Definitions

• Obesity is the most prevalent metabolic disorder in the United States affecting on the order of 35% of adults at an estimated cost of 300,000 lives and $70 billion in direct and indirect costs. As an epidemic, it is growing due to the increase in the number of children who can be considered overweight or obese. Obesity is defined as an excess of body fat, frequently resulting in a significant impairment of health. Obesity results when adipocyte size or number in a person's body increases to levels that may result in one or more of a number of physical and psychological disorders. A normal-sized person has between 30 and 35 billion fat cells. When a person gains weight, these fat cells increase in size at first and later in number. Obesity is influenced by genetic, metabolic, biochemical, psychological, and behavioral factors. As such, obesity is a complex disorder that must be addressed on several fronts to achieve a lasting positive clinical outcome (ADAReport, 1997; Perusse and Bouchard, 1999; Pi-Sunjer and Panel, 1998).
• Obese individuals are prone to ailments including: type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea.
• NIDDM type II diabetes mellitus
• Sleep apnea is episodes of not breathing during sleep that correlates with higher incidence of stroke and heart attack, two other factors contributing to obesity-linked morbidity and mortality among the clinically obese (ADAReport, 1997; Pi-Sunjer and Panel, 1998).
• Non-pharmaceutical intervention includes diet, exercise, psychiatric treatment, and surgical treatments to reduce food consumption or remove fat (i.e. liposuction).
• Dexfenfluramine (REDUX ® ) and sibutramine (MERIDIA ® ) are members of the first class and beta3-adrenergic agonists and orlistat (XENICAL ® ) are representative of the . latter (Dunlop and Rosenzweig-Lipson, 1998).
• MSH is derived from adrenocorticotropic hormone (ACTH) a major pituitary hormone that results from the proteolytic processing of the pro-hormone proopiomelanocortin (POMC).
• ACTH adrenocorticotropic hormone
• POMC pro-hormone proopiomelanocortin
• the fat phenotype is the consequence of a mutation in the hypothalamic pro-hormone converting enzyme carboxypeptidase E.
• the least well-characterized obese mouse mutant is tub. tub encodes a cytosolic protein that may influence the processing of hypothalamic neuropeptide hormones such as neuropeptide Y (NPY, an appetite stimulating hormone) and POMC (Aron et al., 1997; Guan et al., 1998; Spiegelman and Flier, 1996; Weigle and Kuijper, 1996).
• the POMC knockout mouse has a phenotype analogous to several mouse models for obesity, particularly that of Ay.
• the POMC knockout has early onset obesity and has yellow hair color as well as adrenal insufficiency due to the apparent morphological absence of their adrenal gland.
• corticosterones increase food intake, it is surprising that they are obese.
• the obese phenotype can be treated with ⁇ - MSH, a peptide hormone derived from POMC (Yaswen et al., 1999).
• fa/fa rats which bear many similarities to the ob/ob and db/db mice, discussed above.
• fa/fa rats are very sensitive to cold, their capacity for non-shivering thermogenesis is normal. It is well established that thermogenesis and metabolism are closely coupled endocrinologically. Torpor, a condition analogous to hibernation and lethargy, seems to play a larger part in the maintenance of obesity in fa/fa rats than in the mice mutants.
• several desert rodents, such as the spiny mouse do not become obese in their natural habitats, but do become so when fed on standard laboratory feed (Tartaglia, 5,861 ,485, 1999).
• Brown Adipose Tissue also known as multilocular adipose tissue, is so called because of the its color due to the large number of capillaries and mitochondria in the cells making up this tissue.
• BAT is primarily found in the shoulder region and flanks of human embryo and newborn infant, it then disappears in the first months of life. In animals, particularly hibernating animals and rodents, it is more abundant.
• BAT has features of an endocrine organ; it is vascularized by capillaries and it receives direct sympathetic innervation. Sympathetic neurotransmission leads to the release of the catecholamines noradrenaline and adrenaline resulting in the activation of a hormone-sensitive lipase.
• Interferons are a part of the group of intercellular messenger proteins known as cytokines.
• IFN ⁇ is the product of a multigene family of at least 16 members, whereas IFN ⁇ is the product of a single gene, ⁇ - and ⁇ - IFNs are also known as type I IFNs.
• Type I IFNs are produced in a variety of cell types. Biosynthesis of type I IFNs is stimulated by viruses and other pathogens and by various cytokines and growth factors. IFN ⁇ , also known as type II IFN, is produced in T-cells and natural killer cells. Biosynthesis of type II IFN is stimulated by antigens to which the organism has been sensitized. Both ⁇ - and ⁇ -IFNs are immunomodulators and anti-inflammatory agents, activating macrophages, T-cells and natural killer cells.
• IFNs are part of the body's natural defense to viruses and tumors. They exert these defenses by affecting the function of the immune system and by direct action on pathogens and tumor cells. IFNs mediate these multiple effects in part by inducing the synthesis of many cellular proteins.
• IFI interferon-inducible
• IFI genes possess antitumor, antiviral and immunomodulatory functions.
• the expression of tumor antigens by cancer cells is increased in the presence of IFN ⁇ , thus rendering the cancer cells more susceptible to immune rejection.
• the IFI proteins synthesized in response to viral infections are known to inhibit viral functions such as cell penetration, uncoating, RNA and protein synthesis, assembly and release (Hardman et al., 1996).
• Type II IFN stimulates expression of major histocompatibility complex (MHC) proteins. For this reason it is thus used in immune response enhancement (De Maeyer and De Maeyer-Guignard, 1998; Janeway and Travers, 1997).
• MHC major histocompatibility complex
• Interferons may be grouped into three categories. IFN ⁇ (leukocyte) interferon is made by white blood cells; IFN ⁇ (fibroblast) interferon is made by skin cells; and IFN ⁇ (immune) interferon is made by lymphocytes after stimulation by antigen. Host response to infection includes changes in metabolic state, for example the regulation of hepatic fatty acid biosynthesis. In response to IFN ⁇ fatty acid biosynthesis is stimulated, but the mechanism appears to be different from that of other cytokines such as interleukin-1 (IL-1) and tumor necrosis factor (TNF) since only treatment with the former in conjunction with either one of the two later cytokines can stimulate lipogenesis.
• IL-1 interleukin-1
• TNF tumor necrosis factor
• IL-1 and TNF cannot act synergistically with each other, but can do so with IFN ⁇ (Grunfeld and Feingold, 1992).
• TNF can affect the thermogenic activity of BAT, the core temperature, the rate of food intake and body weight, and resting oxygen consumption of rats. In this work there seems to be a less robust response to IFN ⁇ (Coombes et al., 1987).
• IFNs and/or other cytokines In addition to changes in fatty acid metabolism and biosynthesis that might be induced by treatment with IFNs and/or other cytokines, it has been observed that treatments can induce the expression of inducible nitric oxide synthase (iNOS) when mice or cell lines (NIH 3T3LI) are treated with multiple cytokines such as IFN ⁇ , TNF ⁇ , and bacterial endotoxin lipopolysaccharide (LPS) together. Alone, these agents do not induce the expression of iNOS (Kapur et al., 1999). Interestingly, IFN ⁇ and IFN ⁇ have been shown to affect the composition of BAT in suckling mice.
• iNOS inducible nitric oxide synthase
• interferon- modulated genes may play a role in the composition and distribution of BAT and WAT. If so, then these IFNresponsive genes may represent targets or agents of therapeutic intervention in metabolic disease, if not excellent markers for the assessment of such compounds.
• the genes that form the interferon-inducible gene cluster contain canonical seven acid repeat regions as well as conserved non-coding regions in the promotor regions. These genes appear to have evolved because of gene duplication then subsequently diverged.
• interferon-inducible genes the founding members of the mouse 200 series genes are 201, 202abc, 203, 204, and 205/D3. The p202 and p204 gene has been localized to the cytoplasm and nucleus of cells.
• p202 binds the cell growth regulatory retinoblastoma protein (pRb) in vitro and in vivo.
• the 202 protein is a 52kD phosphoprotein that can bind to the pRb as well as a number of other transcription factors such as c-Jun, c-Fos, NFKB, and AP-1 (Min et al., 1996).
• the 72kD gene product of the 204 gene is also a phosphoprotein (Choubey and Lengyel, 1992; Choubey and Lengyel, 1993; Choubey et al., 1989; Tannenbau et al., 1993; Wang et al., 1999).
• a human IFI gene known as 6-16 encodes an mRNA that is highly induced by type I IFNs in a variety of human cells (Kelly et al., 1986). After induction, 6-16 mRNA constitutes as much as 0.1% of the total cellular mRNA.
• the 6- 6 mRNA is present at only very low levels in the absence of type I IFN, and is only weakly induced by type II IFN.
• the 6-76 mRNA encodes a hydrophobic protein of 130 amino acids.
• the first 20 to 23 amino acids comprise a putative signal peptide.
• Protein 6- 16 has at least two predicted transmembrane regions culminating in a negatively charged C-terminus.
• the p27 gene encodes a protein with 41% amino acid sequence identity to the 6-16 protein.
• the p27 gene is expressed in some breast tumor cell lines and in a gastric cancer cell line. In other breast tumor cell lines, in the HeLa cervical cancer cell line, and in fetal lung fibroblasts, p27 expression occurs only upon ⁇ -IFN induction. In one breast tumor cell line, p27 is independently induced by estradiol and by IFN (Rasmussen et al., 1993). Expression of p27 was analyzed in 21 primary invasive breast carcinomas, 1 breast cancer bone metastasis, and 3 breast fibroadenomas.
• the small IFI gene products may contribute to viral resistance.
• IFI genes may be important in viral infections, such as in hepatitis, including hepatoxicity induced by inflammation.
• the IFI proteins synthesized in response to viral infections are known to inhibit viral functions such as penetration, uncoating, RNA or protein synthesis, assembly or release.
• the 130-51 protein may inhibit one or more of these functions in HCV.
• a particular virus may be inhibited in multiple functions by IFI proteins.
• the principle inhibitory effect exerted by IFI proteins differs among the virus families (Hardman et al., 1996).
• the IFI proteins of the invention may provide the basis for clinical diagnosis of diseases associated with their induction. These proteins may be useful in the diagnosis and treatment of tumors, viral infections, inflammation, or conditions associated with impaired immunity. Furthermore, these proteins may be used for investigations of the control of gene expression by IFNs and other cytokines in normal and diseased cells.
• interleukin (IL)-12 and IL-18 In murine models of inflammatory bowel disease, systemic administration of interleukin (IL)-12 and IL-18 to wild-type BALB/c mice induces liver injury and intestinal inflammation.
• IL-12 and IL-18 induce striking elevations in serum levels of IFN ⁇ that would be expected to result in the expression of interferon-induced genes.
• the major symptoms of IL-12- and IL-18-induced toxicity are similar to those found in endotoxin-induced septic shock.
• TNF- ⁇ knockout mice induce intestinal mucosal inflammation.
• Liver and spleen lymphocyte populations and hepatic cytokine production were compared in genetically obese, ob/ob mice (a model for obesity-related fatty liver) and lean mice.
• Obese mice have a selective reduction of hepatic CD4+NK T cells.
• Serum IL-18 is also increased basally, and the hepatic mRNA levels of IL-18 and -12 are greater after endotoxin challenge.
• up-regulation of IL-18 and IL-12 in fatty livers may reduce hepatic CD4+NK T cells.
• mononuclear cells from fatty livers have decreased expression of the adhesion molecule, leukocyte factor antigen-1
• LFA-1 which is necessary for the hepatic accumulation of CD4+NK T cells. Consistent with reduced numbers of hepatic CD4+NK T cells, mononuclear cells from fatty livers produce less IL-4. Furthermore, after endotoxin treatment, hepatic induction of IL-10 is inhibited, while that of IFN ⁇ is enhanced. Thus, fatty livers have inherent immunologic alterations that may predispose them to damage from endotoxin and other insults that induce a proinflammatory cytokine response. The role of the IFN-inducible p204 as growth regulator has been investigated by transfecting an expression vector constitutively expressing p204 into several cell lines.
• p204 is a potent growth inhibitor in sensitive cells, as demonstrated by cell focus assays. Since stable transfectants of sensitive lines constitutively overexpressing p204 cannot be established in vitro, investigators have used an inducible promoter to express p204. It has been shown that proliferation of B6MEF fibroblasts lacking endogenous p204 is strongly inhibited by transient p204 expression in the nucleus. p204 delays G1 progression into the S-phase and cells accumulate with a DNA content equivalent to cells arrested in late G1. The role of p204 in the control of cell growth in vivo has been investigated by generating transgenic mice in which the IFI204 gene was constitutively expressed in all tissues.
• the over-expression of the p204 transgene is compatible with embryo development up to the four-cell stage in an in vitro follow-up of 4.5 days. However, no viable animals with an intact copy of the transgene were obtained, suggesting that high and constitutive levels of p204 expression can impair normal embryo development.
• p204 plays a negative role in growth regulation and provide new information about the molecular mechanisms exploited by IFNs to inhibit cell proliferation (Lembo et al., 1998).
• Mutations affecting the expression of interferon-induced proteins may play a role in controlling cellular proliferation as observed in cancer as well as in cellular differentiation. For example, the human interferon induced protein IFI16 has been found to play a role in hematopoiesis.
• IFI16 is expressed in CD34+ and monocytoid daughter cells, but is rapidly and markedly down-regulated at the corresponding stages of polymorphonuclear • anderythroid development. This differential expression of IFI 16 in myeloid precursor subpopulations and its perceived molecular properties are consistent with a possible role in regulating myelopoiesis (Dawson et al., 1998; Landolfo et al., 1998).
• Cachexia is a wasting phenomenon observed in almost half of cancer patients. Cachexia is a result of tumor-induced distant metabolic changes disproportionate to tumor burden. Weight loss by cancer patients is most prevalent in those with pancreatic and gastric cancers, but is not limited to these cancers. Cachexia-induced weight loss may lead to respiratory distress, a major contributing factor to mortality among cancer patients as metabolic changes lead to loss of adipose tissue and skeletal muscle mass, particularly as respiratory muscle is affected. Knowledge about the mechanisms of cachexia may lead to better therapeutic and clinical interventions that complement chemotherapy (DeWys et al., 1980; Tisdale,
• INF ⁇ prevents cancer cachexia in a mouse model, perhaps by the down regulation of the enzyme lipoprotein lipase and/or the up regulation of triglyceride lipase.
• the IFN ⁇ mediated modulation of these genes and/or other indirect regulation of their activity would require the activity of signal transduction and/or transcription factors (Mori et al., 1996a; Tisdale, 1999).
• Interleukin-12's (IL-12) activity in preventing cachexia in a murine model is at least in part due to the ability of IL-12 to down regulate the expression of IL-6 and INF- ⁇ (Mori et al., 1996b). Understanding the mechanisms involved in INF-induced gene expression increases the usefulness of animal models for cachexia.
• Interferon-induced genes act as markers for INF activities, for example in the case of genes that are modulated in response to thermogenic conditions that are known to affect metabolic status. Genes modulated under these conditions, as well as with IFN treatment, make it possible to dissect the roles of multiple proteins in complex pathways that are specific for adipose tissues (WAT and BAT) and skeletal muscle by monitoring the modulation of • INF-affected genes.
• the invention is based in part upon the discovery of novel nucleic acid sequences encoding novel polypeptides.
• Nucleic acids encoding the polypeptides disclosed in the invention, and derivatives and fragments thereof, will hereinafter be collectively designated as "IFI206" nucleic acid or polypeptide sequences.
• the invention provides an isolated IFI206 nucleic acid molecule encoding an IFI206 polypeptide that includes a nucleic acid sequence that has identity to the nucleic acids disclosed in SEQ ID NOS:1 or 3.
• the IFI206 nucleic acid molecule can hybridize under stringent conditions to a nucleic acid sequence complementary to a nucleic acid molecule that includes a protein-coding sequence of an IFI206 sequence.
• the invention also includes an isolated nucleic acid that encodes an IFI206 polypeptide, or a fragment, homolog, analog or derivative thereof.
• the nucleic acid can encode a polypeptide at least 80% identical to a polypeptide comprising the amino acid sequences of SEQ ID NOS:2, 4 or
• the nucleic acid can be, for example, a genomic DNA fragment or a cDNA molecule that includes the nucleic acid sequence of any of SEQ ID NOS: 2, 4 or 15.
• an oligonucleotide e.g., an oligonucleotide which includes at least 6 contiguous nucleotides of an IFI206 ' nucleic acid (e.g., SEQ ID NOS:1 or 3) or a complement of said oligonucleotide.
• substantially purified IFI206 (SEQ ID NO:2, 4 or 15).
• the IFI206 include an amino acid sequence that is substantially identical to the amino acid sequence of a human IFI206.
• the invention also features antibodies that immunoselectively-bind IFI206.
• the invention includes pharmaceutical compositions that include therapeutically- or prophylactically-effective amounts of a therapeutic and a pharmaceutically-acceptable carrier.
• the therapeutic can be, e.g., an IFI206, an IFI206, or an antibody specific for an IFI206.
• the invention includes a kit containing, in one or more containers, a therapeutically- or prophylactically-effective amount of this pharmaceutical composition.
• the invention includes a method of producing a polypeptide by culturing a cell that includes an IFI206, under conditions allowing for expression of the IFI206 encoded by the DNA. If desired, the IFI206 can then be recovered.
• the invention includes a method of detecting the presence of an IFI206 in a sample.
• a sample is contacted with a compound that selectively binds to the polypeptide under conditions allowing for formation of a complex between the polypeptide and the compound.
• the complex is detected, if present, thereby identifying the IFI206 within the sample.
• the invention also includes methods to identify specific cell or tissue types based on their expression of an IFI206.
• Also included in the invention is a method of detecting the presence of an IFI206 molecule in a sample by contacting the sample with an IFI206 probe or primer, and detecting whether the nucleic acid probe or primer bound to an IFI206 molecule in the sample.
• the invention provides a method for modulating the activity of an IFI206 by contacting a cell sample that includes the IFI206 with a compound that binds to the IFI206 in an amount sufficient to modulate the activity of said polypeptide.
• the compound can be, e.g., a small molecule, such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
• a small molecule such as a nucleic acid, peptide, polypeptide, peptidomimetic, carbohydrate, lipid or other organic (carbon containing) or inorganic molecule, as further described herein.
• a medicament for treating or preventing disorders or syndromes related to obesity including, e.g., type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea, as well as those directly related to interferons, such as metabolic disorders, tumors, viral infections, inflammation, cancer (including renal, bladder and ovarian carcinomas, leukemias, and Kaposi's sarcoma), cancer cachexia, infections by viruses or other pathogens (such as HCV and leishmania), and conditions associated with inflammation or immune impairment such as rheumatoid and osteoarthritis and Acquired
• the Therapeutic can be, e.g., an IFI206, an IFI206, or an IFI206 -specific antibody, or biologically-active derivatives or fragments thereof.
• the invention further includes a method for screening for a modulator of disorders or syndromes including, e.g.,. type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea, as well as those directly related to interferons, such as metabolic disorders, tumors, viral infections, inflammation, cancer, cancer cachexia, infections by viruses or other pathogens, and conditions associated with inflammation or immune impairment such as rheumatoid and osteoarthritis and Acquired Immunodeficiency Syndrome (AIDS).
• the method includes contacting a test compound with an IFI206 and determining if the test compound binds to the IFI206. Binding of the test compound to the IFI206 indicates the test compound is a modulator of activity, or of latency or predisposition to the aforementioned disorders or syndromes.
• NIDDM type II diabetes mellitus
• hypertension e.g.
• a modulator of activity, or of latency or predisposition to disorders or syndromes including, e.g., type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea, as well as those directly related to interferons, such as metabolic disorders, tumors, viral infections, inflammation, cancer, cancer cachexia, infections by viruses or other pathogens, and conditions associated with inflammation or immune impairment such as rheumatoid and osteoarthritis and Acquired
• NIDDM type II diabetes mellitus
• hypertension e.g., coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea
• interferons such as metabolic disorders, tumors, viral infections, inflammation, cancer, cancer cachexia, infections by viruses or other pathogens, and conditions associated with inflammation or immune impairment such as rheum
• Immunodeficiency Syndrome by administering a test compound to a test animal at increased risk for the aforementioned disorders or syndromes.
• the test animal expresses a recombinant polypeptide encoded by an IFI206.
• Expression or activity of IFI206 is then measured in the test animal, as is expression or activity of the protein in a control animal which recombinantly- expresses IFI206 and is not at increased risk for the disorder or syndrome.
• the expression of IFI206 in both the test animal and the control animal is compared. A change in the activity of IFI206 in the test animal relative to the control animal indicates the test compound is a modulator of latency of the disorder or syndrome.
• the invention includes a method for determining the presence of or predisposition to a disease associated with altered levels of an IFI206, an IFI206, or both, in a subject (e.g., a human subject).
• the method includes measuring the amount of the IFI206 in a test sample from the subject and comparing the amount of the polypeptide in the test sample to the amount of the IFI206 present in a control sample. An alteration in the level of the IFI206 in the test sample as compared to the control sample indicates the presence of or predisposition to a disease in the subject.
• the predisposition includes, e.g., Type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea, as well as those directly related to interferons, such as metabolic disorders.
• NIDDM Type II diabetes mellitus
• the expression levels of the new polypeptides of the invention can be used in a method to screen for various cancers.
• the invention includes a method of treating or preventing a pathological condition associated with a disorder in a mammal by administering to the subject an IFI206, an IFI206, or an IFI206 -specific antibody to a subject (e.g., a human subject), in an amount sufficient to alleviate or prevent the pathological condition.
• the disorder includes, e.g., Type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea, as well as those directly related to interferons, such as metabolic disorders.
• NIDDM Type II diabetes mellitus
• the invention can be used in a method to identity the cellular components that interact with the IFI206 and polypeptides, including cellular receptors and downstream effectors, by any one of a number of techniques commonly employed in the art. These include but are not limited to the two-hybrid system, affinity purification, co-precipitation with Abs or other specific-interacting molecules.
• FIG 1 shows Global Sequence Similarity (GCG:GAP) (A), Multiple Alignment Analysis (BestFl ' t (Genetics_Computer_Group_(GCG), 1999)) demonstrating the relationship between SEQ_ID_NO_2 and SEQ_ID_NO_4 (B) and PHYLIP Protein Distance Analysis. Neighbor-Joining/UPGMA method version 3.572c, tree is unrooted and negative branch lenghts are allowed.
• FIG 2 shows hydrophobicity plots ((GCG), 1999) for IFI206 (A; SEQ ID NO:1) and its naturally occurring variant (B; SEQ ID NO:3); the X axis reflects amino acid position, and the positive Y axis, hydrophobicity.
• FIG 3 shows the radiation hybrid map of IFI206 (SEQ ID NO:2/SEQ ID NO:4) as generated using Auto-RHMAPPER (Stein et al., 1995).
• FIG 4 shows DOTPLOT and COMPARE analysis of polypeptide sequence from IFI206 variants.
• FIG 5 shows that IFI206 variants are primarily expressed in WAT, BAT, skeletal muscle, and to a much lesser extent in cardiac muscle.
• FIG 6 shows the modulation of expression of the IFI206's expression during the development of NIH3T3LI cells in culture. Expression of IFI206 family members is variable during maturation of mouse NIH3T3LI pre- adipocytes.
• the inventors have identified a gene and polypeptide that is expressed in response to interferon stimulation, IFI206.
• IFI206 or IFI206 refers to the nucleotide sequence that encodes IFI206.
• Isolated when referred to a molecule, refers to a molecule that has been identified and separated and/or recovered from a component of its natural environment. Contaminant components of its natural environment are materials that interfere with diagnostic or therapeutic use.
• Container is used broadly to mean any receptacle for holding material or reagent.
• Containers may be fabricated of glass, plastic, ceramic, metal, or any other material that can hold reagents. Acceptable materials will not react adversely with the contents. 1. Nucleic acid-related definitions
• Control sequence are DNA sequences that enable the expression of an operably-linked coding sequence in a particular host organism.
• Prokaryotic control sequences include promoters, operator sequences, and ribosome binding sites.
• Eukaryotic cells utilize promoters, polyadenylation signals, and enhancers.
• Nucleic acid is operably-linked when it is placed into a functional relationship with another nucleic acid sequence.
• a promoter or enhancer is operably-linked to a coding sequence if it affects the transcription of the sequence, or a ribosome-binding site is operably-linked to a coding sequence if positioned to facilitate translation.
• "operably-linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. However, enhancers do not have to be contiguous. Linking is accomplished by conventional recombinant DNA methods.
• isolated nucleic acids An isolated nucleic acid molecule is purified from the setting in which it is found in nature and is separated from at least one contaminant nucleic acid molecule. Isolated IFI206 molecules are distinguished from the specific IFI206 molecule, as it exists in cells. However, an isolated IFI206 molecule includes IFI206 molecules contained in cells that ordinarily express the IFI206 where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells.
• Protein-related definitions (a) purified polypeptide When the molecule is a purified polypeptide, the polypeptide will be purified (1 ) to obtain at least 15 residues of N-terminal or internal amino acid sequence using a sequenator, or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or silver stain.
• Isolated polypeptides include those expressed heterologously in genetically- engineered cells or expressed in vitro, since at least one component of the IFI206 natural environment will not be present. Ordinarily, isolated polypeptides are prepared by at least one purification step.
• An active IFI206 or IFI206 fragment retains a biological and/or an immunological activity of native or naturally-occurring IFI206.
• Immunological activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native IFI206;
• biological activity refers to a function, either inhibitory or stimulatory, caused by a native IFI206 that excludes immunological activity.
• a biological activity of IFI206 includes, for example, binding of nucleic acids, such as binding mRNA expressed in BAT.
• Abs Antibody may be single anti-IFI206 monoclonal Abs (including agonist, antagonist, and neutralizing Abs), anti-IFI206 antibody compositions with polyepitopic specificity, single chain anti-IFI206 Abs, and fragments of anti- IFI206 Abs.
• a "monoclonal antibody” refers to an antibody obtained from a population of substantially homogeneous Abs, i.e., the individual Abs comprising the population are identical except for naturally-occurring mutations that may be present in minor amounts
• An epitope tagged polypeptide refers to a chimeric polypeptide fused to a "tag polypeptide". Such tags provide epitopes against which Abs can be made or are available, but do not interfere with polypeptide activity. To reduce anti-tag antibody reactivity with endogenous epitopes, the tag polypeptide is preferably unique. Suitable tag polypeptides generally have at least six amino acid residues and usually between about 8 and 50 amino acid residues, preferably between 8 and 20.amino acid residues). Examples of epitope tag sequences include HA from Influenza A virus and FLAG. The invention is based, in part, upon the discovery of novel nucleic acid sequences that encode novel polypeptides, particularly interferon-inducible proteins.
• the nucleic acids, and their encoded polypeptides, are collectively designated herein as "IFI206".
• the novel IFI206 of the invention include the nucleic acids whose sequences are provided in Tables 1 and 3, or a fragment thereof.
• the invention also includes a mutant or variant IFI206, any of whose bases may be changed from the corresponding base shown in Tables 1 and 3 while still encoding a protein that maintains the activities and physiological functions of the IFI206 fragment, or a fragment of such a nucleic acid.
• the invention further includes nucleic acids whose sequences are complementary to those just described, including complementary nucleic acid fragments.
• the invention additionally includes nucleic acids or nucleic acid fragments, or complements thereto, whose structures include chemical modifications.
• modifications include, by way of nonlimiting example, modified bases, and nucleic acids whose sugar phosphate backbones are modified or derivatized. These modifications are carried out at least in part to enhance the chemical stability of the modified nucleic acid, such that they may be used, for example, as anti-sense binding nucleic acids in therapeutic applications in a subject. In the mutant or variant nucleic acids, and their complements, up to 20% or more of the bases may be so changed.
• the novel IFI206 of the invention include the protein fragments whose sequences are provided in Tables 2, 4 and 5 inclusive.
• the invention also includes an IFI206 mutant or variant protein, any of whose residues may be changed from the corresponding residue shown in Tables 2, 4 and 5 while still encoding a protein that maintains its native activities and physiological functions, or a functional fragment thereof. In the mutant or variant IFI206, up to 20% or more of the residues may be so changed.
• the invention further encompasses Abs and antibody fragments, such as F ab or (F ab ) 2, that bind immunospecifically to any of the IFI206 of the invention.
• the IFI206 nucleic acid (Table 1) comprises a start codon at nucleotides 290-292 (bold, underline); a stop codon at nucleotides 1708-1710 (bold, dash underline), and a putative polyadenylation site at nucleotides 1770-1777 (bold, double-underlined).
• IFI206 nucleotide fragment (SEQ ID NO:1). cgattcgaat tcggccacac tggccggatc ctctagagat ccctcgacct cgacccacgc 60 gtccgagcac agtgagagac acccagtgct gctcaagaag tgaacaact ctgagagtat 120 cctaaccact ggtgtcttcc ttatacccc atttttcact ttctcagtta ctgaattatc 180 tgcctaccta ctcaaaccaa gcaggccact tctgtgttg aagatctcag 240 ttgctgccga aattccaggg agtataacca acaacttgaa
• a polypeptide encoded by SEQ ID NO:1 is presented in Table 2.
• Table 3 presents an analysis of the physical characteristics of SEQ ID NO:2 (Pace et al., 1995).
• the SEQ ID NO:2 polypeptide consists of 475 amino acids with a calculated molecular weight of 53095.5 Daltons and a predicted isoelectric point of 8.18 ((GCG), 1999).
• the conditions at which this analysis is valid are: pH 6.5, 6.0 M guanidium hydrochloride, 0.02 M phosphate buffer.
• the naturally-occuring variant of interferon-inducible polypeptide 206 (IFI206) nucleic acid comprises a start codon at nucleotides 290-292 (bold, underline); a stop codon at nucleotides 1708-1710 " (bold, dash underline), and a putative polyadenylation site at nucleotides 1770-1777 (bold, double-underlined).
• IFI206b nucleotide fragment, a naturally-occuring variant SEQ ID NO:3, Table 4
• a polypeptide encoded by SEQ ID NO:3 is presented in Table 5.
• SEQ ID NO:4 The SEQ ID NO:4 polypeptide consists of 445 amino acids with a calculated molecular weight of 49899.1 Daltons and a predicted isoelectric point of 8.17 ((GCG), 1999).
• the conditions at which this analysis is valid are: pH 6.5, 6.0 M guanidium hydrochloride, 0.02 M phosphate buffer
• mice IFI206c (SEQ ID NO: 14) derived from a mouse brown adipose tissue (BAT) cDNA library is shown in Table 7. Start codon “ATG” (bold, underlined) and stop codon “TAG” (bold, dash underlined), and the putative polyadenylation sites are (bold, double- underlined) are indicated.
• the cDNA clone was obtained from the library upon PCR amplification cloning methods utilizing specific oligos, followed by further identification of positive clones via common analysis employing a 32P- labeled probe:
• SEQ ID NO:16 (IFI206.snr1 PCR oligo): CATCATGTTAGCAATCTGAAACGTGGTATATTTCT
• SEQ ID NO: 17 (IFI206.snf1 PCR oligo): GTAAAGAAATTTCCAGCTGATGCTGGATTGG
• SEQ ID NO:18 (IFI206.p1 probe): CTTCCTGGGTTGCGGAAGTCTCGCCTCTTTCAGATG
• Table 8 shows the polypeptide sequence (SEQ ID NO:15) of the open reading frame of the polynucleotide sequence shown in Table 7.
• Table 9 presents an analysis of the physical characteristics of SEQ ID NO: 15 (Pace et al., 1995).
• the SEQ ID NO:15 polypeptide consists of 318 amino acids with a calculated molecular weight of 35984.1 Daltons and a predicted isoelectric point of 10.67 ((GCG), 1999).
• the conditions at which this analysis is valid are: pH 6.5, 6.0 M guanidium hydrochloride, 0.02 M phosphate buffer.
• IFI206 function may be assigned by analyzing protein similarity.
• the invention also includes polypeptides having 80-100%, including 81 , 82, 83, 84, 85, 86, 87, 88, 89, 89.2, 90, 91 , 92, 93, 94, 95, 96, 97, 98 and 99%, sequence identity to SEQ ID NOS: 2, 4, and 15, excluding those polypeptides that are identical to SEQ ID NOS:22 and 24, preferably excluding those polypeptides having 80-100%, including 81 , 82, 83, 84, 85,
• SEQ ID NOS:22 and 24 corresponds to IFI204
• SEQ ID NO:24 corresponds to IFI205D3.
• SEQ ID NO:21 and SEQ ID NO:23 are the corresponding nucleotide sequences.
• Table 10 shows the consensus sequence for IFI-induced genes from human and mouse that were generated using the software EMOTIF (Huang and Brutlag, 2001 ; Nevill-Manning et al., 1998) and represented in the single letter abbreviation for amino acids. Residues in []'s indicate that any of those amino acids may be used at that position; a ".” Indicates that any amino acid — or no amino acid — may occupy this position in the motif.
• the nucleic acids and proteins of the invention are potentially useful in the treatment of Type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, reproductive organ cancers, fatty liver, viral infections, inflammation, allergies, steatosis, hepatoxicity, inflammary bowel disease, septic shock, and related conditions and sleep apnea, as well as those directly related to interferons, such as metabolic disorders.
• NIDDM Type II diabetes mellitus
• the IFI206 and proteins of the invention are useful in potential therapeutic applications implicated in Type II diabetes mellitus (NIDDM), hypertension, coronary heart disease, hypercholesterolemia, osteoarthritis, gallstones, cancers of the reproductive organs, and sleep apnea, as well as those directly related to interferons, such as metabolic disorders.
• NIDDM Type II diabetes mellitus
• a cDNA encoding IFI206 may be useful in gene therapy
• IFI206 protein may be useful when administered to a subject in need thereof.
• the novel nucleic acid encoding IFI206, and the IFI206 protein of the invention, or fragments thereof, may further be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein are to be assessed.
• nucleic acid molecules that encode IFI206 or biologically-active portions thereof. Also included in the invention are nucleic acid fragments sufficient for use as hybridization probes to identify IFI206-encoding nucleic acids (e.g., IFI206 mRNAs) and fragments for use as polymerase chain reaction (PCR) primers for the amplification and/or mutation of IFI206 molecules.
• a "nucleic acid molecule” includes DNA molecules (e.g., cDNA or genomic DNA), RNA molecules (e.g., mRNA), analogs of the DNA or RNA generated using nucleotide analogs, and derivatives, fragments and homologs.
• the nucleic acid molecule may be single-stranded or double-stranded, but preferably comprises double-stranded
• Probes are nucleic acid sequences of variable length, preferably between at least about 10 nucleotides (nt), 100 nt, or many (e.g., 6,000 nt) depending on the specific use. Probes are used to detect identical, similar, or complementary nucleic acid sequences. Longer length probes can be obtained from a natural or recombinant source, are highly specific, and much slower to hybridize than shorter-length oligomer probes. Probes may be single- or double-stranded and designed to have specificity in PCR, membrane-based hybridization technologies, or ELISA-like technologies. Probes are substantially purified oligonucleotides that will hybridize under stringent conditions to at least optimally12, 25, 50, 100, 150, 200, 250, 300,
• the full- or partial length native sequence IFI206 may be used to "pull out" similar (homologous) sequences (Ausubel et al., 1987; Sambrook, 1989), such as: (1) full-length or fragments of IFI206 cDNA from a cDNA library from any species (e.g. human, murine, feline, canine, bacterial, viral, retroviral, yeast), (2) from cells or tissues, (3) variants within a species, and (4) homologues and variants from other species.
• the probe may be designed to encode unique sequences or degenerate sequences. Sequences may also be genomic sequences including promoters, enhancer elements and introns of native sequence IFI206.
• IFI206 coding region in another species may be isolated using such probes.
• a probe of about 40 bases is designed, based on IFI206, and made.
• probes are labeled using, for example, radionuclides such as 32 P or 35 S, or enzymatic labels such as alkaline phosphatase coupled to the probe via avidin-biotin systems. Labeled probes are used to detect nucleic acids having a complementary sequence to that of IFI206 in libraries of cDNA, genomic DNA or mRNA of a desired species.
• Such probes can be used as a part of a diagnostic test kit for identifying cells or tissues which mis-express an IFI206, such as by measuring a level of an IFI206 in a sample of cells from a subject e.g., detecting IFI206 mRNA levels or determining whether a genomic IFI206 has been mutated or deleted.
• isolated nucleic acid An isolated nucleic acid molecule is separated from other nucleic acid molecules which are present in the natural source of the nucleic acid. Preferably, an isolated nucleic acid is free of sequences that naturally flank the nucleic acid (i.e., sequences located at the 5'- and 3'-termini of the nucleic acid) in the genomic DNA of the organism from which the nucleic acid is derived.
• isolated IFI206 molecules can contain less than about 5 kb, 4 kb, 3 kb, 2 kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA of the cell/tissue from which the nucleic acid is derived (e.g., brain, heart, liver, spleen, etc.).
• an isolated nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material * or culture medium when produced by recombinant techniques, or of chemical precursors or other chemicals when chemically synthesized.
• a nucleic acid molecule of the invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NOS: 2, 4 or 15, or a complement of this aforementioned nucleotide sequence, can be isolated using standard molecular biology techniques and the provided sequence information. Using all or a portion of the nucleic acid sequence of SEQ ID NOS: 2, 4 or 15 as a hybridization probe, IFI206 molecules can be isolated using standard hybridization and cloning techniques (Ausubel et al., 1987; Sambrook, 1989). PCR amplification techniques can be used to amplify IFI206 using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers.
• nucleic acids can be cloned into an appropriate vector and characterized by DNA sequence analysis.
• oligonucleotides corresponding to IFI206 sequences can be prepared by standard synthetic techniques, e.g., an automated DNA synthesizer.
• oligonucleotide comprises a series of linked nucleotide residues, which oligonucleotide has a sufficient number of nucleotide bases to be used in a PCR reaction or other application.
• a short oligonucleotide sequence may be based on, or designed from, a genomic or cDNA sequence and is used to amplify, confirm, or reveal the presence of an identical, similar or complementary DNA or RNA in a particular cell or tissue.
• Oligonucleotides comprise portions of a nucleic acid sequence having about 10 nt, 50 nt, or 100 nt in length, preferably about 15 nt to 30 nt in length.
• an oligonucleotide comprising a nucleic acid molecule less than 100 nt in length would further comprise at least 6 contiguous nucleotides of SEQ ID NOS:1 or 3, or a complement thereof. Oligonucleotides may be chemically synthesized and may also be used as probes.
• an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule that is a complement of the nucleotide sequence shown in SEQ ID NOS: 2, 4 or 15, or a portion of this nucleotide sequence (e.g., a fragment that can be used as a probe or primer or a fragment encoding a biologically-active portion of an IFI206).
• a nucleic acid molecule that is complementary to the nucleotide sequence shown in SEQ ID NOS:1 or 3, is one that is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOS:1 or 3, that it can hydrogen bond with little or no mismatches to the nucleotide sequence shown in SEQ ID NOS:1 or 3, thereby forming a stable duplex.
• Binding means the physical or chemical interaction between two polypeptides or compounds or associated polypeptides or compounds or combinations thereof. Binding includes ionic, non-ionic, van der Waals, hydrophobic interactions, and the like.
• a physical interaction can be either direct or indirect. Indirect interactions may be through or due to the effects of another polypeptide or compound. Direct binding refers to interactions that do not take place through, or due to, the effect of another polypeptide or compound, but instead are without other substantial chemical intermediates.
• Nucleic acid fragments are at least 6 (contiguous) nucleic acids or at least 4 (contiguous) amino acids, a length sufficient to allow for specific hybridization in the case of nucleic acids or for specific recognition of an epitope in the case of amino acids, respectively, and are at most some portion less than a full-length sequence. Fragments may be derived from any contiguous portion of a nucleic acid or amino acid sequence of choice.
• derivatives, and analogs Derivatives are nucleic acid sequences or amino acid sequences formed from the native compounds either directly or by modification or partial substitution.
• Analogs are nucleic acid sequences or amino acid sequences that have a structure similar to, but not identical to, the native compound but differ from it in respect to certain components or side chains. Analogs may be synthetic or from a different evolutionary origin and may have a similar or opposite metabolic activity compared to wild type.
• Homologs are nucleic acid sequences or amino acid sequences of a particular gene that are derived from different species.
• Derivatives and analogs may be full length or other than full length, if the derivative or analog contains a modified nucleic acid or amino acid, as described below.
• Derivatives or analogs of the nucleic acids or proteins of the invention include, but are not limited to, molecules comprising regions that are substantially homologous to the nucleic acids or proteins of the invention, in various embodiments, by at least about 70%, 80%, or 95% identity (with a preferred identity of 80-95%) over a nucleic acid or amino acid sequence of identical size or when compared to an aligned sequence in which the alignment is done by a computer homology program known in the art, or whose encoding nucleic acid is capable of hybridizing to the complement of a sequence encoding the aforementioned proteins under stringent, moderately stringent, or low stringent conditions (Ausubel et al., 1987).
• homology refers to sequences characterized by a homology at the nucleotide level or amino acid level as discussed above. Homologous nucleotide sequences encode those sequences coding for isoforms of IFI206. Isoforms can be expressed in different tissues of the same organism as a result of, for example, alternative splicing of RNA. Alternatively, different genes can encode isoforms.
• homologous nucleotide sequences include nucleotide sequences encoding for an IFI206 of species other than humans, including, but not limited to: vertebrates, and thus can include, e.g., frog, mouse, rat, rabbit, dog, cat cow, horse, and other organisms.
• homologous nucleotide sequences also include, but are not limited to, naturally occurring allelic variations and mutations of the nucleotide sequences set forth herein.
• a homologous nucleotide sequence does not, however, include the exact nucleotide sequence encoding human IFI206.
• Homologous nucleic acid sequences include those nucleic acid sequences that encode conservative amino acid substitutions (see below) in SEQ ID NOS:2, 4 or 15, as well as a polypeptide possessing IFI206 biological activity. Various biological activities of the IFI206 are described below. 7. open reading frames The open reading frame (ORF) of an IFI206 gene encodes I FI206.
• ORF is a nucleotide sequence that has a start codon (ATG) and terminates with one of the three "stop" codons (TAA, TAG, or TGA).
• ATG start codon
• TAA stop codon
• an ORF may be any part of a coding sequence that may or may not comprise a start codon and a stop codon.
• preferable IFI206 ORFs encode at least 50 amino acids.
• An IFI206 can encode a mature IFI206.
• a "mature" form of a polypeptide or protein disclosed in the present invention is the product of a naturally occurring polypeptide or precursor form or proprotein.
• the naturally" occurring polypeptide, precursor or proprotein includes, by way of nonlimiting example, the full-length gene product, encoded by the corresponding gene. Alternatively, it may be defined as the polypeptide, precursor or proprotein encoded by an open reading frame described herein.
• the product "mature" form arises, again by way of nonlimiting example, as a result of one or more naturally occurring processing steps as they may take place within the cell, or host cell, in which the gene product arises.
• Examples of such processing steps leading to a "mature" form of a polypeptide or protein include the cleavage of the N-terminal methionine residue encoded by the initiation codon of an open reading frame, or the proteolytic cleavage of a signal peptide or leader sequence.
• a mature form arising from a precursor polypeptide or protein that has residues 1 to N, where residue 1 is the N-terminal methionine would have residues 2 through N remaining after removal of the N-terminal methionine.
• a mature form arising from a precursor polypeptide or protein having residues 1 to N, in which an N-terminal signal sequence from residue 1 to residue M is cleaved would have the residues from residue
• a "mature" form of a polypeptide or protein may arise from a step of post-translational modification other than a proteolytic cleavage event.
• additional processes include, by way of non-limiting example, glycosylation, myristoylation or phosphorylation.
• a mature polypeptide or protein may result from ' the operation of only one of these processes, or a combination of any of them.
• An active IFI206 polypeptide or IFI206 polypeptide fragment retains a biological and/or an immunological activity similar, but not necessarily identical, to an activity of a naturally-occuring (wild-type) IFI206 polypeptide of the invention, including mature forms.
• a particular biological assay, with or without dose dependency, can be used to determine IFI206 activity.
• a nucleic acid fragment encoding a biologically-active portion of IFI206 can be prepared by isolating a portion of SEQ ID NOS: 2, 4 or 15 that encodes a polypeptide having an IFI206 biological activity (the biological activities of the IFI206 are described below), expressing the encoded portion of IFI206 (e.g., by recombinant expression in vitro) and assessing the activity of the encoded portion of IFI206.
• Immunological activity refers to the ability to induce the production of an antibody against an antigenic epitope possessed by a native IFI206; biological activity refers to a function, either inhibitory or stimulatory, caused by a native IFI206 that excludes immunological activity.
• IFI206 nucleic acid variants and hybridization
• variant polynucleotides, genes and recombinant genes The invention further encompasses nucleic acid molecules that differ from the nucleotide sequences shown in SEQ ID NOS:1 or 3 due to degeneracy of the genetic code and thus encode the same IFI206 as that encoded by the nucleotide sequences shown in SEQ ID NOS: 1 or 3.
• An isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NOS:2, 4 or 15. In addition to the IFI206 sequences shown in SEQ ID NOS:2, 4 or 15,
• DNA sequence polymorphisms that change the amino acid sequences of the IFI206 may exist within a population. For example, allelic variation among individuals will exhibit genetic polymorphism in IFI206.
• the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame (ORF) encoding IFI206, preferably a vertebrate IFI206. Such natural allelic variations can typically result in 1-5% variance in IFI206. Any and all such nucleotide variations and resulting amino acid polymorphisms in the IFI206, which are the result of natural allelic variation and that do not alter the functional activity of the IFI206 are within the scope of the invention.
• IFI206 from other species that have a nucleotide sequence that differs from the human sequence of SEQ ID NOS:1 or 3, are contemplated.
• Nucleic acid molecules corresponding to natural allelic variants and homologues of the IFI206 cDNAs of the invention can be isolated based on their homology to the IFI206 of SEQ ID NOS:1 or 3 using cDNA- derived probes to hybridize to homologous IFI206 sequences under stringent conditions.
• IFI206 variant polynucleotide or "IFI206 variant nucleic acid sequence” means a nucleic acid molecule which encodes an active IFI206 that (1) has at least about 80% nucleic acid sequence identity with a nucleotide acid sequence encoding a full-length native IFI206, (2) a full-length native IFI206 lacking the signal peptide, (3) an extracellular domain of an
• an IFI206 variant polynucleotide will have at least about 80% nucleic acid sequence identity, more preferably at least about 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% nucleic acid sequence identity and yet more preferably at least about 99% nucleic acid sequence identity with the nucleic acid sequence encoding a full-length native IFI206.
• An IFI206 variant polynucleotide may encode full-length native IFI206 lacking the signal peptide, an extracellular domain of an IFI206, with or without the signal sequence, or any other fragment of a full-length IFI206. Variants do not encompass the native nucleotide sequence.
• IFI206 variant polynucleotides are at least about 30 nucleotides in length, often at least about 60, 90, 120, 150, 180, 210, 240, 270, 300, 450, 600 nucleotides in length, more often at least about 900 nucleotides in length, or more.
• Percent (%) nucleic acid sequence identity with respect to IFI206- encoding nucleic acid sequences identified herein is defined as the percentage of nucleotides in a candidate sequence that are identical with the nucleotides in the IFI206 sequence of interest, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining % nucleic acid sequence identity can be achieved in various ways that are within the skill in the art, for instance, using publicly available computer software such as BLAST, BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms needed to achieve maximal alignment over the full length of the sequences being compared.
• the % nucleic acid sequence identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can alternatively be phrased as a given nucleic acid sequence C that has or comprises a certain % nucleic acid sequence identity to, with, or against a given nucleic acid sequence D) can be calculated as follows:
• W is the number of nucleotides cored as identical matches by the sequence alignment program's or algorithm's alignment of C and D and
• Z is the total number of nucleotides in D.
• the % nucleic acid sequence identity of C to D will not equal the % nucleic acid sequence identity of D to C.
• Homologs ⁇ i.e., nucleic acids encoding IFI206 derived from species other than human) or other related sequences (e.g., paralogs) can be obtained by low, moderate or high stringency hybridization with all or a portion of the particular human sequence as a probe using methods well known in the art for nucleic acid hybridization and cloning.
• hybridization stringency increases as the propensity to form DNA duplexes decreases.
• stringency can be chosen to either favor specific hybridizations (high stringency), which can be used to identify, for example, full-length clones from a library. Less-specific hybridizations (low stringency) can be used to identify related, but not exact, DNA molecules (homologous, but not identical) or segments.
• DNA duplexes are stabilized by: (1 ) the number of complementary base pairs, (2) the type of base pairs, (3) salt concentration (ionic strength) of the reaction mixture, (4) the temperature of the reaction, and (5) the presence of certain organic solvents, such as formamide which decreases DNA duplex stability.
• the longer the probe the higher the temperature required for proper annealing.
• a common approach is to vary the temperature: higher relative temperatures result in more stringent reaction conditions. (Ausubel et al., 1987) provide an excellent explanation of stringency of hybridization reactions.
• stringent conditions To hybridize under “stringent conditions” describes hybridization protocols in which nucleotide sequences at least 60% homologous to each other remain hybridized.
• stringent conditions are selected to be about 5°C lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength and pH.
• Tm is the temperature (under defined ionic strength, pH and nucleic acid concentration) at which 50% of the probes ' complementary to the target sequence hybridize to the target sequence at equilibrium. Since the target sequences are generally present at excess, at Tm, 50% of the probes are occupied at equilibrium,
• Stringent hybridization conditions enable a probe, primer or oligonucleotide to hybridize only to its target sequence. Stringent conditions are sequence-dependent and will differ. Stringent conditions comprise: (1 ) low ionic strength and high temperature washes (e.g. 15 mM sodium chloride, 1.5 mM sodium citrate, 0.1 % sodium dodecyl sulfate at 50°C); (2) a denaturing agent during hybridization (e.g.
• washes typically also comprise 5X
• SSC (0.75 M NaCI, 75 mM sodium citrate), 50 mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5 x Denhardt's solution, sonicated salmon sperm DNA (50 ⁇ g/ml), 0.1% SDS, and 10% dextran sulfate at 42°C, with washes at 42°C in 0.2 x SSC (sodium chloride/sodium citrate) and 50% formamide at 55°C, followed by a high-stringency wash consisting of 0.1 x
• the conditions are such that sequences at least about 65%, 70%, 75%, 85%, 90%, 95%, 98%, or 99% homologous to each other typically remain hybridized to each other. These conditions are presented as examples and are not meant to be limiting.
• Modely stringent conditions use washing solutions and hybridization conditions that are less stringent (Sambrook, 1989), such that a polynucleotide will hybridize to the entire, fragments, derivatives or analogs of SEQ ID NOS:1 or 3.
• One example comprises hybridization in 6X SSC, 5X Denhardt's solution, 0.5% SDS and 100 mg/ml denatured salmon sperm DNA at 55°C, followed by one or more washes in 1X SSC, 0.1% SDS at 37°C.
• the temperature, ionic strength, etc. can be adjusted to accommodate experimental factors such as probe length.
• Other moderate stringency conditions are described in (Ausubel et al., 1987; Kriegler, 1990).
• Low stringent conditions use washing solutions and hybridization conditions that are less stringent than those for moderate stringency
• a non-limiting example of low stringency hybridization conditions are hybridization in 35% formamide, 5X SSC, 50 mM Tris-HCI (pH 7.5), 5 mM EDTA, 0.02% PVP, 0.02% Ficoll, 0.2% BSA, 100 mg/ml denatured salmon sperm DNA, 10%
• allelic variants of IFI206 changes can be introduced by mutation into SEQ ID NO NOS:1 or 3 sequences that incur alterations in the amino acid sequences of the encoded IFI206 that do not alter IFI206 function.
• nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NOS:2, 4 or 15.
• a "non-essential" amino acid residue is a residue that can be altered from the wild-type sequences of the IFI206 without altering their biological activity, whereas an "essential" amino acid residue is required for such biological activity.
• amino acid residues that are conserved among the IFI206 of the invention are predicted to be particularly non-amenable to alteration. Amino acids for which conservative substitutions can be made are well-known in the art.
• Non-conservative substitutions that effect (1) the structure of the polypeptide backbone, such as a ⁇ -sheet or ⁇ -helical conformation, (2) the charge or (3) hydrophobicity, or (4) the bulk of the side chain of the target site can modify IFI206 polypeptide function or immunological identity.
• Residues are divided into groups based on common side-chain properties as denoted in Table B.
• Non-conservative substitutions entail exchanging a member of one of these classes for another class. Substitutions may be introduced into conservative substitution sites or more preferably into non-conserved sites.
• the variant polypeptides can be made using methods known in the art such as oligonucleotide-mediated (site-directed) mutagenesis, alanine scanning, and PCR mutagenesis.
• Site-directed mutagenesis Carter, 1986; Zoller and Smith, 1987
• cassette mutagenesis restriction selection mutagenesis
• restriction selection mutagenesis Wells et al., 1985
• other known techniques can be performed on the cloned DNA to produce the IFI206 variant DNA (Ausubel et al., 1987; Sambrook, 1989).
• the isolated nucleic acid molecule comprises a nucleotide sequence encoding a protein, wherein the protein comprises an amino acid sequence at least about 45%, preferably 60%, more preferably 70%, 80%, 90%, and most preferably about 95% homologous to SEQ ID NOS:2, 4 or 15.
• a mutant IFI206 can be assayed for blocking adipocyte differentiation in vitro.
• antisense and sense IFI206 oligonucleotides can prevent IFI206 polypeptide expression. These oligonucleotides bind to target nucleic acid sequences, forming duplexes that block transcription or translation of the target sequence by enhancing degradation of the duplexes, terminating prematurely transcription or translation, or by other means.
• Antisense or sense oligonucleotides are singe-stranded nucleic acids, either RNA or DNA, which can bind target IFI206 mRNA (sense) or IFI206
• Anti-sense nucleic acids can be designed according to Watson and Crick or Hoogsteen base pairing rules.
• the anti- sense nucleic acid molecule can be complementary to the entire coding region of IFI206 mRNA, but more preferably, to only a portion of the coding or noncoding region of IFI206 mRNA.
• the anti-sense oligonucleotide can be complementary to the region surrounding the translation start site of IFI206 mRNA.
• Antisense or sense oligonucleotides may comprise a fragment of the IFI206 DNA coding region of at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
• antisense RNA or DNA molecules can comprise at least 5, 10, 15, 20, 25, 30,
• modified nucleotides that can be used to generate the anti-sense nucleic acid include: 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5- iodouracil, hypoxanthine, xanthine, 4-acetylcytosine, 5- (carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2-thiouridine, 5- carboxymethylaminomethyluracil, dihydrouracil, beta-D-galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2- dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- meth
• the anti-sense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been sub-cloned in an anti-sense orientation such that the transcribed RNA will be complementary to a target nucleic acid of interest.
• any gene transfer method may be used.
• gene transfer methods include (1) biological, such as gene transfer vectors like Epstein-Barr virus or conjugating the exogenous DNA to a ligand-binding molecule, (2) physical, such as electroporation and injection, and (3) chemical, such as CaPO precipitation and oligonucleotide- lipid complexes.
• An antisense or sense oligonucleotide is inserted into a suitable gene transfer retroviral vector.
• a cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo.
• retroviral vectors examples include those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (WO 90/13641 , 1990).
• vector constructs in which the transcription of the anti-sense nucleic acid molecule is controlled by a strong pol II or pol III promoter are preferred.
• ligand-binding molecules As described in (WO 91/04753, 1991).
• Ligands are chosen for receptors that are specific to the target cells. Examples of suitable ligand-binding molecules include cell surface receptors, growth factors, cytokines, or other ligands that bind to cell surface receptors or molecules.
• conjugation of the ligand-binding molecule does not substantially interfere with the ability of the receptors or molecule to bind the ligand-binding molecule conjugate, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
• Liposomes efficiently transfer sense or an antisense oligonucleotide to cells (WO 90/10448, 1990).
• the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
• the anti-sense nucleic acid molecule of the invention may be an ⁇ - anomeric nucleic acid molecule.
• An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al., 1987).
• the anti-sense nucleic acid molecule can also comprise a 2'-o- methylribonucleotide (Inoue et al., 1987a) or a chimeric RNA-DNA analogue (Inoue et al., 1987b).
• an anti-sense nucleic acid of the invention is a ribozyme.
• Ribozymes are catalytic RNA molecules with ribonuclease activity that are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
• ribozymes such as hammerhead ribozymes (Haseloff and Gerlach, 1988) can be used to catalytically cleave IFI206 mRNA transcripts and thus inhibit translation.
• a ribozyme specific for an IFI206-encoding nucleic acid can be designed based on the nucleotide sequence of an IFI206 cDNA (i.e., SEQ ID NOS:1 or 3).
• a derivative of a Tetrahymena L-19 IVS RNA can be constructed in which the nucleotide sequence of the active site is complementary to the nucleotide sequence to be cleaved in an IFI206-encoding mRNA (Cech et al., U.S. Patent No. 5,116,742, 1992; Cech et al., U.S. Patent No. 4,987,071, 1991 ).
• IFI206 mRNA can also be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules (Bartel and Szostak, 1993).
• IFI206 expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region of the IFI206 (e.g., the IFI206 promoter and/or enhancers) to form triple helical structures that prevent transcription of the IFI206 in target cells (Helene, 1991 ; Helene et al., 1992; Maher, 1992).
• nucleotide sequences complementary to the regulatory region of the IFI206 e.g., the IFI206 promoter and/or enhancers
• Modifications of antisense and sense oligonucleotides can augment their effectiveness. Modified sugar-phosphodiester bonds or other sugar linkages (WO 91/06629, 1991), increase in vivo stability by conferring resistance to endogenous nucleases without disrupting binding specificity to target sequences. Other modifications can increase the affinities of the oligonucleotides for their targets, such as covalently linked organic moieties (WO 90/10448, 1990) or poly-(L)-lysine. Other attachments modify binding . specificities of the oligonucleotides for their targets, including metal complexes or intercalating (e.g. ellipticine) and alkylating agents.
• the deoxyribose phosphate backbone of the nucleic acids can be modified to generate peptide nucleic acids (Hyrup and Nielsen, 1996).
• Peptide nucleic acids or “PNAs” refer to nucleic acid mimics (e.g., DNA mimics) in that the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
• the neutral backbone of PNAs allows for specific hybridization to DNA and RNA under conditions of low ionic strength.
• the synthesis of PNA oligomers can be performed using standard solid phase peptide synthesis protocols
• PNAs of IFI206 can be used in therapeutic and diagnostic applications.
• PNAs can be used as anti-sense or antigene agents for sequence-specific modulation of gene expression by inducing transcription or* translation arrest or inhibiting replication.
• IFI206 PNAs may also be used in the analysis of single base pair mutations (e.g., PNA directed PCR clamping; as artificial restriction enzymes when used in combination with other enzymes, e.g., Si nucleases (Hyrup and Nielsen, 1996); or as probes or primers for DNA sequence and hybridization (Hyrup and Nielsen, 1996; Perry- O'Keefe et al., 1996).
• PNAs of IFI206 can be modified to enhance their stability or cellular uptake. Lipophilic or other helper groups may be attached to PNAs, PNA- DNA dimmers formed, or the use of liposomes or other drug delivery techniques.
• PNA-DNA chimeras can be generated that may combine the advantageous properties of PNA and DNA. Such chimeras allow DNA recognition enzymes (e.g., RNase H and DNA polymerases) to interact with the DNA portion while the PNA portion provides high binding affinity and specificity.
• PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup and Nielsen, 1996).
• PNA-DNA chimeras can be performed (Finn et al., 1996; Hyrup and Nielsen, 1996).
• a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry, and modified nucleoside analogs, e.g., 5'-(4-methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used between the PNA and the 5' end of DNA (Finn et al., 1996; Hyrup and Nielsen, 1996).
• PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment
• chimeric molecules can be synthesized with a 5' DNA segment and a 3' PNA segment (Petersen et al., 1976).
• the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (Lemaitre et al., 1987; Letsinger et al.,
• oligonucleotides can be modified with hybridization-triggered cleavage agents (van der Krol et al., 1988b) or intercalating agents (Zon, 1988).
• the oligonucleotide may be conjugated to another molecule, e.g., a peptide, a hybridization triggered cross-linking agent, a transport agent, a hybridization-triggered cleavage agent, and the like.
• IFI206 polypeptides One aspect of the invention pertains to isolated IFI206, and biologically-active portions derivatives, fragments, analogs or homologs thereof. Also provided are polypeptide fragments suitable for use as immunogens to raise anti-IFI206 Abs.
• native IFI206 can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
• IFI206 are produced by recombinant DNA techniques. Alternative to recombinant expression, an IFI206 or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
• An IFI206 polypeptide includes the amino acid sequence of IFI206 whose sequences are provided in SEQ ID NOS:2, 4 or 15.
• the invention also includes a mutant or variant protein any of whose residues may be changed from the corresponding residues shown in SEQ ID NOS:2, 4 or 15, while still encoding a protein that maintains its IFI206 activities and physiological functions, or a functional fragment thereof.
• an IFI206 variant that preserves IFI206-like function and includes any variant in which residues at a particular position in the sequence have been substituted by other amino acids, and further includes the possibility of inserting an additional residue or residues between two residues of the parent protein as well as the possibility of deleting one or more residues from the parent sequence. Any amino acid substitution, insertion, or deletion is encompassed by the invention. In favorable circumstances, the substitution is a conservative substitution as defined above.
• IFI206 polypeptide variant means an active IFI206 polypeptide having at least: (1 ) about 80% amino acid sequence identity with a full-length native sequence IFI206 polypeptide sequence, (2) a IFI206 polypeptide sequence lacking the signal peptide, (3) an extracellular domain of a IFI206 polypeptide, with or without the signal peptide, or (4) any other fragment of a full-length IFI206 polypeptide sequence.
• IFI206 polypeptide variants include IFI206 polypeptides wherein one or more amino acid residues are added or deleted at the N- or C- terminus of the full-length native amino acid sequence.
• a IFI206 polypeptide variant will have at least about 80% amino acid sequence identity, preferably at least about 81 % amino acid sequence identity, more preferably at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and most preferably at least about 99% amino acid sequence identity with a full-length native sequence IFI206 polypeptide sequence.
• a IFI206 polypeptide variant may have a sequence lacking the signal peptide, an extracellular domain of a IFI206 polypeptide, with or without the signal peptide, or any other fragment of a full-length IFI206 polypeptide sequence.
• IFI206 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids in length, or more.
• Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues that are identical with amino acid residues in the disclosed IFI206 polypeptide sequence in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art. Often publicly available computer software such as BLAST, BLAST2, ALIGN2 or
• % amino acid sequence ' identity of a given amino acid sequence A to, with, or against a given amino acid sequence B can be calculated as:
• the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
• Isolated/purified polypeptides An "isolated” or “purified” polypeptide, protein or biologically active fragment is separated and/or recovered from a component of its natural environment. Contaminant components include materials that would typically interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous - materials. Preferably, the polypeptide is purified to a sufficient degree to obtain at least 15 residues of N-terminal or internal amino acid sequence. To be substantially isolated, preparations having less than 30% by dry weight of non-IFI206 contaminating material (contaminants), more preferably less than 20%, 10% and most preferably less than 5% contaminants.
• contaminants non-IFI206 contaminating material
• An isolated, recombinantly-produced IFI206 or biologically active portion is preferably substantially free of culture medium, i.e., culture medium represents less than 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the IFI206 preparation.
• culture medium represents less than 20%, more preferably less than about 10%, and most preferably less than about 5% of the volume of the IFI206 preparation.
• contaminants include cell debris, culture media, and substances used and produced during in vitro synthesis of IFI206.
• Biologically active portions of IFI206 include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequences of the IFI206 (SEQ ID NOS:2 or 4) that include fewer amino acids than the full-length IFI206, and exhibit at least one activity of an IFI206.
• Biologically active portions comprise a domain or motif with at least one activity of native IFI206.
• a biologically active portion of an IFI206 can be a polypeptide that is, for example, 10, 25, 50, 100 or more amino acid residues in length.
• Other biologically active portions, in which other regions of the protein are deleted, can be prepared by recombinant techniques and evaluated for one or more of the functional activities of a native IFI206.
• Biologically active portions of IFI206 may have an amino acid sequence shown in SEQ ID NOS:2 or 4, or substantially homologous to SEQ ID NOS:2 or 4, and retains the functional activity of the protein of SEQ ID NOS:2 or 4, yet differs in amino acid sequence due to natural allelic variation or mutagenesis.
• Other biologically active IFI206 may comprise an amino acid sequence at least 45% homologous to the amino acid sequence of SEQ ID
• IFI206 variant means an active IFI206 having at least: (1) about 80% amino acid sequence identity with a full-length native sequence IFI206 sequence, (2) an IFI206 sequence lacking the signal peptide, (3) an extracellular domain of an IFI206, with or without the signal peptide, or (4) any other fragment of a full-length IFI206 sequence.
• IFI206 variants include IFI206 wherein one or more amino acid residues are added or deleted at the N- or C- terminus of the full-length native amino acid sequence.
• An IFI206 variant will have at least about 80% amino acid sequence identity, preferably at least about 81% amino acid sequence identity, more preferably at least about 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% amino acid sequence identity and most preferably at least about 99% amino acid sequence identity with a full-length native sequence IFI206 sequence.
• An IFI206 variant may have a sequence lacking the signal peptide, an extracellular domain of an IFI206, with or without the signal peptide, or any other fragment of a full-length IFI206 sequence.
• IFI206 variant polypeptides are at least about 10 amino acids in length, often at least about 20 amino acids in length, more often at least about 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, or 300 amino acids in length, or more.
• Percent (%) amino acid sequence identity is defined as the percentage of amino acid residues that are identical with amino acid residues in the disclosed IFI206 sequence in a candidate sequence when the two sequences are aligned. To determine % amino acid identity, sequences are aligned and if necessary, gaps are introduced to achieve the maximum % sequence identity; conservative substitutions are not considered as part of the sequence identity. Amino acid sequence alignment procedures to determine percent identity are well known to those of skill in the art.
• % amino acid sequence identity of a given amino acid sequence A to, with, or against a given amino acid sequence B can be calculated as:
• X is the number of amino acid residues scored as identical matches by the sequence alignment program's or algorithm's alignment of A and B and
• Y is the total number of amino acid residues in B. If the length of amino acid sequence A is not equal to the length of amino acid sequence B, the % amino acid sequence identity of A to B will not equal the % amino acid sequence identity of B to A.
• Chimeric and fusion proteins Fusion polypeptides are useful in expression studies, cell-localization, bioassays, and IFI206 purification.
• An IFI206 "chimeric protein" or “fusion protein” comprises IFI206 fused to a non-IFI206 polypeptide.
• a non-IFI206 polypeptide is not substantially homologous to IFI206 (SEQ ID NOS:2 or 4).
• An IFI206 fusion protein may include any portion to the entire IFI206, including any number of the biologically active portions.
• IFI206 may be fused to the C-terminus of the GST (glutathione S-transf erase) sequences.
• Such fusion proteins facilitate the purification of recombinant IFI206.
• heterologous signal sequences fusions may ameliorate IFI206 expression and/or secretion. Additional exemplary fusions are presented in Table C.
• IFI206 immunoglobulin
• Fusions with members of the immunoglobulin (Ig) protein family are useful in therapies that inhibit IFI206 ligand or substrate interactions, consequently suppressing IFI206-mediated signal transduction in vivo.
• Such fusions, incorporated into pharmaceutical compositions, may be used to treat proliferative and differentiation disorders, as well as modulating cell survival.
• IFI206-lg fusion polypeptides can also be used as immunogens to produce anti-IFI206 Abs in a subject, to purify IFI206 ligands, and to screen for molecules that inhibit interactions of IFI206 with other molecules.
• Fusion proteins can be easily created using recombinant methods.
• a nucleic acid encoding IFI206 can be fused in-frame with a non-IFI206 encoding nucleic acid, to the IFI206 NH 2 - or COO- -terminus, or internally. Fusion genes may also be synthesized by conventional techniques, including automated DNA synthesizers. PCR amplification using anchor primers that give rise to complementary overhangs between two consecutive gene fragments that can subsequently be annealed and reamplified to generate a chimeric gene sequence (Ausubel et al., 1987) is also useful. Many vectors are commercially available that facilitate sub-cloning IFI206 in-frame to a fusion moiety.
• Antagonist includes any molecule that partially or fully blocks, inhibits, or neutralizes a biological activity of endogenous IFI206.
• agonist includes any molecule that mimics a biological activity of endogenous IFI206.
• Molecules that can act as agonists or antagonists include Abs or antibody fragments, fragments or variants of endogenous IFI206, peptides, antisense oligonucleotides, small organic molecules, etc.
• IFI206 is added to, or expressed in, a cell along with the compound to be screened for a particular activity. If the compound inhibits the activity of interest in the presence of the IFI206, that compound is an antagonist to the IFI206; if IFI206 activity is enhanced, the compound is an agonist.
• Any molecule that alters IFI206 cellular effects is a candidate antagonist or agonist. Screening techniques well known to those skilled in the art can identify these molecules. Examples of antagonists and agonists include: (1) small organic and inorganic compounds, (2) small peptides, (3) Abs and derivatives, (4) polypeptides closely related to IFI206, (5) antisense DNA and RNA, (6) ribozymes, (7) triple DNA helices and (8) nucleic acid aptamers. Small molecules that bind to the IFI206 active site or other relevant part of the polypeptide and inhibit the biological activity of the IFI206 are antagonists. Examples of small molecule antagonists include small peptides, peptide-like molecules, preferably soluble, and synthetic non-peptidyl organic or inorganic compounds. These same molecules, if they enhance IFI206 activity, are examples of agonists.
• antibody antagonists include polyclonal, monoclonal, single-chain, anti-idiotypic, chimeric Abs, or humanized versions of such Abs or fragments. Abs may be from any species in which an immune response can be raised. Humanized Abs are also contemplated.
• a potential antagonist or agonist may be a closely related protein, for example, a mutated form of the IFI206 that recognizes an IFI206- interacting protein but imparts no effect, thereby competitively inhibiting IFI206 action.
• a mutated IFI206 may be constitutively activated and may act as an agonist.
• Antisense RNA or DNA constructs can be effective antagonists. Antisense RNA or DNA molecules block function by inhibiting translation by hybridizing to targeted mRNA. Antisense technology can be used to control gene expression through triple-helix formation or antisense DNA or RNA, both of which depend on polynucleotide binding to DNA or RNA. For example, the 5' coding portion of the IFI206 sequence is used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
• a DNA oligonucleotide is designed to be complementary to a region of the gene involved in transcription (triple helix) (Beal and Dervan, 1991 ; Cooney et al., 1988; Lee et al., 1979), thereby preventing transcription and the production of the IFI206.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into the IFI206 (antisense) (Cohen, 1989; Okano et al., 1991).
• These oligonucleotides can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the IFI206.
• antisense DNA is used, oligodeoxyribonucleotides derived from the translation-initiation site, e.g., between about -10 and +10 positions of the target gene nucleotide sequence, are preferred.
• Ribozymes are enzymatic RNA molecules capable of catalyzing the specific cleavage of RNA. Ribozymes act by sequence-specific hybridization to the complementary target RNA, followed by endonucleolytic cleavage. Specific ribozyme cleavage sites within a potential RNA target can be identified by known techniques (WO 97/33551 , 1997; Rossi, 1994). To inhibit transcription, triple-helix nucleic acids that are single- stranded and comprise deoxynucleotides are useful antagonists. These oligonucleotides are designed such that triple-helix formation via Hoogsteen base-pairing rules is promoted, generally requiring stretches of purines or pyrimidines (WO 97/33551 , 1997).
• an IFI206 activity may include nucleic acid binding, such as BAT mRNA
• molecules that compete for IFI206 nucleic acid binding site(s) can be effective intracellular competitors.
• Aptamers are short oligonucleotide sequences that can be used to recognize and specifically bind almost any molecule.
• the systematic evolution of ligands by exponential enrichment (SELEX) process (Ausubel et al., 1987; Ellington and Szostak, 1990; Tuerk and Gold, 1990) is powerful and can be used to find such aptamers.
• Aptamers have many diagnostic and clinical uses; almost any use in which an antibody has been used clinically or diagnostically, aptamers too may be used. In addition, are cheaper to make once they have been identified, and can be easily applied in a variety of formats, including administration in pharmaceutical compositions, in bioassays, and diagnostic tests (Jayasena,
• the invention encompasses Abs and antibody fragments, such as F ab ' or (F ab ) 2 , that bind immunospecifically to any IFI206 epitopes.
• Antibody comprises single Abs directed against IFI206 (anti- IFI206 Ab; including agonist, antagonist, and neutralizing Abs), anti-IFI206 Ab compositions with poly-epitope specificity, single chain anti-IFI206 Abs, and fragments of anti-IFI206 Abs.
• a "monoclonal antibody” is obtained from a population of substantially homogeneous Abs, i.e., the individual Abs comprising the population are identical except for possible naturally-occurring mutations that may be present in minor amounts.
• Exemplary Abs include polyclonal (pAb), monoclonal (mAb), humanized, bi-specific (bsAb), and heteroconjugate Abs.
• Polyclonal Abs can be raised in a mammalian host, for example, by one or more injections of an immunogen and, if desired, an adjuvant.
• the immunogen and/or adjuvant are injected in the mammal by multiple subcutaneous or intraperitoneal injections.
• the immunogen may include IFI206 or a fusion protein.
• adjuvants include Freund's complete and monophosphoryl Lipid A synthetic-trehalose dicorynomycolate (MPL-TDM).
• MPL-TDM monophosphoryl Lipid A synthetic-trehalose dicorynomycolate
• an immunogen may be conjugated to a protein that is immunogenic in the host, such as keyhole limpet hemocyanin (KLH), serum albumin, bovine thyroglobulin, and soybean trypsin inhibitor. Protocols for antibody production are described by (Ausubel et al., 1987; Harlow and Lane, 1988).
• KLH keyhole limpet hemocyanin
• serum albumin serum albumin
• bovine thyroglobulin bovine thyroglobulin
• soybean trypsin inhibitor Protocols for antibody production are described by (Aus
• Anti-IFI206 mAbs may be prepared using hybridoma methods (Milstein and Cuello, 1983). Hybridoma methods comprise at least four steps: (1 ) immunizing a host, or lymphocytes from a host; (2) harvesting the mAb secreting (or potentially secreting) lymphocytes, (3) fusing the lymphocytes to immortalized cells, and (4) selecting those cells that secrete the desired (anti- IFI206) mAb.
• lymphocytes A mouse, rat, guinea pig, hamster, or other appropriate host is immunized to elicit lymphocytes that produce or are capable of producing Abs that will specifically bind to the immunogen.
• the lymphocytes may be immunized in vitro.
• peripheral blood lymphocytes PBLs
• spleen cells or lymphocytes from other mammalian sources are preferred.
• the immunogen typically includes IFI206 or a fusion protein.
• the lymphocytes are then fused with an immortalized cell line to form hybridoma cells, facilitated by a fusing agent such as polyethylene glycol (Goding, 1996).
• a fusing agent such as polyethylene glycol
• Rodent, bovine, or human myeloma cells immortalized by transformation may be used, or rat or mouse myeloma cell lines.
• the cells after fusion are grown in a suitable medium that contains one or more substances that inhibit the growth or survival of unfused, immortalized cells.
• a common technique uses parental cells that lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT).
• hypoxanthine, aminopterin and thymidine are added to the medium (HAT medium) to prevent the growth of HGPRT-deficient cells while permitting hybridomas to grow.
• HAT medium a medium such as HAT
• Preferred immortalized cells fuse efficiently, can be isolated from mixed populations by selecting in a medium such as HAT, and support stable and high-level expression of antibody after fusion.
• Preferred immortalized cell lines are murine myeloma lines, available from the American Type Culture Collection (Manassas, VA). Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human mAbs (Kozbor et al., 1984; Schook, 1987).
• the culture media can be assayed for the presence of mAbs directed against IFI206 (anti- IFI206 mAbs).
• Immunoprecipitation or in vitro binding assays such as radio immunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA), measure the binding specificity of mAbs (Hariow and Lane, 1988; Hariow and Lane, 1999), including Scatchard analysis (Munson and Rodbard, 1980).
• Anti-IFI206 mAb secreting hybridoma cells may be isolated as single clones by limiting dilution procedures and sub-cultured (Goding, 1996). Suitable culture media include Dulbecco's Modified Eagle's Medium, RPMI-
• a protein-free or -reduced or serum-free medium e.g., Ultra DOMA PF or HL-1 ; Biowhittaker; Walkersville, MD.
• the hybridoma cells may also be grown in vivo as ascites.
• the mAbs may be isolated or purified from the culture medium or ascites fluid by conventional Ig purification procedures such as protein A- Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, ammonium sulfate precipitation or affinity chromatography (Hariow and Lane, 1988; Hariow and Lane, 1999).
• the mAbs may also be made by recombinant methods (U.S. Patent No. 4166452, 1979).
• DNA encoding anti-IFI206 mAbs can be readily isolated and sequenced using conventional procedures, e.g., using oligonucleotide probes that specifically bind to murine heavy and light antibody chain genes, to probe preferably DNA isolated from anti-IFI206-secreting mAb hybridoma cell lines. Once isolated, the isolated DNA fragments are sub-cloned into expression vectors that are then transfected into host cells such as simian COS-7 cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce Ig protein, to express mAbs.
• host cells such as simian COS-7 cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce Ig protein, to express mAbs.
• the isolated DNA fragments can be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains in place of the homologous murine sequences (U.S. Patent No. 4816567, 1989; Morrison et al., 1987), or by fusing the Ig coding sequence to all or part of the coding sequence for a non-lg polypeptide.
• a non-lg polypeptide can be substituted for the constant domains of an antibody, or can be substituted for the variable domains of one antigen-combining site to create a chimeric bivalent antibody.
• Monovalent Abs may be monovalent Abs that consequently do not cross-link with each other. For example, one method involves recombinant expression of Ig light chain and modified heavy chain.
• Abs can be digested to produce fragments, such as F ab fragments (Hariow and Lane, 1988; Hariow and Lane, 1999).
• Anti-IFI206 Abs may further comprise humanized or human Abs.
• Humanized forms of non-human Abs are chimeric Igs, Ig chains or fragments
• a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an
• “import" variable domain Humanization is accomplished by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody (Jones et al., 1986; Riechmann et al., 1988; Verhoey en et al., 1988).
• Such "humanized” Abs are chimeric Abs (U.S. Patent No. 4816567, 1989), wherein substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species.
• humanized Abs are typically human Abs in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent Abs.
• Humanized Abs include human Igs (recipient antibody) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit, having the desired specificity, affinity and capacity. In some instances, corresponding non-human residues replace F v framework residues of the human Ig. Humanized Abs may comprise residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. In general, the humanized antibody comprises substantially all of at least one, and typically two, variable domains, in which most if not all of the CDR regions correspond to those of a non- human Ig and most if not all of the FR regions are those of a human Ig consensus sequence.
• the humanized antibody optimally also comprises at least a portion of an Ig constant region (F c ), typically that of a human Ig (Jones et al., 1986; Presta, 1992; Riechmann et al., 1988).
• Human Abs can also be produced using various techniques, including phage display libraries (Hoogenboom et al., 1991 ; Marks et al., 1991) and the preparation of human mAbs (Boerner et al., 1991 ; Reisfeld and Sell, 1985).
• phage display libraries Hoogenboom et al., 1991 ; Marks et al., 1991
• human mAbs Boerner et al., 1991 ; Reisfeld and Sell, 1985.
• introducing human Ig genes into transgenic animals in which the endogenous Ig genes have been partially or completely inactivated can be exploited to synthesize human Abs.
• Bi-specific Abs are monoclonal, preferably human or humanized, that have binding specificities for at least two different antigens.
• a binding specificity is IFI206; the other is for any antigen of choice, preferably a cell-surface protein or receptor or receptor subunit.
• bi-specific Abs is based on the co-expression of two Ig heavy-chain/light-chain pairs, where the two heavy chains have different specificities (Milstein and Cuello, 1983). Because of the random assortment of Ig heavy and light chains, the resulting hybridomas (quadromas) produce a potential mixture of ten different antibody molecules, of which only one has the desired bi-specific structure.
• the desired antibody can be purified using affinity chromatography or other techniques (WO 93/08829, 1993; Traunecker et al., 1991).
• variable domains with the desired antibody-antigen combining sites are fused to Ig constant domain sequences.
• the fusion is preferably with an Ig heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions.
• the first heavy-chain constant region (CH1) containing the site necessary for light-chain binding is in at least one of the fusions.
• the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers that are recovered from recombinant cell culture (WO 96/27011 , 1996).
• the preferred interface comprises at least part of the CH3 region of an antibody constant domain.
• one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g. tyrosine or tryptophan).
• Compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule by replacing large amino acid side chains with smaller ones (e.g. alanine or threonine). This mechanism increases the yield of the heterodimer over unwanted end products such as homodimers.
• Bi-specific Abs can be prepared as full length Abs or antibody fragments (e.g. F (ab *) 2 bi-specific Abs).
• F (ab *) 2 bi-specific Abs One technique to generate bi-specific bi-specific antibodies
• Intact Abs exploits chemical linkage. Intact Abs can be proteolytically cleaved to generate F( ab *) 2 fragments (Brennan et al., 1985). Fragments are reduced with a dithiol complexing agent, such as sodium arsenite, to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated F ab ' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the F ab -TNB derivatives is then reconverted to the F a -thiol by reduction with mercaptoethylamine and is mixed with an equimolar amount of the other F ab - TNB derivative to form the bi-specific antibody.
• a dithiol complexing agent such as sodium arsenite
• the produced bi-specific Abs can be used as agents for the selective immobilization of enzymes.
• F ab * fragments may be directly recovered from E. coli and chemically coupled to form bi-specific Abs.
• Fully humanized bi-specific F(ab)2 Abs can be produced (Shalaby et al., 1992).
• Each F ab * fragment is separately secreted from E. coli and directly coupled chemically in vitro, forming the bi-specific antibody.
• Various techniques for making and isolating bi-specific antibody fragments directly from recombinant cell culture have also been described. For example, leucine zipper motifs can be exploited (Kostelny et al., 1992).
• Peptides from the Fos and Jun proteins are linked to the F ab ' portions of two different Abs by gene fusion.
• the antibody homodimers are reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also produce antibody homodimers.
• the "diabody” technology (Holliger et al., 1993) provides an alternative method to generate bi-specific antibody fragments.
• the fragments comprise a heavy- chain variable domain (V H ) connected to a light-chain variable domain (V ) by a linker that is too short to allow pairing between the two domains on the same chain.
• the V H and V L domains of one fragment are forced to pair with the complementary V L and V domains of another fragment, forming two antigen-binding sites.
• bi-specific antibody fragments Another strategy for making bi-specific antibody fragments is the use of single-chain F v (sF v ) dimers (Gruber et al., 1994). Abs with more than two valencies are also contemplated, such as tri-specific Abs (Tutt et al., 1991). Exemplary bi-specific Abs may bind to two different epitopes on a given
• an anti-IFI206 arm may be combined with an arm that binds to a leukocyte triggering molecule, such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7), or to F c receptors for IgG (F c ⁇ R), such as F c ⁇ RI (CD64), F c ⁇ RII (CD32) and F c ⁇ RIII (CD16).
• a leukocyte triggering molecule such as a T-cell receptor molecule (e.g. CD2, CD3, CD28, or B7)
• F c receptors for IgG F c ⁇ R
• Bispecific Abs may also be used to target cytotoxic agents to cells that express a particular IFI206.
• Heteroconjugate Abs consisting of two covalently joined Abs, have been proposed to target immune system cells to unwanted cells (4,676,980, 1987) and for treatment of human immunodeficiency virus (HIV) infection (WO 91/00360, 1991; WO 92/20373, 1992). Abs prepared in vitro using synthetic protein chemistry methods, including those involving cross-linking agents, are contemplated. For example, immunotoxins may be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents include iminothiolate and methyl-4-mercaptobutyrimidate (4,676,980, 1987).
• Immunoconjugates may comprise an antibody conjugated to a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin or fragment of bacterial, fungal, plant, or animal origin), or a radioactive isotope (i.e., a radioconjugate).
• a cytotoxic agent such as a chemotherapeutic agent, toxin (e.g., an enzymatically active toxin or fragment of bacterial, fungal, plant, or animal origin), or a radioactive isotope (i.e., a radioconjugate).
• Useful enzymatically-active toxins and fragments include Diphtheria A chain, non-binding active fragments of Diphtheria toxin, exotoxin A chain from ' Pseudomonas aeruginosa, ricin A chain, abrin A chain, modeccin A chain, ⁇ - sarcin, Aleurites fordii proteins, Dianthin proteins, Phytolaca americana proteins, Momordica charantia inhibitor, curcin, crotin, Sapaona a officinalis inhibitor, gelonin, mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes.
• a variety of radionuclides are available for the production of radioconjugated Abs, such as 212 Bi, 131 l, 131 ln, 90 Y, and 186 Re.
• Conjugates of the antibody and cytotoxic agent are made using a variety of bi-functional protein-coupling agents, such as N-succinimidyl-3-(2- pyridyldithiol) propionate (SPDP), iminothiolane (IT), bi-functional derivatives of imidoesters (such as dimethyl adipimidate HCl), active esters (such as disuccinimidyl suberate), aldehydes (such as glutareldehyde), b/s-azido compounds (such as bis (p-azidobenzoyl) hexanediamine), £>/s-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisqcyanates (such as tolyene 2,6- diisocyanate), and ⁇ /s-active fluorine compounds (such as 1 ,5-difluoro-2,4-dinitrobenzene).
• a ricin immunotoxin can be prepared (Vitetta et al., 1987).
• 14 C-labeled 1- isothiocyanatobenzyl-3-methyldiethylene triaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionuclide to antibody (WO 94/11026, 1994).
• the antibody may be conjugated to a "receptor” (such as streptavidin) for utilization in tumor pre-targeting wherein the antibody-receptor conjugate is administered to the patient, followed by removal of unbound conjugate from the circulation using a clearing agent and then administration of a streptavidin "ligand” (e.g., biotin) that is conjugated to a cytotoxic agent (e.g., a radionuclide).
• a streptavidin "ligand” e.g., biotin
• cytotoxic agent e.g., a radionuclide
• the antibody can be modified to enhance its effectiveness in treating a disease, such as cancer.
• cysteine residue(s) may be introduced into the F c region, thereby allowing interchain disulfide bond formation in this region.
• Such homodimeric Abs may have improved intemalization capability and/or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC) (Caron et al., 1992; Shopes, 1992).
• ADCC antibody-dependent cellular cytotoxicity
• Homodimeric Abs with enhanced anti-tumor activity can be prepared using hetero- bifunctional cross-linkers (Wolff et al., 1993).
• an antibody engineered with dual F c regions may have enhanced complement lysis (Stevenson et al., 1989).
• Liposomes containing the antibody may also be formulated (U.S.
• Useful liposomes can be generated by a reverse-phase evaporation method with a lipid composition comprising phosphatidylcholine, cholesterol, and PEG- derivatized phosphatidylethanolamine (PEG- PE). Such preparations are extruded through filters of defined pore size to yield liposomes with a desired diameter. F ab * fragments of the antibody can be conjugated to the liposomes (Martin and Papahadjopoulos, 1982) via a disulfide-interchange reaction. A chemotherapeutic agent, such as Doxorubicin, may also be contained in the liposome (Gabizon et al., 1989). Other useful liposomes with different compositions are contemplated.
• Anti-IFI206 Abs can be used to localize and/or quantitate IFI206 (e.g., for use in measuring levels of IFI206 within tissue samples or for use in diagnostic methods, etc.).
• Anti-IFI206 epitope Abs can be utilized as pharmacologically-active compounds.
• Anti-IFI206 Abs can be used to isolate IFI206 by standard techniques, such as immunoaffinity chromatography or immunoprecipitation. These approaches facilitate purifying endogenous IFI206 antigen-containing polypeptides from cells and tissues. These approaches, as well as others, can be used to detect IFI206 in a sample to evaluate the abundance and pattern of expression of the antigenic protein.
• Anti-IFI206 Abs can be used to monitor protein levels in tissues as part of a clinical testing procedure; for example, to determine the efficacy of a given treatment regimen. Coupling the antibody to a detectable substance (label) allows detection of Ab-antigen complexes. Classes of labels include fluorescent, luminescent, bioluminescent, and radioactive materials, enzymes and prosthetic groups.
• Useful labels include horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, acetylcholinesterase, streptavidin/biotin, avidin/biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride, phycoerythrin, luminol, luciferase, luciferin, aequorin, and 125 l, 131 l, 35 S or 3 H. 11.
• Antibody therapeutics include horseradish peroxidase, alkaline phosphatase, ⁇ - galactosidase, acetylcholinesterase, streptavidin/biotin, avidin/biotin, umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotria
• Abs of the invention can be used therapeutically. Such agents will generally be employed to treat or prevent a disease or pathology in a subject.
• An antibody preparation preferably one having high antigen specificity and affinity generally mediates an effect by binding the target epitope(s).
• administration of such Abs may mediate one of two effects: (1) the antibody may prevent ligand binding, eliminating endogenous ligand binding and subsequent signal transduction, or (2) the antibody elicits a physiological result by binding an effector site on the target molecule, initiating signal transduction.
• a therapeutically effective amount of an antibody relates generally to the amount needed to achieve a therapeutic objective, epitope binding affinity, administration rate, and depletion rate of the antibody from a subject.
• Common ranges for therapeutically effective doses may be, as a nonlimiting . example, from about 0.1 mg/kg body weight to about 50 mg/kg body weight. Dosing frequencies may range, for example, from twice daily to once a week.
• Anti-IFI206 Abs as well as other IFI206 interacting molecules (such as aptamers) identified in other assays, can be administered in pharmaceutical compositions to treat various disorders. Principles and considerations involved in preparing such compositions, as well as guidance in the choice of components can be found in (de Boer, 1994; Gennaro, 2000; Lee, 1990).
• Liposomes may also be used as a delivery vehicle for intracellular introduction. Where antibody fragments are used, the smallest inhibitory fragment that specifically binds to the epitope is preferred.
• peptide molecules can be designed that bind a preferred epitope based on the variable-region sequences of a useful antibody. Such peptides can be synthesized chemically and/or produced by recombinant DNA technology (Marasco et al., 1993).
• Formulations may also contain more than one active compound for a particular treatment, preferably those with activities that do not adversely affect each other.
• the composition may comprise an agent that enhances function, such as a cytotoxic agent, cytokine, chemotherapeutic agent, or growth-inhibitory agent.
• the active ingredients can also be entrapped in microcapsules prepared by coacervation techniques or by interfacial polymerization; for example, hydroxymethylcellulose or gelatin-microcapsules and poly- (methylmethacrylate) microcapsules, respectively, in colloidal drug delivery systems (for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules) or in macroemulsions.
• colloidal drug delivery systems for example, liposomes, albumin microspheres, microemulsions, nano-particles, and nanocapsules
• the formulations to be used for in vivo administration are highly preferred to be sterile. This is readily accomplished by filtration through sterile filtration membranes or any of a number of techniques.
• Sustained-release preparations may also be prepared, such as semi- permeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g., films, or microcapsules.
• sustained-release matrices include polyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)), polylactides (Boswell and Scribner, U.S. Patent No.
• copolymers of L-glutamic acid and ⁇ ethyl-L-glutamate non- degradable ethylene-vinyl acetate
• degradable lactic acid-glycolic acid copolymers such as injectable microspheres composed of lactic acid-glycolic acid copolymer
• poly-D-(-)-3-hydroxybutyric acid While polymers such as ethylene-vinyl acetate and lactic acid-glycolic acid enable release of molecules for over 100 days, certain hydrogels release proteins for shorter time periods and may be preferred.
• IFI206 recombinant expression vectors and host cells
• Vectors are tools used to shuttle DNA between host cells or as a means to express a nucleotide sequence. Some vectors function only in prokaryotes, while others function in both prokaryotes and eukaryotes, enabling large-scale DNA preparation from prokaryotes for expression in eukaryotes. Inserting the DNA of interest, such as IFI206 nucleotide sequence or a fragment, is accomplished by ligation techniques and/or mating protocols well-known to the skilled artisan. Such DNA is inserted such that its integration does not disrupt any necessary components of the vector. In the case of vectors that are used to express the inserted DNA protein, the introduced DNA is operably-linked to the vector elements that govern its transcription and translation.
• Vectors can be divided into two general classes: Cloning vectors are replicating plasmid or phage with regions that are non-essential for propagation in an appropriate host cell, and into which foreign DNA can be inserted; the foreign DNA is replicated and propagated as if it were a component of the vector.
• An expression vector (such as a plasmid, yeast, or animal virus genome) is used to introduce foreign genetic material into a host cell or tissue in order to transcribe and translate the foreign DNA.
• the introduced DNA is operably-linked to elements, such as promoters, that signal to the host cell to transcribe the inserted DNA.
• Some promoters are exceptionally useful, such as inducible promoters that control gene transcription in response to specific factors.
• Operably-linking IFI206 or anti-sense construct to an inducible promoter can control the expression of IFI206 or fragments, or anti-sense constructs.
• Examples of classic inducible promoters include those that are responsive to ⁇ -interferon, heat-shock, heavy metal ions, and steroids such as glucocorticoids (Kaufman, 1990) and tetracycline.
• Other desirable inducible promoters include those that are not endogenous to the cells in which the construct is being introduced, but, however, is responsive in those cells when the induction agent is exogenously supplied.
• Vectors have many difference manifestations.
• a "plasmid” is a circular double stranded DNA molecule into which additional DNA segments can be introduced.
• Viral vectors can accept additional DNA segments into the viral genome.
• Certain vectors are capable of autonomous replication in a host cell (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors).
• Other vectors e.g., non-episomal mammalian vectors
• useful expression vectors are often plasmids.
• expression vectors such as viral vectors (e.g., replication defective retrovi ruses, adenoviruses and adeno-associated viruses) are contemplated.
• "Operably-linked" indicates that a nucleotide sequence of interest is linked to regulatory sequences such that expression of the nucleotide sequence is achieved.
• Vectors can be introduced in a variety of organisms and/or cells (Table D). Alternatively, the vectors can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase. Table D Examples of hosts for cloning or expression
• Vector choice is dictated by the organism or cells being used and the desired fate of the vector.
• Vectors may replicate once in the target cells, or may be "suicide" vectors.
• vectors comprise signal sequences, origins of replication, marker genes, enhancer elements, promoters, and transcription termination sequences. The choice of these elements depends on the organisms in which the vector will be used and are easily determined. Some of these elements may be conditional, such as an inducible or conditional promoter that is turned “on” when conditions are appropriate. Examples of inducible promoters include those that are tissue-specific, which relegate expression to certain cell types, steroid-responsive, or heat-shock reactive.
• Vectors often use a selectable marker to facilitate identifying those cells that have incorporated the vector. Many selectable markers are well known in the art for the use with prokaryotes, usually antibiotic-resistance genes or the use of autotrophy and auxotrophy mutants.
• antisense and sense IFI206 oligonucleotides can prevent IFI206- polypeptide expression. These oligonucleotides bind to target nucleic acid sequences, forming duplexes that block transcription or translation of the target sequence by enhancing degradation of the duplexes, terminating prematurely transcription or translation, or by other means.
• Antisense or sense oligonucleotides are singe-stranded nucleic acids, either RNA or DNA, which can bind target IFI206 mRNA (sense) or IFI206 DNA (antisense) sequences.
• antisense or sense oligonucleotides comprise a fragment of the IFI206 DNA coding region of at least about 14 nucleotides, preferably from about 14 to 30 nucleotides.
• antisense RNA or DNA molecules can comprise at least 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 bases in length or more.
• Step and Cohen, 1988; van der Krol et al. are singe-stranded nucleic acids, either RNA or DNA, which can bind target IFI206 mRNA (sense) or IFI206 DNA (antisense) sequences.
• antisense or sense oligonucleotides comprise a fragment of the IFI206 DNA coding
• Modifications of antisense and sense oligonucleotides can augment their effectiveness. Modified sugar-phosphodiester bonds or other sugar linkages (WO 91/06629, 1991), increase in vivo stability by conferring resistance to endogenous nucleases without disrupting binding specificity to target sequences. Other modifications can increase the affinities of the oligonucleotides for their targets, such as covalently linked organic moieties (WO 90/10448, 1990) or poly-(L)-lysine. Other attachments modify binding specificities of the oligonucleotides for their targets, including metal complexes or intercalating (e.g. ellipticine) and alkylating agents.
• any gene transfer method may be used and are well known to those of skill in the art.
• gene transfer methods include 1 ) biological, such as gene transfer vectors like
• Epstein-Barr virus or conjugating the exogenous DNA to a ligand-binding molecule (WO 91/04753, 1991), 2) physical, such as electroporation, and 3) chemical, such as CaPO 4 precipitation and oligonucleotide-lipid complexes (WO 90/10448, 1990).
• host cell and "recombinant host cell” are used interchangeably. Such terms refer not only to a particular subject cell but also to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term.
• Vectors often use a selectable marker to facilitate identifying those cells that have incorporated the vector.
• selectable markers are well known in the art for the use with prokaryotes, usually antibiotic-resistance genes or the use of autotrophy and auxotrophy mutants.
• Table F lists often- used selectable markers for mammalian cell transfection.
• a host cell of the invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce IFI206. Accordingly, the invention provides methods for producing IFI206 using the host cells of the invention. In one embodiment, the method comprises culturing the host cell of the invention (into which a recombinant expression vector encoding IFI206 has been introduced) in a suitable medium, such that IFI206 is produced. In another embodiment, the method further comprises isolating IFI206 from the medium or the host cell.
• Transgenic animals are useful for studying the function and/or activity of IFI206 and for identifying and/or evaluating modulators of IFI206 activity.
• Transgenic animals are non-human animals, preferably mammals, more preferably a rodents such as rats or mice, in which one or more of the cells include a transgene. Other transgenic animals include primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
• a "transgene” is exogenous DNA that is integrated into the genome of a cell from which a transgenic animal develops, and that remains in the genome of the mature animal.
• Transgenes preferably direct the expression of an encoded gene product in one or more cell types or tissues of the transgenic animal with the purpose of preventing expression of a naturally encoded gene product in one or more cell types or tissues (a "knockout" transgenic animal), or serving as a marker or indicator of an integration, chromosomal location, or region of recombination (e.g. cre/loxP mice).
• a "homologous recombinant animal” is a non-human animal, such as a rodent, in which endogenous IFI206 has been altered by an exogenous DNA molecule that recombines homologously with endogerious IFI206 in a (e.g. embryonic) cell prior to development the animal.
• Host cells with exogenous IFI206 can be used to produce non-human transgenic animals, such as fertilized oocytes or embryonic stem cells into which IFI206- coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals or homologous recombinant animals.
• a transgenic animal can be created by introducing IFI206 into the male pronuclei of a fertilized oocyte (e.g., by microinjection, retroviral infection) and allowing the oocyte to develop in a pseudopregnant female foster animal (pffa).
• the IFI206 cDNA sequences (SEQ ID NO: 1) can be introduced as a transgene into the genome of a non-human animal.
• a homologue of IFI206 such as the naturally-occuring variant of IFI206 (SEQ ID NO:3), can be used as a transgene.
• Intronic sequences and polyadenylation signals can also be included in the transgene to increase transgene expression.
• Tissue-specific regulatory sequences can be operably-linked to the IFI206 transgene to direct expression of IFI206 to particular cells.
• Methods for generating transgenic animals via embryo manipulation and microinjection, particularly animals such as mice, have become conventional in the art, e.g. (Evans et al., U.S. Patent No. 4,870,009, 1989; Hogan, 0879693843, 1994; Leder and Stewart, U.S. Patent No. 4,736,866, 1988;
• transgenic founder animal which can be used to breed additional transgenic animals, can be identified based upon the presence of the transgene in its genome and/or expression of the transgene mRNA in tissues or cells of the animals.
• Transgenic animals can be bred to other transgenic animals carrying other transgenes.
• IFI206 can be a murine gene (SEQ ID NO:1), or other IFI206 homologue, such as the naturally occurring variant (SEQ ID NO:3).
• a knockout vector functionally disrupts the endogenous IFI206 gene upon homologous recombination, and thus a non-functional IFI206 protein, if any, is expressed.
• the vector can be designed such that, upon homologous recombination, the endogenous IFI206 is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression of endogenous IFI206).
• the altered portion of the IFI206 is flanked at its 5'- and 3'-termini by additional nucleic acid of the IFI206 to allow for homologous recombination to occur between the exogenous IFI206 carried by the vector and an endogenous IFI206 in an embryonic stem cell.
• the additional flanking IFI206 nucleic acid is sufficient to engender homologous . recombination with endogenous IFI206.
• flanking DNA both at the 5'- and 3'-termini
• the vector is then introduced into an embryonic stem cell line (e.g., by electroporation), and cells in which the introduced IFI206 has homologously-recombined with the endogenous IFI206 are selected (Li et al., 1992).
• IFI206 transgene cells during development Selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (Bradley, 1987). A chimeric embryo can then be implanted into a suitable pffa and the embryo brought to term. Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene.
• an animal e.g., a mouse
• a chimeric embryo can then be implanted into a suitable pffa and the embryo brought to term.
• Progeny harboring the homologously-recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously-recombined DNA by germline transmission of the transgene.
• transgenic animals that contain selected systems that allow for regulated expression of the transgene can be produced.
• An example of such a system is the cre/loxP recombinase system of bacteriophage P1 (Lakso et al., 1992).
• Another recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al., 1991). If a cre/loxP recombinase system is used to regulate expression of the transgene, animals containing transgenes encoding both the Cre recombinase and a selected protein are required. Such animals can be produced as "double" transgenic animals, by mating an animal containing a transgene encoding a selected protein to another containing a transgene encoding a recombinase.
• Clones of transgenic animals can also be produced (Wilmut et al., 1997).
• a cell from a transgenic animal can be isolated and induced to exit the growth cycle and enter Go phase.
• the quiescent cell can then be fused to an enucleated oocyte from an animal of the same species from which the quiescent cell is isolated.
• the reconstructed oocyte is then cultured to develop to a morula or blastocyte and then transferred to a pffa.
• the offspring borne of this female foster animal will be a clone of the "parent" transgenic animal.
• compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
• pharmaceutically acceptable carrier includes any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like, compatible with pharmaceutical administration
• compositions include, but are not limited to, water, saline, finger's solutions, dextrose solution, and 5% human serum albumin. Liposomes and non-aqueous vehicles such as fixed oils may also be used. Except when a conventional media or agent is incompatible with an active compound, use of these compositions is contemplated. Supplementary active compounds can also be incorporated into the compositions. 1. General considerations
• a pharmaceutical composition of the invention is formulated to be compatible with its intended route of administration, including intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (i.e., topical), transmucosal, and rectal administration.
• Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid (EDTA); buffers such as acetates, citrates or phosphates, and agents for the adjustment of tonicity such as sodium chloride or dextrose.
• the pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
• the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
• compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
• suitable carriers include physiological saline, bacteriostatic water, CREMOPHOR EL TM (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS).
• the composition must be sterile and should be fluid so as to be administered using a syringe.
• Such compositions should be stable during manufacture and storage and must be preserved against contamination from microorganisms such as bacteria and fungi.
• the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (such as glycerol, propylene glycol, and liquid polyethylene glycol), and suitable mixtures.
• Proper fluidity can be maintained, for example, by using a coating such as lecithin, by maintaining the required particle size in the case of dispersion and by using surfactants.
• Various antibacterial and antifungal agents for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal, can contain microorganism contamination.
• Isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, and sodium chloride can be included in the composition.
• Compositions that can delay absorption include agents such as aluminum monostearate and gelatin.
• Sterile injectable solutions can be prepared by incorporating the active compound (e.g., an IFI206 or anti-IFI206 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients as required, followed by sterilization.
• the active compound e.g., an IFI206 or anti-IFI206 antibody
• dispersions are prepared by incorporating the active compound into a sterile vehicle that contains a basic dispersion medium, and the other required ingredients as discussed.
• Sterile powders for the preparation of sterile injectable solutions, methods of preparation include vacuum drying and freeze-drying that yield a powder containing the active ingredient and any desired ingredient from a sterile solutions. 3.
• Oral compositions e.g., an IFI206 or anti-IFI206 antibody
• Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients arid used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included.
• Tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch; a lubricant such as magnesium stearate or STEROTES; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
• a binder such as microcrystalline cellulose, gum tragacanth or gelatin
• an excipient such as starch or lactose, a disintegrating agent such as alginic acid, PRIMOGEL, or corn starch
• a lubricant such as magnesium stearate or STEROTES
• a glidant such as colloidal silicon dioxide
• compositions for inhalation For administration by inhalation, the compounds are delivered as an aerosol spray from a a nebulizer or a pressurized container that contains a suitable propellant, e.g., a gas such as carbon dioxide.
• a suitable propellant e.g., a gas such as carbon dioxide.
• Systemic administration can also be transmucosal or transdermal.
• penetrants that can permeate the target barrier(s) are selected.
• Transmucosal penetrants include, detergents, bile salts, and fusidic acid derivatives.
• Nasal sprays or suppositories can be used for transmucosal administration.
• the active compounds are formulated into ointments, salves, gels, or creams.
• the compounds can also be prepared in the form of suppositories (e.g., with bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
• suppositories e.g., with bases such as cocoa butter and other glycerides
• retention enemas for rectal delivery.
• the active compounds are prepared with carriers that protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
• a controlled release formulation including implants and microencapsulated delivery systems.
• Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Such materials can be obtained commercially from ALZA Corporation (Mountain View, CA) and NOVA Pharmaceuticals, Inc. (Lake Elsinore, CA), or prepared by one of skill in the art.
• Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, such as in (Eppstein et al., US Patent No. 4,522,81 1 , ' 1985).
• Unit dosage form refers to physically discrete units suited as single dosages for the subject to be treated, containing a therapeutically effective quantity of active compound in association with the required pharmaceutical carrier.
• the specification for the unit dosage forms of the invention are dictated by, and directly dependent on, the unique characteristics of the active compound and the particular desired therapeutic effect, and the inherent limitations of compounding the active compound.
• Gene therapy compositions The nucleic acid molecules of the invention can be inserted into vectors and used as gene therapy vectors.
• Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (Nabel and Nabel, US Patent No. 5,328,470, 1994), or by stereotactic injection (Chen et al., 1994).
• the pharmaceutical preparation of a gene therapy vector can include an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
• the pharmaceutical preparation can include one or more cells that produce the gene delivery system.
• compositions can be included in a kit, container, pack, or dispenser together with instructions for administration.
• the different components of the composition may be packaged in separate containers and admixed immediately before use. Such packaging of the components separately may permit long-term storage without losing the active components' functions.
• Kits may also include reagents in separate containers that facilitate the * execution of a specific test, such as diagnostic tests or tissue typing.
• reagents in separate containers that facilitate the * execution of a specific test, such as diagnostic tests or tissue typing.
• IFI206 DNA templates and suitable primers may be supplied for internal controls.
• the reagents included in the kits can be supplied in containers of any sort such that the life of the different components are preserved, and are not adsorbed or altered by the materials of the container.
• sealed glass ampules may contain lyophilized luciferase or buffer that have been packaged under a neutral, non-reacting gas, such as nitrogen.
• Ampoules may consist of any suitable material, such as glass, organic polymers, such as polycarbonate, polystyrene, etc., ceramic, metal or any other material typically employed to hold reagents.
• suitable containers include simple bottles that may be fabricated from similar substances as ampules, and envelopes, that may consist of foil-lined interiors, such as aluminum or an alloy.
• Containers include test tubes, vials, flasks, bottles, syringes, or the like.
• Containers may have a sterile access port, such as a bottle having a stopper that can be pierced by a hypodermic injection needle.
• Other containers may have two compartments that are separated by a readily removable membrane that upon removal permits the components to mix.
• Removable membranes may be glass, plastic, rubber, etc.
• Kits may also be supplied with instructional materials. Instructions may be printed on paper or other substrate, and/or may be supplied as an electronic-readable medium, such as a floppy disc, CD-ROM, DVD-ROM, Zip disc, video tape, audio tape, etc. Detailed instructions may not be physically associated with the kit; instead, a user may be directed to an internet web site specified by the manufacturer or distributor of the kit, or supplied as electronic mail.
• the isolated nucleic acid molecules of the invention can be used to express IFI206 (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect IFI206 mRNA (e.g., in a biological sample) or a genetic lesion in an IFI206, and to modulate IFI206 activity, as described below.
• IFI206 polypeptides can be used to screen drugs or compounds that modulate the IFI206 activity or expression as well as to treat disorders characterized by insufficient or excessive production of IFI206 or production of IFI206 forms that have decreased or aberrant activity compared to IFI206 wild-type protein, or modulate biological function that involve IFI206 (e.g. obesity).
• the anti-IFI206 Abs of the invention can be used to detect and isolate IFI206 and modulate IFI206 activity.
• Screening assays The invention provides a method (screening assay) for identifying modalities, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drugs), foods, combinations thereof, etc., that effect IFI206, a stimulatory or inhibitory effect, inlcuding translation, transcription, activity or copies of the gene in cells.
• the invention also includes compounds identified in screening assays.
• a compound may modulate IFI206 activity by affecting: (1) the number of copies of the gene in the cell (amplifiers and deamplifiers); (2) increasing or decreasing transcription of the IFI206 (transcription up- regulators and down-regulators); (3) by increasing or decreasing the translation of IFI206 mRNA into protein (translation up-regulators and down- regulators); or (4) by increasing or decreasing the activity of IFI206 itself (agonists and antagonists). (a) effects of compounds
• RNA or protein is assessed (Ausubel et al., 1987).
• DNA amplifiers and deamplifiers the amount of IFI206 DNA is measured, for those compounds that are transcription up-regulators and down-regulators the amount of IFI206 mRNA is determined; for translational up- and down-regulators, the amount of IFI206 polypeptides is measured.
• Compounds that are agonists or antagonists may be identified by contacting cells or organisms with the compound, and then measuring, for example, adipocyte differentiation in vitro.
• Ttest compounds can be obtained using any of the numerous approaches in combinatorial library methods, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
• biological libraries include: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the "one-bead one-compound” library method; and synthetic library methods using affinity chromatography selection.
• the biological library approach is limited to peptides, while the other four approaches encompass peptide, non-peptide oligomer or small molecule libraries of compounds (Lam, 1997).
• small molecules refers to a composition that has a molecular weight of less than about 5 kD and most preferably less than about 4 kD. Small molecules can be, nucleic acids, peptides, polypeptides, peptidomimetics, carbohydrates, lipids or other organic or inorganic molecules. Libraries of chemical and/or biological mixtures, such as fungal, bacterial, or algal extracts, are known in the art and can be screened with any of the assays of the invention.
• Libraries of compounds may be presented in solution (Houghten et al., 1992) or on beads (Lam et al., 1991), on chips (Fodor et al., 1993), bacteria, spores (Ladner et al., US Patent No. 5,223,409, 1993), plasmids (Cull et al., 1992) or on phage (Cwirla et al., 1990; Devlin et al., 1990; Felici et al., 1991 ; Ladner et al., US Patent No. 5,223,409, 1993; Scott and Smith, 1990).
• a cell-free assay comprises contacting IFI206 or biologically-active fragment with a known compound that binds IFI206 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability of the test compound to interact with IFI206, where determining the ability of the test compound to interact with IFI206 comprises determining the ability of the IFI206 to preferentially bind to or modulate the activity of an IFI206 target molecule.
• the cell-free assays of the invention may be used with both soluble or a membrane-bound forms of IFI206.
• a solubilizing agent to maintain IFI206 in solution.
• solubilizing agents include non-ionic detergents such as n-octylglucoside, n-dodecylglucoside, n-dodecylmaltoside, octanoyl- N-methylglucamide, decanoyl-N-methylglucamide, TRITON ® X-100 and others from the TRITON ® series, THESIT ® , lsotridecypoly(ethylene glycol ether) n , N-dodecyl-N,N-dimethyl-3-ammonio-1 -propane sulfonate, 3-(3- cholamidopropyl) dimethylamminiol-1 -propane s
• immobilizing either IFI206 or its partner molecules can facilitate separation of complexed from uncomplexed forms of one or both of the proteins, as well as to accommodate high throughput assays.
• Binding of a test compound to IFI206, or interaction of IFI206 with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants, such as microtiter plates, test tubes, and micro-centrifuge tubes.
• a fusion protein can be provided that adds a domain that allows one or both of the proteins to be bound to a matrix. For example,
• GST-IFI206 fusion proteins or GST-target fusion proteins can be adsorbed onto glutathione sepharose beads (SIGMA Chemical, St. Louis, MO) or glutathione derivatized microtiter plates that are then combined with the test compound or the test compound and either the non-adsorbed target protein or IFI206, and the mixture is incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH). Following incubation, the beads or microtiter plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described. Alternatively, the complexes can be dissociated from the matrix, and the level of IFI206 binding or activity determined using standard techniques.
• glutathione sepharose beads SIGMA Chemical, St. Louis, MO
• glutathione derivatized microtiter plates that are then combined with the test compound or the test compound and either the non-adsorbed target protein or I
• IFI206 or its target molecule can be immobilized using biotin-avidin or biotin-streptavidin systems. Biotinylation can be accomplished using many reagents, such as biotin-NHS
• Methods for detecting such complexes include immunodetection of complexes using Abs reactive with IFI206 or its target, as well as enzyme-linked assays that rely on detecting an enzymatic activity associated with the IFI206 or target molecule, (e) screens to identify modulators
• Modulators of IFI206 expression can be identified in a method where a cell is contacted with a candidate compound and the expression of IFI206 mRNA or protein in the cell is determined. The expression level of IFI206 mRNA or protein in the presence of the candidate compound is compared to IFI206 mRNA or protein levels in the absence of the candidate compound.
• the candidate compound can then be identified as a modulator of IFI206 mRNA or protein expression based upon this comparison. For example, when expression of IFI206 mRNA or protein is greater (i.e., statistically significant) in the presence of the candidate compound than in its absence, the candidate compound is identified as a stimulator of IFI206 mRNA or protein expression. Alternatively, when expression of IFI206 mRNA or protein is less (statistically significant) in the presence of the candidate compound than in its absence, the candidate compound is identified as an inhibitor of IFI206 mRNA or protein expression.
• the level of IFI206 mRNA or protein expression in the cells can be determined by methods described for detecting IFI206 mRNA or protein.
• IFI206 can be used as "bait" in two-hybrid or three hybrid assays [Saifer, 1994 #38; Zervos, 1993 #382; Madura, 1993 #383; Bartel, 1993 #384; Iwabuchi, 1993 #385; Brent, 1994 #386] to identify other proteins that bind or interact with IFI206 (FI206-binding proteins (IFI206-bps)) and modulate IFI206 activity.
• IFI206-bps FI206-binding proteins
• the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains.
• the assay utilizes two different DNA constructs.
• the gene that codes for IFI206 is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL4).
• a DNA sequence from a library of DNA sequences that encodes an unidentified protein (“prey" or "sample” is fused to a gene that codes for the activation domain of the known transcription factor.
• the DNA-binding and activation domains of the transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) that is operably-linked to a transcriptional regulatory site responsive to the transcription factor. Expression of the reporter gene can be detected, and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene that encodes the IFI206- interacting protein.
• a reporter gene e.g., LacZ
• the invention further pertains to novel agents identified by the aforementioned screening assays and uses thereof for treatments as described herein.
• IFI206 cDNA sequences identified herein are useful in themselves.
• these sequences can be used to: (1) identify an individual from a minute biological sample (tissue typing); and (2) aid in forensic identification of a biological sample, (a) Tissue typing
• the IFI206 sequences of the invention can be used to identify individuals from minute biological samples.
• an individual's genomic DNA is digested with one or more restriction enzymes and probed on a Southern blot to yield unique bands.
• the sequences of the invention are useful as additional DNA markers for "restriction fragment length polymorphisms" (RFLP; (Smulson et al., US Patent No. 5,272,057, 1993)).
• the IFI206 sequences can be used to determine the actual base-by-base DNA sequence of targeted portions of an individual's genome. IFI206 sequences can be used to prepare two PCR primers from the 5'- and 3'-termini of the sequences that can then be used to amplify an the corresponding sequences from an individual's genome and then sequence the amplified fragment. Panels of corresponding DNA sequences from individuals can provide • unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences. The sequences of the invention can be used to obtain such identification sequences from individuals and from tissue. The IFI206 sequences of the invention uniquely represent portions of an individual's genome.
• allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions.
• the allelic variation between individual humans occurs with a frequency of about once ever 500 bases.
• Much of the allelic variation is due to single nucleotide polymorphisms (SNPs), which include RFLPs.
• SNPs single nucleotide polymorphisms
• Each of the sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification purposes. Because greater numbers of polymorphisms occur in noncoding regions, fewer sequences are necessary to differentiate individuals.
• Noncoding sequences can positively identify individuals with a panel of 10 to 1 ,000 primers that each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NOS:1 or 3 are used, a more appropriate number of primers for positive individual identification would be 500-2,000.
• the invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, pharmacogenomics, and monitoring clinical trials are used for prognostic (predictive) purposes to treat an individual prophylactically. Accordingly, one aspect of the invention relates to diagnostic assays for determining IFI206 and/or nucleic acid expression as well as IFI206 activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant IFI206 expression or activity, including obesity.
• a biological sample e.g., blood, serum, cells, tissue
• Pharmacogenomics allows for the selection of modalities (e.g., drugs, foods) for therapeutic or prophylactic treatment of an individual based on the individual's genotype (e.g., the individual's genotype to determine the individual's ability to respond to a particular agent).
• Another aspect of the invention pertains to monitoring the influence of modalities (e.g., drugs, foods) on the expression or activity of IFI206 in clinical trials.
• An exemplary method for detecting the presence or absence of IFI206 in a biological sample involves obtaining a biological sample from a subject and contacting the biological sample with a compound or an agent capable of detecting IFI206 or IFI206 nucleic acid (e.g., mRNA, genomic DNA) such that the presence of IFI206 is confirmed in the sample.
• a compound or an agent capable of detecting IFI206 or IFI206 nucleic acid e.g., mRNA, genomic DNA
• IFI206 mRNA or genomic DNA is a labeled nucleic acid probe that can hybridize to IFI206 mRNA or genomic DNA.
• the nucleic acid probe can be, for example, a full-length IFI206 nucleic acid, such as the nucleic acid of SEQ ID NOS: 1 or 3, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to IFI206 mRNA or genomic DNA.
• An agent for detecting IFI206 polypeptide is an antibody capable of binding to IFI206, preferably an antibody with a detectable label. Abs can be polyclonal, or more preferably, monoclonal.
• An intact antibody, or a fragment e.g., F ab or F(ab') 2
• a labeled probe or antibody is coupled (i.e., physically linking) to a detectable substance, as well as indirect detection of the probe or antibody by reactivity with another reagent that is directly labeled.
• Examples of indirect labeling include detection of a primary antibody using a fluorescently-labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently-labeled streptavidin.
• biological sample includes tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject.
• the detection method of the invention can be used to detect IFI206 mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo.
• in vitro techniques for detection of IFI206 mRNA include Northern hybridizations and in situ hybridizations.
• In vitro techniques for detection of IFI206 polypeptide include enzyme linked immunosorbent assays
• IFI206 In vitro techniques for detection of IFI206 genomic DNA include Southern hybridizations and fluorescence in situ hybridization (FISH). Furthermore, in vivo techniques for detecting IFI206 include introducing into a subject a labeled anti-IFI206 antibody.
• the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
• the biological sample from the subject contains protein molecules, and/or mRNA molecules, and/or genomic DNA molecules.
• a preferred biological sample is blood.
• the methods further involve obtaining a biological sample from a subject to provide a control, contacting the sample with a compound or agent to detect IFI206, mRNA, or genomic DNA, and comparing the presence of IFI206, mRNA or genomic DNA in the control sample with the presence of IFI206, mRNA or genomic DNA in the test sample.
• the invention also encompasses kits for detecting IFI206 in a biological sample.
• the kit can comprise: a labeled compound or agent capable of detecting IFI206 or IFI206 mRNA in a sample; reagent and/or equipment for determining the amount of IFI206 in the sample; and reagent and/or equipment for comparing the amount of IFI206 in the sample with a standard.
• the compound or agent can be packaged in a suitable container.
• the kit can further comprise instructions for using the kit to detect IFI206 or nucleic acid.
• the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant IFI206 expression or activity.
• the assays described herein can be used to identify a subject having or at risk of developing a disorder associated with IFI206, nucleic acid expression or activity.
• the prognostic assays can be used to identify a subject having or at risk for developing a disease or disorder.
• Tthe invention provides a method for identifying a disease or disorder associated with aberrant IFI206 expression or activity in which a test sample is obtained from a subject and IFI206 or nucleic acid (e.g., mRNA, genomic DNA) is detected.
• a test sample is a biological sample obtained from a subject.
• a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
• Pognostic assays can be used to determine whether a subject can be administered a modality (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, food, etc.) to treat a disease or disorder associated with aberrant IFI206 expression or activity. Such methods can be used to determine whether a subject can be effectively treated with an agent for a disorder.
• a modality e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, food, etc.
• the invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant IFI206 expression or activity in which a test sample is obtained and IFI206 or nucleic acid is detected (e.g., where the presence of IFI206 or nucleic acid is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant IFI206 expression or activity).
• the methods of the invention can also be used to detect genetic lesions in an IFI206 to determine if a subject with the genetic lesion is at risk for a disorder characterized by aberrant cell proliferation, differentiation or obesity.
• Methods include detecting, in a sample from the subject, the presence or absence of a genetic lesion characterized by at an alteration affecting the integrity of a gene encoding an IFI206 polypeptide, or the mis- expression of IFI206.
• Such genetic lesions can be detected by ascertaining: (1 ) a deletion of one or more nucleotides from IFI206; (2) an addition of one or more nucleotides to IFI206; (3) a substitution of one or more nucleotides in IFI206, (4) a chromosomal rearrangement of an IFI206 gene; (5) an alteration in the level of a IFI206 mRNA transcripts, (6) aberrant modification of an
• IFI206 such as a change genomic DNA methylation, (7) the presence of a non-wild-type splicing pattern of an IFI206 mRNA transcript, (8) a non-wild- type level of IFI206, (9) allelic loss of IFI206, and/or (10) inappropriate post- translational modification of IFI206 polypeptide.
• assay techniques that can be used to detect lesions in IFI206. Any biological sample containing nucleated cells may be used.
• lesion detection may use a probe/primer in a polymerase chain reaction (PCR) (e.g., (Mullis, US Patent No. 4,683,202, 1987; Mullis et al., US Patent No. 4,683,195, 1987), such as anchor PCR or rapid amplification of cDNA ends (RACE) PCR, or, alternatively, in a ligation chain reaction (LCR) (e.g., (Landegren et al., 1988; Nakazawa et al., 1994), the latter is particularly useful for detecting point mutations in /F/206-genes (Abravaya et al., 1995).
• PCR polymerase chain reaction
• LCR ligation chain reaction
• This method may include collecting a sample from a patient, isolating nucleic acids from the sample, contacting the nucleic acids with one or more primers that specifically hybridize to IFI206 under conditions such that hybridization and amplification of the IFI206 (if present) occurs, and detecting the presence or absence of an amplification product, or detecting the size of the amplification product and comparing the length to a control sample. It is anticipated that PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any of the techniques used for detecting mutations described herein.
• Alternative amplification methods include: self sustained sequence replication (Guatelli et al., 1990), transcriptional amplification system (Kwoh et al., 1989); Q ⁇ Replicase (Lizardi et al., 1988), or any other nucleic acid amplification method, followed by the detection of the amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules present in low abundance.
• Mutations in IFI206 from a sample can be identified by alterations in restriction enzyme cleavage patterns.
• sample and control DNA * is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
• sequence specific ribozymes can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
• Hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high-density arrays containing hundreds or thousands of oligonucleotides probes can identify genetic mutations in IFI206 (Cronin et al., 1996; Kozal et al., 1996).
• genetic mutations in IFI206 can be identified in two- dimensional arrays containing light-generated DNA probes as described in Cronin, et al., supra.
• a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential overlapping probes. This step allows the identification of point mutations.
• Each mutation array is composed of parallel probe sets, one complementary to the wild-type gene and the other complementary to the mutant gene.
• any of a variety of sequencing reactions known in the art can be used to directly sequence the IFI206 and detect mutations by comparing the sequence of the sample IFI206-with the corresponding wild-type (control) sequence.
• Examples of sequencing reactions include those based on classic techniques (Maxam and Gilbert, 1977; Sanger et al. , 1977).
• Any of a variety of automated sequencing procedures can be used when performing diagnostic assays (Naeve et al., 1995) including sequencing by mass spectrometry (Cohen et al., 1996; Griffin and Griffin, 1993; Koster, WO94/16101 , 1994).
• RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the IFI206 include those in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al., 1985).
• the technique of "mismatch cleavage" starts by providing heteroduplexes formed by hybridizing (labeled) RNA or DNA containing the wild-type IFI206 sequence with potentially mutant RNA or DNA obtained from a sample.
• the double-stranded duplexes are treated with an agent that cleaves single- stranded regions of the duplex such as those that arise from base pair mismatches between the control and sample strands. For instance,
• RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with Si nuclease to enzymatically digest the mismatched regions.
• either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. The digested material is then separated by size on denaturing polyacrylamide gels to determine the mutation site (Grompe et al., 1989; Saleeba and Cotton, 1993). The control DNA or RNA can be labeled for detection.
• Mismatch cleavage reactions may employ one or more proteins that recognize mismatched base pairs in double-stranded DNA (DNA mismatch repair) in defined systems for detecting and mapping point mutations in IFI206 cDNAs obtained from samples of cells.
• DNA mismatch repair the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al., 1994).
• a probe based on a wild-type IFI206 sequence is hybridized to a cDNA or other DNA product from a test cell(s).
• the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be detected from electrophoresis protocols or the like (Modrich et al., US Patent No. 5,459,039, 1995).
• Electrophoretic mobility alterations can be used to identify mutations in IFI206.
• single strand conformation polymorphism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Cotton, 1993; Hayashi, 1992; Orita et al., 1989).
• Single-stranded DNA fragments of sample and control IFI206 nucleic acids are denatured and then renatured.
• the secondary structure of single- stranded nucleic acids varies according to sequence; the resulting alteration in electrophoretic mobility allows detection of even a single base change.
• DNA fragments may be labeled or detected with labeled probes.
• the sensitivity of the assay may be enhanced by using RNA (rather than DNA), in which the secondary structure is more sensitive to a sequence changes.
• the subject method may use heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility
• DGGE denaturing gradient gel electrophoresis
• a temperature gradient may also be used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rossiter and Caskey, 1990).
• oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions that permit hybridization only if a perfect match is found (Saiki et al., 1986; Saiki et al., 1989).
• Such allele- specific oligonucleotides are hybridized to PCR-amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
• Oligonucleotide primers for specific amplifications may carry the mutation of interest in the center of the molecule (so that amplification depends on differential hybridization (Gibbs et al., 1989)) or at the extreme 3'-terminus of one primer where, under appropriate conditions, mismatch can prevent, or reduce polymerase extension (Prosser, 1993). Novel restriction site in the region of the mutation may be introduced to create cleavage-based detection (Gasparini et al., 1992). Certain amplification may also be performed using Taq ligase for amplification (Barany, 1991). In such cases, ligation occurs only if there is a perfect match at the 3'-terminus of the 5' sequence, allowing detection of a known mutation by scoring for amplification.
• kits comprising at least one probe (nucleic acid or antibody) that may be conveniently used, for example, in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving IFI206.
• probe nucleic acid or antibody
• Agents, or modulators that have a stimulatory or inhibitory effect on IFI206 activity or expression, as identified by a screening assay can be administered to individuals to treat, prophylactically or therapeutically, disorders, including obesity.
• the pharmacogenomics i.e., the study of the relationship between a subject's genotype and the subject's response to a foreign modality, such as a food, compound or drug
• Metabolic differences of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration of the pharmacologically active drug.
• the pharmacogenomics of the individual permits the selection of effective agents (e.g., drugs) for prophylactic or therapeutic treatments based on a consideration of the individual's genotype. Pharmacogenomics can further be used to determine appropriate dosages and therapeutic regimens. Accordingly, the activity of IFI206, expression of IFI206 nucleic acid, or IFI206 mutation(s) in an individual can be determined to guide the selection of appropriate agent(s) for therapeutic or prophylactic treatment. Pharmacogenomics deals with clinically significant hereditary variations in the response to modalities due to altered modality disposition and abnormal action in affected persons (Eichelbaum and Evert, 1996; Under et al., 1997).
• two pharmacogenetic conditions can be differentiated: (1) genetic conditions transmitted as a single factor altering the interaction of a modality with the body (altered drug action) or (2) genetic conditions transmitted as single factors altering the way the body acts on a modality (altered drug metabolism).
• These pharmacogenetic conditions can occur either as rare defects or as nucleic acid polymorphisms.
• glucose-6-phosphate dehydrogenase (G6PD) deficiency is a common inherited enzymopathy in which the main clinical complication is hemolysis after ingestion of oxidant drugs (anti-malarials, sulfonamides, analgesics, nitrofurans) and consumption of fava beans.
• the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drug action.
• drug metabolizing enzymes e.g., N-acetyltransferase 2 (NAT 2) and cytochrome P450 enzymes CYP2D6 and CYP2C19
• NAT 2 N-acetyltransferase 2
• CYP2D6 and CYP2C19 cytochrome P450 enzymes
• CYP2D6 and CYP2C19 cytochrome P450 enzymes CYP2D6 and CYP2C19
• the CYP2D6 gene is highly polymorphic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers due to mutant CYP2D6 and CYP2C19 frequently experience exaggerated drug responses and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM shows no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its CYP2D6-formed metabolite morphine. At the other extreme are the so-called ultra-rapid metabolizers who are unresponsive to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
• the activity of IFI206, expression of IFI206 nucleic acid, or mutation content of IFI206 in an individual can be determined to select appropriate agent(s) for therapeutic or prophylactic treatment of the individual.
• pharmacogenetic studies can be used to apply genotyping of polymorphic alleles encoding drug-metabolizing enzymes to the identification of an individual's drug responsiveness phenotype. This knowledge, when applied to dosing or drug selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with an IFI206 modulator, such as a modulator identified by one of the described exemplary screening assays. 4. Monitoring effects during clinical trials
• IFI206 Monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of IFI206 (e.g., the ability to modulate aberrant cell proliferation and/or differentiation) can be applied not only in basic drug screening, but also in clinical trials.
• agents e.g., drugs, compounds
• the effectiveness of an agent determined by a screening assay to increase IFI206 expression, protein levels, or up-regulate IFI206 activity can be monitored in clinical trails of subjects exhibiting decreased IFI206 expression, protein levels, or down- regulated IFI206 activity.
• the effectiveness of an agent determined to decrease IFI206 expression, protein levels, or down-regulate IFI206 activity can be monitored in clinical trails of subjects exhibiting increased IFI206 expression, protein levels, or up-regulated IFI206 activity.
• the expression or activity of IFI206 and, preferably, other genes that have been implicated in, for example, obesity can be used as a "read out" or markers for a particular cell's responsiveness.
• genes including IFI206, that are modulated in cells by treatment with a modality (e.g., food, compound, drug or small molecule) can be identified.
• a modality e.g., food, compound, drug or small molecule
• cells can be isolated and RNA prepared and analyzed for the levels of expression of IFI206 and other genes implicated in the disorder.
• the gene expression pattern can be quantified by Northern blot analysis, nuclear run-on or RT-PCR experiments, or by measuring the amount of protein, or by measuring the activity level of IFI206 or other gene products.
• the invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, protein, peptide, peptidomimetic, nucleic acid, small molecule, food or other drug candidate identified by the screening assays described herein) comprising the steps of (1) obtaining a pre-administration sample from a subject; (2) detecting the level of expression of an IFI206, mRNA, or genomic DNA in the preadministration sample; (3) obtaining one or more post-administration samples from the subject; (4) detecting the level of expression or activity of the IFI206, mRNA, or genomic DNA in the post-administration samples; (5) comparing the level of expression or activity of the IFI206, mRNA, or genomic DNA in the pre-administration sample with the IFI206, mRNA, or genomic
• DNA in the post administration sample or samples may be altered by altering the administration of the agent to the subject accordingly.
• increased administration of the agent may be desirable to increase the expression or activity of IFI206 to higher levels than detected, i.e., to increase the effectiveness of the agent.
• decreased administration of the agent may be desirable to decrease expression or activity of IFI206 to lower levels than detected, i.e., to decrease the effectiveness of the agent. 5.
• the invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant IFI206 expression or activity.
• the disorders include obesity.
• these same methods of treatment may be used to induce weight loss, or enhance weight loss, by changing the level of expression or activity of IFI206.
• IFI206 Intravascular endothelial fibroblasts
• diseases and disorders that are characterized by increased IFI206 levels or biological activity may be treated with therapeutics that antagonize .
• Antognists may be administered in a therapeutic or prophylactic manner.
• Therapeutics that may be used include:
• IFI206 peptides or analogs, derivatives, fragments or homologs thereof;
• Therapeutics that upregulate activity may be administered therapeutically or prophylactically.
• Therapeutics that may be used include peptides, or analogs, derivatives, fragments or homologs thereof; or an agonist that increases bioavailability.
• the same therapeutics used to treat diseases and disorders may also be used to decrease obesity or induce weight gain.
• Increased or decreased levels can be readily detected by quantifying peptide and/or RNA, by obtaining a patient tissue sample (e.g., from biopsy tissue) and assaying in vitro for RNA or peptide levels, structure and/or activity of the expressed peptides (or IFI206 mRNAs).
• Methods include, but are not limited to, immunoassays (e.g., by Western blot analysis, immunoprecipitation followed by sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis, immunocytochemistry, etc.) and/or hybridization assays to detect expression of mRNAs (e.g., Northern assays, dot blots, in situ hybridization, and the like).
• the invention provides a method for preventing, in a subject, a disease or condition associated with an aberrant IFI206 expression or activity, by administering an agent that modulates IFI206 expression or at least one IFI206 activity.
• Subjects at risk for a disease that is caused or contributed to by aberrant IFI206 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays.
• Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic of the IFI206 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
• an IFI206 agonist or IFI206 antagonist can be used to treat the subject.
• the appropriate agent can be determined based on screening assays.
• the modulatory method of the invention involves contacting a cell with an agent that modulates one or more of the activities of IFI206 activity associated with the cell.
• An agent that modulates IFI206 activity can be a nucleic acid or a protein, a naturally occurring cognate ligand of IFI206, a peptide, an IFI206 peptidomimetic, or other small molecule.
• the agent may stimulate IFI206 activity. Examples of such stimulatory agents include active IFI206 and a IFI206 nucleic acid molecule that has been introduced into the cell. In another embodiment, the agent inhibits IFI206 activity. Examples of inhibitory agents include antisense IFI206 nucleic acids and anti-IFI206 Abs.
• Modulatory methods can be performed in vitro (e.g., by culturing the cell with the agent) or, alternatively, in vivo (e.g., by administering the agent to a subject).
• the invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of an IFI206 or nucleic acid molecule.
• the method involves administering an agent (e.g., an agent identified by a screening assay), or combination of agents that modulates (e.g., up-regulates or down-regulates)
• the method involves administering an IFI206 or nucleic acid molecule as therapy to compensate for reduced or aberrant IFI206 expression or activity.
• Stimulation of IFI206 activity is desirable in situations in which IFI206 is abnormally down-regulated and/or in which increased IFI206 activity is likely to have a beneficial effect.
• a subject has a disorder characterized by aberrant cell proliferation and/or differentiation (e.g., cancer or immune associated disorders).
• Another example of such a situation is obesity. 9. Determination of the biological effect of the therapeutic
• Suitable in vitro or in vivo assays can be performed to determine the effect of a specific therapeutic and whether its administration is indicated for treatment of the affected tissue.
• in vitro assays may be performed with representative cells of the type(s) involved in the patient's disorder, to determine if a given therapeutic exerts the desired effect upon the cell type(s).
• Modalities for use in therapy may be tested in suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
• suitable animal model systems including, but not limited to rats, mice, chicken, cows, monkeys, rabbits, and the like, prior to testing in human subjects.
• any of the animal model system known in the art may be used prior to administration to human subjects.
• IFI206 nucleic acids and proteins are useful in potential prophylactic and therapeutic applications implicated in a variety of disorders including, but not limited to obesity.
• a cDNA encoding IFI206 may be useful in gene therapy, and the protein may be useful when administered to a subject in need thereof.
• the compositions of the invention will have efficacy for treatment of patients suffering from infertility.
• IFI206 nucleic acids, or fragments thereof may also be useful in diagnostic applications, wherein the presence or amount of the nucleic acid or the protein is to be assessed.
• a further use could be as an anti-bacterial molecule (i.e., some peptides have been found to possess anti-bacterial properties). These materials are further useful in the generation of Abs that immunospecifically bind to the novel substances of the invention for use in therapeutic or diagnostic methods.
• the KIDNNOT05 cDNA library was constructed from tissue removed from a female infant kidney with anoxia (lot #RU95-04-0274; International Institute of Advanced Medicine, Exton Pa.). The frozen tissue was immediately homogenized and cells lysed with a Brinkmann Homogenizer Polytron PT-3000 (Brinkmann Instruments Inc., Westbury N.Y.) in a guanidinium isothiocyanate solution. Lysates were then loaded on a 5.7 M CsCI cushion and ultracentrifuged in a SW28 swinging bucket rotor for 18 hours at 25,000 rpm at ambient temperature.
• RNA was extracted once with acid phenol at pH 4.0 and precipitated with 0.3 M sodium acetate and 2.5 volumes of ethanol, resuspended in DEPC-treated water and DNAse treated for 25 min at 37°C. The reaction was stopped with an equal volume of pH 8.0 phenol, and the RNA was as above.
• the RNA was isolated using the Qiagen Oligotex kit (QIAGEN Inc, Chatsworth Calif.) and used to construct the cDNA library. The RNA was handled according to the recommended protocols in the
• Miniprep Kit (Catalogue # 77468; Advanced Genetic Technologies Corporation, Gaithersburg Md.). This kit consists of a 96 well block with reagents for 960 purifications. The recommended protocol was employed except for the following changes: 1) the 96 wells were each filled with only 1 ml of sterile Terrific Broth (Catalog # 22711 , LIFE TECHNOLOGIES.TM.,
• the cDNAs were sequenced by the method of Sanger F and AR Coulson (1975; J Mol Biol 94:441 f), using a Hamilton Micro Lab 2200
• Each cDNA was compared to sequences in GenBank using a search algorithm developed by Applied Biosystems and incorporated into the INHERIT- 670 Sequence Analysis System.
• Pattern Specification Language TRW Inc, Los Angeles Calif.
• the three parameters that determine how the sequence comparisons run were window size, window offset, and error tolerance.
• the DNA database was searched for sequences containing regions of homology to the query sequence, and the appropriate sequences were scored with an initial value. Subsequently, these homologous regions were examined using dot matrix homology plots to distinguish regions of homology from chance matches. Smith-Waterman alignments were used to display the results of the homology search.
• Peptide and protein sequence homologies were ascertained using the INHERIT.TM. 670 Sequence Analysis System in a way similar to that used in DNA sequence homologies. Pattern Specification Language and parameter windows were used to search protein databases for sequences containing regions of homology which were scored with an initial value. Dot-matrix homology plots were examined to distinguish regions of significant homology from chance matches.
• BLAST which stands for Basic Local Alignment Search Tool (Altschul S F (1993) J Mol Evol 36:290-300; Altschul, S F et al (1990) J Mol Biol 215:403-10), was used to search for local sequence alignments.
• BLAST produces alignments of both nucleotide and amino acid sequences to determine sequence similarity. Because of the local nature of the alignments, BLAST is especially useful in determining exact matches or in identifying homologs. BLAST is useful for matches which do not contain gaps.
• the fundamental unit of BLAST algorithm output is the High-scoring Segment Pair
• An HSP consists of two sequence fragments of arbitrary but equal lengths whose alignment is locally maximal and for which the alignment score meets or exceeds a threshold or cutoff score set by the user.
• the BLAST approach is to look for HSPs between a query sequence and a database sequence, to evaluate the statistical significance of any matches found, and to report only those matches which satisfy the user-selected threshold of significance.
• the parameter E establishes the statistically significant threshold for reporting database sequence matches. E is interpreted as the upper bound of the expected frequency of chance occurrence of an HSP (or set of HSPS) within the context of the entire database search. Any database sequence whose match satisfies E is reported in the program output. 4.
• Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound (Sambrook et al. supra).
• Analogous computer techniques use BLAST (Altschul SF 1993 and 1990, supra) to search for identical or related molecules in nucleotide databases such as GenBank. This analysis is much faster than multiple, membrane-based hybridizations. In addition, the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or homologous.
• the basis of the search is the product score which is defined as: ##EQU1## and it takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40, the match will be exact within a 1 -2% error; and at 70, the match will be exact. Homologous molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules.
• RNA preparations from liver or pulverized SKM of individual mice were made (Ultraspec reagent; Biotecx Laboratories, Houston TX) and assayed for mRNA abundance using quantitative real-time reverse- transcriptase PCR (RT-PCR) following digestion of samples with DNAse per manufacturer's instructions (GIBCO BRL, Grand Island NY).
• RT-PCR quantitative real-time reverse- transcriptase PCR
• This system employed primers and probes specific to murine IFI206.
• 18S primers/probe were purchased from Perkin-Elmer Applied Biosystems (Foster City, CA).
• Reactions and detection were carried out using a Model 7700 Sequence Detector and TaqMan reagents (PE Applied Biosystems; Boston, MA) in a volume of 50 ⁇ L and containing: 100 ng RNA, 3 mM MgCI 2 , reaction Buffer A (1X), 12.5 U MuLV reverse transcriptase, 1.25 U TaqGold, forward and reverse primers (0.01 O.D. ea.), and 0.1 ⁇ M probe (Note: 18S analyses employed 240 pg RNA, 5.5 mM MgCI 2 , and 0.05 ⁇ M probe/primer). Cycling conditions were: 50°C 15 min and 95°C 10 min, followed by 40 cycles of 95°C 15 sec and 60°C 1 min. 18S mRNA abundance was used as a loading control, and all values reported herein represent 18S-corrected values.
• the nucleic acid sequence encoding full length IFI206 (SEQ ID NO:2) is used to design oligonucleotide primers for extending a partial nucleotide sequence to full length or for obtaining 5' sequences from genomic libraries.
• One primer is synthesized to initiate extension in the antisense direction
• XLR XLR
• XLF sense direction
• Primers allow the extension of the known IFI206 nucleotide sequence "outward" generating amplicons containing new, unknown nucleotide sequence for the region of interest
• the initial primers are designed from the cDNA using OLIGO. RTM. 4.06 Primer Analysis Software (National Biosciences), or another appropriate program, to be 22-30 nucleotides in length, to have a GC content of 50% or more, and to anneal to the target sequence at temperatures about 68.degree.-72°C Any stretch of nucleotides which would result in hairpin structures and primer-primer dimerizations is avoided.
• the original, selected cDNA libraries, or a human genomic library are used to extend the sequence; the latter is most useful to obtain 5' upstream regions. If more extension is necessary or desired, additional sets of primers are designed to further extend the known region.
• PCR is performed using the Peltier Thermal Cycler (PTC200; MJ Research, Watertown Mass.) and the following parameters:
• Step 1 94°C for 1 min (initial denaturation)
• Step 4 94°C for 15 sec
• Step 5 65°C for 1 min
• Step 6 68°C for 7 min
• Step 7 Repeat step 4-6 for 15 additional cycles
• Step 8 94°C for 15 sec
• Step 10 68°C for 7 : 15 min
• Step 11 Repeat step 8-10 for 12 cycles
• Step 13 4°C (and holding) A 5-10 ⁇ l aliquot of the reaction mixture is analyzed by electrophoresis on a low concentration (about 0.6-0.8%) agarose mini-gel to determine which reactions were successful in extending the sequence. Bands thought to contain the largest products were selected and cut out of the gel. Further purification involves using a commercial gel extraction method such as QIAQuick.TM. (QIAGEN Inc). After recovery of the DNA, Klenow enzyme was used to trim single-stranded, nucleotide overhangs creating blunt ends which facilitate religation and cloning.
• the products are redissolved in 13 ⁇ l of ligation buffer, 1 ⁇ l T4-DNA ligase (15 units) and 1 ⁇ l T4 polynucleotide kinase are added, and the mixture is incubated at room temperature for 2-3 hours or overnight at 16°C Competent £. coli cells (in 40 ⁇ l of appropriate media) are . transformed with 3 ⁇ l of ligation mixture and cultured in 80 ⁇ l of SOC medium
• Step 1 94°C for 60 sec
• Step 2 94°C for 20 sec
• Step 3 55°C for 30 sec
• Step 4 72°C for 90 sec
• Step 5 Repeat steps 2-4 for an additional 29 cycles
• Hybridization probes derived from SEQ ID NO:2 are employed to screen cDNAs, genomic DNAs or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base-pairs, is specifically described, essentially the same procedure is used with larger cDNA fragments.
• Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 (National Biosciences), labeled by combining 50 pmol of each oligomer and 250 mCi of ⁇ adenosine triphosphate (Amersham, Chicago III.) and T4 polynucleotide kinase (DuPont NEN; Boston Mass.).
• the labeled oligonucleotides are substantially purified with Sephadex G-25 super fine resin column (Pharmacia).
• a portion containing 10.sup.7 counts per minute of each of the sense and antisense oligonucleotides is used in a typical membrane based hybridization analysis of human genomic DNA digested with one of the following endonucleases (Ase I, Bgl II, Eco Rl, Pst I, Xba 1 , or Pvu II; DuPont NEN).
• DNA from each digest is fractionated on a 0.7 percent agarose gel and transferred to nylon membranes (Nytran Plus, Schleicher & Schuell, Durham N.H.). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under increasingly stringent conditions up to 0.1 .times.saline sodium citrate and
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