WO2010026489A1 - Modulateurs d'immunité innée - Google Patents

Modulateurs d'immunité innée Download PDF

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WO2010026489A1
WO2010026489A1 PCT/IB2009/007001 IB2009007001W WO2010026489A1 WO 2010026489 A1 WO2010026489 A1 WO 2010026489A1 IB 2009007001 W IB2009007001 W IB 2009007001W WO 2010026489 A1 WO2010026489 A1 WO 2010026489A1
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compound
polypeptide
gapdh
peptidomimetic
sequence
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PCT/IB2009/007001
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Neeloffer Mookherjee
Dustin Lippert
Pamela Hamill
Robert E.W. Hancock
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The University Of British Columbia
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/502—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects
    • G01N33/5023—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing non-proliferative effects on expression patterns
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/573—Immunoassay; Biospecific binding assay; Materials therefor for enzymes or isoenzymes
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6863—Cytokines, i.e. immune system proteins modifying a biological response such as cell growth proliferation or differentiation, e.g. TNF, CNF, GM-CSF, lymphotoxin, MIF or their receptors
    • 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
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90—Enzymes; Proenzymes
    • G01N2333/902—Oxidoreductases (1.)
    • G01N2333/90203—Oxidoreductases (1.) acting on the aldehyde or oxo group of donors (1.2)
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • the present invention relates generally to the fields of immunology and microbiology, and more specifically to the identification of compounds that modulate innate immunity and the use of these innate immunity modulators in treating diseases and disorders.
  • Cationic host defense peptides are evolutionarily conserved critical elements of innate immunity. They are widely distributed in Nature, existing in organisms from insects, plants and crustaceans to mammals, indicating their importance in defence against pathogenic challenge and innate immune responses in virtually all organisms.
  • the sole human cathelicidin is LL-37 (LLGDFFRKSKEKIGKEFKRIVQRI-KDFLRNLVPRTES) (Zanetti M. J Leucyte Biology 75: 39-48, 2004; Bowdish, D M e ⁇ al.
  • LL- 37 is widely expressed in a variety of body fluids and tissues, including key immune cell types such as monocytes, neutrophils, epithelial cells and lymphocytes. It displays significant anti-infective and anti-inflammatory properties in vivo and is up-regulated during inflammatory conditions and infections. Conversely, deficiencies in LL-37 expression lead to increased susceptibility to infections in a variety of diseases including Morbus Kostmann, chronic ulcers and atopic dermatitis.
  • the anti-infective properties of LL-37 can be largely attributed to its ability to mediate a diverse array of immunomodulatory activities, since its modest direct microbicidal activity has been shown to be antagonized by physiological ion concentrations.
  • the diverse immunomodulatory functions of LL-37 include pro-inflammatory activities such as direct chemoattraction of mononuclear cells, neutrophils, eosinophils, T-cells and mast cells, and the induction of the expression of chemokines required for cell recruitment, and the like, as well as anti-inflammatory functions including potent neutralization of endotoxin (Bowdish DM, Hancock REW.
  • LL-37 also promotes angiogenesis and wound healing, induces degranulation of mast cells, and influences dendritic cell differentiation and the polarization of T-cells (an adjuvant effect that promotes subsequent immunogenic adaptive immune responses to antigens; Davidson DJ, et al. J of Immunology 172:1146-1156, 2004). Overall, LL-37 plays a critical role in selectively balancing responses to inflammatory stimuli, such as bacterial endotoxin, in human immune cells.
  • Macrophages and monocytes play a key role in the clearance of pathogens. They respond to LL-37 stimulation through changes in gene expression and subsequent release of chemokines. Although certain functions of LL-37, were previously demonstrated to involve a variety of putative cell surface receptors e.g., formyl peptide receptor-like 1 (FPRL-I) for chemoattraction and the transactivator P2X 7 for ILl- ⁇ production respectively (De, Y. et al. J Experimental Medicine 192:1069-1074, 2000; Nagaoka L, et al. J Immunology 176:3044-52, 2006), the receptor(s) through which LL-37 exerts its effects on macrophages remains unidentified.
  • FPRL-I formyl peptide receptor-like 1
  • MAP mitogen activated protein
  • IDR-I 13 amino acid innate defence regulator peptide
  • IDR-I is the first member of a class of innate defence regulators which counter infection by selective modulation of innate immunity. Subsequently our laboratory has created many novel variant peptides that vary from 7 to 12 amino acids long and that show similar activities.
  • the present invention generally relates to methods for identifying compounds that modulate innate immunity, and more specifically to compounds identified by these methods that have an immunomodulatory effect.
  • the therapeutic applications include the treatment of infections by bacteria, viruses, fungi and parasites, sepsis, including endotoxaemia and systemic inflammatory distress syndrome, and any inflammatory-based disease states, including cystic fibrosis, inflammatory bowel disease, asthma, bronchiectasis, cancer, and the like.
  • the present invention also provides pharmaceutical compositions, functional variants, and peptidomimetics thereof that are identified by the methods of the present invention.
  • a method of identifying a compound that modulates innate immunity by binding to glyceraldehyde-3 -phosphate dehydrogenase comprising contacting a cell with a test compound; and measuring gene expression in the presence and absence of the test compound, wherein a change in gene expression indicates an immunomodulatory effect.
  • the measuring of gene expression comprises measuring expression of a cytokine or a chemokine.
  • the cytokine or chemokine is MIP- l ⁇ , MIP-3 ⁇ , Gro- ⁇ , or IL-IO.
  • the change in gene expression pattern(s) is due to the altered activity and nuclear translocation of a transcription factor.
  • the transcription factor is AP-I, AP-2, ARE, CREB, E2F1, GRE, or SP-I.
  • the cells are stimulated by a sepsis or inflammatory inducing agent.
  • the sepsis or inflammatory inducing agent is lipopolysaccharide (LPS).
  • the change in gene expression pattern(s) does not occur when GAPDH expression is significantly reduced.
  • GAPDH expression is significantly reduced by a small inhibitory RNA targeted to GAPDH.
  • the change in gene expression pattern(s) does not occur when AP-I, AP-2, ARE, CREB, E2F1, GRE, or SP-I expression is significantly reduced.
  • the compound identified by the method modulates adaptive immunity.
  • the compound is a polypeptide or peptidomimetic.
  • the polypeptide or peptidomimetic is amphipathic.
  • the polypeptide or peptidomimetic can adopt a predominantly ⁇ -helical conformation.
  • the polypeptide or peptidomimetic comprises about 5 to about 50 residues.
  • the polypeptide or peptidomimetic has a net cationic charge.
  • the compound is cell permeable.
  • a method for treating an immune disorder or infection comprising administering to a subject in need thereof an effective amount of the compounds identified by the methods of the invention described herein.
  • a method for modulating innate immunity in a subject comprising administering to a subject in need thereof an effective amount of the compound identified by the methods of the invention described herein.
  • a method for treating an inflammatory disorder in a subject comprising administering to a subject in need thereof an effective amount of the compound identified by the methods of the invention described herein.
  • a method of identifying a compound that modulates innate immunity comprising adding a test compound to a preparation comprising glyceraldehyde-3 -phosphate dehydrogenase (GAPDH); and determining whether the test compound binds GAPDH.
  • GAPDH is recombinant GAPDH.
  • GAPDH is endogenous GAPDH.
  • the preparation comprises a peripheral blood mononuclear cell (PMBC) cell lysate.
  • the preparation comprises peripheral blood mononuclear cells.
  • the test compound is a polypeptide or peptidomimetic. In some such aspects, the polypeptide or peptidomimetic is amphipathic.
  • the polypeptide or peptidomimetic can adopt a predominantly ⁇ -helical conformation. In some such aspects, the polypeptide or peptidomimetic comprises about 5 to about 50 residues. In other such aspects, the polypeptide or peptidomimetic has a net cationic charge. In some such aspects, the test compound is cell permeable. In one aspect, the compound identified by the method modulates adaptive immunity. In some such aspects, the compound is a polypeptide or peptidomimetic. In some such aspects, the polypeptide or peptidomimetic is amphipathic. In some such aspects, the polypeptide or peptidomimetic can adopt a predominantly ⁇ -helical conformation. In some such aspects, the polypeptide or peptidomimetic comprises about 5 to about 50 residues. In some such aspects, the polypeptide or peptidomimetic has a net cationic charge. In some such aspects, the compound is cell permeable.
  • a method of identifying a test compound that modulates innate immunity comprising adding a test compound to a preparation comprising peripheral blood mononuclear cells, measuring cellular uptake of the test compound, and determining if the test compound binds to GAPDH.
  • the test compound is added in the presence of a sepsis or inflammatory inducing agent.
  • the sepsis or inflammatory inducing agent is lipopolysaccharide (LPS).
  • the test compound is a polypeptide.
  • the compound identified by the method modulates adaptive immunity.
  • the compound is a polypeptide or peptidomimetic.
  • the polypeptide or peptidomimetic is amphipathic. In some such aspects, the polypeptide or peptidomimetic can adopt a predominantly ⁇ -helical conformation. In some such aspects, the polypeptide or peptidomimetic comprises about 5 to about 50 residues. In some such aspects, the polypeptide or peptidomimetic has a net cationic charge. In some such aspects, the compound is cell permeable.
  • compositions comprising the compound(s) identified by the methods of the invention described above and a pharmaceutically acceptable carrier are also provided.
  • FIG. 1 LL-37-induced chemokine response was independent of FPRL-I receptor and P2X 7 transactivator.
  • Human PBMC were pre-treated with the specific (A) FPRLl -antagonist WRW4 (10 ⁇ M) or (B) P2X7-antagonist KN62 for 1 hr, followed by stimulation with LL-37 (20 ⁇ g/ml) or FPRLl-agonist W-peptide (5 ⁇ M) for 24 hr.
  • Tissue culture supernatants were monitored for chemokine MCP-I production by capture ELISA.
  • Results represent average of at least three biological replicates from human PBMC isolated from independent donors +/- standard error (*p ⁇ 0.05).
  • FIG. 1 Cellular uptake of LL-37 in macrophages.
  • Murine macrophages RAW 264.7 (ATCC TIB-71) were stimulated with biotinylated LL-37 (LL-37B) for (A) 0 min, or (B) 30 min.
  • the cells were washed and fixed with 3% formaldehyde, permeabilized using PBS containing 0.05% saponin and 3 % BSA.
  • AF488-streptavidin conjugate was used to visualise uptake of the peptide LL-37. Images displayed are taken from the mid-point of Z- stacks acquired by confocal laser scanning microscopy (Nikon) using a 60 X oil immersion lens.
  • FIG. 3 LL-37-induced chemokine responses were dependent on cytoskeletal integrity.
  • Human PBMC were pre-treated with 0.5 ⁇ g/ml each of nocodazole (for microtubulin depolymerization) or cytochalasin D (for actin disruption) followed by stimulation with LL-37 (20 ⁇ g/ml) for 24 hr.
  • Tissue culture supernatants were monitored for MCP-I production by capture ELISA. Results represent the average of at least three biological replicates from human PBMC isolated from independent donors ⁇ standard error (*p ⁇ 0.05). Fluorescent activated cell sorting demonstrated that monocytes were the producers of MCP-I in PBMC (data not shown).
  • FIG. 4 GAPDH was quantitatively enriched in samples treated with biotinylated LL-37 after avidin affinity purification indicating that it selectively bound to LL-37.
  • Protein extracts from unlabeled RAW cells were treated with LL-37, while extracts from cells metabolically labeled with heavy amino acids ( 13 C 6 -LyS and 13 C 6 -Arg) were treated with LL-7B as described. After crosslinking and avidin affinity purification, equal quantities of these samples were mixed for simultaneous proteomic analysis using mass spectrometry.
  • FIG. 5 Evaluation of macrophage GAPDH as a direct interacting protein partner of cathelicidin LL-37. Immunoblots were performed using samples obtained following affinity tag pull-down experiments using either LL-37B or unlabeled LL-37 using both murine RAW macrophages and human primary CD 14+ monocytes. Immunoblots with samples obtained from SILAC-labeled RAW cells were probed with (A) HRP-conjugated anti-biotin antibody, (B) rabbit anti-LL-37 polyclonal antibody, or (C) rabbit anti-GAPDH. Results are representative of three independent biological replicates.
  • FIG. 1 In-vitro binding assays for peptide interactions.
  • GAPDH (2 ⁇ g/ml) was immobilized on polystyrene microtitre plates followed by incubation with LL- 37B (0-50 mg/ml) and the in-vitro interaction of GAPDH with LL-37B was detected employing HRP-conjugated anti-biotin antibody.
  • Lane 1 20 ⁇ g GAPDH which was shown as expected to migrate towards the positive pole (NB the disperse nature of the GAPDH band is expected of a protein with weak net negative charge given the lack of a stacking gel).
  • Lane 2 20 ⁇ g GAPDH + 10 ⁇ g IDR-I.
  • Lane 3 20 ⁇ g GAPDH + 20 ⁇ g IDR-I.
  • Lane 4 20 ⁇ g GAPDH + 100 ⁇ g IDR-I.
  • Lane 5 20 ⁇ g GAPDH + 200 ⁇ g IDR-I.
  • FIG. 1 The structure of LL-37 that was used for modeling (NMR coordinates obtained from the authors; Porcelli F et al. Biochemistry 47:5565, 2008. [0024]
  • FIG. 10 Evaluation of LL-37-induced MAPK p38 phosphorylation in GAPDH knock-down cells. Phosphorylation of MAPK p38 in wild-type (WT) and GAPDH- knockdown (KD) cells were evaluated with either LL37 (50 ⁇ g/ml), LPS (100 ng/ml) or LTA (2 ⁇ g/ml) after 30min.
  • WT wild-type
  • KD GAPDH- knockdown
  • A Histograms of FL4 fluorescence of wild type and GAPDH-knock down cells, stained with anti-phospho-p38 3D7 rabbit monoclonal (Cell Signaling technology) and anti-rabbit AF647 secondary antibody. The data is representative of three independent experiments.
  • the relative signal intensities (proportional to amount of activated TF in each nuclear extract) were measured using a Chemigenius imaging system. Values shown are representative of results from 2 biological replicates. Examples of TFs whose activity is increased, decreased or unchanged are given (total number TF 's per array was 54).
  • Figure 12 Model describing the mechanism of peptide immunomodulatory activity due to its interaction with GAPDH.
  • LL-37 or IDR-I peptide enters cells using pathways typical of cell penetrating peptides involving the cell cytoskeletal machinery ( Figures 1-3; NB this may be preceded by interaction with a surface receptorl).
  • LL-37 then interacts with GAPDH and through its interacting partners MAP3K3, MAP3K14, TRAF-I, TXN and YWHAZ and others (Figure 13) increases signaling through the p38 MAPK pathway24. This causes the migration of certain transcription factors into the nucleus, e.g.
  • API, SPl and MYC:MAX which are all downstream of the p38 pathway and activated by LL-37 and IDR-I (unpublished data), and the resultant transcription of chemokine genes (Fig 8-10).
  • the induced chemokines including MCP-I and others lead to cell recruitment that aids resolution of the infection.
  • FIG. 13 A summary of GAPDH-interacting proteins found in InnateDB.
  • InnateDB (www.innatedb.ca) is a database and analysis platform that has been developed to facilitate systems level analyses of the mammalian innate immune response.
  • InnateDB incorporates information on the whole human and mouse interactomes by integrating molecular interaction and pathway information from several of the major publicly available databases and captures improved coverage of the innate immunity interactome through detailed manual curation.
  • InnateDB was used to identify the known interacting partners of GAPDH through the combination of manually curated data and from data integrated into InnateDB from the BIND, BIOGRID, MINT, IntAct, and DIP interaction databases.
  • Pathways associated with the interacting partners of GAPDH were identified using InnateDB, which integrates pathway information on more than 2,500 pathways from the KEGG, NCI- Nature PID, INOH, NetPath and Reactome databases. For mouse interactions, pathway information was inferred from human orthologs. Absence of pathway information for the corresponding genes has been denoted as "-”. HUGO Gene Nomenclature Committee symbols are used to represent the interaction partners.
  • Figure 14 Summary of primers used for quantitative real-time PCR in the examples.
  • the present invention provides methods for identifying compounds that modulate innate immunity.
  • Examples of pharmaceutical compositions comprising these compounds that modulate innate immunity, and are capable of being identified by these methods, are also provided.
  • Human cationic host defense peptide LL-37 has a broad range of immunomodulatory, anti-infective functions.
  • a synthetic innate defense regulator peptide, IDR-I, based conceptually on LL-37, was recently shown to selectively modulate innate immunity to protect against a wide range of bacterial infections.
  • the present invention is based on the discovery, as disclosed herein, that glyceraldehyde-3 -phosphate dehydrogenase (GAPDH) is a direct binding partner for LL-37 and IDR-I in monocytes using advanced proteomic techniques, ELISA and Western blotting procedures. Enzyme kinetics and mobility shift studies also indicated LL-37 as well as IDR-I binding to GAPDH. The functional relevance of GAPDH in peptide -induced responses was demonstrated by showing that gene silencing of GAPDH resulted in impaired chemokine and cytokine transcriptional responses and downstream p38 signaling induced by LL-37 and IDR-I. As described herein, GAPDH is a mononuclear cell receptor for human cathelicidin LL-37 and immunomodulatory IDR-I, and conclusively demonstrates its relevance in the functioning of cationic host defense peptides.
  • GAPDH glyceraldehyde-3 -phosphate dehydrogena
  • the present invention provides a number of methods, reagents, and compounds that can be used for modulating innate immunity. It is to be understood that this invention is not limited to particular methods, reagents, compounds, compositions, or biological systems, which can, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular aspects only, and is not intended to be limiting. As used in this specification and the appended claims, the singular forms "a”, “an”, and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a peptide” includes a combination of two or more peptides, and the like.
  • Innate immunity refers to the natural ability of an organism to defend itself against invasion by pathogens.
  • Pathogens or microbes as used herein can include, but are not limited to bacteria, fungi, parasite, and viruses.
  • Innate immunity is contrasted with acquired/adaptive immunity in which the organism develops a defensive mechanism based substantially on antibodies and/or immune lymphocytes that is characterized by specificity, amplifiability and self vs. non-self discrimination. With innate immunity, broad, nonspecific immunity is provided and there is no immunologic memory of prior exposure.
  • innate immunity The hallmarks of innate immunity are effectiveness against a broad variety of potential pathogens, independence of prior exposure to a pathogen, and immediate effectiveness (in contrast to the specific immune response which takes days to weeks to be elicited).
  • the system of innate immunity can be amplified and together with amplified protective responses, there is an amplification of pro-inflammatory cytokines including tumour necrosis factor- ⁇ (TNF- ⁇ ) and interleukin 1- ⁇ ) that can potentially cause harm if overstimulated.
  • TNF- ⁇ tumour necrosis factor- ⁇
  • interleukin 1- ⁇ interleukin 1- ⁇
  • innate immunity includes immune responses that affect other diseases, such as cancer, inflammatory diseases, multiple sclerosis, various viral infections, and the like.
  • the innate and adaptive immune responses both function to protect against invading organisms, but they differ in a number of ways.
  • the innate immune system is constitutively present and reacts immediately to infection.
  • the adaptive immune response to an invading organism takes some time (days to weeks) to develop.
  • the innate immune system is not specific in its response and reacts equally well to a variety of organisms, whereas the adaptive immune system is antigen-specific and reacts only with the organism that induced the response.
  • the adaptive immune system exhibits immunological memory. It "remembers" that it has encountered an invading organism (antigen) and reacts more rapidly on subsequent exposure to the same organism.
  • the innate immune system does not possess a memory.
  • innate immnity instructs adaptive immunity
  • one of the roles of innate immunity is to prepare for a subsequent adaptive immune response (a phenomenon that is part of the adjuvant effect); furthermore the effector mechanisms of adaptive immunity overlap with those of innate immunity.
  • cationic peptide refers to a sequence of amino acids from about 5 to about 50 amino acids in length. In one aspect, the cationic peptide of the invention is from about 10 to about 35 amino acids in length. A peptide is "cationic” if it possesses sufficient positively charged amino acids to have a pKa greater than 8.0. Typically, at least two of the amino acid residues of the cationic peptide will be positively charged, for example, lysine or arginine. "Positively charged” refers to the side chains of the amino acid residues which have a net positive charge at pH 7.0.
  • Examples of naturally occurring cationic antimicrobial peptides that can be recombinantly produced according to the invention include defensins, cathelicidins, magainins, melittin, and cecropins, bactenecins, indolicidins, polyphemusins, tachyplesins, and analogs thereof.
  • defensins cathelicidins, magainins, melittin, and cecropins
  • bactenecins indolicidins
  • polyphemusins tachyplesins
  • analogs thereof A variety of organisms make cationic peptides, molecules used as part of a non-specific defense mechanism against microorganisms. When isolated, these peptides are often toxic to a wide variety of microorganisms, including bacteria, fungi, and certain enveloped viruses. While cationic peptides act against many pathogens, notable exceptions and varying degrees of toxicity exist.
  • innate immunity the immune response is not dependent upon antigens.
  • the innate immunity process can include the production of secretory molecules and cellular components as set forth above.
  • the pathogens are recognized by pattern recognition receptors encoded in the germline. These Toll-like (TLR) and nucleotide oligomerization domain (NOD) family receptors have broad specificity and are capable of recognizing many pathogens.
  • TLR Toll-like
  • NOD nucleotide oligomerization domain
  • cationic peptides When cationic peptides are present in the immune response, they aid in the host response to pathogens. This change in the immune response induces the selective release of chemokines, which promote the recruitment of immune cells to the site of infection.
  • Chemokines are a subgroup of immune factors that mediate chemotactic and other pro-inflammatory phenomena (See, Schall, Cytokine 3:165- 183, 1991). Chemokines are small molecules of approximately 70-80 residues in length and can generally be divided into 4 subgroups, CXC, which have two N-terminal cysteines separated by a single amino acid, CC, which have two adjacent cysteines at the N terminus,and the C which have a single cysteine residue at the N terminus and CX3C which have two cysteines at the N terminus separated by 3 residues (reviewed by Horuk, R., Trends Pharmacol.
  • the amino termini of the chemokines have been implicated in the mediation of cell migration induced by these chemokines. This involvement has been proven by intensive studies, such as the observation that the deletion of the amino terminal 8 residues of MCP-I, amino terminal 9 residues of MCP-3, and amino terminal 8 residues of RANTES and the addition of a methionine to the amino terminus of RANTES, antagonize the chemotaxis, calcium mobilization and/or enzyme release stimulated by their native counterparts (Gong et ah, J. Biol. Chem.
  • Inflammation refers to an innate immune response that occurs when tissues are injured by bacteria, trauma, toxins, heat, or any other cause. The damaged tissue releases compounds including histamine, bradykinin, and serotonin. Inflammation refers to both acute responses (i.e., responses in which the inflammatory processes are active) and chronic responses (i.e., responses marked by slow progression and formation of new connective tissue). Acute and chronic inflammation can be distinguished by the cell types involved. Acute inflammation often involves polymorphonuclear neutrophils; whereas chronic inflammation is normally characterized by a lymphohistiocytic and/or granulomatous response. Inflammation includes reactions of both the specific and nonspecific defense systems.
  • a specific defense system reaction is a specific immune system reaction response to an antigen (possibly including an autoantigen).
  • a non-specific defense system reaction is an inflammatory response mediated by leukocytes incapable of immunological memory. Such cells include granulocytes, macrophages, neutrophils and eosinophils. Examples of specific types of inflammation are diffuse inflammation, focal inflammation, croupous inflammation, interstitial inflammation, obliterative inflammation, parenchymatous inflammation, reactive inflammation, specific inflammation, toxic inflammation and traumatic inflammation.
  • Sepsis or "systemic inflammatory response syndrome” is the systemic inflammatory response to infection. Sepsis is the result of the interaction between the microorganism and their products and the host factors released on response (cytokines and other mediators). This host response is an innate mechanism developed to protect the organism from harm but in sepsis the response is in excess, with negative effects, leading to organ dysfunction and frequently to death. The amplitude of the host defense depends on the amount of the invading microorganism present. The septic response then involves complex interactions among microbial signal molecules, leukocytes, humoral mediators and vascular endothelium. Inflammatory cytokines amplify and diversify the overall response.
  • Microbial toxins stimulate the production of cytokines like TNF- ⁇ and IL-I ⁇ , which in turn promote endothelial cell-leukocyte adhesion, release of proteases and arachidonic acid metabolites and activation of clotting.
  • IL-8 a neutrophil chemotaxin
  • IL-IO and to some extent IL-6 which are counter- regulatory, inhibit the generation of TNF- ⁇ , augment the action of acute phase reactants and immunoglobulins, and inhibit T-lymphocyte and macrophage function.
  • IL-6 along with other mediators can also promote intravascular coagulation.
  • Glyceraldehyde-3 -phosphate dehydrogenase (abbreviated as “GAPDH” or less commonly as “G3PDH”) is an enzyme that catalyzes the sixth step of glycolysis (conversion of D-glyceraldehyde-3 -phosphate to 1,3-bisphosphoglycerate) and thus serves to break down glucose for energy and carbon molecules.
  • GAPDH has recently been implicated in several non-metabolic processes, including transcriptional activation, initiation of apoptosis (Tarze et al. Oncogene 26, 2606-2620, 2007, and ER to Golgi vesicle shuttling.
  • GAPDH can be, for example, recombinant or endogenous GAPDH.
  • Receptor denotes a cell-associated protein that binds to a bioactive molecule termed a "ligand.” This interaction mediates the effect of the ligand on the cell.
  • Receptors can be membrane bound, cytosolic or nuclear; monomeric (e.g. , thyroid stimulating hormone receptor, beta-adrenergic receptor) or multimeric (e.g., TNF receptor I, TNF receptor II, PDGF receptor, growth hormone receptor, IL-3 receptor, GM-CSF receptor, G-CSF receptor, erythropoietin receptor and IL-6 receptor).
  • Membrane-bound receptors are characterized by a multi-domain structure comprising an extracellular ligand-binding domain and an intracellular effector domain that is typically involved in signal transduction.
  • the extracellular ligand-binding domain and the intracellular effector domain are located in separate polypeptides that comprise the complete functional receptor.
  • the binding of ligand to receptor results in a conformational change in the receptor that causes an interaction between the effector domain and other molecule(s) in the cell, which in turn leads to an alteration in the metabolism of the cell.
  • Metabolic events that are often linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, increase in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids.
  • Signaling in cells refers to the interaction of a ligand, such as an endogenous or exogenous ligand with receptors, involving GAPDH for example, resulting in cell signaling to produce a response, for example, an anti-inflammatory response.
  • a ligand such as an endogenous or exogenous ligand with receptors, involving GAPDH for example, resulting in cell signaling to produce a response, for example, an anti-inflammatory response.
  • amino acid residues identified herein are in the natural L-configuration. In keeping with standard polypeptide nomenclature, J. Biol. Chem., 243: 3557-59, 1969, abbreviations for amino acid residues are as shown in the following table.
  • a "gene” is a region on the genome that is capable of being transcribed to an RNA that either has a regulatory function, a catalytic function, and/or encodes a protein.
  • a eukaryotic gene typically has introns and exons, which can organize to produce different RNA splice variants that encode alternative versions of a mature protein.
  • a "full-length" gene or RNA therefore encompasses any naturally occurring splice variants, allelic variants, other alternative transcripts, splice variants generated by recombinant technologies which bear the same function as the naturally occurring variants, and the resulting RNA molecules.
  • a "fragment" of a gene, including an oncogene can be any portion from the gene, which can or can not represent a functional domain, for example, a catalytic domain, a DNA binding domain, and the like.
  • a fragment can include nucleotide sequences that encode for at least 5 to about 50 residues.
  • a fragment can also preferably include nucleotide sequences that encode for at least 25 contiguous amino acids, and preferably at least about 30, 40, 50, 60, 65, 70, 75 or more contiguous amino acids or any integer thereabout or therebetween.
  • a fragment can also preferably include nucleotide sequences that encode for at least 10 contiguous amino acids, and preferably at least about 10, 20, 30, 40, 50, 60, 70, 80 or more contiguous amino acids or any integer thereabout or therebetween.
  • Gene and “recombinant gene” refer to nucleic acid molecules which include an open reading frame encoding an innate immunity modulator protein of the invention, preferably a mammalian innate immunity modulator protein, and can further include non- coding regulatory sequences, and introns.
  • a "detectable" RNA expression level means a level that is detectable by standard techniques currently known in the art or those that become standard at some future time, and include for example, differential display, RT (reverse transcriptase)- coupled polymerase chain reaction (PCR), Northern Blot, and/or RNase protection analyses. The degree of differences in expression levels need only be large enough to be visualized or measured via standard characterization techniques.
  • Cell Cell
  • cell line cell line
  • cell culture are used interchangeably and all such designations include progeny.
  • the words “transformants” and “transformed cells” include the primary subject cell and cultures derived therefrom without regard for the number of transfers. It is also understood that all progeny cannot be precisely identical in DNA content, due to deliberate or inadvertent mutations. Mutant progeny that have the same function or biological activity as screened for in the originally transformed cell are included. Where distinct designations are intended, it will be clear from the context.
  • a "label” is a composition detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means.
  • useful labels include 32 P, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in an ELISA), biotin, digoxigenin, or haptens and proteins for which antisera or monoclonal antibodies are available (e.g., the polypeptides of the invention can be made detectable, e.g., by incorporating a radiolabel into the peptide, and used to detect antibodies specifically reactive with the peptide).
  • Sorting in the context of cells as used herein to refers to both physical sorting of the cells, as can be accomplished using, e.g., a fluorescence activated cell sorter, as well as to analysis of cells based on expression of cell surface markers, e.g., FACS analysis in the absence of sorting.
  • Nucleic acid or “nucleic acid molecule” refer to a deoxyribonucleotide or ribonucleotide polymer in either single- or double-stranded form, and unless otherwise limited, can encompass known analogs of natural nucleotides that can function in a similar manner as naturally occurring nucleotides.
  • a polynucleotide probe is a single stranded nucleic acid capable of binding to a target nucleic acid of complementary sequence through one or more types of chemical bonds, usually through complementary base pairing, usually through hydrogen bond formation.
  • a polynucleotide probe can include natural (i.e., A, G, C, or T) or modified bases (e.g., 7- deazaguanosine, inosine). Therefore, polynucleotide probes can 5-10,000, 10-5,000, 10-500, 10-50, 10-25, 10-20, 15-25, and 15-20 bases long. Probes are typically about 10-50 bases long, and are often 15-20 bases.
  • the array includes test probes (also referred to as polynucleotide probes) more than 5 bases long, preferably more than 10 bases long, and some more than 40 bases long.
  • the probes can also be less than 50 bases long.
  • these polynucleotide probes can range from about 5 to about 45 or 5 to about 50 nucleotides long, or from about 10 to about 40 nucleotides long, or from about 15 to about 40 nucleotides in length.
  • the probes can also be about 20 or 25 nucleotides in length.
  • the bases in a polynucleotide probe can be joined by a linkage other than a phosphodiester bond, so long as it does not interfere with hybridization.
  • polynucleotide probes can be peptide nucleic acids in which the constituent bases are joined by peptide bonds rather than phosphodiester linkages.
  • the length of probes used as components of pools for hybridization to distal segments of a target sequence often increases as the spacing of the segments increased thereby allowing hybridization to be conducted under greater stringency to increase discrimination between matched and mismatched pools of probes.
  • the polynucleotide probes can be less than 50 nucleotides in length, generally less than 46 nucleotides, more generally less than 41 nucleotides, most generally less than 36 nucleotides, preferably less than 31 nucleotides, more preferably less than 26 nucleotides, and most preferably less than 21 nucleotides in length.
  • the probes can also be less than 16 nucleotides, less than 13 nucleotides in length, less than 9 nucleotides in length and less than 7 nucleotides in length.
  • arrays can have polynucleotides as short as 10 nucleotides or 15 nucleotides. In addition, 20 or 25 nucleotides can be used to specifically detect and quantify nucleic acid expression levels. Where ligation discrimination methods are used, the polynucleotide arrays can contain shorter polynucleotides. Arrays containing longer polynucleotides are also suitable. High density arrays can comprise greater than about 100, 1000, 16,000, 65,000, 250,000 or even greater than about 1,000,000 different polynucleotide probes.
  • Target nucleic acid refers to a nucleic acid (often derived from a biological sample), to which the polynucleotide probe is designed to specifically hybridize. It is either the presence or absence of the target nucleic acid that is to be detected, or the amount of the target nucleic acid that is to be quantified.
  • the target nucleic acid has a sequence that is complementary to the nucleic acid sequence of the corresponding probe directed to the target.
  • the term target nucleic acid can refer to the specific subsequence of a larger nucleic acid to which the probe is directed or to the overall sequence (e.g., gene or mRNA), the expression level of which it is desired to detect. The difference in usage can be apparent from context.
  • “Subsequence” refers to a sequence of nucleic acids that comprise a part of a longer sequence of nucleic acids.
  • substantially pure or isolated means an object species (e.g., an antibody of the invention) has been identified and separated and/or recovered from a component of its natural environment such that the object species is the predominant species present (i.e., on a molar basis it is more abundant than any other individual species in the composition); a "substantially pure” or “isolated” composition also means where the object species comprises at least about 50 percent (on a molar basis) of all macromolecular species present.
  • a substantially pure or isolated composition can also comprise more than about 80 to 90 percent by weight of all macromolecular species present in the composition.
  • an isolated object species e.g., antibodies of the invention
  • an isolated antibody to GAPDH can be substantially free of other antibodies that lack binding to human GAPDH and bind to a different antigen.
  • an isolated antibody that specifically binds to an epitope, isoform or variant of human GAPDH can, however, have cross-reactivity to other related antigens, e.g., from other species (e.g., GAPDH species homologs).
  • an isolated antibody of the invention can be substantially free of other cellular material (e.g., non-immunoglobulin associated proteins) and/or chemicals.
  • Specific binding refers to preferential binding of an antibody to a specified antigen relative to other non-specified antigens.
  • the phrase “specifically (or selectively) binds” to an antibody refers to a binding reaction that is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
  • the antibody binds with an association constant (K a ) of at least about 1 x 10 6 M “1 or 10 7 M “1 , or about 10 8 M “1 to 10 9 M “1 , or about 10 10 M “1 to 10 11 M “1 or higher, and binds to the specified antigen with an affinity that is at least two-fold greater than its affinity for binding to a non-specific antigen (e.g., BSA, casein) other than the specified antigen or a closely-related antigen.
  • a non-specific antigen e.g., BSA, casein
  • a predetermined antigen is an antigen that is chosen prior to the selection of an antibody that binds to that antigen.
  • bind(s) or "bind(s) specifically” when referring to a peptide refers to a peptide molecule which has intermediate or high binding affinity, exclusively or predominately, to a target molecule.
  • the phrase "specifically binds to” refers to a binding reaction which is determinative of the presence of a target protein in the presence of a heterogeneous population of proteins and other biologies.
  • the specified binding moieties bind preferentially to a particular target protein and do not bind in a significant amount to other components present in a test sample. Specific binding to a target protein under such conditions can require a binding moiety that is selected for its specificity for a particular target antigen.
  • Nucleic acid molecule includes DNA molecules (e.g., a cDNA or genomic DNA) and RNA molecules (e.g., an mRNA) and analogs of the DNA or RNA generated, e.g., by the use of nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double- stranded, but preferably is double-stranded DNA.
  • isolated or purified nucleic acid molecule includes nucleic acid molecules which are separated from other nucleic acid molecules which are present in the natural source of the nucleic acid.
  • isolated includes nucleic acid molecules which are separated from the chromosome with which the genomic DNA is naturally associated.
  • an "isolated" nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and/or 3' ends of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
  • the isolated nucleic acid molecule can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of 5' and/or 3' nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular materials, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • An "isolated” or “purified” polypeptide or protein is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language "substantially free” means a preparation of an innate immunity modulator protein having less than about 30%, 20%, 10% and more preferably 5% (by dry weight), of non- innate immunity modulator protein (also referred to herein as a "contaminating protein"), or of chemical precursors or non- innate immunity modulator chemicals.
  • the an innate immunity modulator protein, or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the protein preparation.
  • the invention includes isolated or purified preparations of at least 0.01, 0.1, 1.0, and 10 milligrams in dry weight.
  • the terms “subject” or “patient” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • "Patient”, “subject” or “mammal” are used interchangeably and refer to mammals such as human patients and non-human primates, as well as experimental animals such as rabbits, rats, and mice, and other animals.
  • Animals include all vertebrates, e.g., mammals and non-mammals, such as sheep, dogs, cows, chickens, amphibians, birds and reptiles. Accordingly, the term “subject” or “patient” as used herein means any mammalian patient or subject to which the compositions of the invention can be administered.
  • accepted screening methods are employed to determine the status of an existing disease or condition in a subject or risk factors associated with a targeted or suspected disease or condition. These screening methods include, for example, examinations to determine whether a subject is suffering from sepsis or an inflammatory-based disease or disorder. These and other routine methods allow the clinician to select subjects in need of therapy.
  • Treating includes the administration of the compositions, compounds or agents of the present invention to prevent or delay the onset of the symptoms, complications, or biochemical indicia of a disease, alleviating or ameliorating the symptoms or arresting or inhibiting further development of the disease, condition, or disorder. "Treating” further refers to any indicia of success in the treatment or amelioration or prevention of the disease, condition, or disorder, including any objective or subjective parameter such as abatement; remission; diminishing of symptoms or making the disease condition more tolerable to the patient; slowing in the rate of degeneration or decline; or making the final point of degeneration less debilitating.
  • the treatment or amelioration of symptoms can be based on objective or subjective parameters; including the results of an examination by a physician.
  • treating includes the administration of the compounds or agents of the present invention to prevent or delay, to alleviate, or to arrest or inhibit development of the symptoms or conditions associated with a disease or disorder.
  • therapeutic effect refers to the reduction, elimination, or prevention of the disease or disorder, symptoms of the disease or disorder, or side effects of the disease or disorder in the subject.
  • Treating” or “treatment” using the methods of the present invention includes preventing the onset of symptoms in a subject that can be at increased risk of a disease or disorder or but does not yet experience or exhibit symptoms, inhibiting the symptoms of a isease or disorder (slowing or arresting its development), providing relief from the symptoms or side-effects of a disease or disorder (including palliative treatment), and relieving the symptoms of a disease or disorder (causing regression).
  • Treatment can be prophylactic (to prevent or delay the onset of the disease, or to prevent the manifestation of clinical or subclinical symptoms thereof) or therapeutic suppression or alleviation of symptoms after the manifestation of the disease or condition.
  • a chronic disease or condition is a disease or condition that is long-lasting or recurrent.
  • the term chronic describes the course of the disease, or its rate of onset and development.
  • a chronic course is distinguished from a recurrent course; recurrent diseases or conditions relapse repeatedly, with periods of remission in between.
  • Treatment of recurrent diseases and conditions with the proteins disclosed herein are also contemplated.
  • a chronic disease or condition can have one or more of the following characteristics: a chronic disease or condition is permanent, leaves residual disability, can be caused by nonreversible pathological alteration, requires special training of the patient for rehabilitation, or can be expected to require a long period of supervision, observation, or care.
  • the phrase “well tolerated” refers to the absence of adverse changes in health status that occur as a result of the treatment and would affect treatment decisions.
  • “In combination with”, “combination therapy” and “combination products” refer, in certain aspects, to the concurrent administration to a patient of a first therapeutic and the compounds as used herein.
  • each component can be administered at the same time or sequentially in any order at different points in time.
  • each component can be administered separately but sufficiently closely in time so as to provide the desired therapeutic effect.
  • Concomitant administration of a known drug with a compound of the present invention means administration of the drug and the compound at such time that both the known drug and the compound will have a therapeutic effect or diagnostic effect. Such concomitant administration can involve concurrent (i.e. at the same time), prior, or subsequent administration of the drug with respect to the administration of a compound of the present invention.
  • a person of ordinary skill in the art would have no difficulty in determining the appropriate timing, sequence and dosages of administration for particular drugs and compounds of the present invention.
  • Modemator includes inhibitors and activators.
  • Inhibitors are agents that, e.g., bind to, partially or totally block stimulation, decrease, prevent, delay activation, inactivate, desensitize, or down regulate the activity of glyceraldehyde-3-phaosphate dehydrogenase (GAPDH), e.g., antagonists.
  • GPDH glyceraldehyde-3-phaosphate dehydrogenase
  • Antagonists can be proteins, nucleic acids, carbohydrates, antibodies, small molecules, or any other molecule which decrease the activity of the target molecule (e.g., GAPDH).
  • Activators are agents that, e.g., bind to, stimulate, increase, open, activate, facilitate, enhance activation, sensitize or up regulate the activity of GAPDH, e.g., agonists.
  • Agonists can be proteins, nucleic acids, carbohydrates, small molecules, or any other molecule which activate GAPDH.
  • Modulators include agents that, e.g., alter the interaction of GAPDH: proteins that bind activators or inhibitors, receptors, including proteins, peptides, lipids, carbohydrates, polysaccharides, or combinations of the above, e.g., lipoproteins, glycoproteins, and the like.
  • Modulators include genetically modified versions of naturally-occurring receptor ligands, e.g., with altered activity, as well as naturally occurring and synthetic ligands, antagonists, agonists, small chemical molecules and the like.
  • modulate refers to a change in the activity of GAPDH polypeptide. For example, modulation can cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of GAPDH.
  • Cell-based assays for inhibitors and activators include, e.g., applying putative innate immunity modulator compounds to a cell expressing a receptor, e.g.
  • Cell based assays include, but are not limited to, in vivo tissue or cell samples from a mammalian subject or in vitro cell-based assays comprising GAPDH are treated with a potential activator, inhibitor, or modulator are compared to control samples without the inhibitor, activator, or modulator to examine the extent of inhibition. Control samples
  • GAPDH is achieved when the GAPDH activity value relative to the control is about 80%, optionally 50% or 25-0%. Activation of GAPDH or is achieved when the B-lymphocyte receptor activity value relative to the control is 110%, optionally 150%, optionally 200-
  • Contacting refers to mixing a test compound in a soluble form into an assay system, for example, a cell-based assay system, such that an effect can be measured, such as receptor-mediated signaling or an immunomodulatory event (innate immunity modulating event).
  • an effect such as receptor-mediated signaling or an immunomodulatory event (innate immunity modulating event).
  • “Signaling responsiveness” or “effective to activate signaling” or “stimulating a cell-based assay system” refers to the ability of innate immunity modulators of the invention to inhibit or enhance an immune response, or treat infection, inflammation, sepsis, autoimmune disease, or a related disease or disorder.
  • Detecting an effect refers to an effect measured in a cell-based assay system.
  • the effect detected can be modulation of innate immunity in an assay system, for example, a cellular assay, ligand receptor binding assay.
  • Assay being indicative of modulation refers to results of a cell-based assay system indicating that modulation by an innate immunity modulator of the invention induces a response in cells, for example, to sepsis or inflammation.
  • Biological activity and “biologically active” with regard to a modulator of innate immunity of the present invention refer to the ability of the modulator to specifically bind to and signal through a native or recombinant receptor, or to block the ability of a native or recombinant receptor to participate in signal transduction.
  • the (native and variant) ligands of the receptor of the present invention can include agonists and antagonists of a native or recombinant receptor.
  • solid phase is meant a non-aqueous matrix to which a reagent of interest can adhere.
  • solid phases encompassed herein include those formed partially or entirely of glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrene, polyvinyl alcohol and silicones.
  • the solid phase can comprise the well of an assay plate; in others it is a purification column (e.g., an affinity chromatography column). This term also includes a discontinuous solid phase of discrete particles, such as those described in U.S. Pat. No. 4,275,149.
  • Test compound refers to a nucleic acid, DNA, RNA, protein, polypeptide, or small chemical entity that is determined to affect an increase or decrease in a gene expression as a result the activity of modulators of innate immunity.
  • the test compound can be an antisense RNA, ribozyme, polypeptide, or small molecular chemical entity.
  • the term "test compound” can be any small chemical compound, or a biological entity, such as a protein, sugar, nucleic acid or lipid. Typically, test compounds will be small chemical molecules and polypeptides.
  • a "test compound specific for GAPDH" is determined to be a modulator of innate immunity. Typically, test compounds will be peptides or peptidemimetics.
  • Receptor refers to any macromolecule that functions to bind a ligand in a host organism.
  • the receptor can be either a membrane-bound receptor, such as an extracellular or intracellular membrane-bound protein receptor, or can be a soluble receptor.
  • Receptor ligand refers to a ligand that interacts in a host organism with a receptor as this term is defined above. Examples of such ligands include hormones, neurotransmitters, drugs, vasoactive amines (such as histamine and serotonin), cytokines and intracellular messengers.
  • biological function is meant to signify that the receptor or ligand has some bioactive function in the host organism.
  • bioactive functions include metabolic functions in regulating the level of a particular metabolite or group of metabolites, binding functions in titrating the amount of a compound in a biological fluid such as blood, messenger functions, functions as mediators of inflammatory processes and recruiters of defence mechanisms, immunomodulators such as cytokines and chemokines, functions in dilating or contracting blood vessels, modulators of antibody-related and other immune responses and so on.
  • a biological fluid such as blood, messenger functions, functions as mediators of inflammatory processes and recruiters of defence mechanisms
  • immunomodulators such as cytokines and chemokines
  • isolated when used in reference to a peptide, refers to a peptide substantially free of proteins, lipids, nucleic acids, for example, with which it can be naturally associated.
  • the invention includes peptides, analogs or derivatives thereof, as long as the bioactivity ⁇ e.g., innate immunity modulating) of the peptide remains.
  • Minor modifications of the primary amino acid sequence of the peptides of the invention can result in peptides that have substantially equivalent activity as compared to the specific peptides described herein. Such modifications can be deliberate, as by site-directed mutagenesis, or can be spontaneous. All of the peptides produced by these modifications are included herein as long as the biological activity of the original peptide still exists.
  • deletion of one or more amino acids can also result in a modification of the structure of the resultant molecule without significantly altering its biological activity. This can lead to the development of a smaller active molecule that would also have utility.
  • amino or carboxy terminal amino acids that are not required for biological activity of the particular peptide can be removed.
  • Peptides of the invention include any analog, homo log, mutant, isomer or derivative of the peptides disclosed in the present invention, so long as the bioactivity as described herein remains. All peptides were synthesized using L amino acids, however, all D forms of the peptides can be synthetically produced and can have similar activity.
  • C-terminal derivatives can be produced, such as C-terminal methyl esters and C-terminal amidates, in order to increase the innate immunity modulating activity of a peptide of the invention.
  • the peptide can be synthesized such that the sequence is reversed whereby the last amino acid in the sequence becomes the first amino acid, and the penultimate amino acid becomes the second amino acid, and so on. It is well known that such reversed peptides usually have similar innate immunity modulating activities to the original sequence.
  • the peptides of the invention include peptide analogs and peptide mimetics. Indeed, the peptides of the invention include peptides having any of a variety of different modifications, including those described herein.
  • Peptide analogs of the invention are generally designed and produced by chemical modifications of a lead peptide, including, e.g., any of the particular peptides described herein, such as any of the following sequences disclosed in the tables.
  • the present invention clearly establishes that these peptides in their entirety and derivatives created by modifying any side chains of the constituent amino acids have the ability to inhibit, prevent, or destroy the growth or proliferation of microbes such as bacteria, fungi, viruses, parasites or the like.
  • the present invention further encompasses polypeptides up to about 50 amino acids in length that include the amino acid sequences and functional variants or peptide mimetics of the sequences described herein.
  • a peptide of the present invention is a pseudopeptide.
  • Pseudopeptides or amide bond surrogates refers to peptides containing chemical modifications of some (or all) of the peptide bonds. The introduction of amide bond surrogates not only decreases peptide degradation but also can significantly modify some of the biochemical properties of the peptides, particularly the conformational flexibility and hydrophobicity.
  • polypeptides of the present invention protein engineering can be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or muteins including single or multiple amino acid substitutions, deletions, additions, or fusion proteins.
  • modified polypeptides can show, e.g. , increased/decreased biological activity or increased/decreased stability.
  • they can be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • the polypeptides of the present invention can be produced as multimers including dimers, trimers and tetramers. Multimerization can be facilitated by linkers, introduction of cysteines to permit creation of interchain disulphide bonds, or recombinantly though heterologous polypeptides such as F c regions.
  • Another method of improving or altering the characteristics of polypeptides of the present invention involves the use of peptide arrays in which numerous variants of peptides can be made using spot synthesis by robot on cellulose or other substrates (Hilpert K. et ah, Nature Biotechnology 23: 1008-1012, 2005; Hilpert K, D Winkler, REW Hancock. Nature Protocols 2: 1333-1349, 2007).
  • This can be assisted using quantative structure activity relationship modeling techniques to simply identify enhanced peptides using computational means (Jenssen H et al. J Peptide Science 14: 110-114, 2008).
  • the present invention provides polypeptides having one or more residues deleted from the amino terminus.
  • many examples of biologically functional C-terminal deletion mutants are known (see, e.g., Dobeli et al., J. Biotechnology 7: 199-216, 1988).
  • the present invention provides polypeptides having one or more residues deleted from the carboxy terminus.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini as described below.
  • mutants in addition to N- and C-terminal deletion forms of the protein discussed above are included in the present invention.
  • the invention further includes variations of the polypeptides which show substantial chaperone polypeptide activity.
  • Such mutants include deletions, insertions, inversions, repeats, and substitutions selected according to general rules known in the art so as to have little effect on activity.
  • There are two main approaches for studying the tolerance of an amino acid sequence to change see, Bowie et ah, Science 247: 1306-1310, 1994.
  • the first method relies on the process of evolution, in which mutations are either accepted or rejected by natural selection.
  • the second approach uses genetic or protein engineering to introduce amino acid changes at specific positions of a cloned gene and selections or screens to identify sequences that maintain functionality.
  • substitutions are the replacements, one for another, among the aliphatic amino acids Ala, VaI, Leu and Phe; interchange of the hydroxyl residues Ser and Thr, exchange of the acidic residues Asp and GIu, substitution between the amide residues Asn and GIn, exchange of the basic residues Lys and Arg and replacements among the aromatic residues Phe, Tyr.
  • the polypeptide of the present invention can be, for example: (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue can or cannot be one encoded by the genetic code; or (ii) one in which one or more of the amino acid residues includes a substituent group; or (iii) one in which the polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the above form of the polypeptide, such as an IgG F c fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the above form of the polypeptide or a pro-protein sequence.
  • a conserved or non-conserved amino acid residue preferably a conserved amino acid residue
  • substituted amino acid residue can or cannot
  • polypeptides of the present invention can include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation.
  • changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein.
  • the following groups of amino acids represent equivalent changes: (1) Ala, Pro, GIy, GIu, Asp, GIn, Asn, Ser, Thr; (2) Cys, Ser, Tyr, Thr; (3) VaI, He, Leu, Met, Ala, Phe; (4) Lys, Arg, His; (5) Phe, Tyr, Trp, His.
  • polypeptides of the present invention can include one or more amino acid substitutions that mimic modified amino acids.
  • An example of this type of substitution includes replacing amino acids that are capable of being phosphorylated (e.g., serine, threonine, or tyrosine) with a negatively charged amino acid that resembles the negative charge of the phosphorylated amino acid (e.g., aspartic acid or glutamic acid).
  • substitution of amino acids that are capable of being modified by hydrophobic groups e.g., arginine
  • amino acids carrying bulky hydrophobic side chains such as tryptophan or phenylalanine.
  • a specific aspect of the invention includes polypeptides that include one or more amino acid substitutions that mimic modified amino acids at positions where amino acids that are capable of being modified are normally positioned. Further included are polypeptides where any subset of modifiable amino acids is substituted. For example, a polypeptide that includes three serine residues can be substituted at any one, any two, or all three of said serines. Furthermore, any polypeptide amino acid capable of being modified can be excluded from substitution with a modification-mimicking amino acid. [0098] The present invention is further directed to fragments of the polypeptides of the present invention.
  • the present invention embodies purified, isolated, and recombinant polypeptides comprising at least any one integer between 6 and 504 (or the length of the polypeptides amino acid residues minus 1 if the length is less than 1000) of consecutive amino acid residues.
  • the fragments are at least 6, preferably at least 8 to 10, more preferably 12, 15, 20, 25, 30, 35, 40, 50 or more consecutive amino acids of a polypeptide of the present invention.
  • the present invention also provides for the exclusion of any species of polypeptide fragments of the present invention specified by 5' and 3' positions or sub- genuses of polypeptides specified by size in amino acids as described above. Any number of fragments specified by 5' and 3' positions or by size in amino acids, as described above, can be excluded.
  • the peptides of the present invention include two or more modifications, including, but not limited to those described herein.
  • modifications including, but not limited to those described herein.
  • Polypeptide “peptide” and “protein” are used interchangeably herein to refer to a polymer of amino acid residues.
  • the terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non- naturally occurring amino acid polymer.
  • Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but which functions in a manner similar to a naturally occurring amino acid.
  • Non-natural residues are well described in the scientific and patent literature; a few exemplary non-natural compositions useful as mimetics of natural amino acid residues and guidelines are described below.
  • Mimetics of aromatic amino acids can be generated by replacing by, e.g., D- or L- naphylalanine; D- or L-phenylglycine; D- or L-2 thieneylalanine; D- or L-I, -2,3-, or 4- pyreneylalanine; D- or L-3 thieneylalanine; D- or L-(2-pyridinyl)-alanine; D- or L-(3- pyridinyl)-alanine; D- or L-(2-pyrazinyl)-alanine; D- or L-(4-isopropyl)-phenylglycine; D- (trifluoromethyl)-phenylglycine; D-(trifluoromethyl)-phenylalanine; D-p-fluoro- phenylalanine; D- or L-p-biphenylphenylalanine; K- or L-p-methoxy-biphenyl
  • Aromatic rings of a non-natural amino acid include, e.g., thiazolyl, thiophenyl, pyrazolyl, benzimidazolyl, naphthyl, furanyl, pyrrolyl, and pyridyl aromatic rings.
  • Peptide as used herein includes peptides that are conservative variations of those peptides specifically exemplified herein.
  • Constant variation as used herein denotes the replacement of an amino acid residue by another, biologically similar residue.
  • conservative variations include, but are not limited to, the substitution of one hydrophobic residue such as isoleucine, valine, leucine, alanine, cysteine, glycine, phenylalanine, proline, tryptophan, tyrosine, norleucine or methionine for another, or the substitution of one polar residue for another, such as the substitution of arginine for lysine, glutamic for aspartic acids, or glutamine for asparagine, and the like.
  • Neutral hydrophilic amino acids that can be substituted for one another include asparagine, glutamine, serine and threonine.
  • conservative variation also includes the use of a substituted amino acid in place of an unsubstituted parent amino acid provided that antibodies raised to the substituted polypeptide also immunoreact with the unsubstituted polypeptide. Such conservative substitutions are within the definition of the classes of the peptides of the invention.
  • the biological activity of the peptides can be determined by standard methods known to those of skill in the art, such as “minimal inhibitory concentration (MIC)" assay (Wiegand I et al.
  • the peptides and polypeptides of the invention include all “mimetic” and “peptidomimetic” forms.
  • the terms “mimetic” and “peptidomimetic” refer to a synthetic chemical compound that has substantially the same structural and/or functional characteristics of the polypeptides of the invention.
  • the mimetic can be either entirely composed of synthetic, non-natural analogues of amino acids, or, is a chimeric molecule of partly natural peptide amino acids and partly non-natural analogs of amino acids.
  • the mimetic can also incorporate any amount of natural amino acid conservative substitutions, as long as such substitutions also do not substantially alter the mimetic 's structure and/or activity.
  • conservative substitutions As with polypeptides of the invention that are conservative variants, routine experimentation will determine whether a mimetic is within the scope of the invention, i.e., that its structure and/or function is not substantially altered.
  • a mimetic composition is within the scope of the invention if, when administered to or expressed in a cell, e.g., a polypeptide fragment of an innate immunity modulating protein having innate immunity modulating activity.
  • Polypeptide mimetic compositions can contain any combination of non-natural structural components, which are typically from three structural groups: a) residue linkage groups other than the natural amide bond ("peptide bond") linkages; b) non-natural residues in place of naturally occurring amino acid residues; or c) residues which induce secondary structural mimicry, i.e., to induce or stabilize a secondary structure, e.g., a beta turn, gamma turn, beta sheet, alpha helix conformation, and the like.
  • a polypeptide can be characterized as a mimetic when all or some of its residues are joined by chemical means other than natural peptide bonds.
  • Individual peptidomimetic residues can be joined by peptide bonds, other chemical bonds or coupling means, such as, e.g., glutaraldehyde, N- hydroxysuccinimide esters, bifunctional maleimides, N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC).
  • glutaraldehyde N- hydroxysuccinimide esters
  • bifunctional maleimides N,N'-dicyclohexylcarbodiimide (DCC) or N,N'-diisopropylcarbodiimide (DIC).
  • DCC N,N'-dicyclohexylcarbodiimide
  • DIC N,N'-diisopropylcarbodiimide
  • aminomethylene CH 2 -NH
  • ethylene olefin
  • ether CH 2 -O
  • thioether CH 2 -S
  • Mimetics of acidic amino acids can be generated by substitution by, e.g., non- carboxylate amino acids while maintaining a negative charge; (phosphono)alanine; sulfated threonine.
  • Carboxyl side groups e.g., aspartyl or glutamyl
  • Carboxyl side groups can also be selectively modified by reaction with carbodiimides (R' — N — C — N — R') such as, e.g., l-cyclohexyl-3(2- morpholin-yl-(4-ethyl) carbodiimide or l-ethyl-3(4-azonia-4,4-dimetholpentyl) carbodiimide.
  • Aspartyl or glutamyl can also be converted to asparaginyl and glutaminyl residues by reaction with ammonium ions.
  • Mimetics of basic amino acids can be generated by substitution with, e.g., (in addition to lysine and arginine) the amino acids ornithine, citrulline, guanidino-acetic acid, or (guanidino)alkyl-acetic acid, where alkyl is defined above.
  • Nitrile derivative e.g., containing the CN-moiety in place of COOH
  • Asparaginyl and glutaminyl residues can be deaminated to the corresponding aspartyl or glutamyl residues.
  • Arginine residue mimetics can be generated by reacting arginyl with, e.g., one or more conventional reagents, including, e.g., phenylglyoxal, 2,3-butanedione, 1,2- cyclohexanedione, or ninhydrin, preferably under alkaline conditions.
  • Tyrosine residue mimetics can be generated by reacting tyrosyl with, e.g. , aromatic diazonium compounds or tetranitromethane. N-acetylimidizol and tetranitromethane can be used to form O-acetyl tyrosyl species and 3-nitro derivatives, respectively.
  • Cysteine residue mimetics can be generated by reacting cysteinyl residues with, e.g., alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines; to give carboxymethyl or carboxyamidomethyl derivatives.
  • alpha-haloacetates such as 2-chloroacetic acid or chloroacetamide and corresponding amines
  • Cysteine residue mimetics can also be generated by reacting cysteinyl residues with, e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid; chloroacetyl phosphate, N-alkylmaleimides, 3-nitro-2-pyridyl disulfide; methyl 2-pyridyl disulfide; p-chloromercuribenzoate; 2-chloromercuri-4 nitrophenol; or, chloro-7-nitrobenzo-oxa-l,3-diazole.
  • cysteinyl residues e.g., bromo-trifluoroacetone, alpha-bromo-beta-(5- imidozoyl) propionic acid
  • chloroacetyl phosphate N-alkylmaleimides
  • 3-nitro-2-pyridyl disulfide methyl 2-pyridyl disulfide
  • Lysine mimetics can be generated (and amino terminal residues can be altered) by reacting lysinyl with, e.g., succinic or other carboxylic acid anhydrides. Lysine and other alpha-amino-containing residue mimetics can also be generated by reaction with imidoesters, such as methyl picolinimidate, pyridoxal phosphate, pyridoxal, chloroborohydride, trinitrobenzenesulfonic acid, O-methylisourea, 2,4, pentanedione, and transamidase-catalyzed reactions with glyoxylate. Mimetics of methionine can be generated by reaction with, e.g., methionine sulfoxide.
  • Mimetics of proline include, e.g., pipecolic acid, thiazolidine carboxylic acid, 3- or 4-hydroxy proline, dehydroproline, 3- or 4-methylproline, or 3,3,-dimethylproline.
  • Histidine residue mimetics can be generated by reacting histidyl with, e.g., diethylprocarbonate or para-bromophenacyl bromide.
  • mimetics include, e.g., those generated by hydroxylation of proline and lysine; phosphorylation of the hydroxyl groups of seryl or threonyl residues; methylation of the alpha-amino groups of lysine, arginine and histidine; acetylation of the N-terminal amine; methylation of main chain amide residues or substitution with N-methyl amino acids; or amidation of C-terminal carboxyl groups.
  • a component of a polypeptide of the invention can also be replaced by an amino acid (or peptidomimetic residue) of the opposite chirality.
  • any amino acid naturally occurring in the L-configuration (which can also be referred to as the R or S, depending upon the structure of the chemical entity) can be replaced with the amino acid of the same chemical structural type or a peptidomimetic, but of the opposite chirality, referred to as the D-amino acid, but which can additionally be referred to as the R- or S-form.
  • the invention also provides polypeptides that are "substantially identical" to an exemplary polypeptide of the invention.
  • a "substantially identical" amino acid sequence is a sequence that differs from a reference sequence by one or more conservative or non- conservative amino acid substitutions, deletions, or insertions, particularly when such a substitution occurs at a site that is not the active site of the molecule, and provided that the polypeptide essentially retains its functional properties.
  • a conservative amino acid substitution substitutes one amino acid for another of the same class (e.g., substitution of one hydrophobic amino acid, such as isoleucine, valine, leucine, or methionine, for another, or substitution of one polar amino acid for another, such as substitution of arginine for lysine, glutamic acid for aspartic acid or glutamine for asparagine).
  • One or more amino acids can be deleted, for example, from an innate immunity modulating polypeptide having innate immunity modulating activity of the invention, resulting in modification of the structure of the polypeptide, without significantly altering its biological activity. For example, amino- or carboxyl-terminal, or internal, amino acids that are not required for innate immunity modulating activity can be removed.
  • Modified peptides of the invention can be further produced by chemical modification methods, see, e.g., Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994.
  • Polypeptides and peptides of the invention can be isolated from natural sources, be synthetic, or be recombinantly generated polypeptides. Peptides and proteins can be recombinantly expressed in vitro or in vivo.
  • the peptides and polypeptides of the invention can be made and isolated using any method known in the art. Polypeptide and peptides of the invention can also be synthesized, whole or in part, using chemical methods well known in the art. See e.g., Caruthers, Nucleic Acids Res. Symp. Ser. 215-223, 1980; Horn, Nucleic Acids Res. Symp. Ser.
  • peptide synthesis can be performed using various solid-phase techniques (see e.g., Roberge, Science 269: 202, 1995; Merrifield, Methods Enzymol. 289: 3-13, 1997) and automated synthesis can be achieved, e.g., using the ABI 43 IA Peptide Synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer.
  • Peptides of the invention can be synthesized by such commonly used methods as t-BOC or FMOC protection of alpha-amino groups. Both methods involve stepwise syntheses whereby a single amino acid is added at each step starting from the C terminus of the peptide (See, Coligan et al, Current Protocols in Immunology, Wiley Interscience, 1991, Unit 9). Peptides of the invention can also be synthesized by the well known solid phase peptide synthesis methods described in Merrifield, J. Am. Chem. Soc.
  • Lyophilization of appropriate fractions of the column will yield the homogeneous peptide or peptide derivatives, which can then be characterized by such standard techniques as amino acid analysis, thin layer chromatography, high performance liquid chromatography, ultraviolet absorption spectroscopy, molar rotation, solubility, and quantitated by the solid phase Edman degradation.
  • Analogs polypeptide fragment of innate immunity modulating protein having innate immunity modulating activity, are generally designed and produced by chemical modifications of a lead peptide, including, e.g., any of the particular peptides described herein.
  • nucleic acids or polypeptide sequences refers to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same ⁇ i.e., about 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity over a specified region ⁇ e.g., nucleotide sequence encoding an antibody described herein or amino acid sequence of an antibody described herein), when compared and aligned for maximum correspondence over a comparison window or designated region) as measured using a BLAST or BLAST 2.0 sequence comparison algorithms with default parameters described below, or by manual alignment and visual inspection.
  • sequences are then said to be "substantially identical.”
  • This term also refers to, or can be applied to, the compliment of a test sequence.
  • the term also includes sequences that have deletions and/or additions, as well as those that have substitutions.
  • the preferred algorithms can account for gaps and the like.
  • identity exists over a region that is at least about 25 amino acids or nucleotides in length, or more preferably over a region that is 50-100 amino acids or nucleotides in length.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Preferably, default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a "comparison window" includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol. 48: 443, 1970, by the search for similarity method of Pearson & Lipman, Proc. Nafl. Acad. Sci.
  • Programs for searching for alignments are well known in the art, e.g., BLAST and the like (described in further detail below; see also Section F(7)).
  • BLAST a source of such amino acid sequences or gene sequences (germline or rearranged antibody sequences) can be found in any suitable reference database such as Genbank, the NCBI protein databank (http://ncbi.nlm.nih.gov/BLAST/), VBASE, a database of human antibody genes (http://www.mrc-cpe.cam.ac.uk/imt-doc), and the Kabat database of immunoglobulins (http://www.immuno.bme.nwu.edu) or translated products thereof.
  • the selected genes should be analyzed to determine which genes of that subset have the closest amino acid homology to the originating species antibody. It is contemplated that amino acid sequences or gene sequences which approach a higher degree homology as compared to other sequences in the database can be utilized and manipulated in accordance with the procedures described herein. Moreover, amino acid sequences or genes which have lesser homology can be utilized when they encode products which, when manipulated and selected in accordance with the procedures described herein, exhibit specificity for the predetermined target antigen. In certain aspects, an acceptable range of homology is greater than about 50%. It should be understood that target species can be other than human.
  • a preferred example of algorithm that is suitable for determining percent sequence identity and sequence similarity are the BLAST and BLAST 2.0 algorithms, which are described in Altschul et al, Nuc. Acids Res. 25: 3389-3402, 1977 and Altschul et al, J. MoI. Biol. 215: 403-410, 1990, respectively.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http://www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as the neighborhood word score threshold.
  • HSPs high scoring sequence pairs
  • T is referred to as the neighborhood word score threshold.
  • M forward score for a pair of matching residues; always > 0
  • N penalty score for mismatching residues; always ⁇ 0.
  • a scoring matrix is used to calculate the cumulative score.
  • Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • Polypeptide includes proteins, fusion proteins, oligopeptides and polypeptide derivatives, with the exception that peptidomimetics are considered to be small molecules herein.
  • a "protein” is a molecule having a sequence of amino acids that are linked to each other in a linear molecule by peptide bonds.
  • the term protein refers to a polypeptide that is isolated from a natural source, or produced from an isolated cDNA using recombinant DNA technology; and has a sequence of amino acids having a length of at least about 200 amino acids.
  • a "fusion protein” is a type of recombinant protein that has an amino acid sequence that results from the linkage of the amino acid sequences of two or more normally separate polypeptides.
  • a "protein fragment” is a proteolytic fragment of a larger polypeptide, which can be a protein or a fusion protein.
  • a proteolytic fragment can be prepared by in vivo or in vitro proteolytic cleavage of a larger polypeptide, and is generally too large to be prepared by chemical synthesis.
  • Proteolytic fragments have amino acid sequences having a length from about 200 to about 1,000 amino acids.
  • oligopeptide or "peptide” is a polypeptide having a short amino acid sequence (i.e., 2 to about 200 amino acids).
  • An oligopeptide is generally prepared by chemical synthesis.
  • oligopeptides and protein fragments can be otherwise prepared, it is possible to use recombinant DNA technology and/or in vitro biochemical manipulations.
  • a nucleic acid encoding an amino acid sequence can be prepared and used as a template for in vitro transcription/translation reactions.
  • an exogenous nucleic acid encoding a preselected polypeptide is introduced into a mixture that is essentially depleted of exogenous nucleic acids that contains all of the cellular components required for transcription and translation.
  • One or more radiolabeled amino acids are added before or with the exogenous DNA, and transcription and translation are allowed to proceed.
  • the only nucleic acid present in the reaction mix is the exogenous nucleic acid added to the reaction, only polypeptides encoded thereby are produced, and incorporate the radiolabeled amino acid(s).
  • polypeptides encoded by a preselected exogenous nucleic acid are radiolabeled.
  • the preselected polypeptide is the only one that is produced in the presence of the radiolabeled amino acids and is thus uniquely labeled.
  • polypeptide derivatives include without limitation mutant polypeptides, chemically modified polypeptides, and peptidomimetics.
  • the polypeptides of this invention can generally be prepared following known techniques.
  • synthetic production of the polypeptide of the invention can be according to the solid phase synthetic method.
  • the solid phase synthesis is well understood and is a common method for preparation of polypeptides, as are a variety of modifications of that technique. Merrifield, J. Am. Chem.
  • polypeptides of this invention can be prepared in recombinant systems using polynucleotide sequences encoding the polypeptides.
  • a "variant" or “functional variant” of a polypeptide is a compound that is not, by definition, a polypeptide, i.e., it contains at least one chemical linkage that is not a peptide bond.
  • polypeptide derivatives include without limitation proteins that naturally undergo post-translational modifications such as, e.g., glycosylation. It is understood that a polypeptide of the invention can contain more than one of the following modifications within the same polypeptide.
  • Preferred polypeptide derivatives retain a desirable attribute, which can be biological activity; more preferably, a polypeptide derivative is enhanced with regard to one or more desirable attributes, or has one or more desirable attributes not found in the parent polypeptide. Although they are described in this section, peptidomimetics are taken as small molecules in the present disclosure.
  • a polypeptide having an amino acid sequence identical to that found in a protein prepared from a natural source is a "wild type" polypeptide.
  • Functional variants of polypeptides can be prepared by chemical synthesis, including without limitation combinatorial synthesis.
  • Functional variants of polypeptides larger than oligopeptides can be prepared using recombinant DNA technology by altering the nucleotide sequence of a nucleic acid encoding a polypeptide. Although some alterations in the nucleotide sequence will not alter the amino acid sequence of the polypeptide encoded thereby ("silent" mutations), many will result in a polypeptide having an altered amino acid sequence that is altered relative to the parent sequence. Such altered amino acid sequences can comprise substitutions, deletions and additions of amino acids, with the proviso that such amino acids are naturally occurring amino acids.
  • mutagenesis is one technique that can be used to prepare Functional variants of polypeptides, particularly ones having substitutions of amino acids but no deletions or insertions thereof.
  • a variety of mutagenic techniques are known that can be used in vitro or in vivo including without limitation chemical mutagenesis and PCR-mediated mutagenesis.
  • Such mutagenesis can be randomly targeted ⁇ i.e., mutations can occur anywhere within the nucleic acid) or directed to a section of the nucleic acid that encodes a stretch of amino acids of particular interest. Using such techniques, it is possible to prepare randomized, combinatorial or focused compound libraries, pools and mixtures.
  • Polypeptides having deletions or insertions of naturally occurring amino acids can be synthetic oligopeptides that result from the chemical synthesis of amino acid sequences that are based on the amino acid sequence of a parent polypeptide but which have one or more amino acids inserted or deleted relative to the sequence of the parent polypeptide. Insertions and deletions of amino acid residues in polypeptides having longer amino acid sequences can be prepared by directed mutagenesis.
  • polypeptide includes those having one or more chemical modification relative to another polypeptide, i.e., chemically modified polypeptides.
  • the polypeptide from which a chemically modified polypeptide is derived can be a wild type protein, a functional variant protein or a functional variant polypeptide, or polypeptide fragments thereof; an antibody or other polypeptide ligand according to the invention including without limitation single-chain antibodies, crystalline proteins and polypeptide derivatives thereof; or polypeptide ligands prepared according to the disclosure.
  • the chemical modification(s) confer(s) or improve(s) desirable attributes of the polypeptide but does not substantially alter or compromise the biological activity thereof.
  • Desirable attributes include but are limited to increased shelf-life; enhanced serum or other in vivo stability; resistance to proteases; and the like. Such modifications include by way of non- limiting example N-terminal acetylation, glycosylation, and biotinylation.
  • An effective approach to confer resistance to peptidases acting on the N-terminal or C-terminal residues of a polypeptide is to add chemical groups at the polypeptide termini, such that the modified polypeptide is no longer a substrate for the peptidase.
  • One such chemical modification is glycosylation of the polypeptides at either or both termini. Certain chemical modifications, in particular N-terminal glycosylation, have been shown to increase the stability of polypeptides in human serum.
  • N-terminal D-amino acid increases the serum stability of a polypeptide that otherwise contains L-amino acids, because exopeptidases acting on the N- terminal residue cannot utilize a D-amino acid as a substrate.
  • C- terminal D-amino acid also stabilizes a polypeptide, because serum exopeptidases acting on the C-terminal residue cannot utilize a D-amino acid as a substrate.
  • amino acid sequences of polypeptides with N-terminal and/or C-terminal D-amino acids are usually identical to the sequences of the parent L-amino acid polypeptide.
  • substitution of unnatural amino acids for natural amino acids in a subsequence of a polypeptide can confer or enhance desirable attributes including biological activity. Such a substitution can, for example, confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • the synthesis of polypeptides with unnatural amino acids is routine and known in the art (see, for example, Coller, et al. 1993, cited above).
  • Different host cells will contain different post-translational modification mechanisms that can provide particular types of post-translational modification of a fusion protein if the amino acid sequences required for such modifications is present in the fusion protein.
  • a large number (about 100) of post-translational modifications have been described, a few of which are discussed herein.
  • One skilled in the art will be able to choose appropriate host cells, and design chimeric genes that encode protein members comprising the amino acid sequence needed for a particular type of modification.
  • Glycosylation is one type of post-translational chemical modification that occurs in many eukaryotic systems, and can influence the activity, stability, pharmacogenetics, immunogenicity and/or antigenicity of proteins.
  • specific amino acids must be present at such sites to recruit the appropriate glycosylation machinery, and not all host cells have the appropriate molecular machinery. Saccharomyces cerevisieae and Pichia pastoris provide for the production of glycosylated proteins, as do expression systems that utilize insect cells, although the pattern of glyscoylation can vary depending on which host cells are used to produce the fusion protein.
  • Another type of post-translation modification is the phosphorylation of a free hydroxyl group of the side chain of one or more Ser, Thr or Tyr residues, Protein kinases catalyze such reactions. Phosphorylation is often reversible due to the action of a protein phosphatase, an enzyme that catalyzes the dephosphorylation of amino acid residues.
  • Differences in the chemical structure of amino terminal residues result from different host cells, each of which can have a different chemical version of the methionine residue encoded by a start codon, and these will result in amino termini with different chemical modifications.
  • bacterial proteins are synthesized with an amino terminal amino acid that is a modified form of methionine, i.e., N-formyl-methionine (fMet).
  • fMet N-formyl-methionine
  • E. coli mutants that lack the enzymes (such as, e.g., formylase) that catalyze such post-translational modifications will produce proteins having an amino terminal fMet residue (Guillon et al., J. Bacteriol. 174: 4294-4301, 1992).
  • acetylation of the initiator methionine residue, or the penultimate residue if the initiator methionine has been removed typically occurs co- or post- translationally.
  • the acetylation reactions are catalyzed by N-terminal acetyltransferases (NATs, a.k.a. N-alpha-acetyltransferases), whereas removal of the initiator methionine residue is catalyzed by methionine aminopeptidases (for reviews, see Bradshaw et al, Trends Biochem. ScL 23: 263-267, 1998; and Driessen et al, CRC Crit. Rev. Biochem. 18: 281-325, 1985).
  • Amino terminally acetylated proteins are said to be "N-acetylated,” “N alpha acetylated” or simply "acetylated.”
  • a polypeptide mimetic is a molecule that mimics the biological activity of a polypeptide but is no longer peptidic in chemical nature.
  • a peptidomimetic is a molecule that contains no peptide bonds (that is, amide bonds between amino acids).
  • the term peptidomimetic is sometimes used to describe molecules that are no longer completely peptidic in nature, such as pseudo-peptides, semi-peptides and peptoids. Examples of some peptidomimetics by the broader definition (where part of a polypeptide is replaced by a structure lacking peptide bonds) are described below.
  • peptidomimetics provide a spatial arrangement of reactive chemical moieties that closely resembles the three-dimensional arrangement of active groups in the polypeptide on which the peptidomimetic is based. As a result of this similar active-site geometry, the peptidomimetic has effects on biological systems that are similar to the biological activity of the polypeptide.
  • a mimetic of a given polypeptide rather than the polypeptide itself.
  • polypeptides can exhibit two undesirable attributes, i.e., poor bioavailability and short duration of action.
  • Peptidomimetics are often small enough to be both orally active and to have a long duration of action. There are also problems associated with stability, storage and immunoreactivity for polypeptides that are not experienced with peptidomimetics.
  • Candidate, lead and other polypeptides having a desired biological activity can be used in the development of peptidomimetics with similar biological activities.
  • Techniques of developing peptidomimetics from polypeptides are known. Peptide bonds can be replaced by non-peptide bonds that allow the peptidomimetic to adopt a similar structure, and therefore biological activity, to the original polypeptide. Further modifications can also be made by replacing chemical groups of the amino acids with other chemical groups of similar structure.
  • the development of peptidomimetics can be aided by determining the tertiary structure of the original polypeptide, either free or bound to a ligand, by NMR spectroscopy, crystallography and/or computer-aided molecular modeling.
  • the present invention provides compounds exhibiting enhanced therapeutic activity in comparison to the polypeptides described above.
  • the peptidomimetic compounds obtained by the above methods having the biological activity of the above named polypeptides and similar three-dimensional structure, are encompassed by this invention. It will be readily apparent to one skilled in the art that a peptidomimetic can be generated from any of the modified polypeptides described in the previous section or from a polypeptide bearing more than one of the modifications described from the previous section. It will furthermore be apparent that the peptidomimetics of this invention can be further used for the development of even more potent non-peptidic compounds, in addition to their utility as therapeutic compounds.
  • Proteases act on peptide bonds. It therefore follows that substitution of peptide bonds by pseudopeptide bonds confers resistance to proteolysis. A number of pseudopeptide bonds have been described that in general do not affect polypeptide structure and biological activity. The reduced isosteric pseudopeptide bond is a suitable pseudopeptide bond that is known to enhance stability to enzymatic cleavage with no or little loss of biological activity (Couder et ah, Int. J. Polypeptide Protein Res. 41: 181-184, 1993, incorporated herein by reference).
  • amino acid sequences of these compounds can be identical to the sequences of their parent L-amino acid polypeptides, except that one or more of the peptide bonds are replaced by an isosteric pseudopeptide bond.
  • amino acid sequences of these compounds can be identical to the sequences of their parent L-amino acid polypeptides, except that one or more of the peptide bonds are replaced by an isosteric pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution would confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • peptide bonds can also be substituted by retro- inverso pseudopeptide bonds (Dalpozzo et ah, Int. J. Polypeptide Protein Res. 41: 561-566, 1993, incorporated herein by reference).
  • the amino acid sequences of the compounds can be identical to the sequences of their L-amino acid parent polypeptides, except that one or more of the peptide bonds are replaced by a retro-inverso pseudopeptide bond.
  • the most N-terminal peptide bond is substituted, since such a substitution will confer resistance to proteolysis by exopeptidases acting on the N-terminus.
  • Peptoid derivatives of polypeptides represent another form of modified polypeptides that retain the important structural determinants for biological activity, yet eliminate the peptide bonds, thereby conferring resistance to proteolysis (Simon et ah, Proc. Natl. Acad. Sci. USA 89: 9367-9371, 1992, and incorporated herein by reference).
  • Peptoids are oligomers of N-substituted glycines. A number of N-alkyl groups have been described, each corresponding to the side chain of a natural amino acid.
  • RNA, iRNA, antisense nucleic acid, cDNA, genomic DNA, vectors, viruses or hybrids thereof can be isolated from a variety of sources, genetically engineered, amplified, and/or expressed/generated recombinantly. Recombinant polypeptides generated from these nucleic acids can be individually isolated or cloned and tested for a desired activity. Any recombinant expression system can be used, including bacterial, mammalian, yeast, insect or plant cell expression systems.
  • nucleic acid sample comprising mRNA transcript(s) of the gene or genes, or nucleic acids derived from the mRNA transcript(s) is provided.
  • a nucleic acid derived from an mRNA transcript refers to a nucleic acid for whose synthesis the mRNA transcript or a subsequence thereof has ultimately served as a template.
  • a cDNA reverse transcribed from an mRNA, an RNA transcribed from that cDNA, a DNA amplified from the cDNA, an RNA transcribed from the amplified DNA are all derived from the mRNA transcript and detection of such derived products is indicative of the presence and/or abundance of the original transcript in a sample.
  • suitable samples include mRNA transcripts of the gene or genes, cDNA reverse transcribed from the mRNA, cRNA transcribed from the cDNA, DNA amplified from the genes, RNA transcribed from amplified DNA, and the like.
  • a nucleic acid sample is the total mRNA isolated from a biological sample.
  • biological sample refers to a sample obtained from an organism or from components (e.g., cells) or an organism.
  • the sample can be of any biological tissue or fluid. Frequently the sample is from a patient. Such samples include sputum, blood, blood cells (e.g., white cells), tissue or fine needle biopsy samples, urine, peritoneal fluid, and fleural fluid, or cells therefrom.
  • Biological samples can also include sections of tissues such as frozen sections taken for histological purposes. Often two samples are provided for purposes of comparison.
  • the samples can be, for example, from different cell or tissue types, from different species, from different individuals in the same species or from the same original sample subjected to two different treatments (e.g., drug-treated and control).
  • the invention includes polynucleotides encoding chemokines of the invention.
  • polynucleotide refers to a polymer of deoxyribonucleotides or ribonucleotides, in the form of a separate fragment or as a component of a larger construct.
  • DNA encoding a chemokine of the invention can be assembled from cDNA fragments or from oligonucleotides which provide a synthetic gene which is capable of being expressed in a recombinant transcriptional unit.
  • Polynucleotide sequences of the invention include DNA, RNA and cDNA sequences.
  • polynucleotide sequence can be deduced from the genetic code, however, the degeneracy of the code must be taken into account.
  • Polynucleotides of the invention include sequences which are degenerate as a result of the genetic code. Such polynucleotides are useful for the recombinant production of large quantities of a peptide of interest.
  • Recombinant when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified.
  • recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed or not expressed at all.
  • these nucleic acids can be synthesized in vitro by well-known chemical synthesis techniques, as described in, e.g., Adams, J. Am. Chem. Soc. 105: 661, 1983; Belousov, Nucleic Acids Res. 25: 3440-3444, 1997; Frenkel, Free Radic. Biol. Med. 19: 373-380, 1995; Blommers, Biochemistry 33: 7886-7896, 1994; Narang, Meth. Enzymol. 68: 90, 1979; Brown Meth. Enzymol. 68: 109, 1979; Beaucage, Tetra. Lett. 22: 1859, 1981; U.S. Pat. No. 4,458,066.
  • the invention provides oligonucleotides comprising sequences of the invention, e.g., subsequences of the exemplary sequences of the invention. Oligonucleotides can include, e.g., single stranded poly-deoxynucleotides or two complementary polydeoxynucleotide strands which can be chemically synthesized. [0158] In accordance with the present invention, there can be employed conventional molecular biology, microbiology, immunology, and recombinant DNA techniques within the skill of the art. Such techniques are explained fully in the literature.
  • nucleic acids such as, e.g., subcloning, labeling probes ⁇ e.g., random-primer labeling using Klenow polymerase, nick translation, amplification), sequencing, hybridization and the like are well described in the scientific and patent literature, see, e.g., See, for example, Sambrook, Fitsch & Maniatis, 1989, Molecular Cloning: A Laboratory Manual, 2 nd , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (referred to herein as "Sambrook et al, 1989”); DNA Cloning: A Practical Approach, Volumes I and II (D. N. Glover ed. 1985); Oligonucleotide Synthesis (M. J.
  • Nucleic acids, vectors, capsids, polypeptides, and the like can be analyzed and quantified by any of a number of general means well known to those of skill in the art. These include, e.g., analytical biochemical methods such as NMR, spectrophotometry, radiography, electrophoresis, capillary electrophoresis, high performance liquid chromatography (HPLC), thin layer chromatography (TLC), and hyperdiffusion chromatography, various immunological methods, e.g.
  • Obtaining and manipulating nucleic acids used to practice the methods of the invention can be done by cloning from genomic samples, and, if desired, screening and re- cloning inserts isolated or amplified from, e.g., genomic clones or cDNA clones.
  • Sources of nucleic acid used in the methods of the invention include genomic or cDNA libraries contained in, e.g., mammalian artificial chromosomes (MACs), see, e.g., U.S. Pat. Nos. 5,721,118; 6,025,155; human artificial chromosomes, see, e.g., Rosenfeld, Nat. Genet.
  • MACs mammalian artificial chromosomes
  • yeast artificial chromosomes YAC
  • bacterial artificial chromosomes BAC
  • Pl artificial chromosomes see, e.g., Woon, Genomics 50: 306-316, 1998
  • Pl -derived vectors PACs
  • cosmids recombinant viruses, phages or plasmids.
  • the invention provides fusion proteins and nucleic acids encoding them.
  • a gene product or polypeptide of the invention can be fused to a heterologous peptide or polypeptide, such as N-terminal identification peptides which impart desired characteristics, such as increased stability or simplified purification.
  • Peptides and polypeptides of the invention can also be synthesized and expressed as fusion proteins with one or more additional domains linked thereto for, e.g., producing a more immunogenic peptide, to more readily isolate a recombinantly synthesized peptide, to identify and isolate antibodies and antibody-expressing B cells, and the like.
  • Detection and purification facilitating domains include, e.g., metal chelating peptides such as polyhistidine tracts and histidine -tryptophan modules that allow purification on immobilized metals, protein A domains that allow purification on immobilized immunoglobulin, and the domain utilized in the FLAGS extension/affinity purification system (Immunex Corp, Seattle Wash.).
  • metal chelating peptides such as polyhistidine tracts and histidine -tryptophan modules that allow purification on immobilized metals
  • protein A domains that allow purification on immobilized immunoglobulin
  • the domain utilized in the FLAGS extension/affinity purification system Immunex Corp, Seattle Wash.
  • the inclusion of a cleavable linker sequences such as Factor Xa or enterokinase (Invitrogen, San Diego, CA) between a purification domain and the motif-comprising peptide or polypeptide to facilitate purification.
  • an expression vector can include an epitope-encoding nucleic acid sequence linked to six histidine residues followed by a thioredoxin and an enterokinase cleavage site (see e.g., Williams, Biochemistry 34: 1787-1797, 1995; Dobeli, Protein Expr. PurifXl: 404- 414, 1998).
  • the histidine residues facilitate detection and purification while the enterokinase cleavage site provides a means for purifying the epitope from the remainder of the fusion protein.
  • a nucleic acid encoding a polypeptide of the invention is assembled in appropriate phase with a leader sequence capable of directing secretion of the translated polypeptide or fragment thereof.
  • the nucleic acids of the invention can be operatively linked to a promoter.
  • a promoter can be one motif or an array of nucleic acid control sequences which direct transcription of a nucleic acid.
  • a promoter can include necessary nucleic acid sequences near the start site of transcription, such as, in the case of a polymerase II type promoter, a TATA element.
  • a promoter also optionally includes distal enhancer or repressor elements which can be located as much as several thousand base pairs from the start site of transcription.
  • a "constitutive" promoter is a promoter which is active under most environmental and developmental conditions.
  • An “inducible” promoter is a promoter which is under environmental or developmental regulation.
  • tissue specific promoter is active in certain tissue types of an organism, but not in other tissue types from the same organism.
  • operably linked refers to a functional linkage between a nucleic acid expression control sequence (such as a promoter, or array of transcription factor binding sites) and a second nucleic acid sequence, wherein the expression control sequence directs transcription of the nucleic acid corresponding to the second sequence.
  • the invention provides expression vectors and cloning vehicles comprising nucleic acids of the invention, e.g., sequences encoding the proteins of the invention.
  • Expression vectors and cloning vehicles of the invention can comprise viral particles, baculovirus, phage, plasmids, phagemids, cosmids, fosmids, bacterial artificial chromosomes, viral DNA (e.g., vaccinia, adenovirus, foul pox virus, pseudorabies and derivatives of SV40), Pl -based artificial chromosomes, yeast plasmids, yeast artificial chromosomes, and any other vectors specific for specific hosts of interest (such as bacillus, Aspergillus and yeast). See, for example, 5,707,855.
  • Vectors of the invention can include chromosomal, non-chromosomal and synthetic DNA sequences. Large numbers of suitable vectors are known to those of skill in the art, and are commercially available.
  • the nucleic acids of the invention can be cloned, if desired, into any of a variety of vectors using routine molecular biological methods; methods for cloning in vitro amplified nucleic acids are described, e.g., U.S. Pat. No. 5,426,039. To facilitate cloning of amplified sequences, restriction enzyme sites can be "built into” a PCR primer pair.
  • the invention provides libraries of expression vectors encoding polypeptides and peptides of the invention. These nucleic acids can be introduced into a genome or into the cytoplasm or a nucleus of a cell and expressed by a variety of conventional techniques, well described in the scientific and patent literature.
  • the vectors can be isolated from natural sources, obtained from such sources as ATCC or GenBank libraries, or prepared by synthetic or recombinant methods.
  • the nucleic acids of the invention can be expressed in expression cassettes, vectors or viruses which are stably or transiently expressed in cells ⁇ e.g., episomal expression systems).
  • Selection markers can be incorporated into expression cassettes and vectors to confer a selectable phenotype on transformed cells and sequences. For example, selection markers can code for episomal maintenance and replication such that integration into the host genome is not required.
  • the nucleic acids of the invention are administered in vivo for in situ expression of the peptides or polypeptides of the invention.
  • the nucleic acids can be administered as "naked DNA” (see, e.g., U.S. Pat. No. 5,580,859) or in the form of an expression vector, e.g., a recombinant virus.
  • the nucleic acids can be administered by any route, including peri- or intra-tumorally, as described below.
  • Vectors administered in vivo can be derived from viral genomes, including recombinantly modified enveloped or non- enveloped DNA and RNA viruses, preferably selected from baculoviridiae, parvoviridiae, picornoviridiae, herpesveridiae, poxyiridae, adenoviridiae, or picornnaviridiae. Chimeric vectors can also be employed which exploit advantageous merits of each of the parent vector properties (See e.g., Feng, Nature Biotechnology 15: 866-870, 1997). Such viral genomes can be modified by recombinant DNA techniques to include the nucleic acids of the invention; and can be further engineered to be replication deficient, conditionally replicating or replication competent.
  • vectors are derived from the adenoviral (e.g., replication incompetent vectors derived from the human adenovirus genome, see, e.g., U.S. Pat. Nos. 6,096,718; 6,110,458; 6,113,913; 5,631,236); adeno-associated viral and retroviral genomes.
  • Retroviral vectors can include those based upon murine leukemia virus (MuLV), gibbon ape leukemia virus (GaLV), Simian Immuno deficiency virus (SIV), human immuno deficiency virus (HIV), and combinations thereof; see, e.g., U.S. Pat. Nos.
  • Adeno-associated virus (AAV)-based vectors can be used to adioimmun cells with target nucleic acids, e.g., in the in vitro production of nucleic acids and peptides, and in in vivo and ex vivo gene therapy procedures; see, e.g., U.S. Pat. Nos. 6,110,456; 5,474,935; Okada, Gene Ther. 3: 957-964, 1996.
  • Expression cassette refers to a nucleotide sequence which is capable of affecting expression of a structural gene (i.e., a protein coding sequence, such as a polypeptide of the invention) in a host compatible with such sequences.
  • Expression cassettes include at least a promoter operably linked with the polypeptide coding sequence; and, optionally, with other sequences, e.g., transcription termination signals. Additional factors necessary or helpful in effecting expression can also be used, e.g., enhancers.
  • a nucleic acid is "operably linked” when it is placed into a functional relationship with another nucleic acid sequence.
  • a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence.
  • operably linked means that the DNA sequences being linked are contiguous and, where necessary to join two protein coding regions, contiguous and in reading frame.
  • operably linked indicates that the sequences are capable of effecting switch recombination.
  • expression cassettes also include plasmids, expression vectors, recombinant viruses, any form of recombinant "naked DNA" vector, and the like.
  • Vector is intended to refer to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional DNA segments can be ligated.
  • viral vector Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
  • vectors e.g., non-episomal mammalian vectors
  • vectors can be integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked.
  • Such vectors are referred to herein as "recombinant expression vectors" (or simply, "expression vectors”).
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and vector can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retroviruses, adenoviruses and adeno-associated viruses), which serve equivalent functions.
  • the invention also provides a transformed cell comprising a nucleic acid sequence of the invention, e.g., a sequence encoding a polypeptide of the invention, or a vector of the invention.
  • the host cell can be any of the host cells familiar to those skilled in the art, including prokaryotic cells, eukaryotic cells, such as bacterial cells, fungal cells, yeast cells, mammalian cells, insect cells, or plant cells.
  • Exemplary bacterial cells include E. coli, Streptomyces, Bacillus subtilis, Salmonella typhimurium and various species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
  • Exemplary insect cells include Drosophila S2 and Spodoptera Sf9.
  • Exemplary animal cells include CHO, COS or Bowes melanoma or any mouse or human cell line. The selection of an appropriate host is within the abilities of those skilled in the art.
  • the vector can be introduced into the host cells using any of a variety of techniques, including transformation, transfection, transduction, viral infection, gene guns, or Ti-mediated gene transfer. Particular methods include calcium phosphate transfection, DEAE-Dextran mediated transfection, lipofection, or electroporation.
  • Engineered host cells can be cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants or amplifying the genes of the invention. Following transformation of a suitable host strain and growth of the host strain to an appropriate cell density, the selected promoter can be induced by appropriate means (e.g., temperature shift or chemical induction) and the cells can be cultured for an additional period to allow them to produce the desired polypeptide or fragment thereof.
  • Cells can be harvested by centrifugation, disrupted by physical or chemical means, and the resulting crude extract is retained for further purification.
  • Microbial cells employed for expression of proteins can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents. Such methods are well known to those skilled in the art.
  • the expressed polypeptide or fragment can be recovered and purified from recombinant cell cultures by methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Protein refolding steps can be used, as necessary, in completing configuration of the polypeptide. If desired, high performance liquid chromatography (HPLC) can be employed for final purification steps.
  • HPLC high performance liquid chromatography
  • mammalian cell culture systems can also be employed to express recombinant protein.
  • mammalian expression systems include the COS-7 lines of monkey kidney fibroblasts and other cell lines capable of expressing proteins from a compatible vector, such as the C 127, 3T3, CHO, HeLa and BHK cell lines.
  • the constructs in host cells can be used in a conventional manner to produce the gene product encoded by the recombinant sequence.
  • the polypeptides produced by host cells containing the vector can be glycosylated or can be non-glycosylated.
  • Polypeptides of the invention can or can not also include an initial methionine amino acid residue.
  • the expression vectors can contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells such as dihydro folate reductase or neomycin resistance for eukaryotic cell culture, or such as tetracycline or ampicillin resistance in E. coli.
  • nucleic acids encoding the polypeptides of the invention, or modified nucleic acids can be reproduced by, e.g., amplification.
  • the invention provides amplification primer sequence pairs for amplifying nucleic acids encoding polypeptides of the invention, e.g., primer pairs capable of amplifying nucleic acid sequences comprising the chemokine protein or related protein sequences, or subsequences thereof.
  • Amplification methods include, e.g., polymerase chain reaction, PCR (Per Protocols, A Guide To Methods And Applications, ed. Innis, Academic Press, N. Y., 1990 and PCR STRATEGIES, 1995, ed.
  • LCR ligase chain reaction
  • transcription amplification see, e.g., Kwoh, Proc. Natl. Acad. Sci. USA 86: 1173, 1989
  • self-sustained sequence replication see, e.g., Guatelli, Proc. Natl. Acad. Sci. USA 87: 1874, 1990
  • Q Beta replicase amplification see, e.g., Smith, J. Clin. Microbiol.
  • the invention provides isolated or recombinant nucleic acids that hybridize under stringent conditions to an exemplary sequence of the invention, e.g., a sequence or related sequence, or the complement of any thereof, or a nucleic acid that encodes a polypeptide of the invention.
  • the stringent conditions are highly stringent conditions, medium stringent conditions or low stringent conditions, as known in the art and as described herein. These methods can be used to isolate nucleic acids of the invention.
  • nucleic acids of the invention as defined by their ability to hybridize under stringent conditions can be between about five residues and the full length of nucleic acid of the invention; e.g., they can be at least 5, 10, 15, 20, 25, 30, 35, 40, 50, 55, 60, 65, 70, 75, 80, 90, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800 or more residues in length, or, the full length of a gene or coding sequence, e.g., cDNA. Nucleic acids shorter than full length are also included.
  • nucleic acids can be useful as, e.g., hybridization probes, labeling probes, PCR oligonucleotide probes, iRNA, antisense or sequences encoding antibody binding peptides (epitopes), motifs, active sites and the like.
  • "Selectively (or specifically) hybridizes to” refers to the binding, duplexing, or hybridizing of a molecule to a particular nucleotide sequence under stringent hybridization conditions when that sequence is present in a complex mixture (e.g., total cellular or library DNA or RNA), wherein the particular nucleotide sequence is detected at least at about 10 times background.
  • a nucleic acid can be determined to be within the scope of the invention by its ability to hybridize under stringent conditions to a nucleic acid otherwise determined to be within the scope of the invention (such as the exemplary sequences described herein).
  • Stringent hybridization conditions refers to conditions under which a probe will hybridize to its target subsequence, typically in a complex mixture of nucleic acid, but not to other sequences in significant amounts (a positive signal (e.g., identification of a nucleic acid of the invention) is about 10 times background hybridization). Stringent conditions are sequence-dependent and will be different in different circumstances. Longer sequences hybridize specifically at higher temperatures. An extensive guide to the hybridization of nucleic acids is found in, e.g., Sambrook, ed., 1989; Ausubel, ed. 1997; Tijssen, ed., 1993, supra).
  • stringent conditions are selected to be about 5-10 0 C lower than the thermal melting point I for the specific sequence at a defined ionic strength pH.
  • the T m is the temperature (under defined ionic strength, pH, and nucleic concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30oC for short probes (e.g., 10 to 50 nucleotides) and at least about 60oC for long probes (e.g., greater than 50 nucleotides).
  • Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide as described in Sambrook (cited below).
  • destabilizing agents such as formamide as described in Sambrook (cited below).
  • a positive signal is at least two times background, preferably 10 times background hybridization.
  • Exemplary high stringency or stringent hybridization conditions include: 50% formamide, 5x SSC and 1% SDS incubated at 42° C or 5x SSC and 1% SDS incubated at 65° C, with a wash in 0.2x SSC and 0.1% SDS at 65° C.
  • a positive signal e.g., identification of a nucleic acid of the invention is about 10 times background hybridization.
  • Stringent hybridization conditions that are used to identify nucleic acids within the scope of the invention include, e.g., hybridization in a buffer comprising 50% formamide, 5x SSC, and 1% SDS at 42°C, or hybridization in a buffer comprising 5x SSC and 1% SDS at 65°C, both with a wash of 0.2x SSC and 0.1% SDS at 65°C.
  • genomic DNA or cDNA comprising nucleic acids of the invention can be identified in standard Southern blots under stringent conditions using the nucleic acid sequences disclosed here. Additional stringent conditions for such hybridizations (to identify nucleic acids within the scope of the invention) are those which include hybridization in a buffer of 40% formamide, 1 M NaCl, l% SDS at 37°C.
  • wash conditions used to identify nucleic acids within the scope of the invention include, e.g., a salt concentration of about 0.02 molar at pH 7 and a temperature of at least about 50 0 C or about 55°C to about 60 0 C; or, a salt concentration of about 0.15 M NaCl at 72°C for about 15 minutes; or, a salt concentration of about 0.2X SSC at a temperature of at least about 50 0 C or about 55°C to about 60 0 C for about 15 to about 20 minutes; or, the hybridization complex is washed twice with a solution with a salt concentration of about 2X SSC containing 0.1% SDS at room temperature for 15 minutes and then washed twice by 0.1X SSC containing 0.1% SDS at 68 0 C for 15 minutes; or, equivalent conditions. See Sambrook, Tij
  • the invention also provides nucleic acid probes for identifying nucleic acids encoding a polypeptide which is a modulator immunomodulatory and -signaling activity.
  • the probe comprises at least 10 consecutive bases of a nucleic acid of the invention.
  • a probe of the invention can be at least about 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 110, 120, 130, 150 or about 10 to 50, about 20 to 60 about 30 to 70, consecutive bases of a sequence as set forth in a nucleic acid of the invention.
  • the probes identify a nucleic acid by binding and/or hybridization.
  • the probes can be used in arrays of the invention, see discussion below.
  • the probes of the invention can also be used to isolate other nucleic acids or polypeptides.
  • the invention provides nucleic acids having at least 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an innate immunity modulating polynucleotide or related polynucleotide of the invention.
  • the invention provides polypeptides having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an innate immunity modulatingprotein or related protein.
  • the sequence identities can be determined by analysis with a sequence comparison algorithm or by a visual inspection. Protein and/or nucleic acid sequence identities (homologies) can be evaluated using any of the variety of sequence comparison algorithms and programs known in the art.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • sequence comparison of nucleic acids and proteins the BLAST and BLAST 2.2.2. or FASTA version 3.0t78 algorithms and the default parameters discussed below can be used.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the number of contiguous positions selected from the group consisting of from 20 to 600, usually about 50 to about 200, more usually about 100 to about 150 in which a sequence can be compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • Methods of alignment of sequences for comparison are well-known in the art. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith & Waterman, Adv. Appl. Math. 2: 482, 1981, by the homology alignment algorithm of Needleman & Wunsch, J. MoI. Biol.
  • BLAST and BLAST 2.0 are used, with the parameters described herein, to determine percent sequence identity for the nucleic acids and proteins of the invention.
  • Software for performing BLAST analyses is publicly available through the National Center for Biotechnology Information (http: //www.ncbi.nlm.nih.gov/).
  • This algorithm involves first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive- valued threshold score T when aligned with a word of the same length in a database sequence.
  • T is referred to as the neighborhood word score threshold (Altschul et al, supra).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin & Altschul, Proc. Natl. Acad. Sci. U.S.A. 90: 5873- 5787, 1993).
  • P(N) the smallest sum probability
  • P(N) the smallest sum probability
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.2, more preferably less than about 0.01, and most preferably less than about 0.001.
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendogram showing the clustering relationships used to create the alignment.
  • PILEUP uses a simplification of the progressive alignment method of Feng & Doolittle, J. MoI. Evol. 35: 351-360, 1987.
  • the method used is similar to the method described by Higgins & Sharp, CABIOS 5:151-153, 1989.
  • the program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids.
  • the multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences. This cluster is then aligned to the next most related sequence or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences. The final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters.
  • PILEUP a reference sequence is compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • PILEUP can be obtained from the GCG sequence analysis software package, e.g., version 7.0 (Devereaux et al, Nuc. Acids Res. 12: 387-395, 1984.)
  • Another preferred example of an algorithm that is suitable for multiple DNA and amino acid sequence alignments is the CLUSTALW program (Thompson et al. , Nucl. Acids. Res. 22: 4673-4680, 1994). ClustalW performs multiple pairwise comparisons between groups of sequences and assembles them into a multiple alignment based on homology. Gap open and Gap extension penalties were 10 and 0.05 respectively.
  • the BLOSUM algorithm can be used as a protein weight matrix (Henikoff and Henikoff, Proc. Natl. Acad. Sci. U.S.A. 89: 10915-10919, 1992).
  • Sequence identity refers to a measure of similarity between amino acid or nucleotide sequences, and can be measured using methods known in the art, such as those described below:
  • nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same (i.e., 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more identity over a specified region, when compared and aligned for maximum correspondence over a comparison window, or designated region as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection.
  • substantially identical in the context of two nucleic acids or polypeptides, refers to two or more sequences or subsequences that have at least of at least 60%, often at least 70%, preferably at least 80%, most preferably at least 90% or at least 95% nucleotide or amino acid residue identity, when compared and aligned for maximum correspondence, as measured using one of the following sequence comparison algorithms or by visual inspection.
  • the substantial identity exists over a region of the sequences that is at least about 50 bases or residues in length, more preferably over a region of at least about 100 bases or residues, and most preferably the sequences are substantially identical over at least about 150 bases or residues.
  • the sequences are substantially identical over the entire length of the coding regions.
  • Homology and “identity” in the context of two or more nucleic acids or polypeptide sequences refer to two or more sequences or subsequences that are the same or have a specified percentage of amino acid residues or nucleotides that are the same when compared and aligned for maximum correspondence over a comparison window or designated region as measured using any number of sequence comparison algorithms or by manual alignment and visual inspection.
  • sequence comparison one sequence can act as a reference sequence (an exemplary sequence of an innate immunity modulatinggene product or related gene product or polynucleotide or polypeptide) to which test sequences are compared.
  • test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated.
  • sequence comparison algorithm then calculates the percent sequence identities for the test sequences relative to the reference sequence, based on the program parameters.
  • a “comparison window”, as used herein, includes reference to a segment of any one of the numbers of contiguous residues.
  • contingous residues ranging anywhere from 20 to the full length of an exemplary polypeptide or nucleic acid sequence of the invention, e.g., an innate immunity modulatingpolynucleotide or polypeptide, are compared to a reference sequence of the same number of contiguous positions after the two sequences are optimally aligned.
  • the reference sequence has the requisite sequence identity to an exemplary polypeptide or nucleic acid sequence of the invention, e.g., at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to an innate immunity modulatingpolynucleotide or polypeptide, that sequence is within the scope of the invention.
  • Motifs which can be detected using the above programs include sequences encoding leucine zippers, helix-turn-helix motifs, glycosylation sites, ubiquitination sites, alpha helices, and beta sheets, signal sequences encoding signal peptides which direct the secretion of the encoded proteins, sequences implicated in transcription regulation such as homeoboxes, acidic stretches, enzymatic active sites, substrate binding sites, and enzymatic cleavage sites.
  • the polynucleotide sequence used according to the method of the invention can be isolated from an organism or synthesized in the laboratory.
  • Specific DNA sequences encoding the chemokine of interest can be obtained by: 1) isolation of a double-stranded DNA sequence from the genomic DNA; 2) chemical manufacture of a DNA sequence to provide the necessary codons for the cationic peptide of interest; and 3) in vitro synthesis of a double-stranded DNA sequence by reverse transcription of mRNA isolated from a donor cell. In the latter case, a double-stranded DNA complement of mRNA is eventually formed which is generally referred to as cDNA.
  • the synthesis of DNA sequences is frequently the method of choice when the entire sequence of amino acid residues of the desired peptide product is known.
  • the synthesis of a DNA sequence has the advantage of allowing the incorporation of codons that are more likely to be recognized by a bacterial host, thereby permitting high level expression without difficulties in translation.
  • virtually any peptide can be synthesized, including those encoding natural, variants of the same, or synthetic peptides.
  • the sequence of the invention can be stored, recorded, and manipulated on any medium which can be read and accessed by a computer. Accordingly, the invention provides computers, computer systems, computer readable mediums, computer programs products and the like recorded or stored thereon the nucleic acid and polypeptide sequences of the invention.
  • the words "recorded” and “stored” refer to a process for storing information on a computer medium. A skilled artisan can readily adopt any known methods for recording information on a computer readable medium to generate manufactures comprising one or more of the nucleic acid and/or polypeptide sequences of the invention.
  • Computer readable media include magnetically readable media, optically readable media, electronically readable media and magnetic/optical media.
  • the computer readable media can be a hard disk, a floppy disk, a magnetic tape, CD-ROM, Digital Versatile Disk (DVD), Random Access Memory (RAM), or Read Only Memory (ROM) as well as other types of other media known to those skilled in the art.
  • the invention provides methods (also referred to herein as "screening assays") for identifying compounds which can be used to modulate innate immunity and to use the identified compounds in pharmaceutical compositions to treat related diseases and conditions.
  • Screening assays For identifying compounds which can be used to modulate innate immunity and to use the identified compounds in pharmaceutical compositions to treat related diseases and conditions.
  • Candidate or test compounds or agents which bind to GAPDH and/or modulate the activity or the expression of GAPDH are identified either in assays that employ cells which express GAPDH (cell-based assays) or in assays with isolated GAPDH (cell-free assays).
  • the various assays can employ a variety of variants of GAPDH (e.g., full-length GAPDH, a biologically active fragment of GAPDH, or a fusion protein which includes all or a portion of GAPDH).
  • GAPDH can be derived from any suitable mammalian species (e.g., human GAPDH, rat GAPDH or murine GAPDH).
  • the assay can be a binding assay entailing direct or indirect measurement of the binding of a test compound or a known GAPDH ligand to GAPDH.
  • the assay can also be an activity assay entailing direct or indirect measurement of the activity of GAPDH.
  • the assay can also be an expression assay entailing direct or indirect measurement of the expression of GAPDH mRNA or GAPDH protein.
  • the various screening assays are combined with an in vivo assay entailing measuring the effect of the test compound on the modulation of the innate immune response and diseases associated with regulation of this response.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of GAPDH.
  • Such assays can employ full- length GAPDH, a biologically active fragment of GAPDH, or a fusion protein which includes all or a portion of GAPDH.
  • the test compound can be obtained by any suitable means, e.g., from conventional compound libraries. Determining the ability of the test compound to bind to GAPDH can be accomplished, for example, by coupling the test compound with a radioisotope or enzymatic label such that binding of the test compound to the GAPDH expressing cell can be measured by detecting the labeled compound in a complex.
  • the test compound can be labelled with 125 1, 35 S, 14 C, or H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • the test compound can be enzymatically labelled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • the assay comprises contacting GAPDH expressing cell with a known compound which binds to GAPDH to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with the GAPDH expressing cell, wherein determining the ability of the test compound to interact with the GAPDH expressing cell comprises determining the ability of the test compound to preferentially bind the GAPDH expressing cell as compared to the known compound.
  • Determining the ability of the test compound to modulate the activity of GAPDH can be accomplished, for example, by determining the ability of GAPDH to bind to or interact with a target molecule.
  • the target molecule can be a molecule with which GAPDH binds or interacts with in nature, for example, a molecule that interacts with a cell which expresses GAPDH, a molecule on the surface of a second cell, a molecule in the extracellular milieu, a molecule associated with the internal surface of a cell membrane or a cytoplasmic molecule.
  • the target molecule can be a component of a signal transduction pathway which facilitates transduction of an extracellular signal (e.g., a signal generated by binding of a GAPDH ligand, through the cell membrane and into the cell.
  • the target GAPDH molecule can be, for example, a second intracellular protein which has catalytic activity or a protein which facilitates the association of downstream signaling molecules with GAPDH.
  • Determining the ability of GAPDH to bind to or interact with a target molecule can be accomplished by one of the methods described above for determining direct binding. In one aspect, determining the ability of a polypeptide of the invention to bind to or interact with a target molecule can be accomplished by determining the activity of the target molecule.
  • the activity of the target molecule can be determined by detecting induction of a cellular second messenger of the target (e.g., intracellular Ca2+, diacylglycerol, IP3, and the like), detecting catalytic/enzymatic activity of the target on an appropriate substrate, detecting the induction of a reporter gene (e.g. , a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response.
  • a reporter gene e.g. , a regulatory element that is responsive to a polypeptide of the invention operably linked to a nucleic acid encoding a detectable marker, e.g., luciferase
  • the present invention also includes cell-free assays. Such assays involve contacting a form of GAPDH (e.g.
  • the assay includes contacting GAPDH with a known compound which binds GAPDH to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with GAPDH, wherein determining the ability of the test compound to interact with GAPDH comprises determining the ability of the test compound to preferentially bind to GAPDH as compared to the known compound.
  • GAPDH or a GAPDH target molecule
  • binding of a test compound to GAPDH, or interaction of GAPDH with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and microcentrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both of the proteins to be bound to a matrix.
  • glutathione-S-transferase (GST) fusion proteins or glutathione- S- transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical; St. Louis, Mo.) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or GAPDH, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtitre plate wells are washed to remove any unbound components and complex formation is measured either directly or indirectly, for example, as described above. Alternatively, the complexes can be dissociated from the matrix, and the level of binding or activity of GAPDH can be determined using standard techniques.
  • GAPDH or its target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated polypeptide of the invention or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals; Rockford, 111.), and immobilized in the wells of streptavidin-coated plates (Pierce Chemical).
  • antibodies reactive with GAPDH or target molecules but which do not interfere with binding of the polypeptide of the invention to its target molecule can be derivatized to the wells of the plate, and unbound target or polypeptide of the invention trapped in the wells by antibody conjugation.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with GAPDH or target molecule, as well as enzyme- linked assays which rely on detecting an enzymatic activity associated with GAPDH or target molecule.
  • the screening assay can also involve monitoring the expression of GAPDH.
  • regulators of expression of GAPDH can be identified in a method in which a cell is contacted with a candidate compound and the expression of GAPDH protein or mRNA in the cell is determined. The level of expression of GAPDH protein or mRNA the presence of the candidate compound is compared to the level of expression of GAPDH protein or mRNA in the absence of the candidate compound. The candidate compound can then be identified as a regulator of expression of GAPDH based on this comparison. For example, when expression of GAPDH protein or mRNA protein is greater (statistically significantly greater) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of GAPDH protein or mRNA expression.
  • the candidate compound when expression of GAPDH protein or mRNA is less (statistically significantly less) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of GAPDH protein or mRNA expression.
  • the level of GAPDH protein or mRNA expression in the cells can be determined by methods described below. [0217]
  • the screening assay can also involve reducing the level of expression of GAPDH using either small inhibitory or short hairpin RNA methods (siRNA, shRNA) or by altering its promoter to a regulated promoter.
  • cells with reduced levels of GAPDH are treated with a compound having immunomodulatory activity and the loss of immunomodulatory activity including the decrease of expression of particular cytokines or chemokines and/or signaling responses relative to the situation where unaltered cells are treated with the same compound, is assessed.
  • the screening assay can also assess effects on the activation of particular signalling pathways or transcription factors, that are stimulated by interaction of the compound with GAPDH, as a surrogate for assessing the binding of the compound to GAPDH and modulating its activity. See also Section J entitled "Modulating or Inhibiting Expression of Polypeptides and Transcripts".
  • the test compound is preferably a small molecule or polypeptide which binds to the GAPDH polypeptide.
  • small molecules include, but are not limited to, small peptides or peptide-like molecules.
  • Potential ligands which bind to a polypeptide of the invention include, but are not limited to, the natural ligands of known GAPDH and analogs or derivatives thereof.
  • either the test compound or the GAPDH polypeptide can comprise a detectable label, such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or lucif erase. Detection of a test compound which is bound to GAPDH polypeptide can then be accomplished, for example, by direct counting of radioemmission, by scintillation counting, or by determining conversion of an appropriate substrate to a detectable product. Alternatively, binding of a test compound to a GAPDH polypeptide can be determined without labeling either of the interactants.
  • a detectable label such as a fluorescent, radioisotopic, chemiluminescent, or enzymatic label, such as horseradish peroxidase, alkaline phosphatase, or lucif erase.
  • a microphysiometer can be used to detect binding of a test compound with a GAPDH polypeptide.
  • a microphysiometer e.g., Cytosensor TM
  • a microphysiometer is an analytical instrument that measures the rate at which a cell acidifies its environment using a light-addressable potentiometric sensor (LAPS). Changes in this acidification rate can be used as an indicator of the interaction between a test compound and GAPDH [Haseloff, (1988)].
  • BIA Bimolecular Interaction Analysis
  • a GAPDH-like polypeptide can be used as a "bait protein" in a two-hybrid assay or three-hybrid assay (Szabo, (1995); U.S. Pat. No. 5,283,317), to identify other proteins which bind to or interact with GAPDH and modulate its activity.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
  • the assay utilizes two different DNA constructs.
  • polynucleotide encoding GAPDH can be fused to a polynucleotide encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence that encodes an unidentified protein (“prey" or "sample” can be fused to a polynucleotide that codes for the activation domain of the known transcription factor.
  • the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ), which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the DNA sequence encoding the protein which interacts with GAPDH.
  • a reporter gene e.g., LacZ
  • GAPDH GAPDH-like polypeptide
  • test compound can be bound to a solid support.
  • Suitable solid supports include, but are not limited to, glass or plastic slides, tissue culture plates, microtiter wells, tubes, silicon chips, or particles such as beads (including, but not limited to, latex, polystyrene, or glass beads).
  • Any method known in the art can be used to attach GAPDH-like polypeptide (or polynucleotide) or test compound to a solid support, including use of covalent and non-covalent linkages, passive absorption, or pairs of binding moieties attached respectively to the polypeptide (or polynucleotide) or test compound and the solid support.
  • Test compounds are preferably bound to the solid support in an array, so that the location of individual test compounds can be tracked. Binding of a test compound to GAPDH (or a polynucleotide encoding for GAPDH) can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtiter plates, test tubes, and microcentrifuge tubes.
  • GAPDH is a fusion protein comprising a domain that allows binding of GAPDH to a solid support.
  • glutathione-S-transferase fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, Mo.) or glutathione derivatized microtiter plates, which are then combined with the test compound or the test compound and the non-adsorbed GAPDH; the mixture is then incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components. Binding of the interactants can be determined either directly or indirectly, as described above. Alternatively, the complexes can be dissociated from the solid support before binding is determined.
  • GAPDH or a polynucleotide encoding GAPDH
  • test compounds can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated GAPDH or a polynucleotide encoding biotinylated GAPDH
  • test compounds can be prepared from biotin-NHS (N- hydroxysuccinimide) using techniques well known in the art (e.g.
  • Methods for detecting such complexes include immunodetection of complexes using antibodies which specifically bind to GAPDH polypeptide or test compound, enzyme-linked assays which rely on detecting an activity of GAPDH polypeptide, and SDS gel electrophoresis under non-reducing conditions.
  • Screening for test compounds which bind to a GAPDH polypeptide or polynucleotide also can be carried out in an intact cell. Any cell which comprises a GAPDH polypeptide or polynucleotide can be used in a cell-based assay system. A GAPDH polynucleotide can be naturally occurring in the cell or can be introduced using techniques such as those described above. Binding of the test compound to GAPDH or a polynucleotide encoding GAPDH is determined as described above. I. DETECTING POLYPEPTIDE EXPRESSION
  • marker gene expression suggests that a GAPDH polynucleotide is also present, its presence and expression can need to be confirmed. For example, if a sequence encoding GAPDH is inserted within a marker gene sequence, transformed cells containing sequences which encode GAPDH can be identified by the absence of marker gene function. Alternatively, a marker gene can be placed in tandem with a sequence encoding GAPDH under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of GAPDH polynucleotide.
  • host cells which contain a GAPDH polynucleotide and which express GAPDH can be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • these procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations and protein bioassay or immunoassay techniques which include membrane, solution, or chip-based technologies for the detection and/or quantification of nucleic acid or protein.
  • the presence of a polynucleotide sequence encoding GAPDH can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes or fragments or fragments of polynucleotides encoding GAPDH.
  • Nucleic acid amplification- based assays involve the use of oligonucleotides selected from sequences
  • a variety of protocols for detecting and measuring the expression of GAPDH, using either polyclonal or monoclonal antibodies specific for the polypeptide, are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radio-immunoassay (RIA), and fluorescence activated cell sorting (FACS). A two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on GAPDH can be used, or a competitive binding assay can be employed.
  • ELISA enzyme-linked immunosorbent assay
  • RIA radio-immunoassay
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay using monoclonal antibodies reactive to two non-interfering epitopes on GAPDH can be used, or a competitive binding assay can be employed.
  • a wide variety of labels and conjugation techniques are known by those skilled in the art and can be used in various nucleic acid and
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding GAPDH include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • sequences encoding GAPDH can be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and can be used to synthesize RNA probes in vitro by addition of labeled nucleotides and an appropriate RNA polymerase such as T7, T3, or SP6. These procedures can be conducted using a variety of commercially available kits (Amersham Pharmacia Biotech, Promega, and US Biochemical).
  • Suitable reporter molecules or labels which can be used for ease of detection include radionuclides, enzymes, and fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors, inhibitors, magnetic particles, and the
  • Sequences encoding GAPDH can be synthesized, in whole or in part, using chemical methods well known in the art.
  • GAPDH itself can be produced using chemical methods to synthesize its amino acid sequence, such as by direct peptide synthesis using solid-phase techniques. Protein synthesis can either be performed using manual techniques or by automation. Automated synthesis can be achieved, for example, using Applied Biosystems 43 IA Peptide Synthesizer (Perkin Elmer).
  • fragments of GAPDH can be separately synthesized and combined using chemical methods to produce a full-length molecule.
  • the newly synthesized peptide can be substantially purified by preparative high performance liquid chromatography.
  • the composition of a synthetic GAPDH can be confirmed by amino acid analysis or sequencing. Additionally, any portion of the amino acid sequence of GAPDH can be altered during direct synthesis and/or combined using chemical methods with sequences from other proteins to produce a variant polypeptide or a fusion protein.
  • the invention further provides for the use of nucleic acids complementary to (e.g., antisense sequences to) the nucleic acid sequences of the invention.
  • Antisense sequences are capable of modulating or inhibiting the transport, splicing or transcription of protein- encoding genes, e.g., innate immunity modulating-encoding nucleic acids, differentially expressed nucleic acids, related nucleic acids and the like.
  • the modulation or inhibition can be effected through the targeting of genomic DNA or messenger RNA.
  • the transcription or function of targeted nucleic acid can be inhibited, for example, by hybridization and/or cleavage.
  • One particularly useful set of inhibitors provided by the present invention includes oligonucleotides which are able to either bind gene or message, in either case preventing or inhibiting the production or function of the protein. The association can be through sequence specific hybridization.
  • Another useful class of inhibitors includes oligonucleotides which cause inactivation or cleavage of protein message.
  • the oligonucleotide can have enzyme activity which causes such cleavage, such as ribozymes.
  • the oligonucleotide can be chemically modified or conjugated to an enzyme or composition capable of cleaving the complementary nucleic acid. One can screen a pool of many different such oligonucleotides for those with the desired activity.
  • RNAi RNA interference
  • RNAi encompasses molecules such as short interfering RNA (siRNA), microRNAs (miRNA), small temporal RNA (stRNA).
  • siRNA short interfering RNA
  • miRNA microRNAs
  • stRNA small temporal RNA
  • the invention provides antisense oligonucleotides capable of binding innate immunity modulating messenger RNA which can inhibit polypeptide activity by targeting mRNA.
  • Strategies for designing antisense oligonucleotides are well described in the scientific and patent literature, and the skilled artisan can design such oligonucleotides using the novel reagents of the invention.
  • gene walking/RNA mapping protocols to screen for effective antisense oligonucleotides are well known in the art, see, e.g., Ho, Methods Enzymol. 314: 168-183, 2000, describing an RNA mapping assay, which is based on standard molecular techniques to provide an easy and reliable method for potent antisense sequence selection. See also Smith, Eur. J. Pharm. Sci. 11: 191-198, 2000.
  • Naturally occurring nucleic acids are used as antisense oligonucleotides.
  • the antisense oligonucleotides can be of any length; for example, in alternative aspects, the antisense oligonucleotides are between about 5 to 100, about 10 to 80, about 15 to 60, about 18 to 40. The optimal length can be determined by routine screening.
  • the antisense oligonucleotides can be present at any concentration. The optimal concentration can be determined by routine screening.
  • a wide variety of synthetic, non-naturally occurring nucleotide and nucleic acid analogues are known which can address this potential problem.
  • PNAs peptide nucleic acids
  • non-ionic backbones such as N-(2- aminoethyl) glycine units
  • Antisense oligonucleotides having phosphorothioate linkages can also be used, as described in WO 97/03211; WO 96/39154; Mata, Toxicol Appl Pharmacol. 144: 189-197, 1997; Antisense Therapeutics, ed. Agrawal, Humana Press, Totowa, N.J., 1996.
  • Antisense oligonucleotides having synthetic DNA backbone analogues provided by the invention can also include phosphoro-dithioate, methylphosphonate, phosphoramidate, alkyl phosphotriester, sulfamate, 3'-thioacetal, methylene(methylimino), 3'-N-carbamate, and morpholino carbamate nucleic acids, as described above.
  • Combinatorial chemistry methodology can be used to create vast numbers of oligonucleotides that can be rapidly screened for specific oligonucleotides that have appropriate binding affinities and specificities toward any target, such as the sense and antisense polypeptides sequences of the invention (see, e.g., Gold, J. of Biol. Chem. 270: 13581-13584, 1995).
  • siRNA refers to double-stranded RNA molecules from about 10 to about 30 nucleotides long that are named for their ability to specifically interfere with protein expression through RNA interference (RNAi).
  • RNAi RNA interference
  • siRNA molecules are 12-28 nucleotides long, more preferably 15-25 nucleotides long, still more.
  • RNAi is a two-step mechanism. Elbashir et al, Genes Dev., 15: 188-200, 2001. First, long dsRNAs are cleaved by an enzyme known as Dicer in 21-23 ribonucleotide (nt) fragments, called small interfering RNAs (siRNAs). Then, siRNAs associate with a ribonuclease complex (termed RISC for RNA Induced Silencing Complex) which target this complex to complementary mRNAs. RISC then cleaves the targeted mRNAs opposite the complementary siRNA, which makes the mRNA susceptible to other RNA degradation pathways.
  • RISC RNA Induced Silencing Complex
  • siRNAs of the present invention are designed to interact with a target ribonucleotide sequence, meaning they complement a target sequence sufficiently to bind to the target sequence.
  • the present invention also includes siRNA molecules that have been chemically modified to confer increased stability against nuclease degradation, but retain the ability to bind to target nucleic acids that can be present.
  • the invention provides ribozymes capable of binding message which can inhibit polypeptide activity by targeting mRNA, e.g., inhibition of polypeptides with innate immunity modulating activity, e.g., signaling activity.
  • ribozymes capable of binding message which can inhibit polypeptide activity by targeting mRNA, e.g., inhibition of polypeptides with innate immunity modulating activity, e.g., signaling activity.
  • innate immunity modulating activity e.g., signaling activity.
  • Ribozymes act by binding to a target RNA through the target RNA binding portion of a ribozyme which is held in close proximity to an enzymatic portion of the RNA that cleaves the target RNA.
  • the ribozyme recognizes and binds a target RNA through complementary base-pairing, and once bound to the correct site, acts enzymatically to cleave and inactivate the target RNA. Cleavage of a target RNA in such a manner will destroy its ability to direct synthesis of an encoded protein if the cleavage occurs in the coding sequence.
  • a ribozyme After a ribozyme has bound and cleaved its RNA target, it is typically released from that RNA and so can bind and cleave new targets repeatedly.
  • a ribozyme can be advantageous over other technologies, such as antisense technology (where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule) as the effective concentration of ribozyme necessary to effect a therapeutic treatment can be lower than that of an antisense oligonucleotide.
  • antisense technology where a nucleic acid molecule simply binds to a nucleic acid target to block its transcription, translation or association with another molecule
  • This potential advantage reflects the ability of the ribozyme to act enzymatically.
  • a single ribozyme molecule is able to cleave many molecules of target RNA.
  • a ribozyme is typically a highly specific inhibitor, with the specificity of inhibition depending not only on the base pairing mechanism of binding, but also on the mechanism by which the molecule inhibits the expression of the RNA to which it binds. That is, the inhibition is caused by cleavage of the RNA target and so specificity is defined as the ratio of the rate of cleavage of the targeted RNA over the rate of cleavage of non-targeted RNA. This cleavage mechanism is dependent upon factors additional to those involved in base pairing. Thus, the specificity of action of a ribozyme can be greater than that of antisense oligonucleotide binding the same RNA site.
  • the enzymatic ribozyme RNA molecule can be formed in a hammerhead motif, but can also be formed in the motif of a hairpin, hepatitis delta virus, group I intron or RnaseP-like RNA (in association with an RNA guide sequence).
  • hammerhead motifs are described by Rossi, Aids Research and Human Retroviruses 8: 183, 1992; hairpin motifs by Hampel, Biochemistry 28: 4929, 1989, and Hampel, Nuc. Acids Res.
  • RNA molecule of this invention has a specific substrate binding site complementary to one or more of the target gene RNA regions, and has nucleotide sequence within or surrounding that substrate binding site which imparts an RNA cleaving activity to the molecule.
  • the present invention provides novel methods for screening for compositions to treat, for example, inflammation or sepsis, in a mammalian subject.
  • the expression levels of genes are determined for different cellular states of diseased or non-diseased (or viral vs. non- viral) to provide expression profiles.
  • a sepsis expression profile of a particular septic cell state can be a "fingerprint" of the state; while two states can have any particular gene similarly expressed, the evaluation of a number of genes simultaneously allows the generation of a gene expression profile that is unique to the state of the cell.
  • This information can then be used in a number of ways. For example, the evaluation of a particular treatment regime can be evaluated. Similarly, diagnosis can be done or confirmed: does this patient have the gene expression profile of a particular cell state. Furthermore, these gene expression profiles can be used in drug candidate screening to find drugs that mimic a particular expression profile; for example, screening can be done for drugs that treat inflammation as evidenced by a non-inflammatory expression profile. Accordingly, genes are identified and described which are differentially expressed within and among inflammatory or non-inflammatory cells in different states, from which the expression profiles are generated as further described herein. For example, determinations of differentially expressed nucleic acids are provided in the examples.
  • Gene expression profile or “gene expression profile set” refers to the set of genes of a specific tissue or cell type that are transcribed or “expressed” to form RNA molecules. Which genes are expressed in a specific cell line or tissue can depend on factors such as tissue or cell type, stage of development or the cell, tissue, or target organism and whether the cells are normal or diseased cells. For example, a gene can be expressed at the embryonic or fetal stage in the development of a specific target organism and then become non-expressed as the target organism matures. Alternatively, a gene can be expressed in liver tissue but not in brain tissue of an adult human.
  • Specific hybridization refers to the binding, duplexing, or hybridizing of a molecule only to a particular nucleotide sequence under stringent conditions when that sequence is present in a complex mixture (e.g., total cellular) DNA or RNA.
  • Stringent conditions are conditions under which a probe can hybridize to its target subsequence, but to no other sequences. Stringent conditions are sequence-dependent and are different in different circumstances. Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5°C. lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • the T m is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target sequence hybridize to the target sequence at equilibrium. (As the target sequences are generally present in excess, at T m , 50% of the probes are occupied at equilibrium).
  • stringent conditions include a salt concentration of at least about 0.01 to 1.0 M Na+ concentration (or other salts) at pH 7.0 to 8.3 and the temperature is at least about 30 0 C for short probes (e.g., 10 to 50 nucleotides). Stringent conditions can also be achieved with the addition of destabilizing agents such as formamide or tetraalkyl ammonium salts.
  • 5X SSPE 750 mM NaCl, 50 mM Na Phosphate, 5 mM EDTA, pH 7.4
  • a temperature of 25-30 0 C. are suitable for allele-specific probe hybridizations.
  • differential expression refers to both qualitative as well as quantitative differences in the genes' temporal and/or cellular expression patterns within and among, for example, septic cells or non-septic cells.
  • a differentially expressed gene can qualitatively have its expression altered, including an activation or inactivation in, for example, tolerant versus immunosuppressed cells, rested, naive or activated cells, or in a disease cell state vs. non-disease cell state (or viral infected cell state vs. non- viral infected cell state).
  • Genes can be turned on or turned off in a particular state, relative to another state. Any comparison of two or more states can be made.
  • Such a qualitatively regulated gene will exhibit an expression pattern within a state or cell type which can be detectable by standard techniques in one such state or cell type, but can be not detectable in both.
  • the determination can be quantitative in that expression is increased or decreased; that is, the expression of the gene is either upregulated, resulting in an increased amount of transcript, or downregulated, resulting in a decreased amount of transcript.
  • the degree to which expression differs need only be large enough to quantify using standard characterization techniques, for example, by using Affymetrix GeneChipTM expression arrays. Lockhart, Nature Biotechnology 14: 1675-1680, 1996; this reference and all references cited therein are incorporated by reference.
  • Other methods include, but are not limited to, quantitative reverse transcriptase PCR, Northern analysis and RNase protection.
  • the change or modulation in expression is at least about 5%, more preferably at least about 10%, more preferably, at least about 20%, more preferably, at least about 30%, or more preferably by at least about 50%, or at least about 75%, and more preferably at least about 90%.
  • Any one, two, three, four, five, or ten or more genes can be evaluated. These genes include, but are not limited to, the genes discussed herein. Generally, oligonucleotide sequences used in the evaluation of these genes are derived from their 3' untranslated regions.
  • Differentially expressed genes can represent "expression profile genes", which includes “target genes".
  • “Expression profile gene,” as used herein, refers to a differentially expressed gene whose expression pattern can be used in methods for identifying compounds useful in the modulation of diseased cell or non-diseased cell states or activity, or the treatment of diseases or disorders, or alternatively, the gene can be used as part of a prognostic or diagnostic evaluation of immune disorders.
  • the effect of the compound on the expression profile gene normally displayed in connection with a particular state for example, can be used to evaluate the efficacy of the compound to modulate that state, or preferably, to induce or maintain that state.
  • the gene can be used as a diagnostic or in the treatment of immune disorders as also further described below. In some instances, only a fragment of an expression profile gene is used, as further described below.
  • “Expression profile,” as used herein, refers to the pattern of gene expression generated from two up to all of the expression profile genes which exist for a given state. As outlined above, an expression profile is in a sense a "fingerprint” or “blueprint” of a particular cellular state; while two or more states have genes that are similarly expressed, the total expression profile of the state will be unique to that state.
  • the gene expression profile obtained for a given diseased cell or non-diseased cell state can be useful for a variety of applications, including diagnosis of a particular disease or condition and evaluation of various treatment regimes. In addition, comparisons between the expression profiles of different disease cell or non-disease cell states can be similarly informative.
  • An expression profile can include genes which do not appreciably change between two states, so long as at least two genes which are differentially expressed are represented.
  • the gene expression profile can also include at least one target gene, as defined below.
  • the profile can include all of the genes which represent one or more states. Specific expression profiles are described below.
  • Gene expression profiles can be defined in several ways. For example, a gene expression profile can be the relative transcript level of any number of particular set of genes. Alternatively, a gene expression profile can be defined by comparing the level of expression of a variety of genes in one state to the level of expression of the same genes in another state. For example, genes can be either upregulated, downregulated, or remain substantially at the same level in both states.
  • target gene refers to a differentially expressed expression profile gene whose expression is unique for a particular state, such that the presence or absence of the transcript of a target gene(s) can indicate the state the cell is in.
  • a target gene can be completely unique to a particular state; the presence or absence of the gene is only seen in a particular cell state, or alternatively, cells in all other states express the gene but it is not seen in the first state.
  • target genes can be identified as relevant to a comparison of two states, that is, the state is compared to another particular state or standard to determine the uniqueness of the target gene.
  • Target genes can be used in the diagnostic, prognostic, and compound identification methods described herein.
  • a target gene for a first state can be an expression profile gene for a second state.
  • the presence or absence of a particular target gene in one state can be diagnostic of the state; the same gene in a different state can be an expression profile gene.
  • pathway genes are provided herein. "Pathway genes” are defined by the ability of their gene products to interact with expression profile genes. Pathway genes can also exhibit target gene and/or expression profile gene characteristics and can be included as modulators of expression profile genes as further described below.
  • the present invention includes the products of such expression profile, target, and pathway genes to such gene products. Furthermore, the engineering and use of cell- and animal-based models, for example, of various disease cell or non-disease cell states to which such profiles, genes and gene products can contribute, are also described.
  • biochips or arrays for expression monitoring is generally described, for example, WO 97/27317 and WO 97/10365 (these references are herein incorporated by reference in their entirety for all purposes) and in further detail below.
  • nucleic acid arrays There are two principal categories of nucleic acid arrays.
  • One type of array detects the presence and/or levels of particular mRNA sequences that are known in advance.
  • polynucleotide probes can be selected to hybridize to particular preselected subsequences of mRNA gene sequence.
  • Such expression monitoring arrays can include a plurality of probes for each mRNA to be detected.
  • the probes are designed to be complementary to the region of the mRNA that is incorporated into the nucleic acids (i.e., the 3' end).
  • the array can also include one or more control probes.
  • Generic arrays can include all possible nucleotides of a given length; that is, polynucleotides having sequences corresponding to every permutation of a sequence.
  • the polynucleotide probes of this invention preferably include up to 4 bases (A, G, C, T) or (A, G, C, U) or derivatives of these bases, an array having all possible nucleotides of length X contains substantially 4 X different nucleic acids (e.g., 16 different nucleic acids for a 2 mer, 64 different nucleic acids for a 3 mer, 65536 different nucleic acids for an 8 mer). Some small number of sequences can be absent from a pool of all possible nucleotides of a particular length due to synthesis problems, and inadvertent cleavage).
  • An array comprising all possible nucleotides of length X refers to an array having substantially all possible nucleotides of length X. All possible nucleotides of length X includes more than 90%, typically more than 95%, preferably more than 98%, more preferably more than 99%, and most preferably more than 99.9% of the possible number of different nucleotides. Generic arrays are particularly useful for comparative hybridization analysis between two mRNA populations or nucleic acids derived therefrom.
  • the invention provides methods for identifying/screening for innate immunity modulators (e.g., inhibitors, activators, and the like) GAPDH activity, e.g., GAPDH-signaling activity, using arrays.
  • Potential modulators including small molecules, nucleic acids, polypeptides (including antibodies) can be immobilized to arrays.
  • Nucleic acids or polypeptides of the invention can be immobilized to or applied to an array.
  • Arrays can be used to screen for or monitor libraries of compositions (e.g., small molecules, antibodies, nucleic acids, and the like.) for their ability to bind to or modulate the activity of a nucleic acid or a polypeptide of the invention, e.g., a GAPDH activity.
  • a monitored parameter is transcript expression of a gene comprising a nucleic acid of the invention.
  • One or more, or, all the transcripts of a cell can be measured by hybridization of a sample comprising transcripts of the cell, or, nucleic acids representative of or complementary to transcripts of a cell, by hybridization to immobilized nucleic acids on an array, or "biochip.”
  • arrays comprising genomic nucleic acid can also be used to determine the genotype of a newly engineered strain made by the methods of the invention.
  • Polypeptide arrays can be used to simultaneously quantify a plurality of proteins.
  • Small molecule arrays can be used to simultaneously analyze a plurality of GAPDH modulating or binding activities.
  • arrays are generically a plurality of “spots” or “target elements,” each target element comprising a defined amount of one or more biological molecules, e.g., oligonucleotides, immobilized onto a defined area of a substrate surface for specific binding to a sample molecule, e.g., mRNA transcripts.
  • biological molecules e.g., oligonucleotides
  • any known array and/or method of making and using arrays can be incorporated in whole or in part, or variations thereof, as described, for example, in U.S. Pat. Nos. 6,277,628; 6,277,489; 6,261,776; 6,258,606; 6,054,270; 6,048,695; 6,045,996; 6,022,963; 6,013,440; 5,965,452; 5,959,098; 5,856,174; 5,830,645; 5,770,456; 5,632,957; 5,556,752; 5,143,854; 5,807,522; 5,800,992; 5,744,305; 5,700,637; 5,556,752; 5,434,049; see also, e.g., WO 99/51773; WO 99/09217; WO 97/46313; WO 96/17958; see also, e.g., Johnston, Curr.
  • array or “microarray” or “biochip” or “chip” as used herein is a plurality of target elements, each target element comprising a defined amount of one or more polypeptides (including antibodies) or nucleic acids immobilized onto a defined area of a substrate surface.
  • the compounds tested as modulators of immunomodulatory and antiinflammatory chemokine genes or chemokine-receptor dependent and independent signaling can be any small organic molecule, or a biological entity, such as a protein, e.g. , an antibody or peptide, a sugar, a nucleic acid, e.g. , an antisense oligonucleotide, RNAi, or a ribozyme, or a lipid.
  • modulators can be genetically altered versions of immunomodulatory and anti-inflammatory chemokine proteins or a G-protein coupled receptor protein.
  • test compounds will be small organic molecules, peptides, lipids, and lipid analogs.
  • any chemical compound can be used as a potential modulator or ligand in the assays of the invention, although most often compounds can be dissolved in aqueous or organic (especially DMSO-based) solutions are used.
  • the assays are designed to screen large chemical libraries by automating the assay steps and providing compounds from any convenient source to assays, which are typically run in parallel (e.g. , in microtiter formats on microtiter plates in robotic assays). It will be appreciated that there are many suppliers of chemical compounds, including Sigma (St. Louis, MO), Aldrich (St. Louis, MO), Sigma- Aldrich (St. Louis, MO), Fluka Chemika-Biochemica Analytika (Buchs Switzerland) and the like.
  • high throughput screening methods involve providing a combinatorial small organic molecule or peptide library containing a large number of potential therapeutic compounds (potential modulator or ligand compounds). Such "combinatorial chemical libraries” or “ligand libraries” are then screened in one or more assays, as described herein, to identify those library members (particular chemical species or subclasses) that display a desired characteristic activity. The compounds thus identified can serve as conventional "lead compounds" or can themselves be used as potential or actual therapeutics.
  • a combinatorial chemical library is a collection of diverse chemical compounds generated by either chemical synthesis or biological synthesis, by combining a number of chemical "building blocks" such as reagents.
  • a linear combinatorial chemical library such as a polypeptide library is formed by combining a set of chemical building blocks (amino acids) in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can be synthesized through such combinatorial mixing of chemical building blocks.
  • Preparation and screening of combinatorial chemical libraries is well known to those of skill in the art. Such combinatorial chemical libraries include, but are not limited to, peptide libraries (see, e.g., U.S.
  • Other chemistries for generating chemical diversity libraries can also be used. Such chemistries include, but are not limited to: peptoids (e.g., PCT Publication No. WO 91/19735), encoded peptides (e.g., PCT Publication No. WO 93/20242), random bio-oligomers (e.g., PCT Publication No. WO 92/00091), benzodiazepines (e.g., U.S. Pat. No.
  • Patent 5,593,853 small organic molecule libraries (see, e.g., benzodiazepines, Baum C&EN, Jan 18, page 33 (1993); isoprenoids, U.S. Patent 5,569,588; thiazolidinones and metathiazanones, U.S. Patent 5,549,974; pyrrolidines, U.S. Patents 5,525,735 and 5,519,134; morpholino compounds, U.S. Patent 5,506,337; benzodiazepines, 5,288,514, and the like).
  • benzodiazepines Baum C&EN, Jan 18, page 33 (1993)
  • isoprenoids U.S. Patent 5,569,588
  • thiazolidinones and metathiazanones U.S. Patent 5,549,974
  • pyrrolidines U.S. Patents 5,525,735 and 5,519,134
  • morpholino compounds U.S. Patent 5,506,337
  • Candidate compounds are useful as part of a strategy to identify drugs for treating infectious diseases and inflammatory diseases involving innate and specific immune responses.
  • a test compound that binds to, for example, immunomodulatory and antiinflammatory gene products is considered a candidate compound (such as a test compound that binds to GAPDH).
  • test compounds Screening assays for identifying candidate or test compounds that bind to immunomodulatory and anti-inflammatory gene products, or modulate the activity of immunomodulatory and anti-inflammatory proteins or polypeptides or biologically active portions thereof, are also included in the invention.
  • the test compounds can be obtained using any of the numerous approaches in combinatorial library methods known in the art, including, but not limited to, biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
  • the biological library approach can be used for, e.g., peptide libraries, while the other four approaches are applicable to peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, Anticancer Drug Des. 12: 145, 1997).
  • Examples of methods for the synthesis of molecular libraries can be found in the art, for example in: DeWitt et al., Proc. Natl. Acad. ScL U.S.A. 90: 6909, 1993; Erb et al, Proc. Natl. Acad. ScL USA 91: 11422, 1994; Zuckermann et al., J. Med. Chem.
  • test compounds are activating variants of immunomodulatory and antiinflammatory chemokine genes.
  • Libraries of compounds can be presented in solution (e.g. , Houghten, Bio/Techniques 13: 412-421, 1992), or on beads (Lam, Nature 354: 82-84, 1991), chips (Fodor, Nature 364: 555-556, 1993), bacteria (U.S. Pat. No. 5,223,409), spores (U.S. Pat. Nos. 5,571,698, 5,403,484, and 5,223,409), plasmids (Cull et al., Proc. Natl. Acad.
  • the ability of a test compound to modulate the activity of immunomodulatory and anti-inflammatory chemokine genes or related genes or a biologically active portions thereof can be determined, e.g., by monitoring the ability to form complexes in the presence of the test compound.
  • the ability of the test compound to modulate the activity of immunomodulatory and anti-inflammatory chemokine genes, or a biologically active portion thereof, can also be determined by monitoring the ability of proteins binding to these genes.
  • the binding assays can be cell-based or cell-free.
  • test compound In general, the ability of a test compound to bind to immunomodulatory and antiinflammatory genes or their producrts, or otherwise affect induction of cytokines, is compared to a control in which the binding or induction of activity is determined in the absence of the test compound.
  • a predetermined reference value is used. Such reference values can be determined relative to controls, in which case a test sample that is different from the reference would indicate that the compound binds to the molecule of interest or modulates expression.
  • a reference value can also reflect the amount of binding or induction observed with a standard. In this case, a test compound that is similar to (e.g., equal to or less than) the reference would indicate that compound is a candidate compound.
  • This invention further pertains to novel agents identified by the above-described screening assays and uses thereof for treatments as described herein.
  • the invention provides soluble assays using immunomodulatory and anti-inflammatory gene product or protein of interest, or a cell or tissue expressing immunomodulatory and anti-inflammatory gene product or protein or interest, either naturally occurring or recombinant.
  • the invention provides solid phase based in vitro assays in a high throughput format, where immunomodulatory and antiinflammatory chemokine gene product or protein of interest or its ligand is attached to a solid phase substrate via covalent or non-covalent interactions. Any one of the assays described herein can be adapted for high throughput screening.
  • each well of a microtiter plate can be used to run a separate assay against a selected potential modulator, or, if concentration or incubation time effects are to be observed, every 5-10 wells can test a single modulator.
  • a single standard microtiter plate can assay about 100 ⁇ e.g., 96) modulators.
  • 1536 well plates are used, then a single plate can easily assay from about 100- about 1500 different compounds. It is possible to assay many plates per day; assay screens for up to about 6,000, 20,000, 50,000, or more than 100,000 different compounds are possible using the integrated systems of the invention.
  • the protein of interest or a fragment thereof e.g., an extracellular domain, or a cell or membrane comprising the protein of interest or a fragment thereof as part of a fusion protein can be bound to the solid state component, directly or indirectly, via covalent or non covalent linkage e.g., via a tag.
  • the tag can be any of a variety of components. In general, a molecule which binds the tag (a tag binder) is fixed to a solid support, and the tagged molecule of interest is attached to the solid support by interaction of the tag and the tag binder.
  • tags and tag binders can be used, based upon known molecular interactions well described in the literature.
  • a tag has a natural binder, for example, biotin, protein A, or protein G
  • tag binders avidin, streptavidin, neutravidin, the Fc region of an immunoglobulin, and the like
  • Antibodies to molecules with natural binders such as biotin are also widely available and appropriate tag binders; see, SIGMA Immunochemicals 1998 catalogue SIGMA, St. Louis MO).
  • any haptenic or antigenic compound can be used in combination with an appropriate antibody to form a tag/tag binder pair.
  • Thousands of specific antibodies are commercially available and many additional antibodies are described in the literature.
  • the tag is a first antibody and the tag binder is a second antibody which recognizes the first antibody.
  • receptor-ligand interactions are also appropriate as tag and tag-binder pairs.
  • agonists and antagonists of cell membrane receptors ⁇ e.g.
  • cell receptor-ligand interactions such as toll-like receptors, transferrin, c-kit, viral receptor ligands, cytokine receptors, chemokine receptors, interleukin receptors, immunoglobulin receptors and antibodies, the cadherin family, the integrin family, the selectin family, and the like; see, e.g., Pigott & Power, The Adhesion Molecule Facts Book I, 1993.
  • toxins and venoms viral epitopes, hormones ⁇ e.g., opiates, steroids, and the like.
  • intracellular receptors ⁇ e.g.
  • Synthetic polymers such as polyurethanes, polyesters, polycarbonates, polyureas, polyamides, polyethyleneimines, polyarylene sulfides, polysiloxanes, polyimides, and polyacetates can also form an appropriate tag or tag binder. Many other tag/tag binder pairs are also useful in assay systems described herein, as would be apparent to one of skill upon review of this disclosure.
  • Common linkers such as peptides, polyethers, and the like can also serve as tags, and include polypeptide sequences, such as poly gly sequences of between about 5 and 200 amino acids.
  • polypeptide sequences such as poly gly sequences of between about 5 and 200 amino acids.
  • Such flexible linkers are known to persons of skill in the art.
  • polyethylene glycol linkers are available from Shearwater Polymers, Inc. Huntsville, Alabama. These linkers optionally have amide linkages, sulfhydryl linkages, or hetero functional linkages.
  • Tag binders are fixed to solid substrates using any of a variety of methods currently available.
  • Solid substrates are commonly derivatized or functionalized by exposing all or a portion of the substrate to a chemical reagent which fixes a chemical group to the surface which is reactive with a portion of the tag binder.
  • groups which are suitable for attachment to a longer chain portion would include amines, hydroxyl, thiol, and carboxyl groups.
  • Aminoalkylsilanes and hydroxyalkylsilanes can be used to functionalize a variety of surfaces, such as glass surfaces. The construction of such solid phase biopolymer arrays is well described in the literature. See, e.g., Merrifield, J. Am. Chem. Soc.
  • Non-chemical approaches for fixing tag binders to substrates include other common methods, such as heat, cross-linking by UV radiation, and the like.
  • the invention provides isolated or recombinant antibodies that specifically bind to a polypeptide or nucleic acid of the invention, e.g., GAPDH nucleic acids or polypeptides, or related nucleic acids or polypeptides. These antibodies can be used to isolate, identify or quantify a polypeptide of the invention or related polypeptides. These antibodies can be used to isolate other polypeptides within the scope the invention or other related signaling activity polypeptides.
  • antibody includes a peptide or polypeptide derived from, modeled after or substantially encoded by an immunoglobulin gene or immunoglobulin genes, or fragments thereof, capable of specifically binding an antigen or epitope, see, e.g. Fundamental Immunology, Third Edition, W. E. Paul, ed., Raven Press, N.Y., 1993; Wilson, J. Immunol. Methods 175: 267-273, 1994; Yarmush, J. Biochem. Biophys. Methods 25: 85- 97, 1992.
  • antibody includes antigen-binding portions, i.e., "antigen binding sites," ⁇ e.g., fragments, subsequences, complementarity determining regions (CDRs)) that retain capacity to bind antigen, including (i) a Fab fragment, a monovalent fragment consisting of the VL, VH, CL and CHl domains; (ii) a F(ab')2 fragment, a bivalent fragment comprising two Fab fragments linked by a disulfide bridge at the hinge region; (iii) a Fd fragment consisting of the VH and CHl domains; (iv) a Fv fragment consisting of the VL and VH domains of a single arm of an antibody, (v) a dAb fragment (Ward et al., Nature 341: 544- 546, 1989), which consists of a VH domain; and (vi) an isolated complementarity determining region (CDR).
  • antigen binding sites ⁇ e.g., fragments
  • Single chain antibodies are also included by reference in the term "antibody.”
  • the antibodies can be used in immunoprecipitation, staining (e.g., FACS), immunoaff ⁇ nity columns, and the like.
  • nucleic acid sequences encoding for specific antigens can be generated by immunization followed by isolation of polypeptide or nucleic acid, amplification or cloning and immobilization of polypeptide onto an array of the invention.
  • the methods of the invention can be used to modify the structure of an antibody produced by a cell to be modified, e.g., an antibody's affinity can be increased or decreased.
  • the ability to make or modify antibodies can be a phenotype engineered into a cell by the methods of the invention.
  • Antibodies also can be generated in vitro, e.g., using recombinant antibody binding site expressing phage display libraries, in addition to the traditional in vivo methods using animals. See, e.g., Hoogenboom, Trends Biotechnol. 15: 62-70, 1997; Katz, Annu. Rev. Biophys. Biomol. Struct. 26: 27-45, 1997.
  • Polypeptides or peptides can be used to generate antibodies which bind specifically to the polypeptides of the invention.
  • the resulting antibodies can be used in immunoaffmity chromatography procedures to isolate or purify the polypeptide or to determine whether the polypeptide is present in a biological sample.
  • a protein preparation such as an extract, or a biological sample is contacted with an antibody capable of specifically binding to one of the polypeptides of the invention.
  • the antibody is attached to a solid support, such as a bead or other column matrix.
  • the protein preparation is placed in contact with the antibody under conditions in which the antibody specifically binds to one of the polypeptides of the invention. After a wash to remove non-specifically bound proteins, the specifically bound polypeptides are eluted.
  • binding can be determined using any of a variety of procedures familiar to those skilled in the art. For example, binding can be determined by labeling the antibody with a detectable label such as a fluorescent agent, an enzymatic label, or a radioisotope. Alternatively, binding of the antibody to the sample can be detected using a secondary antibody having such a detectable label thereon. Particular assays include ELISA assays, sandwich assays, radioimmunoassay, and Western Blots.
  • Polyclonal antibodies generated against the polypeptides of the invention can be obtained by direct injection of the polypeptides into an animal or by administering the polypeptides to a non-human animal. The antibody so obtained will then bind the polypeptide itself. In this manner, even a sequence encoding only a fragment of the polypeptide can be used to generate antibodies which can bind to the whole native polypeptide. Such antibodies can then be used to isolate the polypeptide from cells expressing that polypeptide. [0292] For preparation of monoclonal antibodies, any technique which provides antibodies produced by continuous cell line cultures can be used.
  • Examples include the hybridoma technique, the trioma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique (see, e.g., Cole (1985) in Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, Inc., pp. 77-96).
  • Antibodies generated against the polypeptides of the invention can be used in screening for similar polypeptides from other organisms and samples. In such techniques, polypeptides from the organism are contacted with the antibody and those polypeptides which specifically bind the antibody are detected. Any of the procedures described above can be used to detect antibody binding.
  • Yet another assay for compounds that modulate receptor or ligand protein activity affecting signaling in innate immunity involves computer assisted drug design, in which a computer system is used to generate a three-dimensional structure of a receptor or ligand protein based on the structural information encoded by its amino acid sequence.
  • the input amino acid sequence interacts directly and actively with a pre-established algorithm in a computer program to yield secondary, tertiary, and quaternary structural models of the protein.
  • the models of the protein structure are then examined to identify regions of the structure that have the ability to bind. These regions are then used to identify compounds that bind to the protein.
  • the three-dimensional structural model of the protein is generated by entering protein amino acid sequences of at least 10 amino acid residues or corresponding nucleic acid sequences encoding a receptor or ligand polypeptide into the computer system.
  • the amino acid sequence represents a primary structure that encodes the information necessary to form the secondary, tertiary and quaternary structure of the protein of interest.
  • the software looks at certain parameters encoded by the primary sequence to generate the structural model. These parameters are referred to as "energy terms,” and primarily include electrostatic potentials, hydrophobic potentials, solvent accessible surfaces, and hydrogen bonding. Secondary energy terms include van der Waals potentials. Biological molecules form the structures that minimize the energy terms in a cumulative fashion.
  • the computer program is therefore using these terms encoded by the primary structure or amino acid sequence to create the secondary structural model.
  • the tertiary structure of the protein encoded by the secondary structure is then formed on the basis of the energy terms of the secondary structure.
  • the user at this point can enter additional variables such as whether the protein is membrane bound or soluble, its location in the body, and its cellular location, e.g. , cytoplasmic, surface, or nuclear. These variables along with the energy terms of the secondary structure are used to form the model of the tertiary structure.
  • the computer program matches hydrophobic faces of secondary structure with like, and hydrophilic faces of secondary structure with like.
  • potential modulator binding regions are identified by the computer system.
  • Three-dimensional structures for potential modulators are generated by entering amino acid or nucleotide sequences or chemical formulas of compounds, as described above. The three-dimensional structure of the potential modulator is then compared to that of the receptor or ligand protein to identify compounds that bind to the protein. Binding affinity between the protein and compound is determined using energy terms to determine which compounds have an enhanced probability of binding to the protein.
  • Computer systems are also used to screen for mutations, polymorphic variants, alleles and interspecies homologs of receptor or ligand induced genes. Such mutations can be associated with disease states or genetic traits.
  • GeneChipTM and related technology can also be used to screen for mutations, polymorphic variants, alleles and interspecies homologs. Once the variants are identified, diagnostic assays can be used to identify patients having such mutated genes. Identification of the mutated receptor or ligand induced genes involves receiving input of a first nucleic acid or amino acid sequence of the naturally occurring receptor or ligand induced gene, respectively, and conservatively modified versions thereof. The sequence is entered into the computer system as described above. The first nucleic acid or amino acid sequence is then compared to a second nucleic acid or amino acid sequence that has substantial identity to the first sequence. The second sequence is entered into the computer system in the manner described above. Once the first and second sequences are compared, nucleotide or amino acid differences between the sequences are identified. Such sequences can represent allelic differences in various receptor or ligand induced genes, and mutations associated with disease states and genetic
  • inflammatory diseases in mammals are can be treated by administering an effective amount of an innate immunity modulator of the invention.
  • inflammatory disease refers to a disease which has inflammation as its etiologic agent.
  • Inflammatory disease or symptoms of the inflammatory disease
  • an innate immunity modulator of the present invention include, but are not limited to, the following: asthma, hypersensitivity, autoimmune disease, chronic inflammation, glomerulonephritis, chronic prostatitis, transplant rejection, allergic disease, neurodegenerative disease, cardiovascular disease, gastrointestinal disease, septic shock, anaphylactic shock, toxic shock syndrome, cachexia, inflammatory bowel diease, pelvic inflammatory disease, reperfusion injury, rheumatoid arthritis, vasculitis, cystic fibrosis, psoriasis, atopic dermatitis, multiple sclerosis, systemic lupus erhthematosus, chronic obstructive pulmonary disease (COPD) and related pulmonary diseases, necrosis, chronic obstruct
  • infectious disease include but are not limited to, viral, bacterial, fungal, or parasitic diseases.
  • the innate immunity modulators of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly increasing the proliferation and differentiation of B and/or T cells, infectious diseases can be treated.
  • the immune response can be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • the innate immunity modulating polypeptide or polynucleotide of the present invention can also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • Exemplary infectious disease include but are not limited to, viral, bacterial, fungal, or parasitic diseases.
  • the innate immunity modulators of the present invention can be used to treat or detect infectious agents. For example, by increasing the immune response, particularly favourably modulating the innate immune response or increasing the proliferation and differentiation of B and/or T cells, infectious diseases can be treated.
  • the immune response can be increased by either enhancing an existing immune response, or by initiating a new immune response.
  • the innate immunity modulators of the present invention can also directly inhibit the infectious agent, without necessarily eliciting an immune response.
  • viral diseases in mammals can be treated by administering an effective amount of an innate immunity modulator of the present invention.
  • the term "viral disease” refers to a disease which has a virus (or viruses) as its etiologic agent.
  • Viral diseases (or symptoms of the viral disease) that can be treated by an innate immunity modulator of the present invention include, but are not limited to, the following.
  • DNA viruses such as hepadnaviridae, adenoviridae, parvoviridae, papovariridae, poxyiridae, iridoviridae, and herpesviridae or any combination thereof can be treated by an innate immunity modulator of the present invention.
  • RNA viruses such as picornaviridae, calciviridae, togaviridae, flaviviridae, coronaviridae, rhabdoviridae, filoviridae, paramyxoviridae, orthomyxoviridae, bunyaviridae, arenaviridae, reoviridae, and bimaviridae or any combination thereof can be treated by an innate immunity modulator of the present invention.
  • Lentiviridae such as HTLV-I, HTLV-II, HTLV-III (HIV-I) and HIV-2 and hepatitis viruses (including hepatitis A, B, C, delta, E and/or G virus) or any combination thereof can be treated by an innate immunity modulator of the present invention.
  • Influenza viral pneumonia, viral bronchitis, herpetic infections (herpes simplex virus, Epstein Barr virus (infectious mononucleosis), herpes zoster (varicella zoster Virus (VZV)), poliomyelitis, adult T-cell leukemia (ATL), papilloma (HPV), measles, rubella, exanthema subitum, erythema infectiosum, viral encephalitis, viral myelitis, Visna (Sheep) and Equestrian Anemia, cytomegalovirus infection, mumps, varicella, rabies, viral enteritis, viral myocarditis, viral pericarditis or any combination thereof can be treated by an innate immunity modulator of the present invention.
  • SARS-associated coronavirus SARS-CoV
  • avian influenza bird flu
  • avian influenza A H5N1
  • Filoviridae such as Ebola-Zaire, Ebola-Sudan, and Ebola-Ivory Coast, and Ebola-Reston, or any combination thereof can be treated by an innate immunity modulator of the present invention. Additional examples viruses and viral disease states are described the most recent edition of Fields Virology, Fields, B. N. et al. (eds.) Lippincott-Raven Publishers, Philadelphia, PA (this reference is incorporated by reference in its entirety for all purposes).
  • bacterial or fungal agents that can cause disease or symptoms and that can be treated by an innate immunity modulators of the present invention include, but not limited to, the following Gram-Negative and Gram-positive bacterial families and f ⁇ ngi: Actinomycetales ⁇ e.g., Corynebacterium, Mycobacterium, Norcardia), Aspergillosis, Bacillaceae ⁇ e.g., Anthrax, Clostridium), Bacteroidaceae, Blastomycosis, Bordetella, Borrelia, Brucellosis, Candidiasis, Campylobacter, Coccidioidomycosis, Cryptococcosis, Dermatocycoses, Enterobacteriaceae (Klebsiella, Salmonella, Serratia, Yersinia), Erysipelothrix, Helicobacter, Legionellosis, Leptospirosis, Listeria, Mycoplasmatales, Neisseriacea
  • bacterial or fungal families can cause the following diseases or symptoms, including, but not limited to: bacteremia, endocarditis, eye infections (conjunctivitis, tuberculosis, uveitis), gingivitis, opportunistic infections ⁇ e.g., AIDS related infections), paronychia, prosthesis-related infections, Reiter's Disease, respiratory tract infections, such as Whooping Cough or Empyema, sepsis, Lyme Disease, Cat-Scratch Disease, Dysentery, Paratyphoid Fever, food poisoning, Typhoid, pneumonia, Gonorrhea, meningitis, Chlamydia, Syphilis, Diphtheria, Leprosy, Paratuberculosis, Tuberculosis, Lupus, Botulism, gangrene, tetanus, impetigo, Rheumatic Fever, Scarlet Fever, sexually transmitted diseases, skin diseases ⁇ e.g.,
  • parasitic agents causing disease or symptoms that can be treated or detected by an innate immunity modulator of the present invention include, but not limited to, the following families: Amebiasis, Babesiosis, Coccidiosis, Cryptosporidiosis, Dientamoebiasis, Dourine, Ectoparasitic, Giardiasis, Helminthiasis, Leishmaniasis, Theileriasis, Toxoplasmosis, Trypanosomiasis, and Trichomonas.
  • These parasites can cause a variety of diseases or symptoms, including, but not limited to: Scabies, Trombiculiasis, eye infections, intestinal disease ⁇ e.g.
  • the method of inhibiting the growth of bacteria can further include the addition of antibiotics for combination or synergistic therapy.
  • the appropriate antibiotic administered will typically depend on the susceptibility of the bacteria such as whether the bacteria is Gram negative or Gram positive, and will be easily discernable by one of skill in the art.
  • antibiotics useful for synergistic therapy with the innate immunity modulators of the invention include aminoglycosides (e.g., tobramycin), penicillins (e.g., piperacillin), cephalosporins (e.g., ceftazidime), fluoroquinolones (e.g., ciprofloxacin), carbapenems (e.g., imipenem), tetracyclines and macro lides (e.g., erythromycin and clarithromycin).
  • aminoglycosides e.g., tobramycin
  • penicillins e.g., piperacillin
  • cephalosporins e.g., ceftazidime
  • fluoroquinolones e.g., ciprofloxacin
  • carbapenems e.g., imipenem
  • tetracyclines e.g., erythromycin and clarithromycin
  • the innate immunity modulating peptides and/or analogs or derivatives or the invention thereof can be administered to any host, including a human or non-human animal, in an amount effective to modulate innate immunity.
  • the immunomodulatory and anti-inflammatory peptides (e.g., innate immunity modulators) of the invention are administered in combination with at least one antibiotic.
  • the antibiotic is selected from the group consisting of aminoglycosides, penicillins, cephalosporins, carbapenems, monobactams, quinolones, tetracyclines, and glycopeptides.
  • typical antibiotics include aminoglycosides (amikacin, gentamicin, kanamycin, netilmicin, t-obramycin, streptomycin), macrolides (azithromycin, clarithromycin, erythromycin, erythromycin estolate/ethylsuccinate/ gluceptate/lactobionate/stearate), beta-lactams such as penicillins (e.g., penicillin G, penicillin V, methicillin, nafcillin, oxacillin, cloxacillin, dicloxacillin, ampicillin, amoxicillin, ticarcillin, carbenicillin, mezlocillin, azlocillin and piperacillin), or cephalosporins (e.g.
  • carbapenems e.g., imipenem, meropenem, panipenem
  • monobactams e.g., aztreonam
  • antibiotics include quinolones (e.g. , fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin), tetracyclines (e.g., doxycycline, minocycline, tetracycline), and glycopeptides (e.g., vancomycin, teicoplanin), for example.
  • quinolones e.g. , fleroxacin, nalidixic acid, norfloxacin, ciprofloxacin, ofloxacin, enoxacin, lomefloxacin and cinoxacin
  • tetracyclines e.g., doxycycline, minocycline, tetracycline
  • glycopeptides e.g., vancomycin, teicoplanin
  • antibiotics include chloramphenicol, clindamycin, trimethoprim, sulfamethoxazole, nitrofurantoin, rifampin, linezolid, synercid, polymyxin B, colisitin, colimycin, methotrexate, daptomycin, phosphonomycin and mupirocin.
  • treatment using an innate immunity modulating polypeptide or polynucleotide of the present invention could either be by administering an effective amount of a polypeptide to the patient, or by removing cells from the patient, supplying the cells with a polynucleotide of the present invention, and returning the engineered cells to the patient (ex vivo therapy).
  • the innate immunity modulating polypeptide or polynucleotide of the present invention can be used as an antigen in a vaccine to raise an immune response against infectious disease.
  • the immunomodulatory and anti-inflammatory compounds, or analogs, derivatives, amidated variations and conservative variations thereof can be administered to any host, including a human or non-human animal, in an amount effective to stimulate innate immunity resulting in the inhibition of growth of a bacterium, but also a virus, parasite or fungus.
  • the innate immunity modulators, or analogs, derivatives, amidated variations and conservative variations thereof can be administered to any host, including a human or non- human animal, in an amount.
  • the innate immunity modulators, or analogs, derivatives, amidated variations and conservative variations thereof can be used as therapeutic compositions either alone or in combination with other therapies, for example, with other drugs, or with radiation or chemotherapies.
  • the invention provides pharmaceutical compositions comprising one or a combination of the innate immunity modulators, or analogs, derivatives, amidated variations and conservative variations thereof of the invention, for example, formulated together with a pharmaceutically acceptable carrier.
  • Some compositions include a combination of multiple (e.g., two or more) analogs, derivatives, amidated variations and conservative variations thereof of the invention.
  • compositions include a combination of an immunomodulatory and anti-inflammatory chemokine (an innate immunity modulator) of the invention together with other drugs or agents (i.e., antimicrobial drugs, antimicrobial agents, antiviral agents, or antifungal agents).
  • an immunomodulatory and anti-inflammatory chemokine an innate immunity modulator
  • other drugs or agents i.e., antimicrobial drugs, antimicrobial agents, antiviral agents, or antifungal agents.
  • the innate immunity modulators and/or analogs or derivatives thereof can be administered to any host, including a human or non-human animal, in an amount effective to stimulate innate immunity resulting in the inhibition of sepsis or inflammation in a patient or subject. These innate immunity modulators are useful by stimulating anti-infective immunity.
  • the innate immunity modulators and/or analogs or derivatives thereof can be administered to any host, including a human or non-human animal, in an amount effective to inhibit not only growth of a bacterium, but also a virus or fungus.
  • pharmaceutically acceptable carrier and “pharmaceutically acceptable excipient” include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.
  • the carrier is suitable for parenteral administration.
  • the carrier can be suitable for intravenous, intraperitoneal or intramuscular administration.
  • the carrier is suitable for oral administration.
  • Pharmaceutically acceptable carriers include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion. The use of such media and agents for pharmaceutically active substances is well known in the art.
  • “Pharmaceutically acceptable salts and esters” means salts and esters that are pharmaceutically acceptable and have the desired pharmacological properties. Such salts include salts that can be formed where acidic protons present in the compounds are capable of reacting with inorganic or organic bases. Suitable inorganic salts include those formed with the alkali metals, e.g., sodium and potassium, magnesium, calcium, and aluminum.
  • Suitable organic salts include those formed with organic bases such as the amine bases, e.g., ethanolamine, diethanolamine, triethanolamine, tromethamine, N-methylglucamine, and the like. Such salts also include acid addition salts formed with inorganic acids (e.g., hydrochloric and hydrobromic acids) and organic acids (e.g., acetic acid, citric acid, maleic acid, and the alkane- and arene-sulfonic acids such as methanesulfonic acid and benzenesulfonic acid).
  • Pharmaceutically acceptable esters include esters formed from carboxy, sulfonyloxy, and phosphonoxy groups present in the compounds, e.g., C 1-6 alkyl esters.
  • a pharmaceutically acceptable salt or ester can be a mono-acid-mono-salt or ester or a di-salt or ester; and similarly where there are more than two acidic groups present, some or all of such groups can be salif ⁇ ed or esterified.
  • Compounds named in this invention can be present in unsalif ⁇ ed or unesterif ⁇ ed form, or in salif ⁇ ed and/or esterified form, and the naming of such compounds is intended to include both the original (unsalified and unesterified) compound and its pharmaceutically acceptable salts and esters.
  • certain compounds named in this invention can be present in more than one stereoisomeric form, and the naming of such compounds is intended to include all single stereoisomers and all mixtures (whether racemic or otherwise) of such stereoisomers.
  • “Pharmaceutically acceptable”, “physiologically tolerable” and grammatical variations thereof, as they refer to compositions, carriers, diluents and reagents, are used interchangeably and represent that the materials are capable of administration to or upon a human without the production of undesirable physiological effects to a degree that would prohibit administration of the composition.
  • compositions or medicaments are administered to a patient susceptible to, or otherwise at risk of a disease or condition (i.e., as a result of bacteria, fungi, viruses, parasites or the like) in an amount sufficient to eliminate or reduce the risk, lessen the severity, or delay the outset of the disease, including biochemical, histologic and/or behavioral symptoms of the disease, its complications and intermediate pathological phenotypes presenting during development of the disease.
  • compositions or medicants are administered to a patient suspected of, or already suffering from such a disease or condition in an amount sufficient to cure, or at least partially arrest, the symptoms of the disease or condition (e.g., biochemical and/or histologic), including its complications and intermediate pathological phenotypes in development of the disease or condition.
  • An amount adequate to accomplish therapeutic or prophylactic treatment is defined as a therapeutically- or prophylactically-effective dose.
  • agents are usually administered in several dosages until a sufficient response has been achieved. Typically, the response is monitored and repeated dosages are given if the response starts to wane.
  • the pharmaceutical composition of the present invention should be sterile and fluid to the extent that the composition is deliverable by syringe.
  • the carrier can be an isotonic buffered saline solution, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion and by use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition.
  • the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.
  • an agent which delays absorption for example, aluminum monostearate or gelatin.
  • compositions of the invention also can be administered in combination therapy, i.e., combined with other agents.
  • the combination therapy can include a composition of the present invention with at least one agent or other conventional therapy.
  • a composition of the present invention can be administered by a variety of methods known in the art.
  • the route and/or mode of administration vary depending upon the desired results.
  • the phrases "parenteral administration” and “administered parenterally” mean modes of administration other than enteral and topical administration, usually by injection, and includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, epidural and intrasternal injection and infusion.
  • the chemokine of the invention can be administered parenterally by injection or by gradual infusion over time.
  • the chemokine can also be prepared with carriers that protect the compound against rapid release, such as a controlled release formulation, including implants, transdermal patches, and microencapsulated delivery systems Further methods for delivery of the chemokine include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation. To administer a chemokine of the invention by certain routes of administration, it can be necessary to coat the compound with, or coadminister the compound with, a material to prevent its inactivation.
  • the method of the invention also includes delivery systems such as microencapsulation of innate immunity modulators into liposomes or a diluent.
  • Microencapsulation also allows co-entrapment of molecules along with the antigens, so that these molecules, such as antibiotics, can be delivered to a site in need of such treatment in conjunction with the innate immunity modulators of the invention.
  • Liposomes in the blood stream are generally taken up by the liver and spleen.
  • Pharmaceutically acceptable diluents include saline and aqueous buffer solutions.
  • Liposomes include water-in-oil-in- water CGF emulsions as well as conventional liposomes. Strejan et ah, J. Neuroimmunol. 7: 27, 1984.
  • the method of the invention is particularly useful for delivering innate immunity modulators to such organs.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Many methods for the preparation of such formulations are described by e.g., Robinson, 1978, Sustained and Controlled Release Drug Delivery Systems. Other methods of administration will be known to those skilled in the art.
  • the chemokines of the invention can be administered parenterally by injection or by gradual infusion over time.
  • the chemokines can be administered intravenously, intraperitoneally, intramuscularly, subcutaneously, intracavity, or transdermally.
  • Further methods for delivery of the chemokines include orally, by encapsulation in microspheres or proteinoids, by aerosol delivery to the lungs, or transdermally by iontophoresis or transdermal electroporation.
  • the method of the invention also includes delivery systems such as microencapsulation of chemokines into liposomes.
  • Microencapsulation also allows co-entrapment of innate immunity modulator molecules along with the antigens, so that these molecules, such as antibiotics, can be delivered to a site in need of such treatment in conjunction with the chemokines of the invention.
  • Liposomes in the blood stream are generally taken up by the liver and spleen.
  • the method of the invention is particularly useful for delivering innate immunity modulators to such organs.
  • Other methods of administration will be known to those skilled in the art.
  • Preparations for parenteral administration of a chemokine of the invention include sterile aqueous or non-aqueous solutions, suspensions, and emulsions.
  • nonaqueous solvents examples include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate.
  • Aqueous carriers include water, alcoholic/aqueous solutions, emulsions or suspensions, including saline and buffered media.
  • Parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, or fixed oils.
  • Intravenous vehicles include fluid and nutrient replenishers, electrolyte replenishers (such as those based on Ringer's dextrose), and the like. Preservatives and other additives can also be present such as, for example, antimicrobials, anti-oxidants, chelating agents, and inert gases and the like.
  • compositions typically must be sterile, substantially isotonic, and stable under the conditions of manufacture and storage.
  • the composition can be formulated as a solution, microemulsion, liposome, or other ordered structure suitable to high drug concentration.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyethylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants.
  • isotonic agents for example, sugars, polyalcohols such as mannitol, sorbitol, or sodium chloride in the composition.
  • Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent that delays absorption, for example, monostearate salts and gelatin.
  • Sterile injectable solutions can be prepared by incorporating the active compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by sterilization microf ⁇ ltration.
  • dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying (lyophilization) that yield a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Therapeutic compositions can also be administered with medical devices known in the art.
  • a therapeutic composition of the invention can be administered with a needleless hypodermic injection device, such as the devices disclosed in, e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • a needleless hypodermic injection device such as the devices disclosed in, e.g., U.S. Patent Nos. 5,399,163, 5,383,851, 5,312,335, 5,064,413, 4,941,880, 4,790,824, or 4,596,556.
  • implants and modules useful in the present invention include: U.S. Patent No. 4,487,603, which discloses an implantable micro-infusion pump for dispensing medication at a controlled rate; U.S. Patent No. 4,486,194, which discloses a therapeutic device for administering medicants through the skin; U.S. Patent No.
  • the compounds of the present invention are administered as pharmaceuticals, to humans and animals, they can be given alone or as a pharmaceutical composition containing, for example, 0.01 to 99.5% (or 0.1 to 90%) of active ingredient in combination with a pharmaceutically acceptable carrier.
  • compositions of the invention can be administered in a variety of unit dosage forms depending upon the method of administration. Dosages for typical nucleic acid, peptide and polypeptide pharmaceutical compositions are well known to those of skill in the art. Such dosages are typically advisorial in nature and are adjusted depending on the particular therapeutic context, patient tolerance, and the like.
  • the amount of nucleic acid, peptide or polypeptide adequate to accomplish this is defined as a "therapeutically effective dose.”
  • the dosage schedule and amounts effective for this use i.e., the "dosing regimen” will depend upon a variety of factors, including the stage of the disease or condition, the severity of the disease or condition, the general state of the patient's health, the patient's physical status, age, pharmaceutical formulation and concentration of active agent, and the like.
  • the mode of administration also is taken into consideration.
  • the dosage regimen must also take into consideration the pharmacokinetics, i.e., the pharmaceutical composition's rate of absorption, bioavailability, metabolism, clearance, and the like.
  • Dosage regimens of the pharmaceutical compositions of the present invention are adjusted to provide the optimum desired response ⁇ e.g., a therapeutic response). For example, a single bolus can be administered, several divided doses can be administered over time or the dose can be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms of the invention are dictated by and directly dependent on (a) the unique characteristics of the active compound and the particular therapeutic effect to be achieved, and (b) the limitations inherent in the art of compounding such an active compound for the treatment of sensitivity in individuals.
  • Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient which is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient.
  • the selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, or the ester, salt or amide thereof, the route of administration, the time of administration, the rate of excretion of the particular compound being employed, the duration of the treatment, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.
  • a physician or veterinarian can start doses of the compounds of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved.
  • a suitable daily dose of a compound of the invention is that amount of the compound which is the lowest dose effective to produce a therapeutic effect. Such an effective dose generally depends upon the factors described above. It is preferred that administration be intravenous, intramuscular, intraperitoneal, or subcutaneous, or administered proximal to the site of the target. If desired, the effective daily dose of a therapeutic composition can be administered as two, three, four, five, six or more sub-doses administered separately at appropriate intervals throughout the day, optionally, in unit dosage forms.
  • an effective dose of each of the innate immunity modulating compounds disclosed herein as potential therapeutics for use in treating microbial diseases and conditions is from about 1 ⁇ g to 500 mg/kg body weight, per single administration, which can readily be determined by one skilled in the art. As discussed above, the dosage depends upon the age, sex, health, and weight of the recipient, kind of concurrent therapy, if any, and frequency of treatment. Other effective dosage range upper limits are 100 mg/kg body weight, 50 mg/kg body weight, 25 mg/kg body weight, and 10 mg/kg body weight.
  • the dosage and frequency of administration can vary depending on whether the treatment is prophylactic or therapeutic.
  • a relatively low dosage is administered at relatively infrequent intervals over a long period of time. Some patients continue to receive treatment for the rest of their lives.
  • a relatively high dosage at relatively short intervals is sometimes required until progression of the disease is reduced or terminated, and preferably until the patient shows partial or complete amelioration of symptoms of disease. Thereafter, the patent can be administered a prophylactic regime.
  • innate immunity modulating compositions are administered to a patient suffering from an infectious disease, an inflammatory disease, or a related disease or disorder in an amount sufficient to at least partially arrest the condition or a disease and/or its complications.
  • a soluble peptide pharmaceutical composition dosage for intravenous (IV) administration would be about 0.01 mg/hr to about 1.0 mg/hr administered over several hours (typically 1, 3, or 6 hours), which can be repeated for weeks with intermittent cycles.
  • CSF cerebrospinal fluid
  • Some innate immunity modulating compounds of the invention can be formulated to ensure proper distribution in vivo.
  • the blood-brain barrier (BBB) excludes many highly hydrophilic compounds.
  • the therapeutic innate immunity modulating compounds of the invention cross the BBB (if desired)
  • they can be formulated, for example, in liposomes.
  • liposomes For methods of manufacturing liposomes, See, e.g., U.S. Patents 4,522,811; 5,374,548; and 5,399,331.
  • the liposomes can comprise one or more moieties which are selectively transported into specific cells or organs, thus enhance targeted drug delivery (See, e.g., Ranade, J. Clin. Pharmacol. 29: 685, 1989).
  • Exemplary targeting moieties include folate or biotin (See, e.g., U.S. Patent 5,416,016 to Low et al); mannosides (Umezawa et ah, Biochem. Biophys. Res. Commun. 153: 1038, 1988); antibodies (Bloeman et al, FEBS Lett. 357: 140, 1995; Owais et al., Antimicrob. Agents Chemother. 39: 180, 1995); surfactant protein A receptor (Briscoe et al, Am. J. Physiol.
  • the therapeutic innate immunity modulating compounds of the invention are formulated in liposomes; in a more preferred aspect, the liposomes include a targeting moiety.
  • the therapeutic innate immunity modulating compounds in the liposomes are delivered by bolus injection to a site proximal to the tumor or infection.
  • the composition should be fluid to the extent that easy syringability exists. It should be stable under the conditions of manufacture and storage and should be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the relative effectiveness of the innate immunity modulators of the invention for the applications described can be readily determined by one of skill in the art by determining the sensitivity of any organism to one of the innate immunity modulating compounds of the invention.
  • compositions are prepared as injectables, either as liquid solutions or suspensions; solid forms suitable for solution in, or suspension in, liquid vehicles prior to injection can also be prepared.
  • the preparation also can be emulsified or encapsulated in liposomes or micro particles such as polylactide, polyglycolide, or copolymer for enhanced adjuvant effect, as discussed above. Langer, Science 249: 1527, 1990 and Hanes, Advanced Drug Delivery Reviews 28: 97-119, 1997.
  • the agents of this invention can be administered in the form of a depot injection or implant preparation which can be formulated in such a manner as to permit a sustained or pulsatile release of the active ingredient.
  • binders and carriers include, for example, polyalkylene glycols or triglycerides; such suppositories can be formed from mixtures containing the active ingredient in the range of 0.5% to 10%, preferably l%-2%.
  • Oral formulations include excipients, such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, and magnesium carbonate. These compositions take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain 10%-95% of active ingredient, preferably 25%-70%.
  • Topical application can result in transdermal or intradermal delivery. Topical administration can be facilitated by co-administration of the agent with cholera toxin or detoxified derivatives or subunits thereof or other similar bacterial toxins. Glenn et al, Nature 391: 851, 1998. Co-administration can be achieved by using the components as a mixture or as linked molecules obtained by chemical crosslinking or expression as a fusion protein.
  • transdermal delivery can be achieved using a skin patch or using transferosomes. Paul et al, Eur. J. Immunol. 25: 3521-24, 1995; Cevc et al, Biochem. Biophys. Acta 1368: 201-15, 1998.
  • compositions are generally formulated as sterile, substantially isotonic and in full compliance with all Good Manufacturing Practice (GMP) regulations of the U.S. Food and Drug Administration.
  • GMP Good Manufacturing Practice
  • Innate immunity modulators or their homologs are useful tools for examining expression and regulation of signaling in innate immunity.
  • Reagents that specifically hybridize to nucleic acids encoding receptor proteins or ligand proteins (including probes and primers of the proteins), and reagents that specifically bind to the proteins, e.g., antibodies, are used to examine expression and regulation.
  • Nucleic acid assays for the presence of innate immunity modulators in a sample include numerous techniques are known to those skilled in the art, such as Southern analysis, northern analysis, dot blots, RNase protection, Sl analysis, amplification techniques such as PCR and LCR, high density oligonucleotide array analysis, and in situ hybridization.
  • in situ hybridization for example, the target nucleic acid is liberated from its cellular surroundings in such as to be available for hybridization within the cell while preserving the cellular morphology for subsequent interpretation and analysis.
  • receptor proteins or ligand proteins can be detected with the various immunoassay techniques described above.
  • the test sample is typically compared to both a positive control ⁇ e.g. , a sample expressing recombinant receptor protein or ligand protein) and a negative control.
  • Kits are provided for screening innate immunity modulators. Such kits can be prepared from readily available materials and reagents are provided. For example, such kits can comprise any one or more of the following materials: the receptor proteins or ligand proteins, agonists, or antagonists, reaction tubes, and instructions for testing the activities of receptor protein genes or ligand protein genes. A wide variety of kits and components can be prepared depending upon the intended user of the kit and the particular needs of the user. For example, the kit can be tailored for in vitro or in vivo assays for measuring the activity of receptor proteins or ligand proteins proteins or modulators of signaling in innate immunity. [0347] Kits comprising probe arrays as described above are provided.
  • kits include, for example, other restriction enzymes, reverse- transcriptase or polymerase, the substrate nucleoside triphosphates, means used to label (for example, an avidin-enzyme conjugate and enzyme substrate and chromogen if the label is biotin), and the appropriate buffers for reverse transcription, PCR, or hybridization reactions.
  • the kits also contain instructions for carrying out the methods.
  • Human PBMC were isolated from healthy volunteers under the approval and ethics guidelines of the University of British Columbia.
  • CD 14+ human monocytes were isolated from PBMC using the Dynal negative isolation kit according to the manufacturer's instructions (Invitrogen, Burlington ON).
  • Murine RAW 264.7 ATCC TIB-71TM were cultured and differentially labeled with heavy amino acids employing SILACTM D-MEM Flex-media kit (Invitrogen) as described below.
  • Human THP-I (ATCC TIB-202) were differentiated with phorbol 12-myristate 13 -acetate (PMA; Sigma- Aldrich, Oakville ON) as previously described to induce plastic-adherent macrophage-like cells, and maintained in RPMI- 1640 media, supplemented with 10% (v/v) heat inactivated fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM sodium pyruvate (all from Invitrogen). Cultures were maintained at 37 0 C in a humidified 5% (v/v) CO2 incubator.
  • PMA phorbol 12-myristate 13 -acetate
  • FBS heat inactivated fetal bovine serum
  • 2 mM L-glutamine 2 mM L-glutamine
  • 1 mM sodium pyruvate all from Invitrogen
  • Murine RAW 264.7 (ATCC TIB-71TM) were cultured and differentially labeled with heavy amino acids ( 13 C6-L-Lysine and 13 C6-L-Arginine) employing SILACTM D-MEM Flex-media kit (Invitrogen) as per the manufacturer's instructions. Briefly, one cell population was cultured in media with light (normal) amino acids and the second cell population was grown in parallel in media supplemented with heavy ( 13 C6-L-Lysine and 13 C 6 - L-Arginine) amino acids. All the media components including dialyzed FBS, media supplements and amino acids were obtained from Invitrogen.
  • the cells were maintained at 37°C in a humidified 5% (v/v) CO2 incubator, and grown for 10 doublings. Once incorporated into cellular protein, the mass differences of heavy amino acids, which differed from unlabelled amino acids by 6-Da, were utilized to quantitatively compare samples.
  • LL-37 was biotinylated with a carboxy-terminal biotin tag as described previously. LL37 without biotin was used as a control.
  • RAW 264.7 cells (1 X 108 for each treatment) were washed with cold PBS and were lysed on ice for 30 min in TNE lysis buffer (10 mM Tris-HCl, pH 7.5, 150 mM NaCl, 2 mM EDTA) containing a protease inhibitor cocktail (Sigma- Aldrich) and 1% NP-40.
  • the RAW cell lysates from the cells labeled with heavy amino acids were treated with LL-37B (50 ⁇ g/ml) and the lysates from unlabeled cells were either untreated or treated with LL37 (50 ⁇ g/ml) to be used as paired controls, for 30 min at 37°C.
  • peptide-protein crosslinking was accomplished using 1% paraformaldehyde in PBS containing protease inhibitor cocktail for 15 min at 37°C, followed by quenching with 2M glycine for 5 min at 23°C.
  • the samples were diluted to a concentration of 1 M guanidinium hydrochloride by the addition of 20 mM ammonium bicarbonate prior to digestion.
  • the reduced and alkylated samples were digested for 16 hours with 2 ⁇ g of modified porcine trypsin (Promega) at 37°C.
  • Aliquots of the peptide mixtures (5 ⁇ g of total peptide) were subsequently acidified by dilution with an equal volume of 5% acetic acid.
  • the samples were then passed over a over a STop And Go Extraction stage tip50 and loaded onto tips in the acidified digest buffer, washed with 50 ⁇ L of 0.5% acetic acid and eluted with 20 ⁇ L of 80% acetonitrile in 0.5% acetic acid.
  • Samples were lyophilized in a centrifugal vacuum concentrator for 20 minutes to evaporate the acetonitrile and resuspended to a volume of 10 ⁇ L with 0.5% acetic acid. Samples were analyzed via nanoflow liquid chromatography coupled to tandem mass spectrometry. The analysis was accomplished using an LTQ Orbitrap (Thermo Fisher Scientific, Bremen, Germany) coupled to an Agilent 1100 Series nanoflow HPLC via a nanospray ionization source (Proxeon Biosystems, Odense, Denmark).
  • a reverse phase column consisting of ReproSil-Pur C18 resin packed into a fused silica emitter (15 cm x 75 ⁇ m internal diameter) was used for sample separation, and was developed with a gradient of 0.5% acetic acid (Buffer A) and 80% acetonitrile in 0.5% acetic acid (Buffer B). The gradient was run from 6% B to 30% B over 60 min, followed by 30% to 80% B over 10 min, 80% B for 5 min and finally from 80% B back to 6% B for 15 minutes to recondition the column. Full-range scans were collected from 350-1500 Th at 60,000 resolution and the five most intense ions in each cycle were selected for fragmentation in a data dependent manner.
  • the database search criteria chosen were trypsin cleavage allowing one missed cleavage, carbamidomethyl cysteine fixed modification, 13 C6-L-Lysine and 13 C6-L-Arginine as variable modifications, parent ion mass tolerance of 5 ppm, MS/MS mass tolerance of 0.6 Da and the ESI-TRAP scoring scheme. MSQuant was used for extracting quantitative information from the raw data for all of the proteins identified during the Mascot search.
  • Binding of the peptide was further evaluated using rabbit anti-LL-37 polyclonal, anti-biotin antibody or goat anti-rabbit HRP-conjugated antibodies.
  • LL-37-protein interaction was detected employing ImmunoPure ® TMB substrate (Pierce) by monitoring absorbance at 450 nm.
  • PMA-differentiated THP-I cells were treated with Dharmacon ON-Target/?/ra SMARTpool siRNA for human GAPDH (D-OO 1830-01-20, Fisher, Lafayette, CO), using a pool of four different designed siRNA against GAPDH, according to the manufacturer's instructions.
  • PMA-differentiated plastic adherent THP-I cells in the presence and absence of GAPDH siRNA were stimulated with either LL-37 (20 and 50 ⁇ g/ml), IDR-I (200 ⁇ g/ml), LPS (100 ng/ml), or LTA (2 ⁇ g/ml) for the indicated times.
  • Wild type (WT) and GAPDH-knockdown (KD) THP-I cells were stimulated with either LL-37 (20 or 50 ⁇ g/ml) or, as a paired control, bacterial LPS (100 ng/ml), for 4 hr.
  • RNA was isolated and analyzed for gene expression by qRT-PCR using Superscript III Platinum Two-Step qRT-PCR Kit with SYBR Green (Invitrogen), according to the manufacturer's instructions, in the ABI PRISM 7000 sequence detection system (Applied Biosystems). Fold changes were calculated using the comparative Ct method51, after normalization using 18S rRNA primers (Ambion). The list of primers employed is shown in Figure 14.
  • Wild type (WT) and GAPDH-knockdown (KD) THP-I cells were stimulated for 30 min with either LL-37 (50 ⁇ g/ml) or, as paired controls, bacterial the Toll-like receptor- ligands LPS (100 ng/ml) or LTA (2 ⁇ g/ml). The cells were detached using trypsin, and stained for p38 activation using an anti-phospho-P38 (Thrl80/Tyrl82) 3D7 rabbit monoclonal from Cell Signaling Technology (Danvers, MA), according to the manufacturer's protocol.
  • WT Wild type
  • KD GAPDH-knockdown
  • the cells were fixed in 2 % (w/v) paraformaldehyde for 15 min at 23 0 C, permeabilized in ice-cold 90 % (v/v) methanol for 20 min, washed twice in 0.5 %BSA in PBS (w/v), and stained for 1 hr at room temperature with the anti-phospho-P38 3D7 rabbit monoclonal antibody diluted 1/100 in 0.5 % BSA in PBS. The cells were further stained with goat-anti rabbit IgG AF647 (Invitrogen-Molecular Probes) at 2 ⁇ g/ml for 30 min at room temperature. The data was collected and analysed using BD FACSCalibur System and BD CellQuest Pro software (BD Biosciences). EXAMPLE 2:
  • FPRL-I is known to be a receptor for LL-37, mediating direct leukocyte chemotaxis.
  • PBMC peripheral blood mononuclear cells
  • WR W4 monocyte chemoattractant protein-1
  • MCP-I monocyte chemoattractant protein-1
  • LL-37-induced MCP-I production in PBMC was not suppressed by the P2X 7 inhibitor KN-62 (Fig. IB).
  • LL-37B C-terminally biotinylated LL-37
  • Inhibitors of cellular uptake/endocytic mobilization of LL-37 led to the significant (p ⁇ 0.05) inhibition of downstream chemokine MCP-I induction by LL-37 (Fig. 3), with the actin inhibitor cytochalasin D causing almost complete inhibition, and the tubulin inhibitor nocadazole causing 45% inhibition.
  • biotinylated LL- 37B was employed for SILAC and affinity tag pull-down experiments in the murine macrophage cell line RAW264.7 (ATCC TIB-71TM), in which cell LL-37 induces immunomodulatory and anti-endotoxin activities analagous to those observed in human monocytic cells.
  • the RAW264.7 cells were differentially labeled by growing the cells either with the heavy amino acids 13C6-L-Lys and 13 C 6 -L- Arg or corresponding unlabelled amino acids.
  • LL-37B peptide was used as a bait to selectively bind putative protein receptors from cell lysates labeled with heavy amino acids, while parallel lysates from unlabeled cells were either untreated or treated with LL-37 without biotin, to provide paired controls.
  • the mixed pull-down complexes were analyzed via nanoflow liquid chromatography coupled to tandem mass spectrometry.
  • Peptide binding assays demonstrating LL-37 -G ⁇ PDH interaction in vitro The LL-37-GAPDH interaction was monitored in-vitro using modified ELISA- based capture assays. Purified human GAPDH was immobilized on polystyrene plates as confirmed by probing with a rabbit anti-human GAPDH (Cedarlane Laboratories). The interaction of GAPDH with a broad range of LL-37B concentrations (0 - 50 ⁇ g/ml) was demonstrated using HRP-conjugated anti-biotin antibodies (Fig. 6A). In this assay the concentration of LL-37B giving half maximal binding (Kd) was 0.4 ⁇ g/ml, i.e. 0.1 ⁇ M.
  • chemokines e.g. CXCL- 1/Gro- ⁇
  • anti-inflammatory responses e.g. IL-IO
  • HH2 VQLRIRVAVIRA-NH 2
  • 1002 VQRWLIVWRIRK-NH 2
  • WT and GAPDH knockdown cells were also stimulated with bacterial LPS (100 ng/ml) for 4 hours.
  • LL-37 induces the phosphorylation of the MAP kinase p38 in human monocytes, and this is essential for LL-37-mediated chemokine production and other downstream responses.
  • LL-37-induced phosphorylation of p38 MAP kinase was compared in WT and GAPDH knock-down THP-I cells by flow cytometry to measure intracellular phosphorylation of p38 (at Tl 80/Yl 82) employing an anti-phospho-p38 rabbit monoclonal antibody 3D7. Knock-down of GAPDH completely abolished p38 phosphorylation in response to LL-37, while having no significant effect on responses to bacterial LPS or lipoteichoic acid (LTA) (Fig. 10).
  • HB activator proteins- 1 and -2 (AP-I and AP -2), cAMP response element-binding (CREB) protein, E2F1, early growth response factor (EGR), nuclear factor-kappa B (NF -KB), the zinc finger protein motif containing transcription factor SP-I, and the androgen and glucocorticod response elements ARE and GRE (data not shown).
  • TF activities that remained unchanged following LL-37 stimulation, as evaluated by the arrays, were RXR (retinoid X-receptor) and SIE (Sis- inducible element), and in contrast TFIID (transcription factor II D) activity was decreased after LL-37 treatment.
  • the transcription factor array analysis was consistent with the computational TFBS analysis in demonstrating the activity of AP-I, AP -2, E2F1, GRE, NF- KB and SPl (Fig. 11) on stimulation with LL-37 in human monocytic cells; and the activation of the nuclear receptors AR and GR as well as AP-I and NF -KB. Similarly almost identical results were obtained for IDR-I .
  • LL-37 is a modulator of apoptosis in epithelial cells and neutrophils and causes degranulation of mast cells whereas IDR-I does not.
  • Other LL-37 functions such as direct chemotaxis (which tends to involve diverse receptors for different cationic host defense peptides), angiogenesis and wound healing have not been described for IDR-I. We anticipate that this diversity of responses reflects the possibility of multiple receptors for LL-37 as suggested previously.
  • GAPDH While classically considered to be a glycolytic enzyme, mammalian GAPDH has been implicated in several non-metabolic processes including membrane transport, microtubule assembly, phosphotransferase/kinase activity and apoptosis (Sirover M. A., Biochimica Biophysica Acta 1432: 159-184, 1999).
  • GAPDH associates with the cell surface in both murine and human macrophages, and is a functional transferrin receptor involved in endosomal trafficking.
  • GAPDH was also shown to directly interact with the cytoplasmic domain of the macrophage scavenger receptor and therefore was hypothesized to be involved in macrophage scavenger receptor -related functions.
  • a key feature of host defense peptides in their activity as immunomodulators is an absolute requirement for uptake into cells. It has been shown previously that LL-37 is taken up into epithelial cells and dendritic cells, as also shown here for mononuclear cells. This likely reflects the similarity of host defense peptides to at least one class of so-called cell penetrating peptides that tend to carry strong cationic charge.
  • LL-37 has been demonstrated to be capable of carrying cargoes into cells (Sandgren S et al, J Biological Chemistry 279: 17951, 2004) while analogies have been drawn between viral nuclear localization signal proteins like HIV Tat and the cationic innate immunity modulating peptides (a subset of host defense peptides) (Kobayashi N et al, Antimicrobial Agents Chemotherapy 50: 1118, 2006. We propose therefore that the property of cell penetration is an essential requirement for immunomodulatory cationic peptides, but can not be sufficient to enable their immunomodulatory properties, as interaction with GAPDH is also required.

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Abstract

L'invention porte sur des procédés pour identifier des composés qui modulent l'immunité innée. Ces procédés sont basés sur la découverte que GAPDH est un partenaire de liaison directe dans des monocytes pour la cathélicidine LL-37 et les peptides immunomodulateurs cationiques IDR-1, et que cette liaison est requise pour le fonctionnement immunomodulateur inné desdits peptides, y compris l'induction de réponses transcriptionnelles des chimiokines et des cytokines et la signalisation en aval de p38. L'invention porte également sur des composés modulant l'immunité innée, sur des compositions pharmaceutiques comprenant ces composés et sur des méthodes de traitement utilisant ces composés.
PCT/IB2009/007001 2008-09-05 2009-09-02 Modulateurs d'immunité innée WO2010026489A1 (fr)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011156903A3 (fr) * 2010-06-15 2012-02-02 University Of Manitoba Compositions de peptide régulateur de l'immunité innée pour le traitement de l'arthrite
US20120177689A1 (en) * 2010-09-30 2012-07-12 Zhifang Zhang Generation of novel bone forming cells (monoosteophils) from ll-37 treated monocytes
WO2013034982A2 (fr) * 2011-09-09 2013-03-14 The University Of British Columbia Peptides immunomodulateurs utilisables en vue du traitement de maladies neurodégénératives évolutives
WO2015077888A1 (fr) * 2013-11-28 2015-06-04 University Of Manitoba Compositions de peptides idr et leur utilisation pour traiter des maladies inflammatoires avec dysrégulation des lymphocytes th2
EP3038638A1 (fr) * 2013-08-27 2016-07-06 The University Of British Columbia Peptides idr et anti-biofilm cationiques de petite taille
CN112695045A (zh) * 2021-01-26 2021-04-23 郑州轻工业大学 一种鳞翅目昆虫Hpx12基因及应用

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WO2003048383A2 (fr) * 2001-12-03 2003-06-12 University Of British Columbia Effecteurs d'immunite innee

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MOOKHERJEE, N. ET AL.: "Cationic host defence peptides: Innate immune regulatory peptides as a novel approach for treating infections", CELLULAR AND MOLECULAR LIFE SCIENCES., vol. 64, no. 7-8, April 2007 (2007-04-01), pages 922 - 933 *
MOOKHERJEE, N. ET AL.: "Intracellular Receptor for Human Host Defense Peptide LL-37 in Monocytes", THE JOURNAL OF IMMUNOLOGY, vol. 183, no. 4, 15 August 2009 (2009-08-15), pages 2688 - 2696 *
MOOKHERJEE, N. ET AL.: "Systems biology evaluation of immune responses induced by human host defence peptide LL-37 in mononuclear cells", MOLECULAR BIOSYSTEMS, vol. 5, no. 5, May 2009 (2009-05-01), pages 483 - 496 *
SCOTT, M. ET AL.: "An anti-infective peptide that selectively modulates the innate immune response", NATURE BIOTECHNOLOGY, vol. 25, no. 4, April 2007 (2007-04-01), pages 465 - 472 *
YU, J. ET AL.: "Host defense peptide LL-37, in synergy with inflammatory mediator IL-1beta, augments immune responses by multiple pathways", THE JOURNAL OF IMMUNOLOGY, vol. 179, no. 11, 1 December 2007 (2007-12-01), pages 7684 - 7691 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2011156903A3 (fr) * 2010-06-15 2012-02-02 University Of Manitoba Compositions de peptide régulateur de l'immunité innée pour le traitement de l'arthrite
US20120177689A1 (en) * 2010-09-30 2012-07-12 Zhifang Zhang Generation of novel bone forming cells (monoosteophils) from ll-37 treated monocytes
US8513013B2 (en) * 2010-09-30 2013-08-20 City Of Hope Generation of novel bone forming cells (monoosteophils) from LL-37 treated monocytes
WO2013034982A2 (fr) * 2011-09-09 2013-03-14 The University Of British Columbia Peptides immunomodulateurs utilisables en vue du traitement de maladies neurodégénératives évolutives
WO2013034982A3 (fr) * 2011-09-09 2013-05-30 The University Of British Columbia Peptides immunomodulateurs utilisables en vue du traitement de maladies neurodégénératives évolutives
EP3038638A1 (fr) * 2013-08-27 2016-07-06 The University Of British Columbia Peptides idr et anti-biofilm cationiques de petite taille
EP3038638A4 (fr) * 2013-08-27 2017-09-13 The University Of British Columbia Peptides idr et anti-biofilm cationiques de petite taille
WO2015077888A1 (fr) * 2013-11-28 2015-06-04 University Of Manitoba Compositions de peptides idr et leur utilisation pour traiter des maladies inflammatoires avec dysrégulation des lymphocytes th2
US20170158745A1 (en) * 2013-11-28 2017-06-08 University Of Manitoba Idr peptide compositions and use thereof for treatment of th2-dysregulated inflammatory conditions
CN112695045A (zh) * 2021-01-26 2021-04-23 郑州轻工业大学 一种鳞翅目昆虫Hpx12基因及应用
CN112695045B (zh) * 2021-01-26 2022-11-11 郑州轻工业大学 一种鳞翅目昆虫Hpx12基因及应用

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