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WO2005068497A1 - Iis: nouvel proteine inductible par ifn-gamma, element de la famille des secretoglobines, modulant l'invasion et la migration cellulaire - Google Patents

Iis: nouvel proteine inductible par ifn-gamma, element de la famille des secretoglobines, modulant l'invasion et la migration cellulaire Download PDF

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WO2005068497A1
WO2005068497A1 PCT/US2004/043717 US2004043717W WO2005068497A1 WO 2005068497 A1 WO2005068497 A1 WO 2005068497A1 US 2004043717 W US2004043717 W US 2004043717W WO 2005068497 A1 WO2005068497 A1 WO 2005068497A1
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iis
polypeptide
seq
residues
sequence
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Anil Mukherjee
Moonsuk S. Choi
Zhongjian Zhang
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US Department of Health and Human Services
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4718—Cytokine-induced proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4721—Lipocortins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to the discovery of IIS, a novel member of the secretoglobin protein family that is inducible by interferon- ⁇ and that has modulatory effects on cellular migration and invasion.
  • Interferons are cytokines that play a complex and central role in the resistance of mammalian hosts to pathogens.
  • Type II interferon or interferon- ⁇ IFN- ⁇
  • T thymus-derived cells under certain conditions of activation and by natural killer (NK) cells.
  • IFN- ⁇ The properties of IFN- ⁇ include the regulation of several aspects of the immune response, the stimulation of bactericidal activity of phagocytes, the stimulation of antigen presentation through class I and class II major histocompatability complex (MHC) molecules, the orchestration of leukocyte- endothelium interactions, and effects on cell proliferation and apoptosis (Boehm et al., 1997, Cellular Responses to Interferon- ⁇ , Anna. Rev. Immunol. 15:749-95).
  • MHC major histocompatability complex
  • uteroglobin the founding member of the secretoglobin (SCGB) superfamily of genes.
  • SCGB secretoglobin
  • the isolation and characterization of cDNAs encoding UG have been reported for the following organisms: the mouse (Ray et al., 1993, Cloning and characterization of the mouse Clara cell specific 10 kDa protein gene: comparison of the 5 '-flanking region with the human rat and rabbit gene, Biochem. Biophys. Res. Commun. 197:163), the pig (Gutierrez, et al., 1998, Cloning and sequencing of the cDNA coding for pig pre-uteroglobin/Clara cell 10 kDa protein,
  • paralogous proteins in different mammalian species include the rat prostatic binding protein (Heyns et al., 1977, Prostatic binding protein: a steroid-binding protein secreted by rat prostate, Eur. J. Biochem. 78:221) also known as "Prostatein” (Lea et al., 1979, Prostatein: a major secretory protein of the rat ventral prostate, J. Biol. Chem. 254:6196) or prostate-alpha protein (Chen et al., 1982, Prostate alpha-protein: isolation and characterization of the polypeptide components and cholesterol binding. J. Biol. Chem. 257:116).
  • rat prostatic binding protein Heyns et al., 1977, Prostatic binding protein: a steroid-binding protein secreted by rat prostate, Eur. J. Biochem. 78:2211
  • Prostatein Lea et al., 1979, Prostatein: a major secretory protein of the
  • Prostatein forms oligomers among its subunits C1/C3 and C2/C3 and has been reported to inhibit microtubule assembly (Heyns et al., 1978, Purification and characterization of prostatic binding protein and its subunits, Eur. J. Biochem. 89: 181). Several homologous proteins to rat prostatein have been described in other species.
  • the rat prostatein C3 shows 34 and 41% sequence identity with human lacryglobin (Molloy et al., 1997, Establishment of the human reflex tear two-dimensional polyacrylamide gel electrophoresis reference map: new proteins of potential diagnostic value, Electrophoresis 18:2811) and mammaglobin (MGB1) (Watson et al., 1996, Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer, Cancer Res. 56:860), respectively.
  • lacryglobin has been reported to be identical to mammaglobin 2 (MGB2) (Zhao et al., 1999, Lipophilins: human peptides homologous to rat prostatein, Biochem. Biophys. Res. Commun. 256:147) and lipophilin C (LIP-C) (Lehrer et al., 1988, Lipophilin, a novel heterodimeric protein of human tears, FEBSLett. 432:163).
  • MGB2 mammaglobin 2
  • LIP-C lipophilin C
  • UG has been recognized as a multifunctional protein with potent anti- inflammatory, anti-chemotactic and tumor suppressor-like activities (Mukherjee et al., 1999, Uteroglobin: a novel cytokine?, Cell. Mol. Life Sci. 55:771). MGB1 is reported to be expressed at high levels in mammary tumors (Watson et al., 1999,
  • the present invention relates to the identification and characterization of a novel member of secretoglobin (SCGB) family and to the discovery that this novel protein is inducible by interferon- ⁇ .
  • SCGB secretoglobin
  • This protein was named interferon- ⁇ -inducible SCGB ("IIS").
  • IIS interferon- ⁇ -inducible SCGB
  • the amino acid sequence of the IIS protein is shown in SEQ ID NO:l.
  • Trp Trp Lys 83 The protein is synthesized as precursor protein with a signal sequence (amino acid residues 1-21 of SEQ ID NO:l) contiguous with the amino residues of the mature IIS polypeptide (amino acid residues 22-83 of SEQ ID NO:l).
  • the sequence of the cDNA encoding the IIS protein is shown in SEQ ID NO:2.
  • the precursor IIS polypeptide is encoded by residues 22-270 of SEQ ID NO:2.
  • the mature IIS protein is encoded by residues 85-270 of SEQ ID NO:2.
  • the IIS protein bears 30% amino acid sequence identity with UG, a multifunctional protein with potent anti-inflammatory / immunomodulatory properties.
  • the expression of IIS-mRNA was found to be markedly elevated in activated CD8 + T cells and CD19 + B cells. Additionally, it was found that treatment of lymphoblast cells with an IIS antisense-s-oligonucleotide markedly inhibited cellular migration and cellular invasion, thus demonstrating an immunomodulatory function for the IIS protein.
  • the invention relates to an isolated IIS polypeptide selected from the group consisting of: (a) a polypeptide comprising the sequence of IIS (SEQ ID NO: 1) residues 22 through 83; (b) a polypeptide comprising an amino acid sequence at least 90% identical to the amino acid sequence of (IIS) (SEQ ID NO:l) residues 22 through 83; (c) a polypeptide comprising a biologically active fragment of IIS (SEQ ID NO:l) residues 22 through 83; and (d) a polypeptide comprising an immunologically active fragment of IIS (SEQ DDNO.I).
  • the invention relates to an isolated antibody that is specific for the IIS polypeptide, including, for example, antibodies that are specific the mature IIS polypeptide (residues 22 through 83 of SEQ ID NO:l)or the precursor IIS polypeptide (SEQ ID NO:l).
  • the invention relates to an isolated US polypeptide-encoding nucleic acid molecule selected from the group consisting of: (a) a nucleic acid molecule consisting essentially of a polynucleotide that encodes an IIS polypeptide having the sequence of IIS (SEQ DD NO:l) resiudes 22 through 83; (b) a nucleic acid molecule consisting essentially of a polynucleotide that encodes an IIS polypeptide having a sequence at least 90% identical to that of IIS (SEQ ID NO: 1) residues 22 through 83 ; and (c) a nucleic acid molecule consisting essentially of a polynucleotide that encodes a biologically active fragment of IIS (SEQ D3 NO:l) residues 22 through 83.
  • the invention in another aspect, relates to a method of inhibiting cellular migration or cellular invasion in a cell population comprising administering an IIS inhibitor to the cell population, wherein the IIS inhibitor is selected from the group consisting of: (a) an antibody selective for IIS (SEQ ID NO:l); and (b) an anti-sense nucleic acid molecule that inhibits the expression of the IIS polypeptide in the cell population.
  • the IIS inhibitor is selected from the group consisting of: (a) an antibody selective for IIS (SEQ ID NO:l); and (b) an anti-sense nucleic acid molecule that inhibits the expression of the IIS polypeptide in the cell population.
  • the invention in another aspect, relates to a method of stimulating cellular migration or invasion in a cell population comprising administering an IIS polypeptide or an IIS nucleic acid to the cell population.
  • the invention in another aspect, relates to a method for screening for agents that modulate the effects of interferon- ⁇ comprising the steps of: (a) exposing a cell population to interferon- ⁇ in the presence of a test agent; (b) determining the effects of the test agent on the interferon- ⁇ induction of IIS expression in the cell population; and (c) identifying the test agent as a modulator of the effects of interferon- ⁇ if the induction of expression of IIS in the cell population was increased or decreased.
  • Figure 1 shows characteristics of the interferon- ⁇ -inducible (IIS) gene and gene expression.
  • Panel A shows the mRNA expression levels of secretoglobin
  • SCGB family members in human lymphoblast cells by semi-quantitative RT-PCR: lane 1 - uteroglobin (UG); lane 2 - lipophilin A (LIP- A); lane 3 - mammaglobin 1 (MGB1); lane 4 - mammaglobin 2 (MGB2); lane 5 - lipophilin B (LIP-B); lane 6 - interferon- ⁇ -inducible SCGB (US).
  • Panel B shows the schematic structure of the human IIS gene with the exons shown by boxes and the introns shown by horizontal lines. The coding region of the mature protein is shown by solid boxes.
  • a short tandem repeat polymorphism (STRP) in intron 2 and a single nucleotide polymorphism (SNP) in the 3' region of the gene are indicated by arrows in Panel B.
  • Panel C shows the exon-intron boundaries of the human IIS gene. The consensus nucleotides of exon-intron boundaries are in boldface.
  • Figure 2 shows polymorphisms of the IIS gene.
  • Panel A shows a single strand conformational polymorphism (SSCP) analysis of IIS genomic DNA. Each lane shows different SSCP pattern from normal individuals.
  • Panel B shows an SNP (G- ⁇ A substitution) in the 3 'region of the IIS gene, with three different genotypes shown: GG (left panel); GA (middle panel); and AA (right panel). Arrows indicate SNP sites.
  • SSCP single strand conformational polymorphism
  • Panel C shows STRPs of the IIS gene in intron 2 [c(t) 8 (SEQ ID NO:27), c(t) 9 (SEQ ID NO:28), or (cttt) 2 (ctttt) (SEQ D3 NO:29), with three different genotypes shown: c(t) 8 (SEQ ED NO:27) / c(t) 8 (SEQ H> NO:27) (left panel), c(t) 9 (SEQ ID NO:28) / c(t) 9 (SEQ D NO:28) (middle panel), and (cttt) 2 (ctttt) (SEQ ID NO:29) / c(t) 8 (SEQ DD NO:27) (right panel).
  • Figure 3 shows the mRNA expression levels of IIS in various human tissues and cell lines.
  • the mRNA expression levels were detected by RT-PCR followed by Southern hybridization.
  • the highest level of expression of IIS mRNA was detected in ovary, lymph node, tonsil, and lymphoblasts.
  • the amplification of glyceraldehyde-3- phosphate dehydrogenase (GAPDH) was used to normalize the total RNA added to each RT-PCR reaction.
  • GPDH glyceraldehyde-3- phosphate dehydrogenase
  • Figure 4 shows the expression of IIS protein in E. coli, COS-1 cells, and lymphoblasts.
  • Panel A shows an immunoblot analysis of recombinant IIS protein expressed in E. coli. IIS protein was detected with an S-Tag antibody (left panel) and an anti-US antibody (right panel) following purification using Ni 2+ resin. Lane 1 contained a lysate from cells transfected with vector alone, and lane 2 contained a lysate from cells transfected with a vector containing IIS coding sequence.
  • Panel B shows an immunoblot analysis of IIS expression in COS-1 cells.
  • Cell lysate from the culture of COS-1 cells expressing His-Tagged IIS protein was resolved by SDS- PAGE under reducing (R) and non-reducing (NR) conditions, respectively, and the immunoblotted with anti-X-press antibody.
  • Lane 1 lysate of cells transfected with vector only;
  • lane 2 lysate of cells transfected with plasmid containing IIS-cDNA encoding the mature polypeptide sequence without the leader peptide);
  • lane 3 the lysate from cells transfected with plasmid containing full length IIS cDNA coding sequence.
  • Panel C shows an immunoblot analysis of US-protein expression in lymphoblasts.
  • Figure 5 shows the induction of IIS mRNA expression in cultured lymphoblast cells.
  • Panel A shows the induction of IIS expression by the cytokines TNF- ⁇ , IFN- ⁇ , IL-4, IL-13 for the indicated treatment times.
  • Panel B shows the induction of IIS expression by varying doses of IFN- ⁇ as indicated.
  • the levels of expression are represented as fold induction compared with the expression level in control cells.
  • the experiments were repeated at least three times and the results are expressed as the mean of fold induction + the standard deviation of the mean of three independent experiments. Asterisks indicate significant differences (p ⁇ 0.05).
  • Figure 6 shows the expression levels of IIS mRNA in different types of white blood cells.
  • Panel A shows the expression level in resting cells of each cell type designated; the levels of expression are represented as fold induction compared with that of lymphoblasts.
  • Panel B shows a comparison of the IIS mRNA expression levels between activated WBCs (black bars) and their resting counterparts (gray bars). The results are expressed as the mean of three independent experiments + standard deviation of the mean. Asterisks indicate significant differences (p ⁇ 0.05).
  • Figure 7 shows the effect of IIS antisense s-oligonucleotide treatment of lymphoblasts on cellular migration and invasion.
  • Panel A shows the results of cell migration assays and
  • Panel B shows the results of cell invasion assays.
  • the results are expressed as the total number of migrated or invaded cells per 0.005 mm 3 .
  • the results are expressed as the mean of three independent experiments; * ; the standard deviations of the means. Asterisks indicate significant differences (p ⁇ 0.05).
  • Figure 8 shows a comparison of the amino acid sequences of members of the human SCGB family of proteins and the SCGB phylogenetic tree.
  • Panel A shows the alignment of amino acid sequences of SCGB family members. Accession numbers and references for the sequences are as follows: IIS (AY236538; SEQ ED NO:41); UG (NM_003357; SEQ ED NO:42); YGB (Ni et al., 2000, Ann. N. Y. Acad. Sci.
  • the present invention relates to the discovery of a novel member of the secretoglobin family of proteins that is inducible by interferon- ⁇ and thus has been named Interferon- ⁇ -Inducible Secretoglobin ("IIS").
  • IIS Interferon- ⁇ -Inducible Secretoglobin
  • the amino acid sequence of IIS is set forth in SEQ ED NO:l, wherein amino acid residues 1-21 comprises the signal sequence and amino acid residues 22-83 comprise the mature IIS polypeptide.
  • the cDNA sequence of IIS is GenBank Accession No. AY236538, herein incorporated by reference.
  • the nucleotide sequence of the IIS cDNA is set forth in SED ID NO:2.
  • the present invention relates to all facets of the novel IIS protein and polynucleotides encoding the polypeptide, including antibodies and binding partners thereto, and all applications of the invention such as, for example, the diagnosis or treatment of disease, research, drug discovery, forensic science, etc.
  • isolated refers to material removed from its original environment (e.g., the natural environment if it is naturally occurring), and thus is altered “by the hand of man” from its natural state.
  • an isolated polynucleotide could be part of a vector or a composition of matter, or could be contained within a cell, and still be “isolated” because that vector, composition of matter, or particular cell is not the original environment of the polynucleotide.
  • isolated does not refer to whole genomic or cDNA libraries, whole cell total or mRNA preparations, genomic DNA preparations (including those separated by electrophoresis and transferred onto blots), sheared whole cell genomic DNA preparations or other compositions where the art demonstrates no distinguishing features of the polynucleotide sequences of the present invention.
  • oligonucleotide refers to a nucleic acid molecule of preferably from 10 to 50 nucleotides in length, preferably from 15 to 30 nucleotides in length, and more preferably about 20-25 nucleotides which can be used as a probe or primer.
  • polynucleotide refers to nucleic acid molecules of preferably from 50 to 500 nucleotides in length.
  • nucleic acid molecules may be DNA or RNA, and may be of genomic or synthetic origin. Such molecules may be single- or double-stranded, and represent the sense or antisense strand of nucleic acid molecules that encode the US polypeptides of the present invention.
  • polypeptides and proteins of the present invention can be composed of amino acids joined to each other by peptide bonds or modified peptide bonds, i.e., peptide isoteres, and may contain amino acids other than the 20 gene-encoded amino acids.
  • the polypeptides may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques. Both post- translational modifications and chemical modification techniques are well described in the art. Modifications can occur anywhere in a polypeptide, including the peptide backbone, the amino acid side-chains and the amino or carboxyl termini. The same type of modification may be present in the same or varying degrees at several sites in a given polypeptide, and a given polypeptide may contain many types of modifications.
  • Polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic polypeptides may result from posttranslational natural processes or may be made by synthetic methods.
  • Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross-linking, cyclization, disulfide bond formation, demethylation, formation of covalent crosslinks, formation of cysteine, formation of pyroglutamate, formylation, gamma- carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
  • polypeptides of the invention can be prepared in any suitable manner.
  • Such polypeptides include isolated naturally occurring polypeptides, recombinantly produced polypeptides, synthetically produced polypeptides, or polypeptides produced by a combination of these methods. Means for preparing such polypeptides are well understood in the art.
  • the present invention also encompasses IIS polypeptide variants and nucleic acid molecules encoding those variants.
  • a preferred IIS polypeptide variant is one having at least 80% amino acid sequence similarity to the amino acid sequence of the mature IIS polypeptide (SEQ ID NO:l; amino acid residues 22-83), a more preferred IIS polypeptide variant is one having at least 90% amino acid sequence similarity to the mature IIS polypeptide and a most preferred IIS polypeptide variant is one having at least 95% amino acid sequence similarity to the mature IIS polypeptide.
  • a preferred method for determining the best overall match between a query sequence (a sequence of the present invention) and a subject sequence can be determined using the FASTDB computer program based on the algorithm of Brutlag et al., 1990, Comp. App. Biosci.6:237 '-245.
  • a sequence alignment the query and subject sequences are both amino acid sequences.
  • the result of said global sequence alignment is in percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matchedaligned with a corresponding subject residue, as a percent of the total residues of the query sequence. Whether a residue is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of the present invention.
  • a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N- terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
  • a 90 residue subject sequence is compared with a 100 residue query sequence.
  • deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query.
  • percent identity calculated by FASTDB is not manually corrected.
  • residue positions outside the N- and C-terminal ends of the subject sequence, as displayed in the FASTDB alignment, which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are to be made for the purposes of the present invention.
  • biologically active refers to an IIS polypeptide fragment or an IIS variant polypeptide having one at least one of the activities that have been identified for the IIS polypeptide.
  • IIS expression inhibition studies described herein it has been demonstrated that the IIS polypeptide of has activity in modulating cellular migration or invasion.
  • the biological activity of any IIS variant polypeptide or any IIS polypeptide fragment in modulating cellular invasion or cellular migration activity can be assessed by techniques that are well known in the art, including those techniques that are referenced in Example 7 herein.
  • immunologically active defines the capability of an IIS polypeptide fragment or an IIS variant polypeptide of the invention to induce a specific immune response to the polypeptide of SEQ ED NO:l or to selectively bind with an antibody, or a derivative thereof, that is specific for the polypeptide of SEQ ID NO:l.
  • the present invention also relates to fragments of the polypeptide of SEQ D3
  • Such a fragment comprises a portion of a polypeptide having the amino acid sequence of SEQ ID NO:l.
  • a fragment may be "free-standing", or contained within or contiguous with another polypeptide or protein.
  • Polypeptide fragments of the invention may be at least about 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, or 80 amino acids in length. In this context "about” includes the particularly recited ranges or values, or ranges or values larger or smaller by several (5, 4, 3, 2, or 1) amino acids, at either terminus or at both termini.
  • any polynucleotide that encodes the amino acid sequence of IIS, or a variant thereof, can be used to generate recombinant molecules that express IIS.
  • an "allele” or “allelic sequence” is an alternative form of an IIS nucleotide sequence. Alleles result from a mutation, i.e., a change in the nucleotide sequence, and generally produce altered niRNAs or polypeptides whose structure or function may or may not be altered. Any given gene may have none, one or many allelic forms. Common mutational changes that give rise to alleles are generally ascribed to natural deletions, additions or substitutions of amino acids. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • Methods for DNA sequencing are well known in the art and employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical Corp, Cleveland Ohio)), Taq polymerase (Perkin Elmer, Norwalk Conn.), thermostable T7 polymerase (Amersham, Chicago 111.), or combinations of recombinant polymerases and proofreading exonucleases such as the amplification system marketed by (Gibco BRL, Gaithersburg Md.).
  • the process is automated with machines such as the MICROLAB 2200 (Hamilton, Reno Nev.), Peltier thermal cycler (PTC200; MJ Research, Watertown Mass.), and ABI 377 DNA sequencers (Perkin Elmer).
  • a preferred embodiment of the invention is a nucleic acid consisting essentially of a polynucleotide that encodes the polypeptide of SEQ ID NO:l, or a variant or a biologically active fragment of the polypeptide of SEQ ED NO:l. Consisting essentially of, as used in this context, refers to a polynucleotide as described in the preceding sentence, wherein the polynucleotide contains less than fifteen contiguous nucleotide residues that correspond to the naturally occurring polynucleotide residues at both the 5' or the 3' end of the coding sequence for the specified polypeptide.
  • nucleic acid consisting essentially of a polynucleotide that encodes the polypeptide of amino acid residues of 22-83 of SEQ ED NO:l. Consisting essentially of, as used in this context, refers to a polynucleotide as described in the preceding sentence, wherein the polynucleotide contains less than fifteen contiguous nucleotide residues that correspond to the naturally occurring polynucleotide residues at both the 5' or the 3' end of the coding sequence for the specified polypeptide.
  • nucleotide sequences of the invention may be covalently linked to or contiguous with other nucleic acids such as, for example, heterologous regulatory nucleotide sequences or nucleotide sequences that encode signal polypeptide peptides that direct secretion of the IIS polypeptide from the cell.
  • heterologous regulatory nucleotide sequences or nucleotide sequences that encode signal polypeptide peptides that direct secretion of the IIS polypeptide from the cell may be covalently linked to or contiguous with other nucleic acids such as, for example, heterologous regulatory nucleotide sequences or nucleotide sequences that encode signal polypeptide peptides that direct secretion of the IIS polypeptide from the cell.
  • polynucleotide molecules that encode IIS, fragments of the polypeptide, fusion proteins or functional equivalents thereof may be used in recombinant DNA molecules that direct the expression of IIS in appropriate host cells. Due to the inherent degeneracy of the genetic code, other nucleic acid molecules, which encode substantially the same, or a functionally equivalent amino acid sequence, may be used to clone and express IIS. As will be understood by those of skill in the art, it may be advantageous to produce IIS- encoding polynucleotides possessing non-naturally occurring codons. Codons preferred by a particular prokaryotic or eukaryotic host (Murray E et al (1989) Nucl. Acids Res 17:477-508) can be selected, for example, to increase the rate of IIS expression or to produce recombinant RNA transcripts having desirable properties, such as a longer half-life, than transcripts produced from naturally occurring sequence.
  • polynucleotide molecules of the present invention can be engineered in order to alter an IIS encoding sequence for a variety of reasons, including but not limited to, alterations which modify the cloning, processing and/or expression of the gene product.
  • mutations may be introduced using techniques which are well known in the art, e.g., site-directed mutagenesis to insert new restriction sites, to alter glycosylation patterns, to change codon preference, to produce splice variants, etc.
  • a natural, modified or recombinant polynucleotide encoding IIS may be ligated to a heterologous polynucleotide or oligonucleotide to encode a fusion protein.
  • a heterologous polynucleotide or oligonucleotide to encode a fusion protein.
  • a fusion protein may also be engineered to contain a cleavage site located between an IIS polypeptide and the heterologous polynucleotide, so that the IIS polypeptide may be cleaved and purified away from the heterologous moiety.
  • a polynucleotide encoding an IIS polypeptide is inserted into an appropriate expression vector, i.e., a vector that contains the necessary elements for the transcription and translation of the inserted US-encoding sequence.
  • a variety of expression vector/host systems may be utilized to contain and express an IIS polypeptide. These include but are not limited to microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMN; tobacco mosaic virus, TMV) or transformed with bacterial expression vectors (e.g., Ti or pBR322 plasmid); or animal cell systems.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect cell systems infected with virus expression vectors (e.g., baculovirus); plant cell systems transfected with virus expression vectors (e.g., cauliflower mosaic virus, CaMN; tobacco mosaic virus, T
  • regulatory nucleotide sequences of these systems vary in their strength and specificities and are those untranslated regions of the vector, enhancers, promoters, and 3' untranslated regions, which interact with host cellular proteins to carry out transcription and translation.
  • the gene may be associated with its own regulatory nucleotide sequence (i.e. the endogenous promoter), or the gene may be associated with a regulator nucleic sequence derived from a different gene or organism, herein defined as a “heterologous regulatory nucleotide sequence”.
  • any number of suitable transcription and translation elements including constitutive and inducible promoters, may be used.
  • inducible promoters such as the hybrid lacZ promoter of the BLUESCRIPT phagemid (Stratagene, LaJolla, Calif.) or PSPORT1 (Gibco BRL) and ptrp-lac hybrids and the like may be used.
  • the baculovirus polyhedrin promoter may be used in insect cells.
  • Promoters or enhancers derived from the genomes of plant cells e.g., heat shock, RUBISCO; and storage protein genes
  • plant viruses e.g., viral promoters or leader sequences
  • promoters from the mammalian genes or from mammalian viruses are most appropriate. If it is necessary to generate a cell line that contains multiple copies of IIS, vectors based on SV40 or EBV may be used with an appropriate selectable marker.
  • a number of expression vectors may be selected depending upon the use intended for IIS. For example, when large quantities of IIS polypeptides are needed for the induction of antibodies, vectors that direct high-level expression of fusion proteins that are readily purified may be desirable.
  • Such vectors include, but are not limited to, the multifunctional E. coli cloning and expression vectors such as BLUESCRIPT (Stratagene), in which the IIS coding sequence may be ligated into the vector in frame with sequences for the amino-terminal Met and the subsequent 7 residues of ⁇ -galactosidase so that a hybrid protein is produced; pIN vectors (Van Heeke & Schuster (1989) J Biol. Chem.
  • PGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • GST glutathione S-transferase
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption to glutathione-agarose beads followed by elution in the presence of free glutathione.
  • Proteins made in such systems are designed to include heparin, thrombin or factor XA protease cleavage sites so that the cloned polypeptide of interest can be released from the GST moiety at will.
  • yeast Saccharomyces cerevisiae
  • a number of vectors containing constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH may be used.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase and PGH.
  • the expression of a nucleic acid molecule encoding an IIS polypeptide may be driven by any of a number of promoters.
  • viral promoters such as the 35S and 19S promoters of CaMN (Brisson et al (1984) Nature 310:511-514) may be used alone or in combination with the omega leader sequence from TMV (Takamatsu et al (1987) EMBO J. 3:17-311).
  • plant promoters such as the small subunit of RUBISCO (Coruzzi et al (1984) EMBO J.
  • Autographica californica nuclear polyhedrosis virus (AcNPN) is used as a vector to express foreign genes in Spodoptera frugiperda cells or in Trichoplusia larvae.
  • the IIS polypeptide encoding sequence may be cloned into a nonessential region of the virus, such as the polyhedrin gene, and placed under control of the polyhedrin promoter. Successful insertion of US will render the polyhedrin gene inactive and produce recombinant virus lacking coat protein coat.
  • the recombinant viruses are then used to infect S. frugiperda cells or Trichoplusia larvae in which IIS is expressed (Smith et al (1983) J. Virol. 46:584; Engelhard E K et al (1994) Proc. Natl. Acad. Sci. 91 :3224-7).
  • an IIS polypeptide encoding sequence may be ligated into an adenovirus transcription/translation complex consisting of the late promoter and tripartite leader sequence. Insertion in a nonessential El or E3 region of the viral genome will result in a viable virus capable of expressing IIS in infected host cells (Logan and Shenk (1984) Proc. Natl. Acad. Sci. 81:3655-59).
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • RSV Rous sarcoma virus
  • IIS polypeptide encoding sequence These signals include the ATG initiation codon and adjacent sequences. In cases where IIS, its initiation codon and upstream sequences are inserted into the appropriate expression vector, no additional translational control signals may be needed. However, in cases where only coding sequence, or a portion thereof, is inserted, exogenous transcriptional control signals including the ATG initiation codon must be provided. Furthermore, the initiation codon must be in the correct reading frame to ensure transcription of the entire insert. Exogenous transcriptional elements and initiation codons can be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate to the cell system in use (Scharf D et al (1994) Results Probl. Cell Differ. 20:125-62; Bittner et al (1987) Methods in Enzymol 153:516-544).
  • a host cell strain may be chosen for its ability to modulate the expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, lipidation and acylation.
  • Post-translational processing which cleaves a "prepro" form of the protein, may also be important for correct insertion, folding and or function.
  • Different host cells such as CHO, HeLa, MDCK, 293, WI38, etc have specific cellular machinery and characteristic mechanisms for such post-translational activities and may be chosen to ensure the correct modification and processing of the introduced, foreign protein.
  • cell lines which may be used to stably express IIS, may be transformed using expression vectors containing viral origins of replication or endogenous expression elements and a selectable marker gene.
  • cells may be allowed to grow for 1-2 days in an enriched media before they are switched to selective media.
  • the purpose of the selectable marker is to confer resistance to selection, and its presence allows growth and recovery of cells, which successfully express the introduced polynucleotides or oligonucleotides. Resistant clumps of stably transformed cells can be proliferated using tissue culture techniques appropriate to the cell type.
  • Any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase (Wigler M et al (1977) Cell 11:223-32) and adenine phosphoribosyltransferase (Lowy I et al (1980) Cell 22:817-23) genes, which can be employed in tk- or aprt-cells, respectively.
  • antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection; for example, dhfr, which confers resistance to methotrexate (Wigler M et al (1980) Proc Natl Acad Sci 77:3567-70); npt, which confers resistance to the aminoglycosides neomycin and G-418 (Colbere-Garapin F et al (1981) JMol Biol 150: 1-14); and als or pat, which confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively (Murry, supra).
  • marker gene expression suggests that the gene of interest is also present, its presence and expression should be confirmed.
  • the IIS is inserted within polynucleotide that encodes a marker gene
  • recombinant cells containing IIS can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with an IIS- polypeptide encoding polynucleotide under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem IIS as well.
  • host cells which contain an US-polypeptide encoding polynucleotide and which express IIS, may 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 hybridization and protein bioassay or immunoassay techniques, which include membrane, solution, or chip, based technologies for the detection and/or quantification of the nucleic acid or protein.
  • the presence of a polynucleotide encoding an IIS polypeptide can be detected by DNA-DNA or DNA-RNA hybridization or amplification using probes, portions or fragments of polynucleotides encoding IIS.
  • Nucleic acid amplification based assays involve the use of oligonucleotides or oligomers based on the sequence of US polypeptide-encoding polynucleotide to detect transformants containing DNA or RNA encoding IIS.
  • a variety of protocols for detecting and measuring the expression of IIS, using either polyclonal or monoclonal antibodies specific for the protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non- interfering epitopes on IIS is preferred, but a competitive binding assay may be employed. These and other assays are described, among other places, in Hampton R et al (1990, Serological Methods, a Laboratory Manual, APS Press, St Paul Minn.) and Maddox D E et al (1983, JExp Med 158:1211).
  • Means for producing labeled hybridization or PCR probes for detecting nucleic acid molecules that are related to US-encoding polynucleotides include oligolabeling, nick translation, end-labeling or PCR amplification using a labeled nucleotide.
  • an IIS polypeptide-encoding polynucleotide, or any portion of it may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commercially available, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as 17, T3 or SP6 and labeled nucleotides.
  • reporter molecules or labels include those radionuclides, enzymes, fluorescent, chemiluminescent, or chromogenic agents as well as substrates, cofactors, inhibitors, magnetic particles and the like. Patents teaching the use of such labels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149 and 4,366,241. Also, recombinant immunoglobulins may be produced as shown in U.S. Pat. No.4,816,567, incorporated herein by reference. Purification of IIS
  • Host cells transformed with an IIS polypeptide-encoding polynucleotide may be cultured under conditions suitable for the expression and recovery of the encoded protein from cell culture.
  • the protein produced by a recombinant cell may be secreted or contained intracellularly depending on the polynucleotide and/or the vector used.
  • expression vectors containing polynucleotides encoding IIS can be designed to encode signal sequences that direct secretion of IIS through a prokaryotic or eukaryotic cell membrane.
  • secretory signal sequence refers to a signal sequence as described in the above sentence .
  • IIS may also be expressed as a recombinant protein with one or more additional polypeptide domains added to facilitate protein purification.
  • purification facilitating domains include, but are not limited to, metal chelating peptides such as 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 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 an oligonucleotide that encodes a cleavable linker such as Factor XA or enterokinase (Invitrogen, San Diego CA), etc.
  • One such expression vector provides for expression of a fusion protein compromising an IIS and contains nucleic acid encoding 6 histidine residues followed by thioredoxin and an enterokinase cleavage site.
  • the histidine residues facilitate purification on IMIAC (immobilized metal ion affinity chromatography as described in Porath et al (1992) Protein Expression and Purification 3: 263-281) while the enterokinase cleavage site provides a means for purifying IIS from the fusion protein.
  • fragments of IIS may be produced by direct peptide synthesis using the solid-phase techniques (cf Stewart et al (1969) Solid-Phase Peptide Synthesis, W H Freeman Co, San Francisco; Merrifield J (1963) J Am Chem Soc 85:2149-2154).
  • In vitro protein synthesis may be performed using manual techniques or by automation. Automated synthesis may be achieved, for example, using the ABI 431 A peptide synthesizer (Perkin Elmer) in accordance with the instructions provided by the manufacturer. Narious fragments of IIS may be chemically synthesized separately and combined using chemical methods to produce the full length molecule.
  • anti-IIS antibodies are useful for the diagnosis of conditions and diseases associated with expression of IIS.
  • Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, single chain, Fab fragments and fragments produced by a Fab expression library.
  • Neutralizing antibodies i.e., those that inhibit dimer formation, are especially preferred for diagnostics and therapeutics.
  • IIS for antibody induction does not require biological activity; however, the protein fragment, or oligopeptide must be antigenic.
  • Peptides used to induce specific antibodies preferably comprise at least 5 amino acids in length, more preferably at least 10 amino acids in length.
  • such molecules should mimic the two- dimensional sequence or three-dimensional conformation of an IIS polypeptide and may contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of IIS amino acids may be fused with those of another protein such as keyhole limpet hemocyanin and antibody produced against the chimeric molecule. Procedures well known in the art can be used for the production of antibodies to US.
  • various hosts including goats, rabbits, rats, mice, etc may be immunized by injection with IIS or any portion, fragment or oligopeptide that retains immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanin, and dinitrophenol.
  • BCG Bacilli Calmette- Guerin
  • Corynebacterium parvum are potentially useful human adjuvants.
  • Monoclonal antibodies to IIS may be prepared using any technique that provides for the production of antibody molecules by continuous cell lines in culture. These include but are not limited to the hybridism technique originally described by Koehler and Millstone (1975 Nature 256:495-497), the human B-cell hybridism technique (Osborn et al (1983) Immunol Today 4:72; Cote et al (1983) Proc. Natl. Acad. Sic. 80:2026-2030) and the EBV-hybridism technique (Cole et al (1985) Monoclonal Antibodies and Cancer Therapy, Alan R Less Inc, New York N.Y., pp 77-96).
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening recombinant immunoglobulin libraries or panels of highly specific binding reagents as disclosed in Oriandi et al (1989, Proc Natl Acad Sci 86: 3833-3837), and Winter G and Milstein C (1991; Nature 349:293-299).
  • Antibody fragments that contain specific binding sites for IIS may also be generated.
  • fragments include, but are not limited to, the F(ab) 2 fragments which can be produced by pepsin digestion of the antibody molecule and the Fab fragments which can be generated by reducing the disulfide bridges of the
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse W D et al (1989) Science 256:1275-1281).
  • a variety of protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art. Such immunoassays typically involve the formation of complexes between US and its specific antibody and the measurement of complex formation.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two noninterfering epitopes on a specific IIS protein is preferred, but a competitive binding assay may also be employed. These assays are described in Maddox D E et al (1983, JExpMed 158:1211).
  • IIS antibodies are useful for the diagnosis of conditions or diseases characterized by expression of IIS or in assays to monitor patients being treated with IIS, agonists or inhibitors. Diagnostic assays for US include methods utilizing the antibody and a label to detect IIS in human body fluids or extracts of cells or tissues.
  • the polypeptides and antibodies of the present invention ma be used with or without modification. Frequently, the polypeptides and antibodies will be labeled by joining them, either covalently or noncovalently, with a reporter molecule. A wide variety of reporter molecules are known, several of which were described above.
  • a variety of protocols for measuring IIS, using either polyclonal or monoclonal antibodies specific for the respective protein are known in the art. Examples include enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and fluorescent activated cell sorting (FACS).
  • ELISA enzyme-linked immunosorbent assay
  • RIA radioimmunoassay
  • FACS fluorescent activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on IIS is preferred, but a competitive binding assay may be employed. These assays are described, among other places, in Maddox, D E et al (1983, J Exp Med 158:1211). In order to provide a basis for diagnosis, normal or standard values for IIS expression must be established.
  • the amount of standard complex formation may be quantified by comparing various artificial membranes containing known quantities of IIS with both control and disease samples from biopsied tissues. Then, standard values obtained from normal samples may be compared with values obtained from samples from subjects potentially affected by disease. Deviation between standard and subject values establishes the presence of disease state.
  • IIS and its biologically active or immunologically active fragments thereof, can be used for screening therapeutic compounds in any of a variety of drug screening techniques.
  • the fragment employed in such a test may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes, between IIS and the agent being tested, may be measured.
  • Another technique for drug screening which may be used provides for high throughput screening of compounds having suitable binding affinity to the IIS is described in detail in "Determination of Amino Acid Sequence Antigenicity" by Geysen H N, WO Application 84/03564.
  • large numbers of different small peptide test compounds are synthesized on a solid substrate, such as plastic pins or some other surface.
  • the peptide test compounds are reacted with fragments of IIS and washed. Bound US is then detected by methods well known in the art.
  • Purified IIS can also be coated directly onto plates for use in the aforementioned drug screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the present invention also contemplates the use of competitive drug screening assays in which neutralizing antibodies capable of binding IIS specifically compete with a test compound for binding IIS. In this manner, the antibodies can be used to detect the presence of any peptide that shares one or more antigenic determinants with IIS.
  • the invention relates to methods of screening for agents that modulate the effects of interferon- ⁇ -on a cell population.
  • the method may comprise the steps of (i) exposing a cell population to interferon- ⁇ in the presence of a test agent; (ii) determining the effects of the test agent on interferon- ⁇ induction of IIS expression in the cell population; (iii) identifying the test agent as a modulator of the effects of interferon- ⁇ if the induction of expression of IIS in the cell population was increased or decreased.
  • the cell population comprises cells of the immune system such as, for example, lymphoblasts or lymphocytes.
  • the polynucleotides, polypeptides and antibodies of the invention are useful for the treatment of disease that could benefit from the inhibition or the stimulation of cellular migration or invasion.
  • the modulation of cellular invasion or migration of cells of the immune system including, for example, lymphoblasts or lymphocytes, is contemplated.
  • the modulation of cellular invasion or migration is contemplated both in vitro, ex vivo, and in vivo.
  • Ex vivo refers to the technique wherein a subject's cells are removed from the subject and manipulated in vitro for therapeutic purpose, and then returned to the subject.
  • a subject's lymphoblasts or lymphocytes could be removed and then manipulated ex vivo using the methods and compositions of the invention before the lymphoblasts or lymphocytes are returned to the subject's body for therapeutic purpose.
  • the ex vivo manipulation of lymphocytes has applications in, for example, bone marrow transplants and in techniques for boosting the immune system's ability to responds to infectious organisms, including but not limited to viruses, bacteria and fungi.
  • Polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing diseases, disorders, and or conditions of the immune system, by, for example, activating or inhibiting the proliferation, differentiation, or mobilization (chemotaxis) of immune cells.
  • Immune cells develop through a process called hematopoiesis, producing myeloid (platelets, red blood cells, neutrophils, and macrophages) and lymphoid (B and T lymphocytes) cells from pluripotent stem cells.
  • Polynucleotides, polypeptides, antibodies, and or agonists or antagonists of the present invention may be used to inhibit or enhance an immune response generated by cells associated with the tissue(s) in which the HS gene of the present invention is of the invention is expressed, including but not limited to ovary, lymph node, tonsil, small intestine, colon, bone marrow, and fetal liver.
  • the discoveries of the present invention may be useful in treating, preventing, diagnosing, and/or prognosing immunodeficiencies, including both congenital and acquired immunodeficiencies.
  • B cell immunodeficiencies in which immunoglobulin levels B cell function and/or B cell numbers are decreased include: X-linked agammaglobulinemia (Bruton's disease), X-linked infantile agammaglobulinemia, X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, X-linked lymphoproliferative syndrome (XLP), agammaglobulinemia including congenital and acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia, unspecified hypogammaglobulinemia, recessive agammaglobulinemia (Swiss type), Selective IgM deficiency, selective IgA deficiency, selective IgG subclass deficiencies, IgG subclass deficiency (with or without IgA deficiency), I
  • Examples of congenital immunodeficiencies in which T cell and/or B cell function and/or number is decreased include, but are not limited to: DiGeorge anomaly, severe combined immunodeficiencies (SCID) (including, but not limited to, X-linked SCID, autosomal recessive SCID, adenosine deaminase deficiency, purine nucleoside phosphorylase (PNP) deficiency, Class II MHC deficiency (Bare lymphocyte syndrome), Wiskott-Aldrich syndrome, and ataxia telangiectasia), thymic hypoplasia, third and fourth pharyngeal pouch syndrome, 22ql 1.2 deletion, chronic mucocutaneous candidiasis, natural killer cell deficiency (NK), idiopathic CD4+ T- lymphocytopenia, immunodeficiency with predominant T cell defect (unspecified), and unspecified immunodeficiency of cell mediated immunity.
  • SCID severe combined immunodeficiencies
  • immunodeficiencies that may be treated, prevented, diagnosed, and/or prognosed using polypeptides or polynucleotides of the invention, and/or agonists or antagonists thereof, include, but are not limited to, chronic granulomatous disease, Chdiak-Higashi syndrome, myeloperoxidase deficiency, leukocyte glucose-6- phosphate dehydrogenase deficiency, X-linked lymphoproliferative syndrome (XLP), leukocyte adhesion deficiency, complement component deficiencies (including CI, C2, C3, C4, C5, C6, C7, C8 and/or C9 deficiencies), reticular dysgenesis, thymic alymphoplasia-aplasia, immunodeficiency with thymoma, severe congenital leukopenia, dysplasia with immunodeficiency, neonatal neutropenia, short limbed dwarfism, and Nezelof syndrome-combined immunodeficiency with Ig
  • polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among immunodeficient individuals.
  • polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention could be used as an agent to boost immunoresponsiveness among B cell and/or T cell immunodeficient individuals.
  • the polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention may be useful in treating, preventing, diagnosing and/or prognosing autoimmune disorders.
  • Many autoimmune disorders result from inappropriate recognition of self as foreign material by immune cells. This inappropriate recognition results in an immune response leading to the destruction of the host tissue. Therefore, the administration of polynucleotides, polypeptides, or antibodies of the invention that can inhibit an immune response, particularly the proliferation, differentiation, or chemotaxis of T-cells, may be an effective therapy in preventing autoimmune disorders.
  • Autoimmune diseases or disorders that may be treated, prevented, diagnosed and/or prognosed by polynucleotides, polypeptides, antibodies, and/or agonists or antagonists of the present invention include, but are not limited to, one or more of the following: systemic lupus erythematosus, rheumatoid arthritis, ankylosing spondylitis, multiple sclerosis, autoimmune thyroiditis, Hashimoto's thyroiditis, autoimmune hemolytic anemia, hemolytic anemia, thrombocytopenia, autoimmune thrombocytopenia purpura, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, purpura (e.g., Henloch-Scoenlein purpura), autoimmunocytopenia, Goodpasture's syndrome, Pemphigus vulgaris, myasthenia gravis, Grave's disease (hyperthyroidism), and insulin-resistant diabetes mellitus.
  • Additional disorders that are likely to have an autoimmune component that may be treated, prevented, and/or diagnosed with the compositions of the invention include, but are not limited to, type II collagen-induced arthritis, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, neuritis, uveitis ophthalmia, polyendocrinopathies, Reiter's Disease, Stiff-Man Syndrome, autoimmune pulmonary inflammation, autism, Guillain-Barre Syndrome, insulin dependent diabetes mellitus, autoimmune inflammatory eye disorders, scleroderma with anti-collagen antibodies (often characterized, e.g., by nucleolar and other nuclear antibodies), mixed connective tissue disease (often characterized, e.g., by antibodies to extractable nuclear antigens (e.g., ribonucleoprotein)), polymyositis (often characterized, e.g., by nonhistone
  • the polypeptides, polynucleotide, and antibodies of this invention will be useful in the treatment of viral diseases.
  • the known anti-viral activity of interferon ⁇ and the discovery according to this invention that IIS is inducible by interferon- ⁇ and that IIS appears to have immunostimulatory properties indicate that the HIS polypeptides and nucleic acids of this invention are useful for the treatment of viral diseases, either alone or in combination with interferon- ⁇ .
  • Viral diseases which may be treatable with the methods and compositions of this invention are the following: Retroviridae (e.g.
  • HTN-1 also referred to as HTLN-III, LAV or HTLV-III/LAV, or HIV-III
  • HJN-LP other isolates, such as HJN-LP
  • Picornaviridae e.g. polio viruses, hepatitis A virus; enteroviruses, human Coxsackie viruses, rhinoviruses, echoviruses
  • Calciviridae e.g. strains that cause gastroenteritis
  • Togaviridae e.g. equine encephalitis viruses, rubella viruses
  • Flaviridae e.g. dengue viruses, encephalitis viruses, yellow fever viruses
  • Coronoviridae e.g. coronaviruses
  • Rhabdoviradae e.g. vesicular stomatitis viruses, rabies viruses
  • Coronaviridae e.g. coronaviruses
  • Rhabdoviridae e.g. vesicular stomatitis viruses, rabies viruses
  • Filoviridae e.g. ebola viruses
  • Paramyxoviridae e.g.
  • Orthomyxoviridae e.g. influenza viruses
  • Bungaviridae e.g. Hantaan viruses, bunga viruses, phleboviruses and ⁇ airo viruses
  • Arena viridae hemorrhagic fever viruses
  • Reoviridae e.g.
  • reoviruses reoviruses, orbiviurses and rotaviruses
  • Birnaviridae Hepadnaviridae (Hepatitis B virus); Parvovirida (parvoviruses); Papovaviridae (papilloma viruses, polyoma viruses); Adenoviridae (most adenoviruses); Herpesviridae (herpes simplex virus (HSV) 1 and 2, varicella zoster virus, cytomegalovirus (CMN), herpes virus; Poxviridae (variola viruses, vaccinia viruses, pox viruses); and Iridoviridae (e.g. African swine fever virus); and unclassified viruses (e.g.
  • the findings of the present invention that IIS confers cell migration and cell invasion capabilities on cells indicates that the therapeutic compositions and methods of this invention useful for inhibiting the production of are suitable for the treatment of cancer, including metastatic cancer.
  • Cancers that may be treated using the teachings of the invention include, but are not limited to, bladder cancer, breast cancer, colon cancer, endometrial cancer, head and neck cancer, leukemia, lung cancer, melanoma, non-Hodgkin's lymphoma, ovarian cancer, prostate cancer, and rectal cancer.
  • Expression vectors derived from retroviruses, adenovirus, herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of nucleotide sequences to the targeted organ, tissue or cell population. Methods that are well known to those skilled in the art can be used to construct recombinant vectors that will express antisense polynucleotides of the gene encoding IIS. See, for example, the techniques described in Sambrook et al (supra) and Ausubel et al (supra).
  • antisense molecules DNA, RNA or PNA
  • modify of gene expression can be obtained by designing antisense molecules, DNA, RNA or PNA, to the control regions of gene encoding IIS, i.e., the promoters, enhancers, and introns. Oligonucleotides derived from the transcription initiation site, e.g., between -10 and +10 regions of the leader sequence, are preferred.
  • the antisense molecules may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes. Similarly, inhibition can be achieved using "triple helix" base-pairing methodology. Triple helix pairing compromises the ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules.
  • Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules that can specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding IIS.
  • ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, which include the following sequences, GUA, GUU and GUC. Once identified, short RNA molecules of between 15 and 20 ribonucleotides corresponding to the region of the target gene containing the cleavage site may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ribonuclease protection assays.
  • Antisense molecules and ribozymes of the invention may be prepared by any method known in the art for the synthesis of RNA molecules. These include techniques for chemically synthesizing oligonucleotides such as solid phase phosphoramidite chemical synthesis. Alternatively, RNA molecules may be generated by in vitro or in vivo transcription of US-encoding DNA polynucleotides. Such DNA molecules may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, antisense cDNA constructs that synthesize antisense RNA constitutively or inducibly can be introduced into cell lines, cells or tissues. RNA molecules may be modified to increase intracellular stability and half- life.
  • flanking regions at the 5' and/or 3' ends of the molecule Possible modifications include, but are not limited to, the addition of flanking regions at the 5' and/or 3' ends of the molecule or the use of phosphorothioate or 2'O- methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine and wybutosine as well as acetyl-, methyl-, thio- and similarly modified forms of adenine, cytidine, guanine, thymine, and uridine which are not as easily recognized by endogenous endonucleases.
  • Methods for introducing vectors into cells or tissues include those methods discussed infra and which are equally suitable for in vivo, in vitro and ex vivo therapy.
  • vectors are introduced into stem cells taken from the patient and clonally propagated for autologous transplant back into that same patient is presented in U.S. Patent Nos. 5,399,493 and 5,437,994, disclosed herein by reference. Delivery by transfection and by liposome is quite well known in the art.
  • the present invention relates to pharmaceutical compositions which may comprise oligonucleotides or polynucleotides, proteins, antibodies, agonists, antagonists, or inhibitors, provided alone or in combination with at least one other agent, such as stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier, including, but not limited to, saline, buffered saline, dextrose, and water. Any of these molecules can be administered to a patient alone, or in combination with other agents, drugs or hormones, in pharmaceutical compositions where it is mixed with excipient(s) or pharmaceutically acceptable carriers.
  • the pharmaceutically acceptable carrier is pharmaceutically inert.
  • compositions may be accomplished orally or parenterally.
  • Methods of parenteral delivery include topical, intra-arterial, intramuscular, subcutaneous, intramedullary, intrathecal, intraventricular, intravenous, intraperitoneal, or intranasal administration.
  • these pharmaceutical compositions may contain suitable pharmaceutically acceptable carriers comprising excipients and auxiliaries that facilitate processing of the active compounds into preparations that can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of "Remington's Pharmaceutical Sciences” (Mack Publishing Co, Easton Pa.).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, draggers, capsules, liquids, gels, syrups, slurries, suspensions and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combination of active compounds with solid recipient, optionally grinding a resulting mixture, and processing the mixture of granules, after adding suitable auxiliaries, if desired, to obtain tablets or dragger cores.
  • suitable recipients are carbohydrate or protein fillers such as sugars, including lactose, sucrose, manifold, or orbital; starch from corn, wheat, rice, potato, or other plants; cellulose such as methyl cellulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcellulose; and gums including arabic and tragacanth; and proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • compositions for parenteral administration include aqueous solutions of active compounds.
  • the pharmaceutical compositions of the invention may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution, Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances that increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate or triglycerides, or liposomes.
  • the suspension may also contain suitable stabilizers or agents that increase the solubility of the compounds to allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation. Such penetrants are generally known in the art. Manufacture and Storage
  • compositions of the present invention may be manufactured in a manner that known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to hydrochloric, sulfuric, acetic, lactic, tartaric, malic, succinic, etc. Salts tend to be more soluble in aqueous or other protonic solvents that are the corresponding free base forms.
  • the preferred preparation may be a lyophilized powder in 1 mM-50 mM histidine, 0.1%- 2% sucrose, 2%-7% mannitol at a pH range of 4.5 to 5.5 that is combined with buffer prior to use.
  • compositions comprising a compound of the invention formulated in an acceptable carrier
  • they can be placed in an appropriate container and labeled for treatment of an indicated condition.
  • labeling would include amount, frequency and method of administration.
  • compositions suitable for use in the present invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models, usually mice, rabbits, dogs, or pigs. The animal model is also used to achieve a desirable concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of protein or its antibodies, antagonists, or inhibitors that ameliorate the symptoms or condition. Therapeutic efficacy and toxicity of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., ED50 (the dose therapeutically effective in 50% of the population) and LD50 (the dose lethal to 50% of the population).
  • the dose ratio between therapeutic and toxic effects is the therapeutic index, which can be expressed as the ratio LD50 ED50.
  • Pharmaceutical compositions that exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies is used in formulating a range of dosage for human use.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, sensitivity of the patient, and the route of administration.
  • the exact dosage is chosen by the individual physician in view of the patient to be treated. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Additional factors which may be taken into account include the severity of the disease state, age, weight and gender of the patient; diet, time and frequency of administration, drug combination(s), reaction sensitivities, and tolerance/response to therapy. Long acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or once every two weeks depending on half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from 0.1 to 100,000 micrograms, up to a total dose of about 1 g, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc.
  • IIS polypeptide or an IIS polypeptide- encoding polynucleotides can be delivered in a suitable formulation to stimulate cellular migration or cellular invasion.
  • IIS antagonists such as anti-IIS antibodies or IIS anti-sense oligonucleotides can be delivered in a suitable formulation to inhibit cellular migration or cellular invasion.
  • Immortalized normal human lymphoblast cells were obtained from Dr. Krystyna Wisniewski (Staten Island, N.Y.). These cells were grown in RPMI-1640 (Invitrogen, San Diego, CA) supplemented with 16% heat-inactivated fetal bovine serum (Invitrogen), 1 mM L-glutamine, penicillin (100 units/ml)-streptomycin (100 ⁇ g/ml) and incubated in a humidified incubator in an atmosphere of 5% CO 2 and 95% air at 37°C.
  • RT-PCR Reverse-transcriptase-PCR
  • RNAs were extracted from human lymphoblast using Trizol reagent (Invitrogen) following the manufacturer's instructions. Total RNAs were reverse- transcribed using ThermoScript RT-PCR system (Invitrogen). Semi-quantitative PCR was performed to amplify target fragments of cDNA. Five microliters of the RT-PCR product was used for the PCR. The PCR conditions are as follows: denaturation at 94°C for 1 minute followed by 35 cycles of amplification at 94°C for 30 seconds and at different annealing temperature for 2 minutes, and a final incubation at 68°C for 5 minutes using AdvanTaq Plus DNA polymerase (Clontech, Palo Alto, CA).
  • the primers that amplified LIP-B and the novel IIS polynucleotide were 5'-CTG CTG CTA CCA GGC CAA TG-3' (SEQ ED NO:3) (forward) and 5'-GTC ACA CAC TAG ATT TCT TC-3' (SEQ ED NO:4) (reverse); the primers that amplified only LIP-B were 5'-CCT CTG TTC AAG TTA AGT C-3' (SEQ ID NO:5) (forward) and 5'-CCG CAA TGA GGC TTC GTT TGG-3' (SEQ ID NO:6) (reverse).
  • the annealing temperature was 60°C.
  • the primers that amplified MGB1 were 5'-GAC AAT GCC ACT ACA AAT GCC-3' (SEQ ED NO:7) (forward) and 5'-CAT TGC TCA GAG TTT CAT CCG-3' (SEQ ED NO:8) (reverse), with an annealing temperature of 66°C.
  • the primers that amplified the other cDNAs are as follows: MGB2 - 5'-CTC CTG GAG GAC ATG GTT G-3' (SEQ ID NO:9) (forward) and 5'- CTA TGT GAC TGG TTG AGG-3' (SEQ ID NO:10) (reverse), annealing temperature 66°C; LIP-A - 5'-CAG TGG TCT GCC AAG CTC TTG G-3' (SEQ H) NO:ll) (forward) and 5'-CAT AGG CCA TCG TAT CCA CGC-3' (SEQ ED
  • LIP-B cDNA and the US-encoding polynucleotide were independently amplified by PCR using more specific primers for LIP-B and a separate set of primers for IIS.
  • the PCR products were analyzed by DNA sequencing. The results showed that all members of the SCGB family analyzed (UG, LIP-A, LIP-B, MGB1 and MGB2 ) and the newly identified US-encoding polynucleotide were expressed in human lymphoblasts (Figure 1, Panel A).
  • the IIS cDNA encodes a protein of 83 amino acids that has a calculated molecular mass of 9.2 kDa and a calculated PI of 8.9.
  • a blast search of the human genome database using the IIS cDNA coding sequence identified the genomic sequence containing the IIS gene as being located on chromosome 11, clones RP11-703H8 (GenBank Accession No. AP003306), pDJ741nl5 (Accession o. AC004127), and CTD-253 D15 (Accession No. AP003064).
  • the US gene structure was analyzed and determined to span approximately 3 kb of genomic DNA and to contain a total of three exons and two intions (Figure 1, Panel B). The sequences adjacent to the splice sites were in good conformance with the consensus splice rule (Figure 1, Panel C).
  • the IIS gene promoter sequence was analyzed using NSITE DB program to determine the transcription factors that potentially regulate this gene.
  • Several transcription factor-binding consensus sequences including those of nuclear factor (NF)- ⁇ B, hepatocyte nuclear factor (HNF)-IA, -IB, -1C, SP1, IFN-stimulated response elements (ISRE), and gamma-IFN activated sites (GAS) are present within - 1500 bp of the IIS gene promoter region.
  • NF nuclear factor
  • HNF hepatocyte nuclear factor
  • ISRE IFN-stimulated response elements
  • GAS gamma-IFN activated sites
  • Example 2 Polymorphisms in the IIS gene. Single nucleotide polymorphisms (SNPs) have been identified in the IIS gene. IIS genomic DNAs from 34 normal healthy individuals were analyzed for polymorphisms by SSCP and DNA sequencing.
  • Genomic DNAs of 34 unrelated individuals were obtained from the DNA Polymorphism Discovery resources of Coriell Cell Repositories (Camden, NJ).
  • the fragment (382 bp) of promoter region 1 was amplified using 5'-GTG GCT ACA CAT CAC AGA AAG-3' (SEQ ED NO:17) (forward) and 5'-CAC AGG TGA ATT ATG GCT TC-3' (SEQ ID NO:18) (reverse) primers at annealing temperature 64°C.
  • the fragment (398 bp) of promoter region 2 was amplified using 5'-GAG AAC ACA GCC TTC CAG C-3' (SEQ DD NO:19) (forward) and 5'-CAA TGA GTG ATT TGG ATT CG-3' (SEQ D3 NO:20) (reverse) primers at annealing temperature 63°C.
  • the fragment (160 bp) of exon 1 was amplified using 5'-CTC CAT GAC TAG ACA GGC TC-3' (SEQ ID NO:21) (forward) and 5'-GCT GGA CTC ATG ACT GAT G-3' (SEQ ID NO:22) (reverse) primers at annealing temperature 65°C.
  • the fragment (311 bp) of exon 2 was amplified using 5'-CTG TCT GGT GTA ACC TCA GG-3' (SEQ ED NO:23) (forward) and 5 '-GCT GAG TTG AAT TCT GCC TC-3 ' (SEQ ID NO:24) (reverse) primers at annealing temperature 65°C.
  • the fragment (210 bp) of exon 3 was amplified using 5'-CAG CAG CAG CAT GAC TGA C-3' (SEQ ED NO:25) (forward) and 5'-GAC CAG TGG AGA TGT GCA G-3' (SEQ ID NO:26) (reverse) primers at annealing temperature 66°C.
  • the amplified fragments were resolved on 0.5 X MDE gel (BMA, Rockland, ME) with 0.6 X TBE running buffer at constant 3 W for 12-16 hours and autoradiographs were obtained.
  • Thermo sequenase radiolabeled terminator cycle sequencing kit (USB, Cleveland, OH) was used for the sequencing following the manufacturer's protocol.
  • the samples were resolved by electrophoresis using 6% sequencing gel and autoradiographs were prepared by using BioMax X-ray film (Kodak, Rochester, NY). Five different SSCP patterns were identified in amplicons of the exon 3 region (Figure 2, Panel A). An SNP (G to A substitution) was identified in the 3 '-flanking region near exon 3 ( Figure 2, Panel B).
  • Short tandem repeat polymorphisms such as C(T) 8 (SEQ ED NO :27), C(T) 9 (SEQ ED NO:28), or (CTTT) 2 (CTTTT) (SEQ ID NO:29) in intron 2 were detected and this polymorphism appears to occur at frequencies of 0.662 for C(T) 8 (SEQ ID NO:27), 0.309 for C(T) 9 (SEQ ID NO:28), and 0.029 for (CTTT) 2 (CTTTT) (SEQ ID NO:29) in these individuals ( Figure 2, Panel C).
  • Example 3 IIS-mRNA Expression In Various Tissues Semi-quantitative RT-PCR was employed to determine the expression level of IIS-mRNA in various tissues. The identities of the RT-PCR products were confirmed by Southern hybridization using IIS-cDNA probe as well as by DNA sequencing. Complimentary DNA from various human tissues (MTC panel II, Clontech) were subjected to PCR as described above and the products were resolved by electrophoresis using 1% agarose gels and cDNA bands were transferred on to Hybond-N + nylon membrane (Amersham Biosciences, Buckinghamshire, UK).
  • the blot was hybridized with ⁇ 32 P-dCTP labeled IIS cDNA probe at 42°C overnight using DIG Easy Hyb (Boehringer Manheim, Germany). After prehybridization for 2 hours the blot was washed three times with 2 x SSC, 0.1% SDS solution for 15 minutes each and then three times with 0.1 x SSC, 0.1% SDS solution for 15 minutes each. Autoradiographs were prepared using BioMax X-ray film (Kodak, Rochester, NY).
  • Polyclonal antibody of IIS was raised in the rabbit (Covance Laboratories, Vienna, VA) against a synthetic oligopeptide (NH 2 -SFKKRLSLKKSWWK-COOH) (SEQ ID NO:30) corresponding to the amino acid sequence of IIS (residues 70 to 83) (Peptide Technologies, Gaithersburg, MD). Specificity of this antiserum towards IIS was also tested by pre-adsorption with the oligopeptide that was used as the immunogen for raising this antibody. The results showed that unadsorbed antiserum readily recognized IIS protein band in Western blots, whereas the post-adsorbed antiserum was ineffective in recognizing this protein.
  • Human lymphoblasts were harvested and lysed with lysis buffer containing protease inhibitor cocktail and immunoprecipitated using protein A IP Kit (KPL, Gaithersburg, MD) according to the supplier's instructions.
  • the cell lysate was precleared using 50% resin slurry.
  • the IIS antibody was added to the precleared sample followed by an addition of 50 ⁇ l of 50% resin slurry and incubated overnight at 4°C with gentle agitation.
  • the resin was pelleted and washed three times with lysis buffer and resuspended in 40 ⁇ l of 1 X SDS-PAGE sample buffer. Proteins were resolved by electrophoresis on 18% Tris-glycine gels (Invitrogen).
  • Total proteins from transfected COS-1 cells and transfected E. coli cells were extracted using T- PERTM mammalian and bacterial protein extraction buffers (Pierce, Rockford, IL), respectively, according to the manufacturer's instructions. The concentration of total proteins was determined using Bradford protein assay (Bio-Rad Laboratories, Hercules, CA). One hundred micrograms of total protein from the transfected COS-1 cells were resolved by electrophoresis using 18% Tris-Glycine gel (Invitrogen) followed by electro-transferring onto ImmobilonTM-P transfer membrane (Millipore, Bedford, MA). The blots were incubated in blocking solution containing 4% BSA at 4°C.
  • the blot was washed and incubated with alkaline phosphatase conjugated S-Tag antibody (diluted 1:5000, Novagen) for 15 min.
  • the blots were washed with TBST solution and developed using S-TagTM AP Western Blot Kit (Novagen) following the manufacturer's instruction.
  • S-TagTM AP Western Blot Kit Novagen
  • the blots were incubated with anti-IIS antibody (diluted 1 : 1000) for 1 hour, washed with TBST solution, and incubated with HRP-conjugated goat anti- rabbit IgG (diluted 1:1000, Amersham Biosciences) for 1 hour, washed five times (10 minutes each), and detected by ECL chemiluminescence detection.
  • IIS cDNA containing the entire coding region was amplified using 5'-ATG AGG CTG TCA GTG TGT CTC C-3 ' (SEQ ED NO:31) (forward) and 5 '-CAT TTT TTC ACT ATT TCC ACC AGG ACT-3' (SEQ ID NO:32) (reverse) primers and subcloned into Pshl/EcoRV site of pET-42a (+) vector (Novagen. Madison, WI), which includes an integrated T7 promoter.
  • E. coli strain, BL21 (DE3) was transfected with the cDNA construct and the transformed cells were inoculated into LB broth with kanamycin and grown until it reached an OD reading of 1.0 at 600nm.
  • IJS-protein was induced with 0.4 mM isopropyl- ⁇ -D- thiogalactopyranoside (IPTG) for 2 hours.
  • IlS-protein from the transfected E. coli lysates was purified using BugBuster Ni-NTA His-Bind purification kit and His'Bind columns (Novagen).
  • the transfected E coli cells (1.0 g of cell paste) were harvested and added with 5 ml of BugBuster reagent and 5 ⁇ l of Benzonase followed by incubation for 20 minutes on a shaking platform.
  • the solution was centrifuged and the supernatant was passed through a 0.45 ⁇ syringe-end filter and then loaded onto His Bind column pre-charged with Ni 2+ .
  • the column was washed with 10 volumes of 1 x binding buffer and 6 volumes of IX washing buffer.
  • the bound protein was eluted with 6 volumes of IX elution buffer.
  • the eluted protein (molecular mass 35 kDA) was detected with S-Tag antibody and with US-antibody ( Figure 4, Panel A).
  • the IIS cDNA was also expressed in COS-1 cells.
  • cDNA containing the entire coding sequence of HS was amplified using 5'-ATG AGG CTG TCA GTG TGT CTC C-3 ' (SEQ H) NO:33) (forward) and 5 '-TCA CTA TTT CCA CCA GGA CT-3 ' (SEQ ID NO:34) (reverse) primers and cDNA encoding the mature IIS (without the signal peptide sequence) without signal peptide sequences was amplified using 5'- CTT GTC TGC CCA GCT GTT GCT TC-3' (SEQ H> NO:35) (forward) and 5'- TCA CTA TTT CCA CCA GGA CT-3' (SEQ H> NO:36) (reverse) primers and subcloned into a pcDNA4/HisMax-TOPO TA expression vector (Invitrogen).
  • the recombinant plasmids were linearized with Bgl ⁇ i. and electroporated into COS-1 cells.
  • the transfected cells were culture for 48 hours in DMEM containing 10% fetal bovine serum, 0.8 mM L-glutamine, 100 units/ml of penicillin and 100 ⁇ g/ml streptomycin.
  • the lysates of the transfected cells were resolved by SDA-PAGE and protein bands were analyzed by Western blot. The results show that IIS protein was stably expressed in COS-1 cells, both with and without the signal peptide ( Figure 4, Panel B, lanes 2 and 3, respectively).
  • IIS is inducible by IFN- ⁇
  • IIS mRNA levels were measured by quantitative real-time RT- PCR analysis of total RNA extracted from human lymphoblast cells that were treated with different cytokines and different concentrations of IFN- ⁇ .
  • Cultured human lymphoblast cells were treated with the following cytokines: TNF- ⁇ (10 ng/ml), IFN- ⁇ (10 ng ml), IL-4 (0.5 ng/ml) or IL-13 (10 ngml) for 1 hour, 3 hours, and 6 hours, respectively.
  • cytokines IFN- ⁇ , IL-4, and IL-13
  • TNF- ⁇ Human recombinant cytokines
  • IFN- ⁇ IL-4, and IL-13
  • TNF- ⁇ TNF- ⁇
  • the cells were also treated with different concentrations of IFN- ⁇ (0.1, 1, or 10 ng/ml) for 1 hour to obtain a dose-response curve.
  • Total RNA was isolated from the treated cells using Trizol (Invitrogen) and an RNeasy Mini Kit (Qiagen, Valencia, CA), followed by DNase treatment to eliminate genomic DNA contamination. Quantitative real-time RT-PCR was performed using Smart cycler system (Cepheid, Sunnyvale, CA).
  • First-strand cDNA was synthesized from 1 ⁇ g of total RNA using Superscript III First-strand synthesis system (Invitrogen) following the manufacturer's protocol.
  • Real-time PCR was performed with 2.5 ⁇ l of cDNA and primers (forward: 5'-CTC ACA GCC GAA TAA GCC ACC-3' (SEQ ED NO:37) and reverse: 5'-GTG CAG GGC AAG TGA TTT ATT AAA GC-3 ' (SEQ ED NO:38)) using QuantiTect SYBR Green (Qiagen) following the manufacturer's protocol under the following conditions: denaturation at 94°C for 15 minutes followed by 50 cycles of amplification at 94°C for 15 seconds, 58°C for 30 seconds, 72°C for 30 seconds.
  • Real-time PCR was also performed with first- strand cDNAs from resting and activated blood cells using Human Blood Fractions MTCTM Panel (Clontech). The data from each PCR run was analyzed using Cepheid Smart Cycler Software Program (Cepheid) with FAM as the reference dye. The final data were normalized to ⁇ -actin and are presented as fold induction. Quantitation was performed using at least three separate total RNA samples for each treatment group.
  • IIS- antisense oligonucleotide Inhibition of IIS expression prevents lymphoblast cell migration and invasion.
  • Lymphoblast cells were transfected with IIS- antisense oligonucleotides and used in a chemotaxis assay.
  • a phosphorothioate IIS- antisense oligonucleotide (5'-TTT GGC AAC TTG GAG GTT TA-3') (SEQ ID NO:39) was used to inhibit IIS protein expression.
  • the corresponding US-sense oligonucleotide (5'-TAA ACC TCC AAG TTG CCA AA-3') (SEQ ID NO:40) was used as one of the controls.
  • lymphoblast cells When the lymphoblast cells reached 30-40% confluence, they were rinsed once with serum-free Opti-MEM I (Invitrogen). The s- oligonucleotides were then delivered to the cells by OligofectamineTM (Invitrogen) according to the manufacturer's instructions. Treatment of the cells with Oligofectamine alone was also used as a control. The cells were incubated for 60 hours at 37 °C before using for migration and invasion assays.
  • OligofectamineTM Invitrogen
  • the cells were collected after transfection with IlS-antisense or sense s-oligonucleotides and incubated for 60 hours. Following incubation the cells were rinsed with serum-free RPMI-1640 medium and resuspended in the same serum-free medium.
  • serum-free RPMI-1640 medium One hundred fifty microliters of the conditioned medium from NTH 3T3 cell culture was placed into 96-well feeder tray and 1 x 10 5 cells in 100 ⁇ l serum-free RPMI-1640 medium was placed into cell migration chamber plate and then incubated for 24 hours at 37°C in a CO 2 incubator.
  • the cell invasion assay was performed using QCMTM 96-well cell invasion plate (Chemicon, Temecula, CA).
  • the transfected cells were rinsed with serum-free RPMI medium and then resuspended in serum-free RPMI-1640 medium.
  • the conditions for the invasion assay were identical to those employed for the migration assay, as described above, except that the cell invasion assay kit was used.
  • the migrated and invaded cells were counted using a hemocytometer.

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Abstract

La présente invention concerne la découverte d'un nouvel élément de la famille des protéines sécrétoglobines inductible par l'interféron-? et présentant des effets modulateurs sur la migration et l'invasion cellulaire.
PCT/US2004/043717 2004-01-06 2004-12-30 Iis: nouvel proteine inductible par ifn-gamma, element de la famille des secretoglobines, modulant l'invasion et la migration cellulaire Ceased WO2005068497A1 (fr)

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CN101221185B (zh) * 2007-01-12 2011-08-10 中国科学院化学研究所 一种检测人β干扰素的生物传感器及其专用多肽

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WO1997034997A1 (fr) * 1996-03-21 1997-09-25 Human Genome Sciences, Inc. Facteur i, ii et iii de liaison de steroides specifique a l'endometre humain
WO2000032221A2 (fr) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion et inhibition de l'angiogenese et de la vascularisation cardiaque
WO2000073454A1 (fr) * 1999-06-02 2000-12-07 Genentech, Inc. Polypeptides transmembranaires secretes et acides nucleiques codants pour ceux-ci
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WO1997034997A1 (fr) * 1996-03-21 1997-09-25 Human Genome Sciences, Inc. Facteur i, ii et iii de liaison de steroides specifique a l'endometre humain
WO2000032221A2 (fr) * 1998-12-01 2000-06-08 Genentech, Inc. Promotion et inhibition de l'angiogenese et de la vascularisation cardiaque
WO2000073454A1 (fr) * 1999-06-02 2000-12-07 Genentech, Inc. Polypeptides transmembranaires secretes et acides nucleiques codants pour ceux-ci
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CN101221185B (zh) * 2007-01-12 2011-08-10 中国科学院化学研究所 一种检测人β干扰素的生物传感器及其专用多肽

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