 100%, or at least 200%, or at least 300%, or at least 400%, or at least 500% as compared to an untreated control.
  • a ChTRase antagonist of the invention when administered to a subject in need thereof, improves (e.g., reduces the severity and/or symptoms of) one or more clinical indicator by at least about 2 fold, or at least about 5 fold, or at least about 10 fold, or at least about 20 fold, or at least about 30 fold, or at least about 40 fold, or at least about 50 fold, or at least about 60 fold, or at least about 70 fold, or at least about 80 fold, or at least about 90 fold, or at least about 100 fold, or at least about 200 fold, or at least about 500 fold, or at least about 1000 fold.
  • a ChTRase antagonist of the invention reduces at least one activity of ChTRase by at least 2 fold, or at least 5 fold, or at least 10 fold, or at least 20 fold, or at least 30 fold, or at least 40 fold, or at least 50 fold, or at least 60 fold, or at least 70 fold, or at least 80 fold, or at least 90 fold, or at least 100 fold, or at least 200 fold, or at least 500 fold, or at least 1000 fold as compared to an untreated control.
  • a ChTRase antagonist when administered to a subject in need thereof, reduces inflammation, as compared to an untreated control.
  • a ChTRase antagonist when administered to a subject in need thereof, reduces cellular infiltration of the lung (e.g., reduces the number of eosinophils in bronchioalveolar ravage (BAL) fluid, reduces the number lymphocytes in BAL fluid, increased total cells in BAL fluid) as compared to an untreated control.
  • a ChTRase antagonist when administered to a subject in need thereof, improves airway function as compared to an untreated control. Airway function can be measured using any of a number of methods well known in the art, commonly used pulmonary function tests (PFT) include, for example, spirometry and plethysmography.
  • PFT pulmonary function tests
  • a ChTRase antagonist when administered to a subject in need thereof, reduces airway inflammation.
  • a ChTRase antagonist when administered to a subject in need thereof, reduces airway remodeling (e.g., airway thickening, mucus metaplasia, epithelial hypertrophy and airway fibrosis) as compared to an untreated control.
  • airway remodeling e.g., airway thickening, mucus metaplasia, epithelial hypertrophy and airway fibrosis
  • AHR airway hyperresponsiveness
  • ChTRase antagonist which, when administered to a subject in need thereof, reduces IL- 13 mediated pulmonary disease as compared to an untreated control.
  • a ChTRase antagonist when administered to a subject in need thereof, reduces cellular infiltration of affected joints.
  • a ChTRase antagonist when administered to a subject in need thereof, reduces joint inflammation as compared to an untreated control.
  • a ChTRase antagonist when administered to a subject in need thereof, reduces connective tissue damage as compared to an untreated control.
  • a ChTRase antagonist when administered to a subject in need thereof, reduces bone damage as compared to an untreated control. In another specific embodiment, a ChTRase antagonist, when administered to a subject in need thereof, improves joint mobility as compared to an untreated control.
  • administering leads to decreased cellular infiltration associated with a disease state including but not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis, cystic fibrosis (CF), emphysema, asthma, smoker's cough, allergy, allergic rhinitis and sinusitis, Paget' s disease, abnormal bone remodeling, osteoporosis, Gorham-Stout syndrome, osteoarthritis, rheumatoid arthritis, psoriatic arthritis and brittle bone disease.
  • a disease state including but not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis,
  • administering prevents or reduced tissue damage associated with a disease state including but not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis, cystic fibrosis (CF), emphysema, asthma, smoker's cough, allergy, allergic rhinitis and sinusitis, Paget' s disease, abnormal bone remodeling, osteoporosis, Gorham-Stout syndrome, osteoarthritis, rheumatoid arthritis, psoriatic arthritis and brittle bone disease.
  • a disease state including but not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis, cyst
  • administering leads to an improvement in the reduced airway function associated with a disease state including but not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis, cystic fibrosis (CF), emphysema, asthma, smoker's cough, allergy, allergic rhinitis and sinusitis.
  • a disease state including but not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis, cystic fibrosis (CF), emphysema, asthma, smoker's cough, allergy, allergic rhinitis and sinusitis.
  • ILD interstitial lung disease
  • a ChTRase antagonist of the invention may be administered prophylactically to prevent or reduce the severity of one or more adverse symptom of a disease or disorder.
  • Symptoms which may be prevented including but are not limited to, cellular infiltration of the lung, reduction airway function, airway inflammation, airway remodeling (e.g., airway thickening, mucus metaplasia, epithelial hypertrophy and airway fibrosis), airway hyperresponsiveness (AHR), joint inflammation, connective tissue damage, bone damage, decreased joint mobility.
  • ChTRase antagonists of the invention include, but are not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis, cystic fibrosis (CF), emphysema, asthma, smoker's cough, allergy, allergic rhinitis and sinusitis, Paget's disease, abnormal bone remodeling, osteoporosis, Gorham-Stout syndrome, osteoarthritis, rheumatoid arthritis, psoriatic arthritis and brittle bone disease.
  • ILD interstitial lung disease
  • pulmonary fibrosis bronchitis
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • pneumonia pneumonitis
  • ARDS acute respiratory distress syndrome
  • SARS severe acute respiratory syndrome
  • sarcoidosis cyst
  • a ChTRase antagonist can include, but should not be construed as being limited to a chemical compound, a protein, a peptidomemetic, an antibody, a ribozymes, an siRNA, an aptamer and an antisense nucleic acid molecule.
  • Such antagonists include, among others, those selected from the group consisting of antibodies, Fab 2 fragments, Fab fragments, Fv fragments, scFv molecules, proteins, peptides, non-peptidic agents, small molecule inhibitors, inactive fragments of ChTRases that function as negative regulators (i.e., that bind a ChTRase receptor but do not stimulate down stream signaling pathways), and inactive chitinase-binding fragments of a ChTRase receptor (i.e., that bind chitinase but cannot signal).
  • a ChTRase antagonist is a chemical compound.
  • ChTRase antagonists are well known in the art, and some of the key critical elements of one class of ChTRase antagonists have been defined (Spindler and Spindler-Barth, 1999, Chitin and Chitinases, Birkhauser Verlag Basel, Switzerland). It is contemplated that ChTRase antagonists of the invention encompass already known ChTRase antagonist such as, but not limited to, allosamidin (Carbohydrate Chemistry Industrial Research Limited, Lower Hutt, New Zealand, and Eli Lilly and Co., Greenfield, IN) and its derivatives (see, e.g., U.S. Patent No.
  • ChTRase antagonists include stylogaunidine and its derivatives (Kato et al, 1995, Tetrahedron. Lett. 36:2133 2136), dipeptide cyclo-(L-Arg-D-Pro) (Izumida et al, 1996, J.
  • ChTRase antagonist encompasses a chemically modified compound, and derivatives thereof. Methods for modifying and generating derivatives of a chemical compound are well known to one of skill in the chemical arts.
  • ChTRase antagonist includes such antagonists as discovered in the future, as can be identified by well-known criteria in the art of pharmacology, such as the physiological results of inhibition of a ChTRase as described in detail herein and/or as known in the art. Therefore, the present invention is not limited in any way to any particular ChTRase antagonist as exemplified or disclosed herein; rather, the invention encompasses those ChTRase antagonists that would be understood by the routineer to be useful as are known in the art and as are discovered in the future.
  • ChTRase antagonist can be synthesized chemically. Further, the routineer would appreciate, based upon the teachings provided herein, that a ChTRase antagonist can be obtained from a recombinant organism.
  • compositions and methods for chemically synthesizing ChTRase antagonist and for obtaining them from natural sources are well known in the art and are; described in, among others, Yamada et al., U.S. Patent Nos. 5,413,991, and 5,070,191
  • a ChTRase antagonist is a nucleic acid molecule.
  • a ChTRase antagonist is a nucleic acid molecule that inhibits or reduces the level of messenger RNA encoding a ChTRase including, but not limited to, an aptamer, a ribozyme, an antisense molecule, an siRNA.
  • the invention is directed to ribozymes as antagonists of
  • ChTRases As known in the art ribozymes are single stranded RNA molecules retaining catalytic activities. Their structures are based on naturally occurring site- specific, self- cleaving RNA molecules. Once they have cleaved their target, ribozymes are released from their mRNA target and are free to cleave another mRNA molecule (Usman et al., 2000, J Clin Invest 106:1197-202). It has been shown that unmodified and modified (e.g., 2'-O-methyl and phosphorothioate) synthetic ribozymes can be used to inhibit specifically gene expression in vitro (Usman et al., supra).
  • unmodified and modified (e.g., 2'-O-methyl and phosphorothioate) synthetic ribozymes can be used to inhibit specifically gene expression in vitro (Usman et al., supra).
  • the invention is directed towards RNAi to antagonize ChTRases.
  • siRNA and shRNA RNA interference (RNAi), quelling or post-transcriptional gene silencing (PTGS) designate a phenomenon by which dsRNA specifically suppresses expression of a target gene at post- translational level.
  • RNAi siRNA and shRNA RNA interference
  • PTGS post-transcriptional gene silencing
  • RNAi is initiated by double-stranded RNA molecules (dsRNA) of several thousands of base pair length.
  • dsRNA double-stranded RNA molecules
  • siRNA short interfering RNA
  • the enzyme that catalyzes the cleavage, Dicer, is an endo-RNase that contains RNase III domains (Bernstein et al., 2001, Nature 409:363-6).
  • siRNAs produced by Dicer are 21 23 bp in length, with a 19 or 20 nucleotides duplex sequence, two-nucleotide 3' overhangs and 5 '-triphosphate extremities (Zamore et al. supra; Elbashir et al. Genes Dev 15:188-200; Elbashir et al. ,2001, Embo J 20:6877 88).
  • siRNA are usually designed against a region 50-100 nucleotides downstream the translation initiator codon, whereas 5'UTR
  • siRNA inhibition by siRNA is an efficient process which is at least tenfold more potent as silencing trigger than sense or antisense RNAs alone (Fire et al. supra).
  • RNA interference small double- stranded interfering RNA
  • RNAi small double- stranded interfering RNA
  • RNAi is a post-transcription process, in which double-stranded RNA is introduced, and sequence-specific gene silencing results, though catalytic degradation of the targeted mRNA (see, e.g., Elbashir, S.M. et al, Nature 411 :494- 498 (2001); Lee, N.S., Nature Biotech. 19:500-505 (2002); and Lee, S-K. et al, Nature 7kf ⁇ /zcme 8(7):681-686 (2002).
  • RNAi is used routinely to investigate gene function in a high throughput fashion or to modulate gene expression in human diseases (Chi et al, Proc. Natl Acad. Sci. U.S.A., 100(1 i;:6343-6346 (2003)).
  • Introduction of long double stranded RNA leads to sequence-specific degradation of homologous gene transcripts.
  • the long double stranded RNA is metabolized to small 21-23 nucleotide siRNA (small interfering RNA).
  • siRNA small interfering RNA
  • the siRNA then binds to protein complex RISC (RNA-induced silencing complex) with dual function helicase.
  • the helicase has RNase activity and is able to unwind the RNA.
  • RNAi RNA-binding to a target. This results in sequence dependent degradation of cognate mRNA.
  • exogenous RNAi chemically synthesized or recombinantly produced RNAi can also be used in the compositions and methods of the invention.
  • the invention is directed to aptamers of ChTRase.
  • aptamers are macromolecules composed of nucleic acid (e.g., RNA, DNA) that bind tightly to a specific molecular target (e.g., ChTRase protein, ChTRase domains (e.g., CBP or catalytic domains), ChTRase polypeptide and/or ChTRase epitopes as described herein).
  • a specific molecular target e.g., ChTRase protein, ChTRase domains (e.g., CBP or catalytic domains), ChTRase polypeptide and/or ChTRase epitopes as described herein.
  • a particular aptamer may be described by a linear nucleotide sequence and an aptamer is typically about 15-60 nucleotides in length.
  • aptamers may be obtained for a wide array of molecular targets, including proteins and small molecules.
  • aptamers have very high affinities for their targets (e.g., affinities in the picomolar to low nanomolar range for proteins). Aptamers are chemically stable and can be boiled or frozen without loss of activity.
  • aptamers can be modified to dramatically reduce their sensitivity to degradation by enzymes in the blood.
  • modification of aptamers can also be used to alter their biodistribution or plasma residence time.
  • aptamers that can bind ChTRase or a fragment there of can be achieved through methods known in the art.
  • aptamers can be selected using the SELEX (Systematic Evolution of Ligands by Exponential Enrichment) method (Tuerk, C . , and Gold, L . , Science 249 : 505 -510 ( 1990)) .
  • a large library of nucleic acid molecules (e.g., 10 15 different molecules) is produced and/or screened with the target molecule (e.g., ChTRase protein, ChTRase domain (e.g., CBP or catalytic domain), ChTRase polypeptide and/or ChTRase epitopes as described herein).
  • the target molecule e.g., ChTRase protein, ChTRase domain (e.g., CBP or catalytic domain), ChTRase polypeptide and/or ChTRase epitopes as described herein.
  • the target molecule is allowed to incubate with the library of nucleotide sequences for a period of time.
  • Several methods can then be used to physically isolate the aptamer target molecules from the unbound molecules in the mixture and the unbound molecules can be discarded.
  • the aptamers with the highest affinity for the target molecule can then be purified away from the target molecule and amplified enzymatically to produce a new library of molecules that is substantially enriched for aptamers that can bind the target molecule.
  • the enriched library can then be used to initiate a new cycle of selection, partitioning, and amplification. After 5-15 cycles of this selection, partitioning and amplification process, the library is reduced to a small number of aptamers that bind tightly to the target molecule.
  • Individual molecules in the mixture can then be isolated, their nucleotide sequences determined, and their properties with respect to binding affinity and specificity measured and compared.
  • Isolated aptamers can then be further refined to eliminate any nucleotides that do not contribute to target binding and/or aptamer structure (i.e., aptamers truncated to their core binding domain). See Jayasena, S. D. Clin. Chem. 45:1628-1650 (1999) for review of aptamer technology.
  • the ChTRase antagonists of the invention are aptamers that have the binding specificity and/or functional activity described herein for the antibodies of the invention (see infra).
  • the present invention is drawn to aptamers that have the same or similar binding specificity as described herein for the antibodies of the invention (see, infra) (e.g., binding specificity for a mammalian ChTRase polypeptide, fragments of mammalian ChTRase polypeptide (e.g., ChTRase special domains), epitopic regions of mammalian ChTRase polypeptides (e.g., epitopic regions of ChTRase that are bound by the antibodies of the invention).
  • the aptamers of the invention can bind to a ChTRase polypeptide and inhibit one or more functions of the ChTRase polypeptide as described herein.
  • a ChTRase antagonist of the invention is an antibody.
  • antibody ChTRase antagonists specifically bind ChTRase.
  • antibody ChTRase antagonists that bind more then one chitinase.
  • Antibody antagonists of ChTRase are referred to herein as "ChTRase antibodies of the invention,” “ChTRase antibodies,” and like terms.
  • ChTRase antibodies of the invention include, but are not limited to, monoclonal antibodies, multispecif ⁇ c antibodies, human antibodies, humanized antibodies, camelised antibodies, chimeric antibodies, single-chain Fvs (scFv), disulf ⁇ de-linked Fvs (sdFv), Fab fragments, F (ab') fragments, and anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibodies include immunoglobulin molecules and immunologically active fragments of immunoglobulin molecules, i.e., molecules that contain an antigen binding site, these fragments may or may not be fused to another immunoglobulin domain including but not limited to, an Fc region or fragment thereof.
  • antibody and “antibodies” specifically include the antibodies described herein, full length antibodies and Fc-Fusions comprising Fc regions, or fragments thereof, fused to an immunologically active fragment of an immunoglobulin (e.g., a fragment that specifically binds a ChTRase) or to other proteins as described herein.
  • Fc fusions include but are not limited to, scFv-Fc fusions, variable region (e.g., VL and VH) -Fc fusions, scFv-scFv-Fc fusions.
  • Immunoglobulin molecules can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass.
  • the term "specifically binds to a ChTRase” and analogous terms refer to peptides, polypeptides, proteins, fusion proteins and antibodies or fragments thereof that specifically bind to ChTRase or a fragment thereof.
  • a peptide, polypeptide, protein, or antibody that specifically binds to ChTRase or a fragment thereof may bind to other peptides, polypeptides, or proteins with lower affinity as determined by, e.g., immunoassays, BIAcore, or other assays known in the art.
  • Antibodies or fragments that specifically bind to ChTRase or a fragment thereof may be cross-reactive with related antigens.
  • a peptide, polypeptide, protein, or antibody that specifically binds to the ChTRase may be cross-reactive to other chitinases. It is contemplated that antibodies or fragments that specifically bind to ChTRase or a fragment thereof preferentially bind ChTRase over other antigens.
  • the present invention specifically encompasses antibodies with multiple specificities (e.g., an antibody with specificity for two or more discrete antigens (reviewed in Cao et al., 2003, Adv Drug Deliv Rev 55:171-197; Hudson et al., 2003, Nat Med 1 :129-134)).
  • bispecific antibodies contain two different binding specificities fused together.
  • bispecific antibodies In the simplest case a bispecific antibody would bind to two adjacent epitopes on a single target antigen, such an antibody would not cross-react with other antigens (as described supra). Alternatively, bispecific antibodies can bind to two different antigens. Such an antibody specifically binds to two different molecules, but not to other unrelated molecules. In addition, an antibody that specifically binds ChTRase may cross-react with relatedchitinases. Another class of multispecific antibodies may recognize a shared subunit of multi-subunit complexes in the context of one or more specific complexes. It is also contemplated that ChTRase antibody of the invention may bind to a common epitope shared by more then one chitinase.
  • Antibodies or fragments that specifically bind to ChTRase or a fragment thereof can be identified, for example, by immunoassays, BIAcore, or other techniques known to those of skill in the art.
  • An antibody or fragment thereof binds specifically to ChTRase or a fragment thereof when it binds to ChTRase or a fragment thereof with higher affinity than to any cross-reactive antigen as determined using experimental techniques, such as radioimmunoassays (RIA) and enzyme-linked immunosorbent assays (ELISAs).
  • RIA radioimmunoassays
  • ELISAs enzyme-linked immunosorbent assays
  • ChTRase antagonists are small, engineered protein domains such as, for example, immuno-domains and/or monomer domains (see for example, U.S. Patent Publication Nos. 2003082630 and 2003157561.
  • Immuno-domains contain at least one complementarity determining region (CDR) of an antibody while monomer domains are based upon known naturally-occurring, non-antibody domain families, specifically protein extracellular domains, which contain conserved scaffold and variable binding sites, an example is the LDL receptor A domain which is involved in ligand binding.
  • CDR complementarity determining region
  • Such protein domains can correctly fold independently or with limited assistance from, for example, a chaperonin or the presence of a metal ion.
  • variable binding sites of the protein domains are randomized using various diversity generation methods such as, for example, random mutagenesis, site-specific mutagenesis, as well as by directed evolution methods, such as , for example, recursive error-prone PCR, recursive recombination and the like.
  • diversity generation methods see U.S. Patent Nos.5,811,238; 5,830,721; 5,834,252; PCT Publication Nos.
  • the mutagenized protein domains are then expressed using a display system such as, for example, phage display, which can generate a library of at least 10 10 variants and facilitate isolation of those protein domains with improved affinity and potency for an intended target by subsequent panning and screening.
  • a display system such as, for example, phage display, which can generate a library of at least 10 10 variants and facilitate isolation of those protein domains with improved affinity and potency for an intended target by subsequent panning and screening.
  • Patent Nos. 6,281,344; 6,194,550; 6,207,446; 6,214,553 and 6,258,558 Utilizing these methods a high diversity of engineered protein domains having sub-nM binding affinity (Kd) and blocking function (IC50) can be rapidly generated.
  • Kd binding affinity
  • IC50 blocking function
  • engineered protein domains can be linked together, using natural protein linkers of about 4-15 amino acids in length, to form a binding protein.
  • the individual domains can target a single type of protein or several, depending upon the use/disease indication.
  • the engineered protein domains have several advantages over other types of polypeptide molecules including small size (average 4.5kD per domain) which allows for good tissue penetration, high stability due to the requirement for disulfide bonds and low immunogenicity.
  • the engineered protein domains are non- glycosylated allowing for high yield production via bacterial expression systems and are protease resistant.
  • the serum half-life can readily be extended by binding the designing one domain to bind human serum IgG, human serum albumin or another prevalent target while the remaining domains bind to one or more desired therapeutic target.
  • the invention provides antibodies or fragments thereof which specifically bind ChTRase.
  • an antibody of the invention binds to ChTRase and antagonizes its activity.
  • a ChTRase antibody inhibits the chitinase activity of ChTRase.
  • a ChTRase antibody inhibits the ability of a ChTRase to mediate inflammatory responses including, but not limited to, and those mediated directly by a ChTRase, such as production of IL-8, MCP-I and MMP-9.
  • a ChTRase antibody when administered to a subject in need thereof, improves (e.g., reduces the severity and/or symptoms of) one or more clinical indicator, including but not limited to, cellular infiltration of the lung (e.g., increased eosinophils in bronchioalveolar ravage (BAL) fluid, increased lymphocytes in BAL fluid, increased total cells in BAL fluid), airway function, airway inflammation, airway remodeling (e.g., airway thickening, mucus metaplasia, epithelial hypertrophy and airway fibrosis), airway hyperresponsiveness (AHR), joint inflammation, connective tissue damage, bone damage, joint mobility. Additional, ChTRase activities which may be inhibited by a lung (e.g., increased eosinophils in bronchioalveolar ravage (BAL) fluid, increased lymphocytes in BAL fluid, increased total cells in BAL fluid), airway function, airway inflammation, airway remodeling (e.g.
  • ChTRase antibody of the invention described above. It will be understood by one of skill in the art that a ChTRase antibody may antagonize at least one activity of ChTRase in vivo and/or in vitro. In a specific embodiment, an antibody of the invention will have one or more characteristics of the rabbit polyclonal antibodies designated 208 or 657 as disclosed in PCT publication WO 2007027748.
  • a ChTRase antibody specifically binds the signal sequence of ChTRase.
  • a ChTRase antibody specifically binds the catalytic domain of ChTRase.
  • a ChTRase antibody specifically binds the chitin binding domain of ChTRase.
  • the present invention further encompasses ChTRase antibodies that have a high binding affinity for ChTRase.
  • a ChTRase antibody of the invention specifically binds to ChTRase with an association rate constant or k on rate of at least at least 5X 10 5 MV, at least at least at least IxIO 7 IVrV 1 , at least 5XlO 7 IVrV 1 , or at least IxIO 8 IVrV 1 .
  • a ChTRase antibody of the invention specifically binds to ChTRase with an association rate constant or k on rate of at least about IxIO 5 IVrV 1 , at least about 5X IO 5 JVTS "1 , at least about IxIO 6 IVrV 1 , at least about 5X lO 6 JVTV 1 , at least about Ix IO 7 IVTV 1 , at least about 5XlO 7 M "1 s "1 , or at least about IxIO 8 M 1 S 1 .
  • a ChTRase antibody specifically binds to ChTRase with a k on of at least 2XlO 5 M-V 1 , at least 5XlO 5 M-V 1 , at least IxIO 6 M-V 1 , at least at least Ix IO 7 M-V 1 , at least 5x lO 7 M -1 s -1 , or at least 1 x 10 8 M -1 S "1 .
  • a ChTRase antibody specifically binds to ChTRase with a k on of at least about 2xlO 5 M -1 s -1 , at least about 5XlO 5 M-V 1 , at least about IxIO 6 JVrV 1 , at least about 5X lO 6 JVTy 1 , at least about Ix 1O 7 JVTy 1 , at least about 5XlO 7 IVTy 1 , or at least about IxIO 8 IVTy 1 .
  • a ChTRase antibody of the invention specifically binds to ChTRase with a k o ff rate of less than 1 x 10 1 S -1 , less than 5 ⁇ 10 1 S -1 , less than Ix 10 " V 1 , less than 5xlO "2 s " ⁇ less than Ix 10 " V 1 , less than 5xlO "3 s " ⁇ less than Ix 10 " V 1 , less than less than lxl ⁇ "6 s " ⁇ less than less than Ix 10 " V 1 , less than 5x lO ⁇ 7 s ⁇ less than Ix 10 " V 1 , less than 5X10 ⁇ "1 , less than Ix 10 " V 1 , less than or less than Ix 10 -10-1 s -1 .
  • a ChTRase antibody of the invention specifically binds to ChTRase with a k off rate of less than about 1 x 10 "1 S “1 , less than about 5x 10 "1 S “1 , less than about 1 x 10 "2 S “1 , less than about 5XlO 2 S 1 , less than about Ix 10 "3 S 1 , less than about 5XlO 3 S 1 , less than about Ix 10 "4 S “1 , less than about 5XlO-V 1 , less than about Ix 10 "5 S “1 , less than about less than about less than about 5x less than about IxIO-V 1 , less than about 5xlO "7 s " ⁇ less than about IxIO-V 1 , less than about 5xl0 -8 s -1 , less than about IxIO-V 1 , less than about 5x 1O -9 S -1 , or less than about 1 XlO 10 1 S 1 .
  • a ChTRase antibody specifically binds to ChTRase with a koff, of less than 5XlO -4 S -1 , less than IxIO -5 S -1 , less than less than IxIO-V 1 , less than 5x l0 -6 s -1 , less than IxIO-V 1 , less than less than Ix 10 " V 1 , less than 5X lO-V 1 , less than IxI(TY 1 , less than 5Xl(TY 1 , or less than Ix 10 "10 S 1 .
  • a ChTRase antibody specifically binds to ChTRase with a koff, of less than about 5 ⁇ 10-V 1 , less than about 1x10 -5 S -1 , less than about 5xl0 -5 s -1 , less than about IxIO-V 1 , less than about 5xl0 -6 s -1 , less than about IxIO-V 1 , less than about 5xl0 -7 s -1 , less than about Ix IO-V 1 , less than about 5xl0 -8 s -1 , less than about Ix 10 "9 S "1 , less than about 5XlO-V 1 , or less than about Ix 10 "1 V 1 .
  • a ChTRase antibody of the invention specifically binds to ChTRase with an affinity constant or K a (k on /k off ) of at least 1x10 2 M "1 , at least
  • a ChTRase antibody of the invention specifically binds to ChTRase with an affinity constant or K 3 (k on /k off ) of at least about IxIO 2 M “1 , at least about 5XlO 2 M “1 , at least about IxIO 3 M “1 , at least about 5XlO 3 M “1 , at least about IxIO 4 M “1 , at least about 5XlO 4 M “1 , at least about IxIO 5 M “1 , at least about 5XlO 5 M “1 , at least about IxIO 6 M “1 , at least about at least about IxIO 7 M “1 , at least about 5XlO 7 M “1 , at least about IxIO 8 M “1 , at least about at least about IxIO 9 M “1 , at least about at least about IxIO 10 M “1 , at least about 5XlO 1 M “1 , at least about IxIO 11 M “1 , at least about 5
  • a ChTRase antibody specifically binds to ChTRase with a dissociation constant or K d (k off /k on ) of less than 1x10 2 M, less than 5x10 "2 M, less than 1 x 10 "3 M, less than 5 x 10 "3 M, less than 1 x 10 "4 M, less than 5 x 10 "4 M, less than Ix 10 "5 M, less than 5xlO "5 M, less than Ix 10 "6 M, less than 5x10 "6 M, less than Ix 10 "7 M, less than 5xlO "7 M, less than Ix 10 "8 M, less than 5x lO "8 M, less than Ix 10 "9 M, less than 5xlO "9 M, less than I x 10 "10 M, less than 5xl0 "10 M, less than Ix 10 "11 M, less than 5Xl(T 11 M, less than Ix 10 "12 M, less than 5xlO "
  • a ChTRase antibody specifically binds to ChTRase with a dissociation constant or Kd (koff/kon) of less than about Ix 10 "2 M, less than about 5xlO "2 M, less than about Ix 10 "3 M, less than about 5xlO "3 M, less than about Ix 10 "4 M, less than about 5xlO "4 M, less than about Ix 10 "5 M, less than about 5xlO "5 M, less than about Ix 10 "6 M, less than about 5x10 "6 M, less than about 1 x 10 "7 M, less than about 5 x 10 "7 M, less than about 1 x 10 "8 M, less than about 5xlO "8 M, less than about Ix 10 "9 M, less than about 5xlO "9 M, less than about Ix 10 "10 M, less than about 5xl0 "10 M, less than about Ix 10 "11 M, less than about 5X l(T 11 M, less than
  • a ChTRase antibody specifically binds to ChTRase with a minimal titer of at least 10OK, or at least 200K, or at least 300K, or at least 400 K, or at least 500 K, or at least 600 K, or at least 700 K, or at least 800 K, or at least 1 M, or at least 1.2 M, or at least 1.4 M, or at least 1.6 M, or at least 1.8 M, or at least 2.0 M, or at least 2.2 M, or at least 2.4 M, or at least 2.6 M, or at least 2.8 M, or at least 3.0 M.
  • a ChTRase antibody specifically binds to ChTRase with a minimal titer of at least about 10OK, or at least about 200K, or at least about 300K, or at least about 400 K, or at least about 500 K, or at least about 600 K, or at least about 700 K, or at least about 800 K, or at least about 1 M, or at least about 1.2 M, or at least about 1.4 M, or at least about 1.6 M, or at least about 1.8 M, or at least about 2.0 M, or at least about 2.2 M, or at least about 2.4 M, or at least about 2.6 M, or at least 2.8 about M, or at least 3.0 about M.
  • minimal titer refers to the maximum dilution of an antibody in solution (e.g., polyclonal rabbit serum or hybridoma cell supernatant) for which a signal 2 times background is seen by ELISA.
  • K and M refer to a dilution factor of 1,000 and 1,000,000, respectively (i.e., 400 K is equivalent to 400,000).
  • ChTRase antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homo log of ChTRase are included. Antibodies specific for ChTRase that also bind other polypeptides (and polypeptide fragments) with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity to ChTRase are also included in the present invention.
  • sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared.
  • the molecules are identical at that position.
  • the percent identity between the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity number of identical overlapping positions/total number of positions x 100%). In one embodiment, the two sequences are the same length.
  • the determination of percent identity between two sequences can also be accomplished using a mathematical algorithm.
  • a preferred, non- limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm of Karlin and Altschul, 1990, Proc. Nati. Acad. Sd. US.A.
  • BLAST protein searches can be performed with the XBLAST program parameters set, e.g., to score-50, wordlength3 to obtain amino acid sequences homologous to a protein molecule of the present invention.
  • Gapped BLAST can be utilized as described in Altschul et al, 1997, Nucleic Acids Res. 25: 3389-3402.
  • PSI-BLAST can be used to perform an iterated search which detects distant relationships between molecules (Id.).
  • the default parameters of the respective programs e.g., of XBLAST and NBLAST
  • Another preferred, non-limiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CA BIOS 4: 11-17.
  • the percent identity between two sequences can be determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.
  • the ChTRase antibodies of the present invention bind at least two chitinase-like proteins.
  • antibodies of the present invention bind to AMCase and ChTRase.
  • antibodies of the present invention bind to ChTRase and YLK-40.
  • antibodies of the present invention bind to ChTRase and YKL-39.
  • antibodies of the present invention bind to AMCase, ChTRase and YKL-40.
  • antibodies of the present invention bind to AMCase, ChTRase and YLK-39.
  • antibodies of the present invention bind to ChTRase, YKL-40 and YLK-39. In certain embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homo logs of human ChTRase proteins and the corresponding epitopes thereof.
  • ChTRase antibodies of the invention are chimeric antibodies.
  • the ChTRase antibodies of the invention or fragments thereof are human or humanized antibodies.
  • the present invention also encompasses variants of the ChTRase antibodies of the invention comprising one or more amino acid residue substitutions in the variable light (V L ) domain and/or variable heavy (V H ) domain.
  • the present invention also encompasses variants of the ChTRase antibodies of the invention with one or more additional amino acid residue substitutions in one or more V L CDRS and/or one or more V H CDRS.
  • the antibody generated by introducing substitutions in the V H domain, V H CDRS, V L domain and/or V L CDRs of the ChTRase antibodies of the invention can be tested in vitro and in vivo, for example, for its ability to bind to ChTRase (by, e.g., immunoassays including, but not limited to ELISA and BIAcore), or for its ability to antagonize one or more ChTRase activity.
  • CDRs residue numbers referred to herein are those of Kabat et al. (1991, NIH Publication 91-3242, National Technical Information Service, Springfield, VA). Specifically, residues 24-34 (CDRl), 50-56 (CDR2) and 89-97 (CDR3) in the light chain variable domain and 31-35 (CDRl), 50-65 (CDR2) and 95-102 (CDR3) in the heavy chain variable domain. Note that CDRs vary considerably from antibody to antibody (and by definition will not exhibit homology with the Kabat consensus sequences). Maximal alignment of framework residues frequently requires the insertion of "spacer" residues in the numbering system, to be used for the Fv region. It will be understood that the CDRs referred to herein are those of Kabat et al. supra. In addition, the identity of certain individual residues at any given Kabat site number may vary from antibody chain to antibody chain due to interspecies or allelic divergence.
  • Another embodiment of the present invention includes the introduction of conservative amino acid substitutions in any portion of a ChTRase antibody of the invention.
  • conservative amino acid substitution refers to amino acid substitutions that substitute functionally-equivalent amino acids.
  • Conservative amino acid changes result in silent changes in the amino acid sequence of the resulting peptide.
  • one or more amino acids of a similar polarity act as functional equivalents and result in a silent alteration within the amino acid sequence of the peptide.
  • Substitutions that are charge neutral and which replace a residue with a smaller residue may also be considered “conservative substitutions" even if the residues are in different groups (e.g., replacement of phenylalanine with the smaller isoleucine). Families of amino acid residues having similar side chains have been defined in the art. Several families of conservative amino acid substitutions are shown in Table 3.
  • Standard techniques known to those of skill in the art can be used to introduce mutations (e.g., additions, deletions, and/or substitutions) in the nucleotide sequence encoding a ChTRase antibody of the invention, including, for example, site-directed mutagenesis and PCR-mediated mutagenesis are routinely used to generate amino acid substitutions.
  • the derivatives include less than 25 amino acid substitutions, less than 20 amino acid substitutions, less than 15 amino acid substitutions, less than 10 amino acid substitutions, less than 5 amino acid substitutions, less than 4 amino acid substitutions, less than 3 amino acid substitutions, or less than 2 amino acid substitutions in the relative to the original ChTRase antibody.
  • the derivatives of a ChTRase antibody of the invention have conservative amino acid substitutions (e.g. supra) are made at one or more predicted non-essential amino acid residues (i.e., amino acid residues which are not critical for the antibody to specifically bind to ChTRase).
  • mutations can be introduced randomly along all or part of the coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for biological activity to identify mutants that retain activity. Following mutagenesis, the encoded antibody can be expressed and the activity of the antibody can be determined.
  • conservative amino acid substitution also refers to the use of amino acid analogs or variants.
  • Guidance concerning how to make phenotypically silent amino acid substitutions is provided in Bowie et al., "Deciphering the Message in Protein Sequences: Tolerance to Amino Acid Substitutions,” (1990, Science 247:1306-1310).
  • ChTRase antibodies of the invention may include, but are not limited to, synthetic antibodies, monoclonal antibodies, oligoclonal antibodies recombinantly produced antibodies, intrabodies, multispecific antibodies, bispecif ⁇ c antibodies, human antibodies, humanized antibodies, chimeric antibodies, synthetic antibodies, single-chain FvFcs (scFvFc), single-chain Fvs (scFv), and anti-idiotypic (anti-Id) antibodies.
  • antibodies used in the methods of the present invention include immunoglobulin molecules and immunologically active portions of immunoglobulin molecules.
  • the antibodies of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGi, IgG 2 , IgG 3 , IgG 4 , IgAi and IgA 2 ) or subclass of immunoglobulin molecule.
  • ChTRase antibodies of the invention may be from any animal origin including birds and mammals (e.g., human, murine, donkey, sheep, rabbit, goat, guinea pig, camel, horse, or chicken).
  • the antibodies are human or humanized monoclonal antibodies.
  • "human" antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from mice that express antibodies from human genes.
  • Antibodies like all polypeptides have an Isoelectric Point (pi), which is generally defined as the pH at which a polypeptide carries no net charge. It is known in the art that protein solubility is typically lowest when the pH of the solution is equal to the isoelectric point (pi) of the protein. It is possible to optimize solubility by altering the number and location of ionizable residues in the antibody to adjust the pi. For example the pi of a polypeptide can be manipulated by making the appropriate amino acid substitutions (e.g. , by substituting a charged amino acid such as a lysine, for an uncharged residue such as alanine).
  • amino acid substitutions of an antibody that result in changes of the pi of said antibody may improve solubility and/or the stability of the antibody.
  • amino acid substitutions would be most appropriate for a particular antibody to achieve a desired pi.
  • the pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see for example Bjellqvist et al., 1993, Electrophoresis 14:1023).
  • the pi of the ChTRase antibodies of the invention is higher then about 6.5, about 7.0, about 7.5, about 8.0, about 8.5, or about 9.0.
  • the pi of the ChTRase antibodies of the invention is higher then 6.5, 7.0, 7.5, 8.0, 8.5, or 9.0.
  • substitutions resulting in alterations in the pi of the ChTRase antibody of the invention will not significantly diminish its binding affinity for a ChTRase.
  • substitution(s) of the Fc region that result in altered binding to Fc ⁇ R may also result in a change in the pi.
  • substitution(s) of the Fc region are specifically chosen to effect both the desired alteration in Fc ⁇ R binding and any desired change in pi.
  • the pi value is defined as the pi of the predominant charge form.
  • the pi of a protein may be determined by a variety of methods including but not limited to, isoelectric focusing and various computer algorithms (see, e.g., Bjellqvist et al., 1993, Electrophoresis 14:1023).
  • the Tm of the Fab domain of an antibody can be a good indicator of the thermal stability of an antibody and may further provide an indication of the shelf- life.
  • a lower Tm indicates more aggregation/less stability, whereas a higher Tm indicates less aggregation/ more stability.
  • antibodies having higher Tm are preferable.
  • the Fab domain of a ChTRase antibody has a Tm value higher than at least 50 0 C, 55°C, 60 0 C, 65°C, 70 0 C, 75°C, 80 0 C, 85°C, 90 0 C, 95°C, 100 0 C, 105 0 C, 110 0 C, 115°C or 120 0 C.
  • the Fab domain of an ChTRase antibody has a Tm value higher than at least about 50 0 C, about 55°C, about 60 0 C, about 65°C, about 70 0 C, about 75°C, about 80 0 C, about 85°C, about 90 0 C, about 95°C, about 100 0 C, about 105 0 C, about 110 0 C, about 115°C or about 120 0 C.
  • Thermal melting temperatures (Tm) of a protein domain ⁇ e.g., a Fab domain) can be measured using any standard method known in the art, for example, by differential scanning calorimetry (see, e.g., Vermeer et al., 2000, Biophys. J. 78:394-404; Vermeer et al., 2000, Biophys. J. 79: 2150-2154).
  • ChTRase antibodies of the invention may be monospecific, bispecific, trispecific, or have greater multispecificity. Multispecific antibodies may specifically bind to different epitopes of desired target molecule or may specifically bind to both the target molecule as well as a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g., International Publication Nos. WO 94/04690; WO 93/17715; WO 92/08802; WO 91/00360; and WO 92/05793; Tutt, et al., 1991, J. Immunol. 147:60-69; U.S. Patent Nos.
  • one of the binding specificities is for a ChTRase
  • the other one is for any other antigen ⁇ i.e., another ChTRase, a signaling or effector molecule, etc.).
  • Multispecific antibodies have binding specificities for at least two different antigens. While such molecules normally will only bind two antigens ⁇ i.e. bispecific antibodies, BsAbs), antibodies with additional specificities such as trispecific antibodies are encompassed by the instant invention. Examples of BsAbs include without limitation those with one arm directed against ChTRase and the other arm directed against any other antigen. Methods for making bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, where the two chains have different specificities (Millstein et al.,1983, Nature, 305:537-539).
  • a Di-diabody Methods for producing a Di-diabody are known in the art (see e.g., Lu et al., 2003, J Immunol Methods 279:219-32; Marvin et al., 2005, Acta Pharmacolical Sinica 26:649).
  • antibody variable domains with the desired binding specificities are fused to immunoglobulin constant domain sequences.
  • the fusion preferably is with an immunoglobulin heavy chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
  • the first heavy-chain constant region (CHl) containing the site necessary for light chain binding is present in at least one of the fusions.
  • DNAs encoding the immunoglobulin heavy chain fusions and, if desired, the immunoglobulin light chain are inserted into separate expression vectors, and are co-transfected into a suitable host organism.
  • This provides for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields. It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when, the expression of at least two polypeptide chains in equal ratios results in high yields or when the ratios are of no particular significance.
  • the bispecific antibodies are composed of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm ⁇ e.g. ,
  • a pair of antibody molecules can be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture.
  • the preferred interface comprises at least a part of the CH3 domain of an antibody constant domain.
  • one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
  • Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This provides a mechanism for increasing the yield of the heterodimer over other unwanted end-products such as homodimers.
  • Bispecific antibodies include cross-linked or "heteroconjugate" antibodies.
  • one of the antibodies in the heteroconjugate can be coupled to avidin, the other to biotin.
  • Such antibodies have, for example, been proposed to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for treatment of HIV infection (WO 91/00360, WO 92/200373, and EP 03089.
  • Heteroconjugate antibodies may be made using any convenient cross-linking methods. Suitable cross-linking agents are well known in the art, and are disclosed in U.S. Pat. No. 4,676,980, along with a number of cross-linking techniques.
  • Antibodies with more than two valencies incorporating at least one hinge modification of the invention are contemplated.
  • trispecific antibodies can be prepared. See, e.g., Tutt et al. J. Immunol. 147: 60 (1991).
  • the ChTRase antibodies of the invention encompass single domain antibodies, including camelized single domain antibodies (see e.g., Muyldermans et al., 2001, Trends Biochem. ScL 26:230; Nuttall et al., 2000, Cur. Pharm. Biotech. 1 :253; Reichmann and Muyldermans, 1999, J. Immunol. Meth. 231 :25; International Publication Nos.
  • oligoclonal antibodies refers to a predetermined mixture of distinct monoclonal antibodies. See, e.g., PCT publication WO 95/20401; U.S. Pat. Nos. 5,789,208 and 6,335,163. Preferably oligoclonal antibodies consist of a predetermined mixture of antibodies against one or more epitopes are generated in a single cell.
  • oligoclonal antibodies comprise a plurality of heavy chains capable of pairing with a common light chain to generate antibodies with multiple specificities (e.g., PCT publication WO 04/009618). Oligoclonal antibodies are particularly useful when it is desired to target multiple epitopes on a single target molecule (e.g., ChTRase). Those skilled in the art will know or can determine what type of antibody or mixture of antibodies is applicable for an intended purpose and desired need.
  • antibody-like and antibody-domain fusion proteins may also be ChTRase antagonists of the present invention.
  • An antibody-like molecule is any molecule that has been generated with a desired binding property, see, e.g., PCT Publication Nos. WO 04/044011; WO 04/058821; WO 04/003019 and WO 03/002609.
  • Antibody-domain fusion proteins may incorporate one or more antibody domains such as the Fc domain or the variable domain.
  • heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab) 2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof.
  • Fab fragment, Fd fragment, Fv fragment, F(ab) 2 fragment a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof.
  • a large number of antibody- domain molecules are known in the art including, but not limited to, diabodies (dsFv) 2 (Bera et al, 1998, J. MoI. Biol.
  • minibodies homodimers of scFv-CH3 fusion proteins
  • minibodies homodimers of scFv-CH3 fusion proteins
  • tetravalent di-diabody Li et al., 2003 J. Immunol. Methods 279:219-32
  • tetravalent bi-specific antibodies called Bs(scFv)4-IgG (Zuo et al., 2000, Protein Eng. 13:361-367).
  • Fc domain fusions combine the Fc region of an immunoglobulin with a fusion partner which in general can be an protein, including, but not limited to, a ligand, an enzyme, the ligand portion of a receptor, an adhesion protein, or some other protein or domain.
  • a fusion partner which in general can be an protein, including, but not limited to, a ligand, an enzyme, the ligand portion of a receptor, an adhesion protein, or some other protein or domain.
  • antibodies of the present invention also encompass ChTRase antibodies that have half-lives (e.g., serum half- lives) in a mammal, preferably a human, of greater than 5 days, greater than 10 days, greater than 15 days, preferably greater than 20 days, greater than 25 days, greater than 30 days, greater than 35 days, greater than 40 days, greater than 45 days, greater than 2 months, greater than 3 months, greater than 4 months, or greater than 5 months.
  • ChTRase antibodies that have half-lives e.g., serum half- lives
  • serum half- lives in a mammal, preferably a human, of greater than about 5 days, greater than about 10 days, greater than about 15 days, preferably greater than about 20 days, greater than about 25 days, greater than about 30 days, greater than about 35 days, greater than about 40 days, greater than about 45 days, greater than about 2 months, greater than about 3 months, greater than about 4 months, or greater than about 5 months.
  • the increased half- lives of the antibodies of the present invention in a mammal, preferably a human results in a higher serum titer of said antibodies or antibody fragments in the mammal, and thus, reduces the frequency of the administration of said antibodies or antibody fragments and/or reduces the concentration of said antibodies or antibody fragments to be administered.
  • Antibodies having increased in vivo half- lives can be generated by techniques known to those of skill in the art. For example, antibodies with increased in vivo half- lives can be generated by modifying (e.g., substituting, deleting or adding) amino acid residues identified as involved in the interaction between the Fc domain and the FcRn receptor (see, e.g., International Publication Nos. WO 97/34631; WO 04/029207; U.S. 6,737056 and U.S. Patent Publication No. 2003/0190311).
  • the ChTRase antibodies of the invention may be chemically modified (e.g. , one or more chemical moieties can be attached to the antibody) or be modified to alter its glycosylation, again to alter one or more functional properties of the antibody.
  • the glycosylation of the ChTRase antibodies of the invention is modified.
  • an aglycoslated antibody can be made (i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen.
  • carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • a ChTRase antibody can be made that has an altered type of glycosylation, such as a hypofucosylated antibody having reduced amounts of fucosyl residues or an antibody having increased bisecting GIcNAc structures. Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, RX. et al.
  • the glycosylation of a ChTRase antibody of the invention is modified.
  • an aglycoslated antibody can be made ⁇ i.e., the antibody lacks glycosylation).
  • Glycosylation can be altered to, for example, increase the affinity of the antibody for a target antigen.
  • Such carbohydrate modifications can be accomplished by, for example, altering one or more sites of glycosylation within the antibody sequence.
  • one or more amino acid substitutions can be made that result in elimination of one or more variable region framework glycosylation sites to thereby eliminate glycosylation at that site.
  • Such aglycosylation may increase the affinity of the antibody for antigen.
  • Such an approach is described in further detail in U.S. Patent Nos. 5,714,350 and 6,350,861.
  • a ChTRase antibody can be made that has an altered type of glycosylation, such as a hypofucosylated ChTRase antibody having reduced amounts of fucosyl residues or a ChTRase antibody having increased bisecting GIcNAc structures.
  • Such altered glycosylation patterns have been demonstrated to increase the ADCC ability of antibodies.
  • Such carbohydrate modifications can be accomplished by, for example, expressing the antibody in a host cell with altered glycosylation machinery. Cells with altered glycosylation machinery have been described in the art and can be used as host cells in which to express recombinant antibodies of the invention to thereby produce an antibody with altered glycosylation. See, for example, Shields, R.L.
  • the ChTRase antibodies of the present invention may comprise modifications/substations and/or novel amino acids within their Fc domains such as, for example, those disclosed in Ghetie et al, 1997, Nat Biotech. 15:637-40; Duncan et al, 1988, Nature 332:563-564; Lund et al, 1991, J.
  • ChTRase antibodies of the invention comprising modifications/substitutions and/or novel amino acid residues in their Fc regions can be generated by numerous methods well known to one skilled in the art. Non-limiting examples include, isolating antibody coding regions (e.g., from hybridoma) and making one or more desired substitutions in the Fc region of the isolated antibody coding region. Alternatively, the variable regions of a ChTRase antibody may be subcloned into a vector encoding an Fc region comprising one or modifications/substations and/or novel amino acid residues. 4.3 Antibody Conjugates And Derivatives
  • ChTRase antibodies of the invention include derivatives that are modified (i.e., by the covalent attachment of any type of molecule to the antibody such that covalent attachment).
  • the antibody derivatives include antibodies that have been modified, e.g. , by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to, specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
  • Antibodies or fragments thereof with increased in vivo half-lives can be generated by attaching to said antibodies or antibody fragments polymer molecules such as high molecular weight polyethyleneglycol (PEG).
  • PEG polymer molecules
  • PEG can be attached to said antibodies or antibody fragments with or without a multifunctional linker either through site-specific conjugation of the PEG to the N- or C- terminus of said antibodies or antibody fragments or via epsilon-amino groups present on lysine residues. Linear or branched polymer derivatization that results in minimal loss of biological activity will be used.
  • the degree of conjugation will be closely monitored by SDS-PAGE and mass spectrometry to ensure proper conjugation of PEG molecules to the antibodies.
  • Unreacted PEG can be separated from antibody-PEG conjugates by, e.g., size exclusion or ion-exchange chromatography.
  • antibodies can be conjugated to albumin in order to make the antibody or antibody fragment more stable in vivo or have a longer half life in vivo.
  • the techniques are well known in the art, see e.g. , International Publication Nos. WO 93/15199, WO 93/15200, and WO 01/77137; and European Patent No. EP 413622.
  • the present invention encompasses the use of antibodies or fragments thereof conjugated or fused to one or more moieties, including but not limited to, peptides, polypeptides, proteins, fusion proteins, nucleic acid molecules, small molecules, mimetic agents, synthetic drugs, inorganic molecules, and organic molecules.
  • the present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least 10, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90 or at least 100 amino acids) to generate fusion proteins.
  • the present invention encompasses the use of antibodies or fragments thereof recombinantly fused or chemically conjugated (including both covalent and non-covalent conjugations) to a heterologous protein or polypeptide (or fragment thereof, preferably to a polypeptide of at least about 10, at least about 20, at least about 30, at least about 40, at least about 50, at least about 60, at least about 70, at least about 80, at least about 90 or at least about 100 amino acids) to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • antibodies may be used to target heterologous polypeptides to particular cell types, either in vitro or in vivo, by fusing or conjugating the antibodies to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to heterologous polypeptides may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., International publication No. WO 93/21232; European Patent No. EP 439,095; Naramura et al, 1994, Immunol. Lett. 39:91-99; U.S. Patent No. 5,474,981; Gillies et al, 1992, PNAS 89:1428-1432; and Fell et al., 1991, J. Immunol. 146:2446-2452.
  • the present invention further includes formulations comprising heterologous proteins, peptides or polypeptides fused or conjugated to antibody fragments.
  • the heterologous polypeptides may be fused or conjugated to a Fab fragment, Fd fragment, Fv fragment, F(ab)2 fragment, a VH domain, a VL domain, a VH CDR, a VL CDR, or fragment thereof.
  • Methods for fusing or conjugating polypeptides to antibody portions are well known in the art. See, e.g., U.S. Patent Nos. 5,336,603, 5,622,929, 5,359,046, 5,349,053, 5,447,851, and 5,112,946; European Patent Nos.
  • EP 307,434 and EP 367,166 International publication Nos. WO 96/04388 and WO 91/06570; Ashkenazi et al., 1991, Proc. Natl. Acad. Sci. USA 88: 10535-10539; Zheng et al., 1995, J. Immunol. 154:5590-5600; and ViI et al., 1992, Proc. Natl. Acad. Sci. USA 89:11337- 11341.
  • DNA shuffling may be employed to alter the activities of antibodies of the invention or fragments thereof ⁇ e.g. , antibodies or fragments thereof with higher affinities and lower dissociation rates). See, generally, U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al., 1997, Curr.
  • Antibodies or fragments thereof, or the encoded antibodies or fragments thereof, may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • One or more portions of a polynucleotide encoding an antibody or antibody fragment, which portions specifically bind to a ChTRase may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the antibodies or fragments thereof can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the hemagglutinin "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., 1984, Cell 37:767) and the "flag" tag.
  • ChTRase antibodies of the present invention or analogs or derivatives thereof are conjugated to a diagnostic or detectable agent. Such antibodies can be useful for monitoring or prognosing the development or progression of a cancer as part of a clinical testing procedure, such as determining the efficacy of a particular therapy.
  • Such diagnosis and detection can be accomplished by coupling the antibody to detectable substances including, but not limited to various enzymes, such as but not limited to horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase; prosthetic groups, such as but not limited to streptavidinlbiotin and avidin/biotin; fluorescent materials, such as but not limited to, umbelliferone, fluorescein, fluorescein isothiocynate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin; luminescent materials, such as but not limited to, luminol; bioluminescent materials, such as but not limited to, luciferase, luciferin, and aequorin; radioactive materials, such as but not limited to iodine (1311, 1251, 1231, 1211,), carbon (14C), sulfur (35S),
  • the present invention further encompasses uses of ChTRase antibodies of the invention or fragments thereof conjugated to a therapeutic agent.
  • An antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., alpha-emitters.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
  • Examples include paclitaxel, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, epirubicin, and cyclophosphamide and analogs or homologs thereof.
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6- mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BCNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cisdichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g.
  • a ChTRase antibody or fragment thereof may be conjugated to a therapeutic agent or drug moiety that modifies a given biological response.
  • Therapeutic agents or drug moieties are not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, Onconase (or another cytotoxic RNase), pseudomonas exotoxin, cholera toxin, or diphtheria toxin; a protein such as tumor necrosis factor, ⁇ -interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, an apoptotic agent, e.g. , TNF- ⁇ , TNF- ⁇ , AIM I (see, International Publication No. WO 97/33899), AIM II (see, International Publication No. WO 97/34911), Fas Ligand (Takahashi et al, 1994, J.
  • VEGI vascular endothelial growth factor
  • a thrombotic agent or an anti- angiogenic agent e.g., angiostatin or endostatin
  • a biological response modifier such as, for example, a lymphokine (e.g., interleukin-1 ("IL-I”), interleukin-2 ("IL-2”), interleukin-6 (“IL-6”), granulocyte macrophage colony stimulating factor (“GM-CSF”), and granulocyte colony stimulating factor (“G-CSF”)), or a growth factor (e.g., growth hormone (“GH”)).
  • IL-I interleukin-1
  • IL-2 interleukin-2
  • IL-6 interleukin-6
  • G-CSF granulocyte macrophage colony stimulating factor
  • G-CSF granulocyte colony stimulating factor
  • GH growth hormone
  • a ChTRase antibody can be conjugated to therapeutic moieties such as a radioactive materials or macrocyclic chelators useful for conjugating radiometal ions (see above for examples of radioactive materials).
  • the macrocyclic chelator is 1,4,7, 10-tetraazacyclododecane-N,N',N",N"-tetraacetic acid (DOTA) which can be attached to the antibody via a linker molecule.
  • linker molecules are commonly known in the art and described in Denardo et al., 1998, Clin Cancer Res. 4:2483; Peterson et al, 1999, Bioconjug. Chem. 10:553; and Zimmerman et al., 1999, Nucl. Med. Biol. 26:943.
  • Moieties can be conjugated to antibodies by any method known in the art, including, but not limited to aldehyde/Schiff linkage, sulphydryl linkage, acid-labile linkage, cis-aconityl linkage, hydrazone linkage, enzymatically degradable linkage (see generally Garnett, 2002, Adv Drug Deliv Rev 53:171).
  • Techniques for conjugating therapeutic moieties to antibodies are well known, see, e.g., Arnon et al., "Monoclonal Antibodies For Immunotargeting Of Drugs In Cancer Therapy", in Monoclonal Antibodies And Cancer Therapy, Reisfeld et al. (eds.), pp. 243-56 (Alan R. Liss, Inc. 1985); Hellstrom et al., "Antibodies For Drug
  • an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980.
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the therapeutic moiety or drug conjugated to an antibody or fragment thereof that specifically binds to a ChTRase of the invention should be chosen to achieve the desired prophylactic or therapeutic effect(s) for a particular disorder in a subject.
  • a clinician or other medical personnel should consider the following when deciding on which therapeutic moiety or drug to conjugate to an antibody or fragment thereof that specifically binds to a ChTRase: the nature of the disease, the severity of the disease, and the condition of the subject.
  • ChTRase antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or by recombinant expression techniques.
  • Polyclonal antibodies to a ChTRase can be produced by various procedures well known in the art.
  • a ChTRase or immunogenic fragments thereof can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for a ChTRase.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-CeIl Hybridomas 563-681 (Elsevier, N.Y., 1981).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • mice can be immunized with a ChTRase or a domain thereof (e.g. , the extracellular domain) and once an immune response is detected, e.g. , antibodies specific for a ChTRase are detected in the mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • a ChTRase or a domain thereof e.g. , the extracellular domain
  • an immune response e.g. , antibodies specific for a ChTRase are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • a RIMMS (repetitive immunization, multiple sites) technique can be used to immunize an animal (Kilpatrick et al., 1997, Hybridoma 16:381-9, incorporated herein by reference in its entirety). Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention. Ascites fluid, which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • monoclonal antibodies can be generated by culturing a hybridoma cell secreting an antibody, wherein the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with a ChTRase or immunogenic fragments thereof, with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a ChTRase.
  • Antibody fragments that recognize specific a ChTRase epitopes may be generated by any technique known to those of skill in the art.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHl domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles that carry the polynucleotide sequences encoding them.
  • DNA sequences encoding VH and VL domains are amplified from animal cDNA libraries (e.g., human or murine cDNA libraries of lymphoid tissues).
  • the DNA encoding the VH and VL domains are recombined together with an scFv linker by PCR and cloned into a phagemid vector (e.g., p CANTAB 6 or pComb 3 HSS).
  • the vector is electroporated in E. coli and the E. coli is infected with helper phage.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 and the VH and VL domains are usually recombinantly fused to either the phage gene III or gene VIII.
  • Phage expressing an antigen binding domain that binds to the a ChTRase epitope of interest can be selected or identified with antigen, e.g. , using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman et al., 1995, J. Immunol. Methods 182:41-50; Ames et al., 1995, J. Immunol.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described below.
  • PCR primers including VH or VL nucleotide sequences, a restriction site, and a flanking sequence to protect the restriction site can be used to amplify the VH or VL sequences in scFv clones.
  • VH constant region e.g. , the human gamma constant
  • VL constant region e.g. , human kappa or lamba constant regions.
  • the constant region is an Fc region containing at least one high effector function amino acid.
  • the vectors for expressing the VH or VL domains comprise a promoter, a secretion signal, a cloning site for both the variable and constant domains, as well as a selection marker such as neomycin.
  • the VH and VL domains may also be cloned into one vector expressing the desired constant regions.
  • the heavy chain conversion vectors and light chain conversion vectors are then co-transfected into cell lines to generate stable or transient cell lines that express full-length antibodies, e.g., IgG, using techniques known to those of skill in the art.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different immunoglobulin molecules.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, 1985, Science 229:1202; Oi et al, 1986, BioTechniques 4:214; Gillies et al., 1989, J. Immunol. Methods 125:191-202; and U.S. Patent Nos. 5,807,715, 4,816,567, 4,8 16397, and 6,311,415
  • human or chimeric antibodies For some uses, including in vivo use of antibodies in humans and in vitro detection assays, it may be preferable to use human or chimeric antibodies. Completely human antibodies are particularly desirable for therapeutic treatment of human subjects.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT Publication Nos. WO 98/46645, WO 98/50433, WO 98/24893, WO98/16654, WO 96/34096, WO 96/33735, and WO 91/10741.
  • a humanized antibody is an antibody or its variant or fragment thereof which is capable of binding to a predetermined antigen and which comprises a framework region having substantially the amino acid sequence of a human immunoglobulin and a CDR having substantially the amino acid sequence of a non-human immunoglobulin.
  • a humanized antibody comprises substantially all of at least one, and typically two, variable domains (Fab, Fab', F(ab')2, Fabc, Fv) in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin (i.e., donor antibody) and all or substantially all of the framework regions are those of a human immunoglobulin consensus sequence.
  • a humanized antibody also comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin.
  • Fc immunoglobulin constant region
  • the antibody will contain both the light chain as well as at least the variable domain of a heavy chain.
  • the antibody also may include the CHl, hinge, CH2, CH3, and CH4 regions of the heavy chain.
  • the humanized antibody can be selected from any class of immunoglobulins, including IgM, IgG, IgD, IgA and IgE, and any isotype, including IgGl, IgG2, IgG3 and lgG4.
  • the constant domain is a complement fixing constant domain where it is desired that the humanized antibody exhibit cytotoxic activity, and the class is typically IgG.sub.l. Where such cytotoxic activity is not desirable, the constant domain may be of the IgG.sub.2 class.
  • the humanized antibody may comprise sequences from more than one class or isotype, and selecting particular constant domains to optimize desired effector functions is within the ordinary skill in the art.
  • the framework and CDR regions of a humanized antibody need not correspond precisely to the parental sequences, e.g. , the donor CDR or the consensus framework may be mutagenized by substitution, insertion or deletion of at least one residue so that the CDR or framework residue at that site does not correspond to either the consensus or the import antibody.
  • the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences. In another embodiment at least 90% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences. In a further embodiment, greater than 95% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences. In yet another embodiment, at least about 75% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences. In a further embodiment at least about 90% of the humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences.
  • humanized antibody residues will correspond to those of the parental framework region (FR) and CDR sequences.
  • Humanized antibody can be produced using variety of techniques known in the art, including but not limited to, CDR-grafting (European Patent No. EP 239,400; International Publication No. WO 91/09967; and U.S.
  • framework residues in the framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No. 5,585,089; and Riechmann et al., 1988, Nature 332:323).
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non- functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination.
  • homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g. , a ChTRase or immunogenic fragments thereof.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Lonberg and Huszar (1995, Int. Rev. Immunol.
  • the antibodies of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" a ChTRase using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, 1989, FASEB J. 7(5): 437-444; and
  • antibodies of the invention which bind to and competitively inhibit the binding of a ChTRase (as determined by assays well known in the art and disclosed infra) to its ligands can be used to generate anti-idiotypes that "mimic" a ChTRase binding domains and, as a consequence, bind to and neutralize a ChTRase and/or its ligands.
  • Such neutralizing anti-idiotypes or Fab fragments of such antiidiotypes can be used in therapeutic regimens to neutralize a ChTRase.
  • the invention provides methods employing the use of polynucleotides comprising a nucleotide sequence encoding an antibody of the invention or a fragment thereof.
  • the nucleotide sequence encoding an antibody that specifically binds a ChTRase is obtained and used to generate the ChTRase antibodies of the invention.
  • the nucleotide sequence can be obtained from sequencing hybridoma clone DNA. If a clone containing a nucleic acid encoding a particular antibody or an epitope-binding fragment thereof is not available, but the sequence of the antibody molecule or epitope- binding fragment thereof is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g.
  • the nucleotide sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, Or example, the techniques described in Current Protocols in Molecular Biology, F.M. Ausubel et al, ed., John Wiley & Sons (Chichester, England, 1998); Molecular Cloning: A Laboratory Manual, 3rd Edition, J. Sambrook et al., ed., Cold Spring Harbor Laboratory Press (Cold Spring Harbor, NY, 2001); Antibodies: A Laboratory Manual, E. Harlow and D.
  • one or more of the CDRs is inserted within framework regions using routine recombinant DNA techniques.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al, 1998, J. MoL Biol. 278: 457-479 for a listing of human framework regions).
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds to a ChTRase.
  • one or more amino acid substitutions may be made within the framework regions, and, in yet another embodiment, the amino acid substitutions improve binding of the antibody to its antigen.
  • Such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding a ChTRase antibody of the invention and fragments thereof.
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, to polynucleotides that encode a ChTRase antibody.
  • the antibody binds specifically to a polypeptide having the amino acid sequence of human or mouse chitotriosidase.
  • an anti-ChTRase antibody of the invention binds to ChTRase and also may bind to other polypeptides (and polypeptide fragments) with at least 95%, at least 90%, at least
  • ChTRase 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% amino acid sequence identity to ChTRase.
  • stringent hybridization conditions is intended overnight incubation at 42. degree. C. in a solution comprising: 50% formamide, 5.times.SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5 times. Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.l.times.SSC at about 65. degree. C.
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art.
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al., BioTechniques 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligating of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be chemically synthesized or obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody.
  • a suitable source e.g., an antibody cDNA library
  • nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al., 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
  • the amino acid sequence of the heavy and/or light chain variable domains of the antibodies of the invention may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well known in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability .
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al., J. MoI. Biol. 278: 457-479 (1998) for a listing of human framework regions).
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds. Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art. [0193] In addition, techniques developed for the production of "chimeric antibodies"
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
  • a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof (preferably containing the heavy or light chain variable domain), of the invention has been obtained, the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • Such vectors may include the nucleotide sequence encoding the constant region of the antibody molecule (see, e.g., PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Pat. No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, or a single chain antibody of the invention, operably linked to a heterologous promoter.
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
  • These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast ⁇ e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors ⁇ e.g., baculovirus) containing antibody coding sequences; plant cell systems infected with recombinant virus expression vectors (e.g., cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV) or transformed with recombinant plasmid expression vectors (e.g., Ti plasmid) containing antibody coding sequences; or mammalian cell systems (e.g., COS, CHO, BHK, 293, NSO, 3T3, PerC6 cells) harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g., metallothione
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
  • mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al., Gene 45:101 (1986); Cockett et al., Bio/Technology 8:2 (1990)).
  • U.S. patents 5827739, 5879936, 5981216, and 5658759 are examples of cells.
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
  • vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al., EMBO J. 2:1791 (1983)), in which the antibody coding sequence may be ligated individually into the vector in frame with the lacZ coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, Nucleic Acids Res.
  • pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • AcNPV is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non- essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence.
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a nonessential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g., see Logan & Shenk, Proc. Natl.
  • Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al., Methods in Enzymol. 153:51-544 (1987)).
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g., cleavage) of protein products may be important for the function of the protein.
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed.
  • eukaryotic host cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used.
  • Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeIa, COS, MDCK, 293, 3T3, W138, NSO, Per.C ⁇ and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell lines such as, for example, CRL7030 and Hs578Bst.
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al., Cell 11 :223 (1977)), hypoxanthine- guanine phosphoribosyltransferase (Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA 48:202 (1992)), and adenine phosphoribosyltransferase (Lowy et al., Cell 22:817 (1980)) genes can be employed in tk-, hgprt- or aprt-cells, respectively.
  • antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al., Proc Natl. Acad. Sci. USA 77:357 (1980); O'Hare et al., Proc. Natl. Acad. Sci. USA 78:1527 (1981)); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, Proc. Natl. Acad. Sci.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplif ⁇ able
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et al., MoI. Cell. Biol. 3:257 (1983)).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes, and is capable of expressing, both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, Nature 322:562 (1986); Kohler, Proc. Natl. Acad. Sci. USA 77:2197 (1980)).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography ⁇ e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the antibodies of the present invention or fragments thereof can be fused to heterologous polypeptide sequences described herein or otherwise known in the art, to facilitate purification.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitate purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, Calif, 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al., Cell 11:161 (1984)) and the "flag" tag. 4.7 Methods of Treatment
  • ChTRase antagonists can be utilized for the prevention, management, treatment or amelioration of COPD or one or more symptoms thereof and/or the inhibition of ChTRase mediated inflammation.
  • the present invention provides methods (referred to herein as "method(s) of the invention") for use of the ChTRase antagonists of the invention for the prevention, management, treatment or amelioration of COPD or one or more symptoms thereof and/or the inhibition of ChTRase mediated inflammation.
  • the present invention encompasses a method of treating COPD or one or more symptoms thereof and/or the inhibition of ChTRase mediated inflammation comprising administering to a subject a ChTRase antagonist.
  • the methods of the invention comprise the administration to a subject of an effective amount of one or more ChTRase antagonist alone or in combination with other prophylactic or therapeutic agents.
  • a subject is a mammal. In another embodiment, a subject is a human.
  • the methods of the present invention are useful for the prevention, management, treatment or amelioration of an inflammatory condition, disease or disorder including, but are not limited to, interstitial lung disease (ILD), pulmonary fibrosis, bronchitis, chronic obstructive pulmonary disease (COPD), pneumonia, pneumonitis, acute respiratory distress syndrome (ARDS), severe acute respiratory syndrome (SARS), sarcoidosis, cystic fibrosis (CF), emphysema, asthma, smoker's cough, allergy, allergic rhinitis, sinusitis, Paget's disease, abnormal bone remodeling, osteoporosis, Gorham-Stout syndrome, osteoarthritis, rheumatoid arthritis, psoriatic arthritis and brittle bone disease.
  • ILD interstitial lung disease
  • pulmonary fibrosis bronchitis
  • COPD chronic obstructive pulmonary disease
  • COPD chronic obstructive pulmonary disease
  • pneumonia pneumonitis
  • ARDS acute respiratory distress syndrome
  • the methods of the invention comprise the administration of an effective amount of one or more ChTRase antagonist, to a subject in need thereof, resulting in an improvement (e.g., a reduction in severity) in one or more of the changes associated with inflammatory lung disease including, but not limited to, tissue inflammation, increased lung volume, increased eosinophils in bronchioalveolar ravage (BAL) fluid, increased lymphocytes in BAL fluid, increased total cells in BAL fluid, increased alveolus size, increased deposition of crystals comprising chitinase-like molecules in lung tissue, increased airway resistance, increased mucus metaplasia, increased mucin expression, increased parenchymal fibrosis, increased airway remodeling (e.g., airway thickening, mucus metaplasia, epithelial hypertrophy and airway fibrosis), increased subepithelial fibrosis, increased collagen deposition in airway tissue, epithelial hypertrophy in the lung
  • the methods of the invention comprise the administration of an effective amount of one or more ChTRase antagonist, to a subject in need thereof, resulting in a decrease or inhibition of the ability of a ChTRase to mediated inflammatory responses including, but not limited to, those mediated directly by a ChTRase, such as production of MCP-I, IL-8, and MMP-9.
  • the methods of the invention comprise the administration of an effective amount of one or more ChTRase antagonist resulting in a decrease or inhibition of one or more ChTRase activity including, but not limited to, chitino lytic activity, saccharide (e.g., GIcNAc) binding activity, chemotatic activity, receptor binding activity, signal transduction activity, receptor binding activity.
  • chitino lytic activity e.g., GIcNAc
  • saccharide e.g., GIcNAc
  • the methods detailed herein are not limited to treatment of an inflammatory disease once the disease is established.
  • the symptoms of the disease need not have manifested to the point of detriment to the subject; indeed, the disease need not be detected in a subject before treatment is administered. That is, significant pathology from an inflammatory disease does not have to occur before the present invention may provide benefit. Therefore, the present invention, as described more fully herein, includes a method for preventing an inflammatory disease in a subject, in that a ChTRase antagonist, as detailed herein, can be administered to a subject prior to the onset of an inflammatory disease, thereby preventing the disease as demonstrated by the data disclosed herein.
  • the prevention of inflammatory disease encompasses administering to a subject a ChTRase antagonist as a preventative measure against inflammatory disease.
  • the symptoms and etiologies of ChTRase associated inflammatory disease include tissue inflammation, increased lung volume, increased eosinophils in bronchioalveolar ravage (BAL) fluid, increased lymphocytes in BAL fluid, increased total cells in BAL fluid, increased alveolus size, increased deposition of crystals comprised of ChTRases in lung tissue, increased airway resistance, increased mucus metaplasia, increased mucin expression, increased parenchymal fibrosis, increased airway remodeling, increased subepithelial fibrosis, increased collagen deposition in airway tissue, epithelial hypertrophy in the lung tissue, focal organization of crystalline material into
  • ChTRase antagonists include, but not limited to, the ChTRase antibodies disclosed herein, prevented onset of an inflammatory disease in a subject (e.g., allergen (e.g., cigarette smoke) induced COPD). Accordingly, the skilled artisan would appreciate, based on the disclosure provided elsewhere herein, that the present invention includes a method of preventing disease comprising administering a ChTRase antagonist.
  • methods of antagonizing (e.g. , inhibiting) a ChTRase encompass a wide plethora of techniques for inhibiting not only a ChTRase mediated activity, but also for inhibiting expression of a nucleic acid encoding ChTRase.
  • the present invention encompasses a method of preventing a wide variety of diseases where expression and/or activity of a ChTRase mediates disease. Methods for assessing whether a disease relates to over expression or increased activity of a ChTRase are disclosed elsewhere herein and/or are well-known in the art. Further, the invention encompasses treatment or prevention of such diseases discovered in the future.
  • Chronic obstructive pulmonary disease COPD
  • COPD Chronic obstructive pulmonary disease
  • Symptoms are productive cough and dyspnea that develop over years; common signs include decreased breath sounds, prolonged expiratory phase of respiration, and wheezing. Severe cases may be complicated by weight loss, pneumothorax, right heart failure, and respiratory failure. Diagnosis is based on history, physical examination, chest x-ray, and pulmonary function tests.
  • COPD chronic obstructive pulmonary disease
  • Chronic obstructive bronchitis is chronic bronchitis with airflow obstruction.
  • Chronic bronchitis also called chronic mucous hypersecretion syndrome
  • Chronic bronchitis becomes chronic obstructive bronchitis if spirometric evidence of airflow obstruction develops.
  • Chronic asthmatic bronchitis is a similar, overlapping condition characterized by chronic productive cough, wheezing, and partially reversible airflow obstruction; it occurs predominantly in smokers with a history of asthma. In some cases, the distinction between chronic obstructive bronchitis and chronic asthmatic bronchitis is unclear.
  • Emphysema is destruction of lung parenchyma leading to loss of elastic recoil and loss of alveolar septa and radial airway traction, which increases the tendency for airway collapse. Lung hyperinflation, airflow limitation, and air trapping follow. Airspaces enlarge and may eventually develop bullae.
  • COPD seems to aggregate in families independent of ⁇ l -antitrypsin ( ⁇ l-antiprotease inhibitor) deficiency.
  • Etiology Cigarette smoking is the primary risk factor in most countries, although only about 15% of smokers develop clinically apparent COPD; an exposure history of 40 or more pack-years is especially predictive. Genetic factors also contribute. The best- defined genetic disorder is ⁇ l -antitrypsin deficiency, which is an important cause of emphysema in nonsmokers and influences susceptibility to disease in smokers.
  • Polymorphisms in microsomal epoxide hydrolase, vitamin D-binding protein, IL- l ⁇ , IL-I receptor antagonist, phospholipase A2, matrix metalloproteinase 9, and ADAM-33 genes are all associated with rapid decline in forced expiratory volume in 1 sec (FEVl) in selected populations.
  • Inhalational exposures trigger an inflammatory response in airways and alveoli that leads to disease in genetically susceptible people.
  • the process is thought to be mediated by an increase in protease activity and a decrease in antiprotease activity.
  • Lung proteases such as neutrophil elastase, matrix metalloproteinases, and cathepsins, break down elastin and connective tissue in the normal process of tissue repair.
  • Their activity is balanced by antiproteases, such as ⁇ l -antitrypsin, airway epithelium-derived secretory leukoproteinase inhibitor, elaf ⁇ n, and matrix metalloproteinase tissue inhibitor.
  • neutrophils and other inflammatory cells release proteases as part of the inflammatory process; protease activity exceeds antiprotease activity, and tissue destruction and mucus hypersecretion result.
  • Neutrophil and macrophage activation also leads to accumulation of free radicals, superoxide anions, and hydrogen peroxide, which inhibit antiproteases and cause bronchoconstriction, mucosal edema, and mucous hypersecretion.
  • Neutrophil-induced oxidative damage, release of profibrotic neuropeptides (eg, bombesin), and reduced levels of vascular endothelial growth factor may contribute to apoptotic destruction of lung parenchyma.
  • Infection in conjunction with cigarette smoking, may amplify progression of lung destruction.
  • the inflammation in COPD increases with increasing disease severity, and, in severe (advanced) disease, inflammation may not resolve completely with smoking cessation. Neither does this inflammation appear responsive to corticosteroids.
  • Bacteria especially Haemophilus influenzae, colonize the normally sterile lower airways of about 30% of patients with COPD. In more severely affected patients (eg, those with previous hospitalizations), Pseudomonas aeruginosa colonization is common. Smoking and airflow obstruction may lead to impaired mucus clearance in lower airways, which predisposes to infection. Repeated bouts of infection increase the inflammatory burden that hastens disease progression. There is no evidence, however, that long-term use of antibiotics slows the progression of COPD in susceptible smokers.
  • Symptoms and Signs COPD takes years to develop and progress. Most patients have smoked > 20 cigarettes/day for > 20 yr.
  • Productive cough usually is the initial symptom, developing among smokers in their 40s and 50s.
  • Dyspnea that is progressive, persistent, exertional, or worse during respiratory infection appears years later, by the time patients reach their late 50s or 60s.
  • Symptoms usually progress quickly in patients who continue to smoke and who have higher lifetime tobacco exposure. Morning headache develops in more advanced disease and signals nocturnal hypercapnia or hypoxemia.
  • Acute exacerbations occur sporadically during the course of COPD and are heralded by increased symptom severity. The specific cause of any exacerbation is almost always impossible to determine, but exacerbations are often attributed to viral URIs or acute bacterial bronchitis. As COPD progresses, acute exacerbations tend to become more frequent, averaging about 3 episodes/yr. Those who suffer acute exacerbations are much more likely to have recurrent exacerbations.
  • Signs of COPD include wheezing, increased expiratory phase of breathing, lung hyperinflation manifested as decreased heart and lung sounds, and increased anteroposterior diameter of the thorax (barrel chest).
  • Patients with advanced emphysema lose weight and experience muscle wasting that has been attributed to immobility, hypoxia, or release of systemic inflammatory mediators, such as tumor necrosis factor (TNF)- ⁇ .
  • Signs of advanced disease include pursed-lip breathing, accessory muscle use paradoxical indrawing of the lower intercostal interspaces during inspiration (Hoover's sign), and cyanosis.
  • Signs of cor pulmonale include neck vein distention; splitting of the 2nd heart sound with an accentuated pulmonic component, tricuspid insufficiency murmur, and peripheral edema.
  • Right ventricular heaves are uncommon in COPD because the lungs are hyperinflated.
  • Spontaneous pneumothorax may occur as a result of rupture of bullae and should be suspected in any patient with COPD whose pulmonary status abruptly worsens.
  • the invention encompasses administration of a ChTRase antagonist, for the prevention, management, treatment or amelioration of COPD or one or more symptoms thereof.
  • the present invention provides formulations (e.g., a pharmaceutical composition) comprising one or more ChTRase antagonist (referred to herein as "formulation(s) of the invention,” “compositions of the invention” or simply “formulation(s),” or “compositions”).
  • formulations comprise one or more ChTRase antagonist and a pharmaceutically-acceptable carrier.
  • pharmaceutically-acceptable carrier means a chemical composition with which an appropriate ChTRase antagonist may be combined and which, following the combination, can be used to administer the appropriate ChTRase antagonist to a subject.
  • the formulations of the invention comprise a ChTRase antibody of the invention.
  • formulations comprising one or more ChTRase antagonist and a pharmaceutically-acceptable carrier are liquid formulations, (referred to herein as "liquid formulation(s)" which are specifically encompassed by the more generic terms “formulation(s) of the invention” and “formulation(s)”).
  • the liquid formulations are substantially free of surfactant and/or inorganic salts.
  • the liquid formulations have a pH ranging from about 5.0 to about 7.0, about 5.5 to about 6.5, or about 5.8 to about 6.2, or about 6.0.
  • the liquid formulations have a pH ranging from 5.0 to 7.0, 5.5 to 6.5, or 5.8 to 6.2, or 6.0.
  • the liquid formulations comprise histidine at a concentration ranging from about 1 mM to about 100 mM, or from about 5 mM to about 50 mM, or about 10 mM to about 25 mM.
  • the liquid formulations comprise histidine at a concentration ranging from 1 mM to 10O mM, or from 5 mM to 50 mM, or 1O mM to 25 mM.
  • the liquid formulations may further comprise one or more excipients such as a saccharide, an amino acid (e.g. arginine, lysine, and methionine) and a polyol.
  • excipients such as a saccharide, an amino acid (e.g. arginine, lysine, and methionine) and a polyol. Additional descriptions and methods of preparing and analyzed liquid formulations can be found, for example, in PCT publications WO 03/106644; WO 04/066957; WO 04/091658.
  • the formulations (e.g., liquid formulations) of the invention are pyrogen- free formulations which are substantially free of endotoxins and/or related pyrogenic substances.
  • Endotoxins include toxins that are confined inside a microorganism and are released when the microorganisms are broken down or die.
  • Pyrogenic substances also include fever-inducing, thermostable substances (glycoproteins) from the outer membrane of bacteria and other microorganisms. Both of these substances can cause fever, hypotension and shock if administered to humans. Due to the potential harmful effects, it is advantageous to remove even low amounts of endotoxins from intravenously administered pharmaceutical drug solutions.
  • FDA Food & Drug Administration
  • EU endotoxin units
  • endotoxin and pyrogen levels in the composition are less then 10 EU/mg, or less then 5 EU/mg, or less then 1 EU/mg, or less then 0.1 EU/mg, or less then 0.01 EU/mg, or less then 0.001 EU/mg.
  • compositions described herein When used for in vivo administration, the compositions described herein should be sterile. This is readily accomplished, for example, by filtration through sterile filtration membranes or by other means well known in the art. Sterile compositions for injection can be formulated according to conventional pharmaceutical practice as described in Remington 's Pharmaceutical Sciences (180' ed, Mack Publishing Company, Easton, PA, 1990). Compositions comprising antibodies, such as those disclosed herein, ordinarily will be stored in lyophilized form or in solution.
  • sterile compositions comprising antibodies of the invention are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
  • a sterile access port for example, an intravenous solution bag or vial having an adapter that allows retrieval of the formulation, such as a stopper pierceable by a hypodermic injection needle.
  • the liquid formulations have a concentration of one or more ChTRase antagonist of about 50 mg/ml, about 75 mg/ml, about 100 mg/ml, about 125 mg/ml, about 150 mg/ml, about 175 mg/ml, about 200 mg/ml, about 225 mg/ml, about 250 mg/ml, about 275 mg/ml, or about 300 mg/ml.
  • the liquid formulations have a concentration of one or more ChTRase antagonist of 50 mg/ml, 75 mg/ml, 100 mg/ml, 125 mg/ml, 150 mg/ml, 175 mg/ml, 200 mg/ml, 225 mg/ml, 250 mg/ml, 275 mg/ml, or 300 mg/ml.
  • the liquid formulations of the invention comprise a
  • the liquid formulations should exhibit one, or more of the following characteristics, stability, low to undetectable levels of antibody fragmentation and/or aggregation, very little to no loss of the biological activities of the antibodies or antibody fragments during manufacture, preparation, transportation, and storage.
  • the liquid formulations lose less than 50%, or less than 30%, or less than 20%, or less than 10% or even less than 5% or 1% of the antibody activity within 1 year storage under suitable conditions at about 4°C.
  • the activity of an antibody can be determined by a suitable antigen-binding or effector function assay for the respective antibody.
  • the liquid formulations are of low viscosity and turbidity.
  • the liquid formulations have a viscosity of less than 10.00 cP at any temperature in the range of 1 to 26 0 C.
  • Viscosity can be determined by numerous methods well known in the art.
  • the viscosity of a polypeptide solution can be measured using a ViscoLab 4000 Viscometer System (Cambridge Applied Systems) equipped with a ViscoLab Piston (SN:7497, 0.3055", 1-20 cP) and S6S Reference Standard (Koehler Instrument Company, Inc.) and connected to a water bath to regulate the temperature of the samples being analyzed.
  • the sample is loaded into the chamber at a desired starting temperature (e.g., 2 0 C) and the piston lowered into the sample. After sample was equilibrated to the temperature of the chamber, measurement is initiated.
  • the temperature is increased at a desired rate to the desired final temperature (e.g., > 25 0 C). And the viscosity over time is recorded.
  • compositions are principally directed to pharmaceutical compositions which are suitable for ethical administration to humans, it will be understood by the skilled artisan that such compositions are generally suitable for administration to animals of all sorts. Modification of pharmaceutical compositions suitable for administration to humans in order to render the compositions suitable for administration to various animals is well understood, and the ordinarily skilled veterinary pharmacologist can design and perform such modification with merely ordinary, if any, experimentation. Subjects to which administration of the pharmaceutical compositions of the invention is contemplated include, but are not limited to, humans and other primates, mammals including commercially relevant mammals such as cattle, pigs, horses, sheep, cats and dogs, and birds including commercially relevant birds such as chickens, ducks, geese, and turkeys.
  • a formulation comprising one or more ChTRase antagonist to be administered to a subject should be formulated in a pharmaceutically-acceptable excipient.
  • formulations, pharmaceutical compositions in particular, of the invention include but are not limited to those disclosed in PCT publications WO 02/070007, WO 03/075957 and WO 04/066957.
  • the excipient that is included with the Fc variants and/or variant Fc fusion of the present invention in these formulations can be selected based on the expected route of administration of the formulations in therapeutic applications.
  • the route of administration of the formulations depends on the condition to be treated. For example, intravenous injection may be preferred for treatment of a systemic disorder.
  • the dosage of the formulations to be administered can be determined by the skilled artisan without undue experimentation in conjunction with standard dose-response studies. Relevant circumstances to be considered in making those determinations include the condition or conditions to be treated, the choice of formulations to be administered, the age, weight, and response of the individual patient, and the severity of the patient's symptoms.
  • the actual patient body weight may be used to calculate the dose of the ChTRase antagonist of the present invention in these formulations in milliliters (mL) to be administered. There may be no downward adjustment to "ideal" weight.
  • a formulation of the invention can be administered orally, parenterally, intramuscularly, intranasally, vaginally, rectally, lingually, sublingually, buccally, intrabuccally, intravenously, cutaneously, subcutaneously and/or transdermally to the patient.
  • formulations designed for oral, parenteral, intramuscular, intranasal, vaginal, rectal, lingual, sublingual, buccal, intrabuccal, intravenous, cutaneous, subcutaneous and/or transdermal administration can be made without undue experimentation by means well known in the art, for example, with an inert diluent or with an edible carrier.
  • the formulations may be enclosed in gelatin capsules or compressed into tablets.
  • the formulations of the present invention may be incorporated with excipients and used in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, chewing gums, and the like.
  • Tablets, pills, capsules, troches and the like may also contain binders, recipients, disintegrating agent, lubricants, sweetening agents, and/or flavoring agents.
  • binders include microcrystalline cellulose, gum tragacanth and gelatin.
  • excipients examples include starch and lactose.
  • disintegrating agents include alginic acid, cornstarch, and the like.
  • lubricants include magnesium stearate and potassium stearate.
  • An example of a glidant is colloidal silicon dioxide.
  • sweetening agents include sucrose, saccharin, and the like.
  • flavoring agents include peppermint, methyl salicylate, orange flavoring, and the like.
  • Materials used in preparing these various formulations should be pharmaceutically pure and non-toxic in the amounts used.
  • the formulations of the present invention can be administered parenterally, such as, for example, by intravenous, intramuscular, intrathecal and/or subcutaneous injection.
  • Parenteral administration can be accomplished by incorporating the formulations of the present invention into a solution or suspension.
  • solutions or suspensions may also include sterile diluents, such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol and/or other synthetic solvents.
  • Parenteral formulations may also include antibacterial agents, such as, for example, benzyl alcohol and/or methyl parabens, antioxidants, such as, for example, ascorbic acid and/or sodium bisulfite, and chelating agents, such as EDTA.
  • Buffers such as acetates, citrates and phosphates, and agents for the adjustment of tonicity, such as sodium chloride and dextrose, may also be added.
  • the parenteral preparation can be enclosed in ampules, disposable syringes and/or multiple dose vials made of glass or plastic.
  • Rectal administration includes administering the formulation into the rectum and/or large intestine. This can be accomplished using suppositories and/or enemas.
  • Suppository formulations can be made by methods known in the art.
  • Transdermal administration includes percutaneous absorption of the formulation through the skin. Transdermal formulations include patches, ointments, creams, gels, salves, and the like.
  • the formulations of the present invention can be administered nasally to a patient.
  • nasally administering or nasal administration includes administering the formulations to the mucous membranes of the nasal passage and/or nasal cavity of the patient.
  • compositions of the invention formulated for pulmonary delivery may also provide the active ingredient in the form of droplets of a solution or suspension.
  • Such formulations may be prepared, packaged, or sold as aqueous or dilute alcoholic solutions or suspensions, optionally sterile, comprising the active ingredient, and NO may conveniently be administered using any nebulization or atomization device.
  • Such formulations may further comprise one or more additional ingredients including, but not limited to, a flavoring agent such as saccharin sodium, a volatile oil, a buffering agent, a surface active agent, or a preservative such as methylkydroxybenzoate.
  • the droplets provided by this route of administration preferably have an average diameter in the range from about 0.1 to about 200 nanometers.
  • Pulmonary drug delivery may be achieved by inhalation, and administration by inhalation herein may be oral and/or nasal.
  • Examples of pharmaceutical devices for pulmonary delivery include metered dose inhalers (MDIs) and dry powder inhalers (DPIs).
  • MDIs metered dose inhalers
  • DPIs dry powder inhalers
  • Exemplary delivery systems by inhalation which can be adapted for delivery of the subject antibody and/or active agent are described in, for example, U.S. Patent Nos. 5,756,353; 5,858,784; and PCT applications WO98/31346; WO98/10796;
  • Another formulation suitable for intranasal administration is a coarse powder comprising the active ingredient and having an average particle from about 0.2 to 500 30 micrometers. Such a formulation is administered in the manner in which snuff is taken i.e. by rapid inhalation through the nasal passage from a container of the powder held close to the nares.
  • the formulations are administered to the mammal by subcutaneous (i.e., beneath the skin) administration.
  • the formulations may be injected using a syringe.
  • other devices for administration of the formulations are available such as injection devices (e.g. the Inject- ease_ and Genject_ devices), injector pens (such as the GenPenTM); auto-injector devices, needleless devices (e.g., MediJector and BioJector); and subcutaneous patch delivery systems.
  • a slow release formulation comprises a liquid formulation.
  • Slow release formulations may be formulated from a number of agents including, but not limited to, polymeric nano or microparticles and gels (e.g., a hyaluronic acid gel).
  • slow release formulations offer other advantages for delivery of protein drugs including protecting the protein (e.g., ChTRase antibodies of the invention) over an extended period from degradation or elimination, and the ability to deliver the protein locally to a particular site or body compartment thereby lowering overall systemic exposure.
  • the present invention also contemplates injectable depot formulations in which the protein (e.g., ChTRase antibodies of the invention) is embedded in a biodegradable polymeric matrix.
  • Polymers that may be used include, but are not limited to, the homo- and co-polymers of lactic and glycolic acid (PLGA).
  • PLGA degrades by hydrolysis to ultimately give the acid monomers and is chemically unreactive under the conditions used to prepare, for example, microspheres and thus does not modify the protein.
  • the protein is released by a combination of diffusion and polymer degradation.
  • the hydrolysis rate can be varied thereby allowing release to last from days to months.
  • the present invention provides a nasal spray formulation.
  • a nasal spray formulation comprises the liquid formulation of the present invention.
  • formulations of the invention may be used in accordance with the methods of the invention for the prevention, management, treatment or amelioration of COPD.
  • the formulations of the invention are sterile and in suitable form for a particular method of administration to a subject with COPD.
  • the invention provides methods for preventing, managing, treating or ameliorating COPD or one or more symptoms thereof, said method comprising: (a) administering to a subject in need thereof a dose of a prophylactically or therapeutically effective amount of a formulation comprising one or more ChTRase antagonist, and (b) administering one or more subsequent doses of said formulation, to maintain a plasma concentration of the ChTRase antagonist at a desirable level (e.g., about 0.1 to about 100 ⁇ g/ml), which continuously blocks a ChTRase activity.
  • a desirable level e.g., about 0.1 to about 100 ⁇ g/ml
  • the plasma concentration of the ChTRase antagonist is maintained at 10 ⁇ g/ml, 15 ⁇ g/ml, 20 ⁇ g/ml, 25 ⁇ g/ml, 30 ⁇ g/ml, 35 ⁇ g/ml, 40 ⁇ g/ml, 45 ⁇ g/ml or 50 ⁇ g/ml.
  • said effective amount of ChTRase antagonist to be administered is between at least 1 mg/kg and 100 mg/kg per dose. In another specific embodiment, said effective amount of ChTRase antagonist to be administered is between at least 1 mg/kg and 20 mg/kg per dose.
  • said effective amount of ChTRase antagonist to be administered is between at least 4 mg/kg and 10mg/kg per dose. In yet another specific embodiment, said effective amount of ChTRase antagonist to be administered is between 50 mg and 250 mg per dose. In still another specific embodiment, said effective amount of ChTRase antagonist to be administered is between 100 mg and 200 mg per dose. 4.10 Kits
  • kits relating to antagonizing ChTRases in a mammal which are useful, because, as disclosed elsewhere herein, antagonizing ChTRases provides a method of treating or preventing COPD in a mammal.
  • the invention includes a kit for treating COPD in a mammal.
  • the kit comprises an effective amount of a ChTRase antagonist.
  • the kit further comprises an applicator and an instructional material for the use thereof to be used in accordance with the teachings provided herein.
  • the invention includes various kits which comprise a compound, such as a an antibody that specifically binds a ChTRase, as well as a nucleic acid encoding such an antibody, a nucleic acid complementary to a nucleic acid encoding a ChTRase but in an antisense orientation with respect to transcription, a ribozyme capable of cleaving a single- stranded chitinase-like molecule RNA, an applicator, and instructional materials which describe use of the compound to perform the methods of the invention.
  • the invention further encompasses kits for the diagnosis of COPD in a subject who is at risk of having COPD or an analagous disease.
  • a diagnostic kit of the invention comprises one or more reagents that substantially specifically bind to ChTRase. In another embodiment, a diagnostic kit of the invention comprises one or more reagents that one or more reagents for the detection of ChTRase activity. In a further embodiment, a diagnostic kit of th einvention comprises one or more reagents for the detection of polynucleotide encoding the ChTRase protein or a complementary polynucleotide thereof. In another embodiment, a diagnostic kit of the invention comprises one or more reagents for the detection of the ChTRase protein or a fragment thereof. In some embodiments, a diagnostic kit of the invention further comprises instructions for using the reagent to diagnose COPD, or for using the reagent to assess the severity of COPD in the test subject, or both.
  • kits of the invention have a number of embodiments.
  • a typical embodiment is a kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes an antibody that binds to a ChTRase polypeptide sequence, the label on said container indicates that the composition can be used to evaluate the presence of ChTRase proteins in at least one type of patient sample, and instructions for using the ChTRase antibody for evaluating the level of ChTRase protein present in the patient sample.
  • Another embodiment is a diagnostic kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a polynucleotide that hybridizes to a complement of the ChTRase polynucleotide under stringent conditions, the label on said container indicates that the composition can be used to evaluate the presence of ChTRase in a patient sample, and instructions for using the polynucleotide for evaluating the presence of ChTRase RNA in a patient sample.
  • a further embodiment is a diagnostic kit comprising a container, a label on said container, and a composition contained within said container; wherein the composition includes a substrate for ChTRase, the label on said container indicates that the composition may be used to measure the level of ChTRase activity in a patient sample, and instructions for using the substarte to measure ChTRase activity in a patient sample.
  • the kit may further comprise a set of instructions and materials for preparing a patient sample and applying the reagents to the tissue sample.
  • the invention includes kits for treating or preventing COPD
  • the kit is used pursuant to the methods disclosed in the invention.
  • the kit may be used to contact a mammal with a chemical compound that inhibits ChTRase, or a nucleic acid complementary to a nucleic acid encoding a ChTRase where the nucleic acid is in an antisense orientation with respect to transcription to reduce expression of a ChTRase, or with an antibody that specifically binds with a ChTRase or a nucleic acid encoding the antibody, wherein the decreased expression, amount, or activity of a ChTRase mediates an beneficial effect in the mammal.
  • the kit comprises an applicator and an instructional material for the use of the kit. These instructions simply embody the examples provided herein.
  • the kit includes a pharmaceutically-acceptable carrier.
  • the composition is provided in an appropriate amount as set forth elsewhere herein. Further, the route of administration and the frequency of administration are as previously set forth elsewhere herein.
  • a method for identifying a subject at risk or suspected of having COPD or a f ⁇ brotic lung disease comprising: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is bronchoalveolar lavage (BAL), bronchial biopsy, sputum, blood, serum, plasma, or urine; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD or a fibrotic lung disease.
  • BAL bronchoalveolar lavage
  • a method for differential diagnosis of asthma and COPD comprising: assaying for elevated level of ChTRase or ChTRase activity in a test sample of the subject, wherein the test sample is BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine; and wherein an elevated level of ChTRase or ChTRase activity in the test sample as compared to a control value or to the level of ChTRase or ChTRase activity in a control sample indicates that the subject is at risk of having COPD.
  • 38 The method of embodiment 36 or 37, wherein the subject has one or more symptoms associated with COPD, one or more physiologic parameters associated with COPD, or both.
  • control sample is a corresponding sample from subjects lacking COPD.
  • control value is the level of ChTRase or ChTRase activity in corresponding samples from subjects lacking COPD .
  • control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from subjects lacking COPD.
  • level of ChTRase activity in the control sample is between about 0 and about 2.5 xl03 FU/ml.
  • control value is between about 0 and about 2.5 xl03 FU/ml ChTRase activity.
  • control sample is a corresponding sample from subjects lacking COPD.
  • control value is is between about 0 and about 100 x 103 FU/ml total chitinolytic activity.
  • control sample is a corresponding sample from subjects lacking COPD.
  • control value is between about 0 and about 0.4 ChTRase mRNA to ubiquitin C mRNA ratio.
  • test sample is bronchial biopsy sample.
  • control sample is a corresponding sample from subjects lacking COPD.
  • a method of monitoring the progression of COPD in a subject comprising determining the level of ChTRase or ChTRase activity in a plurality of test samples over time, wherein the test samples are BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine.
  • a method of evaluating the effect of one or more COPD treatment regimens on a subject with COPD comprising: comparing levels of ChTRase or ChTRase activity in BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine of the subject following treatment of the subject to levels of ChTRase or ChTRase activity in the BAL, bronchial biopsy, sputum, blood, serum, plasma, or urine of the subject prior to treatment, and/or comparing levels of ChTRase or ChTRase activity in the BAL, sputum, blood, serum, plasma, or urine of the subject following treatment with a control value or with the levels of ChTRase or ChTRase activity in the BAL, sputum, blood, serum, plasma, or urine, respectively, of a control subject.
  • control sample is a corresponding sample from subjects lacking COPD.
  • control sample is a corresponding sample from the test subject.
  • a diagnostic kit for diagnosing COPD comprising one or more reagents that substantially specifically bind to ChTRase.
  • a diagnostic kit for diagnosing COPD said kit comprising one or more reagents for the detection of ChTRase activity.
  • 114. A diagnostic kit for diagnosing COPD, said kit comprising one or more reagents for the detection of polynucleotide encoding the ChTRase protein or a complementary polynucleotide thereof.
  • a diagnostic kit for diagnosing COPD comprising one or more reagents for the detection of the ChTRase protein or a fragment thereof.
  • a method for treating COPD comprising administering to a subject in need thereof a therapeutically effective amount of a ChTRase antagonist.
  • Stage III denotes severe disease, with an FEVl of 30 to 50 % and stage IV indicates very severe disease, with an FEVl of less than 30 % of the predicted value.
  • Heavy smokers without COPD had chronic cough and sputum but normal measurements on spirometry.
  • 40 non-smoker asthmatic subjects mainly atopies and fulfilling the criteria of the Guidelines for the Diagnosis and Management of Asthma of the National Heart, Lung, and Blood Institute/World Health Organization (Guidelines for the Diagnosis and Management of Asthma: Expert Panareport 2. Atlanta, GA: National Heart, Lung, and Blood Institute, National Institutes of Health. Publication No. 97-405 IA). were recruited (Table 2). None of the subjects had experienced asthma exacerbation within the 2 months preceding the bronchoscopy.
  • Severe asthmatics were defined on the basis of the requirement of high-dose inhaled steroids (> 1000 ⁇ g of fluticasone propionate or equivalent per day) and a long-lasting ⁇ 2-agonist, as add-on therapy to achieve control.
  • Four out of the 15 severe asthmatics were also treated with 10 to 40 mg per day of oral prednisone (Table 2).
  • FEVl Forced Expiratory Volume in 1 second
  • FVC Forced Vital Capacity
  • FRC Functional Residual Capacity
  • RV Residual Volume
  • TLC Total Lung Capacity
  • FEVl Forced Expiratory Volume in 1 second
  • FVC Forced
  • Cytospin preparations from BAL cells were fixed in 4% paraformaldehyde for 20 min at room temperature and stored dried at -20 0 C until use.
  • Four biopsy specimens were taken from the subcarinae in 10 representative never-smokers, in 12 smokers without COPD and in 12 smokers with COPD and snap-frozen in Tissue-Tek compound (Sakura Finetek, Zoeterwoude, The Netherlands).
  • Lung tissue specimens were obtained from 2 never-smokers and from 3 and 4 smokers, respectively with and without COPD, who underwent lung lobectomy for peripheral lung carcinoma. Two specimens were dissected at distance of the tumour (from 7th to 10th bronchi), one specimen was snap-frozen, and the other one was fixed immediately in 10% formaldehyde and processed to paraffin wax.
  • Antibodies and proteins The 293 -F cell line (Invitrogen, Carlsbad, CA) was transfected with plasmids encoding human ChTRase and AMCase. In order to facilitate purification, a His Tag (the sequence of six histidine residues) was expressed at C-terminus of the proteins.
  • Recombinant proteins were purified from supernatant using Ni columns and were characterized by SDS PAGE, western-blot, absorption at 280 nm, mass spectrometry and chitinolytic assay. Preparations were sterilized by passage through 0.2- ⁇ m filters and endotoxin content was below 3 EU/mg of protein (Lymulus Amoebocyte Lysate assay, Associates of Cape Cod, East Falmouth, MA). [0406] Polyclonal sera were generated by immunizing rabbits with recombinant
  • ChTRase or AMCase in complete Freund's adjuvant, and immunoglobulins G (IgG) were subsequently purified on Protein G columns.
  • the antibodies were previously disclosed in PCT publication WO 2007027748 as rabbit polyclonal antibodies 208 or 657.
  • the specificity and titers of the sera and of the purified anti-ChTRase and anti- AMCase IgG were determined by enzyme-linked immunosorbent assay (Elisa) developed in house.
  • the anti-chitinase rabbit polyclonal IgG was used for capture and biotinylated anti- chitinase rabbit polyclonal IgG followed by horseradish peroxidase labeled streptavidin (Amersham, GE Healthcare, Piscataway, NJ) was used for detection.
  • the sensitivity of the assays was 100 pg/ml for human ChTRase and 4 ng/ml for human AMCase.
  • ChTRase-positive alveolar macrophages were enumerated in cytospin preparations from BAL after counting at least 200 cells in randomly-selected fields.
  • BAL cells were resuspended in RPMI- 1640 medium containing 25 mM Hepes, 2 mM glutamine, 2.5 mM sodium pyruvate, non-essential aminoacids, 100 IU/ml penicillin and 100 ⁇ g /ml streptomycin (all from Invitrogen, Cergy-Pontoise, France) and were seeded at the 500,000 macrophages/1 mL in 12-well plates (Corning-Costar, Acton, MA).
  • IL-8 and monocyte-chemoattractant protein-1 were assessed by commercially available Elisa kits (R&D Systems) and MMP-9 was measured by the specific Biotrak activity assay (GE Healthcare, Orsay, France). The sensitivity of these assays was 6.5, 12.5, and 250 pg/ml for IL-8, MCP-I, and MMP-9, respectively.
  • IL-8 a cytokine that has been described to be upregulated in COPD (Barnes PJ. Pharmacol Rev. 56(4): 515-548 (2004)).
  • the levels of IL-8 were slightly increased in smokers without COPD, as compared to never-smokers (median [interquartile range]: 63.0 pg/ml [47.0 to 107.2] and 43.0 pg/ml [22.0 to 77.9], respectively) and they were higher in smokers with COPD (221.6 [108.3 to 494.9]).
  • ChTRase When expressed, ChTRase localized mainly in cells infiltrating the bronchial epithelium and the submucosa and was not detected in the bronchial epithelium, the vascular endothelium and the airway smooth muscle ( Figure 2C and 2D).
  • ChTRase expression was also assessed at the mRNA level in isolated BAL alveolar macrophages. Analysis by quantitative real-time PCR showed that the levels of
  • ChTRase mRNA normalized according to the amount of ubiquitin C, were higher in alveolar macrophages of smokers without COPD than in never-smokers and were further enhanced in smokers with COPD (Figure 4E).
  • alveolar macrophages from smokers with and without COPD that had been cultured for 24 hours in the presence of the medium alone released spontaneously higher amounts of ChTRase than those from never-smokers Figure 4F.
  • Exposure of alveolar macrophages for 24 hours to 10 ng/ml of TNF- ⁇ upregulated the levels of secreted ChTRase in smokers with COPD, but not in smokers without COPD, or in never-smokers (Figure 4F).
  • Alveolar macrophages from COPD subjects, either former or current smokers (n 8 and 6, respectively) showed comparable basal and TNF- ⁇ -stimulated ChTRase production.
  • ChTRase was as potent as 10 ng/ml of TNF- ⁇ in eliciting chemokine release ( Figure 5 A and 5B).
  • Spontaneous production of MMP-9 by medium-exposed alveolar macrophages from smokers with and without COPD was highly variable (between 0.3 and 11.9 ng/ml) and, therefore, the effect of ChTRase and TNF- ⁇ on MMP-9 secretion was calculated as a percentage of release over medium-exposed cells (Figure 5C).
  • ChTRase induced 1.2-, 1.4- and 1.9-fold increase of MMP-9 release from alveolar macrophages from never-smokers, from smokers without COPD and from smokers with COPD, respectively (Figure 5C).
  • the effect of ChTRase was similar to that induced by TNF- ⁇ (2.1 -fold increase, Figure 5C).

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Abstract

La présente invention concerne une nouvelle découverte selon laquelle les fumeurs avec COPD, mais pas les sujets souffrant d'asthme léger, modéré ou grave, présentent une activité BAL chitinolytique supérieure, des niveaux de ChTRase accrus, davantage de cellules ChTRase-positives dans les biopsies des bronches et une proportion élevée de macrophages alvéolaires exprimant la ChTRase par rapport aux fumeurs sans COPD, ou aux non-fumeurs. Par conséquent, la présente invention concerne des antagonistes de la ChTRase, et des compositions et des procédés servant au diagnostic, à la surveillance, à la prévention, à la gestion, au traitement ou à l'amélioration de la COPD.
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2425369C1 (ru) * 2010-05-31 2011-07-27 Государственное образовательное учреждение высшего профессионального образования "Сибирский государственный медицинский университет Федерального агентства по здравоохранению и социальному развитию" (ГОУВПО СибГМУРосздрава) Способ ранней диагностики хронической обструктивной болезни легких
FR2986239A1 (fr) * 2012-01-31 2013-08-02 Univ Strasbourg Biomarqueur pour les maladies inflammatoires pulmonaires chroniques
WO2018191379A3 (fr) * 2017-04-11 2018-12-27 The Regents Of The University Of California Administration de chitinase dans les voies respiratoires pour traiter la fibrose pulmonaire inflammatoire et liée à l'âge

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* Cited by examiner, † Cited by third party
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US20070071746A1 (en) * 2005-08-31 2007-03-29 Medimmune, Inc. C/CLP antagonists and methods of use thereof

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
RU2425369C1 (ru) * 2010-05-31 2011-07-27 Государственное образовательное учреждение высшего профессионального образования "Сибирский государственный медицинский университет Федерального агентства по здравоохранению и социальному развитию" (ГОУВПО СибГМУРосздрава) Способ ранней диагностики хронической обструктивной болезни легких
FR2986239A1 (fr) * 2012-01-31 2013-08-02 Univ Strasbourg Biomarqueur pour les maladies inflammatoires pulmonaires chroniques
WO2013113852A1 (fr) * 2012-01-31 2013-08-08 Universite De Strasbourg Biomarqueur pour les maladies inflammatoires pulmonaires chroniques
WO2018191379A3 (fr) * 2017-04-11 2018-12-27 The Regents Of The University Of California Administration de chitinase dans les voies respiratoires pour traiter la fibrose pulmonaire inflammatoire et liée à l'âge

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