WO2004081197A2 - Proteines associees a une reponse immunitaire - Google Patents

Proteines associees a une reponse immunitaire Download PDF

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WO2004081197A2
WO2004081197A2 PCT/US2004/007716 US2004007716W WO2004081197A2 WO 2004081197 A2 WO2004081197 A2 WO 2004081197A2 US 2004007716 W US2004007716 W US 2004007716W WO 2004081197 A2 WO2004081197 A2 WO 2004081197A2
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seq
polynucleotide
polypeptide
amino acid
sequence
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PCT/US2004/007716
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WO2004081197A3 (fr
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Shanya D. Becha
Amy E. Kable
Reena Khare
Joseph P. Marquis
Uyen K. Tran
Thomas W. Richardson
Narinder K. Chawla
Soo Yeun Lee
David Chien
Jonathan T. Wang
Danniel B. Nguyen
Phillip R. Hawkins
Pei Jin
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Incyte Corporation
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals

Definitions

  • the invention relates to novel nucleic acids, immune response associated proteins encoded by these nucleic acids, and to the use of these nucleic acids and proteins in the diagnosis, treatment, and prevention of immune system, neurological, developmental, muscle, and cell proliferative disorders.
  • the invention also relates to the assessment of the effects of exogenous compounds on the expression of nucleic acids and immune response associated proteins.
  • the cellular components of the immune system include six different types of leukocytes, or white blood cells: monocytes, lymphocytes, polymorphonuclear granulocytes (including neutrophils, eosinophils, and basophils) and plasma cells. Additionally, fragments of megakaryocytes, a seventh type of white blood cell in the bone marrow, occur in large numbers in the blood as platelets.
  • Leukocytes are formed from two stem cell lineages in bone marrow.
  • the myeloid stem cell line produces granulocytes and monocytes and the lymphoid stem cell line produces lymphocytes.
  • Lymphoid cells travel to the thymus, spleen and lymph nodes, where they mature and differentiate into lymphocytes.
  • Leukocytes are responsible for defending the body against invading pathogens. Neutrophils and monocytes attack invading bacteria, viruses, and other pathogens and destroy them by phagocytosis.
  • Monocytes enter tissues and differentiate into macrophages which are extremely phagocytic. Lymphocytes and plasma cells are a part of the immune system which recognizes specific foreign molecules and organisms and inactivates them, as well as signals other cells to attack the invaders.
  • Granulocytes and monocytes are formed and stored in the bone marrow until needed. Megakaryocytes are produced in bone marrow, where they fragment into platelets and are released into the bloodstream. The main function of platelets is to activate the blood clotting mechanism. Lymphocytes and plasma cells are produced in various lymphogenous organs, including the lymph nodes, spleen, thymus, and tonsils.
  • Basophils participate in the release of the chemicals involved in the inflammatory process.
  • the main function of basophils is secretion of these chemicals, to such a degree that they have been referred to as "unicellular endocrine glands.”
  • a distinct aspect of basophilic secretion is that the contents of granules go directly into the extracellular environment, not into vacuoles as occurs with neutrophils, eosinophils, and monocytes.
  • Basophils have receptors for the Fc fragment of immunoglobulin E (IgE) that are not present on other leukocytes. Crosslinking of membrane IgE with anti-IgE or other ligands triggers degranulation.
  • IgE immunoglobulin E
  • Eosinophils arebi- or multi-nucleated white blood cells which contain eosinophilic granules. Their plasma membrane is characterized by Ig receptors, particularly IgG and IgE. Generally, eosinophils are stored in the bone marrow until recruited for use at a site of inflammation or invasion. They have specific functions in parasitic infections and allergic reactions, and are thought to detoxify some of the substances released by mast cells and basophils which cause inflammation. Additionally, Ihey phagocytize antigen-antibody complexes and further help prevent the spread of inflammation. The mononuclear phagocyte system is comprised of precursor cells in the bone marrow, monocytes in circulation, and macrophages in tissues.
  • Macrophages are monocytes that have left the blood stream to settle in tissue. Once monocytes have migrated into tissues, they do not re-enter the bloodstream. They increase several-fold in size and transform into macrophages that are characteristic of the tissue they have entered, surviving in tissues for several months.
  • the mononuclear phagocyte system is capable of very fast and extensive phagocytosis. A macrophage may phagocytize over 100 bacteria, digest them and extrude residues, and then survive for many more months. Macrophages are also capable of ingesting large particles, including red blood cells and malarial parasites.
  • Mononuclear phagocytes are essential in defending the body against invasion by foreign pathogens, particularly intracellular microorganisms such as Mycobacterium tuberculosis, listeria, leishmania and toxoplasma. Macrophages can also control the growth of tumorous cells, via both phagocytosis and secretion of hydrolytic enzymes. Another important function of macrophages is that of processing antigens and presenting them in a biochemically modified form to lymphocytes.
  • the immune system responds to invading microorganisms in two major ways: antibody production and cell mediated responses.
  • Antibodies are immunoglobulin proteins produced by
  • T cells T-lymphocytes
  • T cells T-lymphocytes
  • the T cell either kills the infected cell itself, or secretes signals which activate macrophages and other cells to destroy the infected cell (Paul, supra).
  • T-lymphocytes originate in the bone marrow or liver in fetuses. Precursor cells migrate via the blood to the thymus, where they are processed to mature into T-lymphocytes. This processing is crucial because it involves positive and negative selection of T cells for those that will react with foreign antigen and not with self molecules.
  • T cells After processing, T cells continuously circulate in the blood and secondary lymphoid tissues, such as lymph nodes, spleen, certain epithelium-associated tissues in the gastrointestinal tract, respiratory tract and skin.
  • T-lymphocytes When T-lymphocytes are presented with the complementary antigen, they are stimulated to proliferate and release large numbers of activated T cells into the lymph system and the blood system. These activated T cells can survive and circulate for several days.
  • T memory cells are created, which remain in the lymphoid tissue for months or years. Upon subsequent exposure to that specific antigen, these memory cells will respond more rapidly and with a stronger response than induced by the original antigen. This creates an "immunological memory" that can provide immunity for years.
  • T cells There are two major types of T cells: cytotoxic T cells destroy infected host cells, and helper T cells activate other white blood cells via chemical signals.
  • helper T cells activate other white blood cells via chemical signals.
  • T H 1 activates macrophages to destroy ingested microorganisms, while another, T H 2, stimulates the production of antibodies by B cells.
  • Cytotoxic T cells directly attack the infected target cell. Receptors on the surface of T cells bind to antigen presented by MHC molecules on the surface of the infected cell. Once activated by binding to antigen, T cells secrete ⁇ -interferon, a signal molecule that induces the expression of genes necessary for presenting viral (or other) antigens to cytotoxic T cells. Cytotoxic T cells kill the infected cell by stimulating programmed cell death.
  • Helper T cells constitute up to 75% of the total T cell population. They regulate the immune functions by producing a variety of lymphokines that act on other cells in the immune system and on bone marrow. Among these lymphokines are interleukins 2 through 6, granulocyte-monocyte colony stimulating factor, and ⁇ -interferon.
  • Helper T cells are required for most B cells to respond to antigen.
  • an activated helper cell contacts a B cell, its centrosome and Golgi apparatus become oriented toward the B cell, aiding the directing of signal molecules, such as a transmembrane-bound protein called CD40 ligand, onto the B cell surface to interact with the CD40 transmembrane protein.
  • Secreted signals also help B cells to proliferate and mature and, in some cases, to switch the class of antibody being produced.
  • B-lymphocytes B cells produce antibodies which react with specific antigenic proteins presented by pathogens. Once activated, B cells become filled with extensive rough endoplasmic reticulum and are known as plasma cells.
  • immunoglobulins which together comprise about 20% of total plasma protein. Each class mediates a characteristic biological response after antigen binding. Upon activation by specific antigen B cells switch from making the membrane-bound antibody to the secreted form of that antibody.
  • Antibodies or immunoglobulins, are the founding members of the immunoglobulin (Ig) superfamily and the central components of the humoral immune response. Antibodies are either expressed on the surface of B cells or secreted by B cells into the circulation. Antibodies bind and neutralize blood-borne foreign antigens.
  • the prototypical antibody is a tetramer consisting of two identical heavy polypeptide chains (H-chains) and two identical light polypeptide chains (L-chains) interlinked by disulfide bonds. This arrangement confers the characteristic Y-shape to antibody molecules. Antibodies are classified based on their H-chain composition.
  • the five antibody classes, IgA, IgD, IgE, IgG and IgM, are defined by the , ⁇ , e, ⁇ , and ⁇ H-chain types.
  • L-chains There are two types of L-chains, K and ⁇ , either of which may associate as a pair with any H-chain pair.
  • IgG the most common class of antibody found in the circulation, is tetrameric, while the other classes of antibodies are generally variants or multimers of this basic structure.
  • H-chains and L-chains each contain an N-terminal variable region and a C-terminal constant region.
  • the constant region consists of about 110 amino acids in L-chains and about 330 or 440 amino acids in H-chains.
  • the amino acid sequence of the constant region is nearly identical among H- or L-chains of a particular class.
  • the variable region consists of about 110 amino acids in both H- and L-chains. However, the amino acid sequence of the variable region differs among H- or L-chains of a particular class.
  • Within each H- or L-chain variable region are three hypervariable regions of extensive sequence diversity, each consisting of about 5 to 10 amino acids. In the antibody molecule, the H- and L-chain hypervariable regions come together to form the antigen recognition site.
  • the immune system is capable of recognizing and responding to any foreign molecule that enters the body. Therefore, the immune system must be armed with a full repertoire of antibodies against all potential antigens.
  • antibody diversity is generated by somatic rearrangement of gene segments encoding variable and constant regions. These gene segments are joined together by site- specific recombination which occurs between highly conserved DNA sequences that flank each gene segment. Because there are hundreds of different gene segments, millions of unique genes can be generated combinatorially. In addition, imprecise joining of these segments and an unusually high rate of somatic mutation within these segments further contribute to the generation of a diverse antibody population.
  • Both H-chains and L-chains contain repeated Ig domains.
  • a typical H-chain contains four Ig domains, three of which occur within the constant region and one of which occurs within the variable region and contributes to the formation of the antigen recognition site.
  • a typical L-chain contains two Ig domains, one of which occurs within the constant region and one of which occurs within the variable region.
  • H chains such as ⁇ have been shown to associate with other polypeptides during differentiation of the B-cell.
  • Antibodies canbe described in terms of their two main functional domains. Antigen recognition is mediated by the Fab (antigen binding fragment) region ofthe antibody, while effector functions are mediated by the Fc (crystallizable fragment) region.
  • Binding of antibody to an antigen triggers the destruction of the antigen by phagocytic white blood cells such as macrophages and neutropliils. These cells express surface receptors that specifically bind to the antibody Fc region and allow the phagocytic cells to engulf, ingest, and degrade the antibody-bound antigen.
  • the Fc receptors expressed by phagocytic cells are single-pass transmembrane glycoproteins of about 300 to 400 amino acids (Sears, D.W. et al. (1990) J. Immunol. 144:371-378).
  • the extracellular portion of the Fc receptor typically contains two or three Ig domains.
  • AIDS Abnormal Immunodeficiency Syndrome
  • IgEs have a strong tendency to attach to mast cells and basophils, up to half million each (IgE/mast) which then rupture and release histamine, leukotrienes, eosinophil chemotactic substance, protease, neutrophil chemotactic substance, heparin, and platelet activation factors. Tissues can respond in a number of ways to these substances resulting in what are commonly known as allergic reactions: hay fever, asthma, anaphylaxis, and urticaria (hives).
  • Leukemias are an excess production of white blood cells, to the point where a major portion of the body's metabolic resources are directed solely at proliferation of white blood cells, leaving other tissues to starve.
  • lymphogenous leukemias cancerous lymphogenous cells spread from a lymph node to other body parts. Excess T- and B-lymphocytes are produced.
  • myelogenous leukemias cancerous young myelogenous cells spread from the bone marrow to other organs, especially the spleen, liver, lymph nodes and other highly vascularized regions.
  • the extra leukemic cells released are immature, incapable of function, and undifferentiated.
  • Leukemias maybe caused by exposure to environmental factors such as radiation or toxic chemicals or by genetic aberration.
  • Leukopenia or agranulocytosis occurs when the bone marrow stops producing white blood cells. This leaves the body unprotected against foreign microorganisms, including those which normally inhabit skin, mucous membranes, and gastrointestinal tract. If all white blood cell production stops completely, infection will occur within two days and death may follow only 1 to 4 days later. Acute leukopenia can be caused by exposure to radiation or chemicals containing benzene. Occasionally, drugs such as chloramphenicol and thiouracil can suppress blood cell production by the bone marrow and initiate the onset of agranulocytosis. In cases of monoblastic leukemia, primitive monocytes in blood and bone marrow do not mature.
  • Eosinophilia is an excess of eosinophils commonly observed in patients with allergies (hay fever, asthma), allergic reactions to drugs, rheumatoid arthritis, and cancers (Hodgkins disease, lung, and liver cancer).
  • allergies hay fever, asthma
  • allergic reactions to drugs rheumatoid arthritis
  • cancers Hodgkins disease, lung, and liver cancer.
  • the mechanism for elevated levels of eosinophils in these diseases is unknown (Isselbacher, KJ. et al. (1994) Harrison's Principles of Internal Medicine. McGraw-Hill, Inc., New York, NY).
  • the complement system serves as an effector system and is involved in infectious agent recognition. It can function as an independent immune network or in conjunction with other humoral immune responses.
  • the complement system is comprised of numerous plasma and membrane proteins that act in a cascade of reaction sequences whereby one component activates the next. The result is a rapid and amphfied response to infection through either an inflammatory response or increased phagocytosis.
  • the complement system has more than 30 protein components which can be divided into functional groupings including modified serine proteases, membrane-binding proteins, and regulators of complement activation. Activation occurs through two different pathways, the classical and the alternative. Both pathways serve to destroy infectious agents through distinct triggering mechanisms that eventually merge with the involvement of the component C3.
  • the classical pathway requires antibody binding to infectious agent antigens.
  • the antibodies serve to define the target and initiate the complement system cascade, culminating in the destruction ofthe infectious agent.
  • the complement system can be seen as an effector arm of the humoral immune system.
  • the alternative pathway of the complement system does not require the presence of preexisting antibodies for targeting infectious agent destruction. Rather, this pathway, through low levels of an activated component, remains constantly primed and provides surveillance in the non- immune host to enable targeting and destruction of infectious agents. In this case foreign material triggers the cascade, thereby facilitating phagocytosis or lysis (Paul, supra pp.918-919).
  • Inflammatory responses are divided into four categories on the basis of pathology and include allergic inflammation, cytotoxic antibody mediated inflammation, immune complex mediated inflammation, and monocyte mediated inflammation, Inflammation manifests as a combination of each of these forms with one predominating.
  • Acute inflamation is also mediated by cytotoxic antibodies and can result in the destruction of tissue through the binding of complement-fixing antibodies to cells.
  • the antibodies responsible are of the IgG or IgM types and resultant clinical disorders including autoimmune hemolytic anemia and thrombocytopenia as associated with systemic lupus erythematosis.
  • Immune complex mediated acute inflammation involves the IgG or IgM antibody types which combine with antigen to activate the complement cascade. When such immune complexes bind to neutrophils and macrophages they activate the respiratory burst to form protein and vessel damaging agents such as hydrogen peroxide, hydroxyl radical, hypochlorous acid, and chloramines.
  • Clinical manifestations include rheumatoid arthritis and systemic lupus erythematosus.
  • macrophages are activated and process antigen for presentation to T cells that subsequently produce lymphokines and monokines. This type of inflammatory response is likely important for defense against intracellular parasites and certain viruses.
  • Clinical associations include granulomatous disease, tuberculosis, leprosy, and sarcoidosis (Paul, supra pp.1017-1018).
  • Most cell surface and soluble molecules that mediate functions such as recognition, adhesion or binding have evolved from a common evolutionary precursor (i.e., these proteins have structural homology). A number of molecules outside the immune system that have similar functions are also derived from this same evolutionary precursor.
  • Ig immunoglobulin
  • TCRs T cell receptors
  • MHC major histocompatibility proteins
  • CD2, CD3, CD4, CD8 poly-Ig receptors
  • Fc receptors neural cell-adhesion molecule
  • NCAM neural cell-adhesion molecule
  • PDGFR platelet-derived growth factor receptor
  • Ig domains are regions of conserved amino acid residues that give a polypeptide a globular tertiary structure called an immunoglobulin (or antibody) fold, which consists of two approximately parallel layers of ⁇ -sheets.
  • conserved cysteine residues form an intrachain disulfide- bonded loop, 55-75 amino acid residues in length, which connects the two layers of the ⁇ -sheets.
  • Each ⁇ -sheet has three or four anti-parallel ⁇ -strands of 5-10 amino acid residues. Hydrophobic and hydrophilic interactions of amino acid residues within the ⁇ -strands stabilize the Ig fold
  • a V domain consists of a longer polypeptide than a C domain, with an additional pair of ⁇ -strands in the Ig fold.
  • Ig superfamily genes each sequence of an Ig domain is encoded by a single exon. It is possible that the superfamily evolved from a gene coding for a single Ig domain involved in mediating cell-cell interactions. New members ofthe superfamily then arose by exon and gene duplications. Modern Ig superfamily proteins contain different numbers of V and/or C domains. Another evolutionary feature of this superfamily is the ability to undergo DNA rearrangements, a unique feature retained by the antigen receptor members of the family. Many members of the Ig superfamily are integral plasma membrane proteins with extracellular Ig domains.
  • the hydrophobic amino acid residues of their transmembrane domains and their cytoplasmic tails are very diverse, with little or no homology among Ig family members or to known signal-transducing structures. There are exceptions to this general superfamily description.
  • the cytoplasmic tail of PDGFR has tyrosine kinase activity.
  • Thy- 1 is a glycoprotein found on thymocytes and T cells. This protein has no cytoplasmic tail, but is instead attached to the plasma membrane by a covalent glycophosphatidylinositol linkage.
  • Ig superfamily proteins Another common feature of many Ig superfamily proteins is the interactions between Ig domains which are essential for the function of these molecules. Interactions between Ig domains of a multimeric protein can be either homophilic or heterophilic (i.e., between the same or different Ig domains).
  • Antibodies are multimeric proteins which have both homophilic and heterophilic interactions between Ig domains. Pairing of constant regions of heavy chains forms the Fc region of an antibody and pairing of variable regions of light and heavy chains form the antigen binding site of an antibody. Heterophilic interactions also occur between Ig domains of different molecules. These interactions provide adhesion between cells for significant cell-cell interactions in the immune system and in the developing and mature nervous system. (Reviewed in Abbas, A.K. et al. (1991) Cellular and Molecular Immunology. W.B. Saunders Company, Philadelphia, PA, pp.142-145.)
  • CD21 also called C3d receptor, CR2, Epstein Barr virus receptor or EBV-R
  • EBV-R Epstein Barr virus receptor
  • CD21 is part of a large signal-transduction complex that also involves CD19, CD81, and Leul3.
  • Some of the proteins in this group are responsible for the molecular basis of the blood group antigens, surface markers on the outside of the red blood cell membrane. Most of these markers are proteins, but some are carbohydrates attached to lipids or proteins (for a review see Reid, M.E.
  • Complement decay-accelerating factor belongs to the Cromer blood group system and is associated with Cr(a), Dr(a), Es(a), Tc(a b/c), Wd(a), WES(a/b), IFC and UMC antigens.
  • Complement receptor type 1 (C3b/C4b receptor) (Antigen CD35) belongs to the Knops blood group system and is associated with Kn(a/b), McC(a), Sl(a) and Yk(a) antigens.
  • Human leukocyte-specific transcript 1 (LST1) is a small protein that modulates immune responses and cellular morphogenesis.
  • LST1 is expressed at high levels in dendritic cells.
  • a DNA-binding site and interaction of multiple regulatory elements may be involved in mediating the expression of the various forms of LST1 mRNA (Yu, X. and Weissman, S.M. (2000) J. Biol. Chem. 275:34597-34608).
  • Spalpha is a member of the scavenger receptor cysteine-rich (SRCR) family of proteins.
  • Microarrays are analytical tools used in bioanalysis.
  • a microarray has a plurality of molecules spatially distributed over, and stably associated with, the surface of a solid support.
  • Microarrays of polypeptides, polynucleotides, and/or antibodies have been developed and find use in a variety of applications, such as gene sequencing, monitoring gene expression, gene mapping, bacterial identification, drug discovery, and combinatorial chemistry.
  • array technology can provide a simple way to explore the expression of a single polymorphic gene or the expression profile of a large number of related or unrelated genes.
  • arrays are employed to detect the expression of a specific gene or its variants.
  • arrays provide a platform for identifying genes that are tissue specific, are affected by a substance being tested in a toxicology assay, are part of a signaling cascade, carry out housekeeping functions, or are specifically related to a particular genetic predisposition, condition, disease, or disorder.
  • PBMCs Human peripheral blood mononuclear cells
  • PBMCs can be classified into discrete cellular populations representing the major cellular components of he immune system.
  • PBMCs contain about 52% lymphocytes (12% B lymphocytes, 40% T lymphocytes ⁇ 25% CD4+ and 15% CD8+ ⁇ ), 20% NK cells, 25% monocytes, and 3% various cells that include dendritic cells and progenitor cells.
  • the proportions, as well as the biology of these cellular components tend to vary slightly between healthy individuals, depending on factors such as age, gender, past medical history, and genetic background.
  • LPS lipopolysaccharide
  • LPS elicits a variety of inflammatory responses, and because it activates complement by the alternative (properdin) pathway, it is often part of the pathology of gram-negative bacterial infections.
  • Gram negative bacteria probably release minute amounts of endotoxin while growing. For example, it is known that small amounts of endotoxin may be released in a soluble form, especially by young cultures. For the most part, however, endotoxins remain associated with the cell wall until the bacteria disintegrate. In vivo, disintegration is the result of autolysis ofthe bacteria, external lysis mediated by complement and lysozyme, and phagocytic digestion of bacterial cells.
  • LPS -binding proteins LPS-binding proteins
  • the LPS -binding protein complex interacts with CD14 receptors on monocytes, macrophages, B cells, and other types of receptors on endothelial cells. Activation of human B cells with LPS results in itogenesis as well as imniunoglobulin synthesis.
  • monocytes and macrophages three types of events are triggered during their interaction with LPS: 1) Production of cytokines, including IL-1, IL-6, IL-8, TNF- ⁇ , and platelet-activating factor. These in turn stimulate production of prostaglandins and leukotrienes, powerful mediators of inflammation and septic shock that accompany endotoxin toxemia. 2) Activation of the complement cascade. 3) Activation of the coagulation cascade.
  • Naproxen [22204-53-1] is a nonsteroidal anti-inflammatory drug (NSAID) related to ibuprofen that also possesses antipyretic and analgesic properties.
  • NSAID nonsteroidal anti-inflammatory drug
  • Naproxen inhibits the activity of the enzyme cyclooxygenase, resulting in the peripheral secondary inhibition of prostaglandin.
  • Naproxen has a prominent inhibitory effect on the migration of leukocytes into the inflamed area. When administered orally, peak plasma concentrations of naproxen sodium is achieved after 1-2 hours. Naproxen has a half-life of 10-20 hours.
  • Ibuprofen [15687-27-1] is an oral, nonsteroidal anti-inflammatory drug (NSAID) that is usually used to treat rheumatoid arthritis, osteoarthritis, painful menstruation, and to alleviate mild to moderate pain. Ibuprofen inhibits the production of prostaglandin by inhibiting cyclooxygenase enzymes. In vivo, peak serum concentrations are reached 1-2 hours after oral administration of a single dose. Lung Cancer
  • Lung cancer is the leading cause of cancer death in the United States, affecting more than 100,000 men and 50,000 women each year. Nearly 90% of the patients diagnosed with lung cancer are cigarette smokers. Tobacco smoke contains thousands of noxious substances that induce carcinogen metabolizing enzymes and covalent DNA adduct formation in the exposed bronchial epithelium. In nearly 80% of patients diagnosed with lung cancer, metastasis has already occurred. Most commonly lung cancers metastasize to pleura, brain, bone, pericardium, and liver.
  • Non Small Cell Lung Carcinoma Squamous cell carcinomas
  • Adenocarcinomas typically arise in the peripheral airways and often form mucin secreting glands.
  • Squamous cell carcinomas typically arise in proximal airways.
  • the histogenesis of squamous cell carcinomas may be related to chronic inflammation and injury to the bronchial epithelium, leading to squamous metaplasia.
  • SCLC Small Cell Lung Carcinoma
  • Lung cancer cells accumulate numerous genetic lesions, many of which are associated with cytologically visible chromosomal aberrations.
  • the high frequency of chromosomal deletions associated with lung cancer may reflect the role of multiple tumor suppressor loci in the etiology of this disease. Deletion of the short arm of chromosome 3 is found in over 90% of cases and represents one ofthe earliest genetic lesions leading to lung cancer. Deletions at chromosome arms 9p and 17p are also common.
  • Other frequently observed genetic lesions include overexpression of telomerase, activation of oncogenes such as K-ras and c-myc, and inactivation of tumor suppressor genes such as RB, p53 and CDKN2.
  • thrombospondin-1, fibronectin, intercellular adhesion molecule 1, and cytokeratins 6 and 18 were previously observed to be differentially expressed in lung cancers.
  • Wang et al. 2000; Oncogene 19:1519-1528) used a combination of microarray analysis and subtractive hybridization to identify 17 genes differentially overexpresssed in squamous cell carcinoma compared with normal lung epithelium.
  • the known genes they identified were keratin isoform 6, KOC, SPRC, IGFb2, connexin 26, plakofillin 1 and cytokeratin 13.
  • Familial adenomatous polyposis is caused by mutations in the adenomatous polyposis coli gene (APC), resulting in truncated or inactive forms of the protein.
  • APC adenomatous polyposis coli gene
  • This tumor suppressor gene has been mapped to chromosome 5q.
  • Hereditary nonpolyposis colorectal cancer is caused by mutations in mis-match repair genes.
  • somatic mutations in APC occur in at least 80% of sporadic colon tumors. APC mutations are thought to be the initiating event in the disease. Other mutations occur subsequently.
  • Approximately 50% of colorectal cancers contain activating mutations in ras, while 85% contain inactivating mutations in p53. Changes in all of these genes lead to gene expression changes in colon cancer.
  • compositions including nucleic acids and proteins, for the diagnosis, prevention, and treatment of immune system, neurological, developmental, muscle, and cell prohferative disorders.
  • TRAP' purified polypeptides, immune response associated proteins, referred to collectively as TRAP' and individually as TRAP-1,' TRAP-2,' TRAP-3,' TRAP-4,' TRAP-5,' 'IRAP-6,' TRAP-7,' TRAP-8,' TRAP-9,' TRAP-10,' TRAP-11,' TRAP-12,' TRAP-13,' TRAP-14,' TRAP-15,' TRAP-16,' TRAP-17,' TRAP-18,' TRAP-19,' TRAP-20,' TRAP-21,' TRAP-22,' TRAP-23,' TRAP-24,' TRAP-25,' TRAP-26,' TRAP-27,' TRAP-28,' TRAP-29,' TRAP-30,' TRAP-31,' and TRAP-32' and methods for using these proteins and their encoding polynucleotides for the detection,
  • Embodiments also provide methods for utilizing the purified immune response associated proteins and/or their encoding polynucleotides for facilitating the drug discovery process, including determination of efficacy, dosage, toxicity, and pharmacology.
  • Related embodiments provide methods for utilizing the purified immune response associated proteins and/or their encoding polynucleotides for investigating the pathogenesis of diseases and medical conditions.
  • An embodiment provides an isolated polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l- 32, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32.
  • Another embodiment provides an isolated polypeptide comprising an amino acid sequence of SEQ ID NO: 1-32.
  • Still another embodiment provides an isolated polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32.
  • polynucleotide encodes a polypeptide selected from the group consisting of SEQ ID NO: 1-32. In an alternative embodiment, the polynucleotide is selected from the group consisting of SEQ ID NO:33-64.
  • Still another embodiment provides a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32.
  • Another embodiment provides a cell transformed with the recombinant polynucleotide. Yet another embodiment provides a transgenic organism comprising the recombinant polynucleotide. Another embodiment provides a method for producing a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, b) a polypeptide comprising a naturally occuning amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32.
  • the method comprises a) culturing a cell under conditions suitable for expression of the polypeptide, wherein said cell is transformed with a recombinant polynucleotide comprising a promoter sequence operably linked to a polynucleotide encoding the polypeptide, and b) recovering the polypeptide so expressed.
  • Yet another embodiment provides an isolated antibody which specifically binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32.
  • Still yet another embodiment provides an isolated polynucleotide selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • the polynucleotide can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleot
  • the method comprises a) hybridizing the sample with a probe comprising at least 20 contiguous nucleotides comprising a sequence complementary to said target polynucleotide in the sample, and which probe specifically hybridizes to said target polynucleotide, under conditions whereby a hybridization complex is formed between said probe and said target polynucleotide or fragments thereof, and b) detecting the presence or absence of said hybridization complex.
  • the method can include detecting the amount ofthe hybridization complex.
  • the probe can comprise at least about 20, 30, 40, 60, 80, or 100 contiguous nucleotides.
  • Still yet another embodiment provides a method for detecting a target polynucleotide in a sample, said target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, b) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, c) a polynucleotide complementary to the polynucleotide of a), d) a polynucleotide complementary to the polynucleotide of b), and e) an RNA equivalent of a)-d).
  • a target polynucleotide being selected from the group consisting of a) a polynucleotide comprising a polynucleo
  • the method comprises a) amplifying said target polynucleotide or fragment thereof using polymerase chain reaction amplification, and b) detecting the presence or absence of said amplified target polynucleotide or fragment thereof.
  • the method can include detecting the amount of the amplified target polynucleotide or fragment thereof.
  • compositions comprising an effective amount of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, b) a polypeptide comprising a naturally occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, c) a biologically active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, and a pharmaceutically acceptable excipient
  • the composition can comprise an amino acid sequence selected from the group consisting of SEQ ID NO:l-32.
  • Other embodiments provide a method of treating a disease or condition associated with decreased or abnormal expression of functional IRAP, comprising administering to a patient in need of such treatment the composition.
  • Yet another embodiment provides a method for screening a compound for effectiveness as an agonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, b) a polypeptide comprising a naturaUy occu ⁇ ing amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, c) a biologically active fragment of a polypeptide having an a ino acid sequence selected from the group consisting of SEQ ID NO:l-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32.
  • the method comprises a) contacting a sample comprising the polypeptide with a compound, and b) detecting agonist activity in the sample.
  • Another embodiment provides a composition comprising an agonist compound identified by the method and a pharmaceutically acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with decreased expression of functional IRAP, comprising administering to a patient in need of such treatment the composition.
  • Still yet another embodiment provides a method for screening a compound for effectiveness as an antagonist of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, b) a polypeptide comprising a naturaUy occu ⁇ ing amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO:l-32.
  • the method comprises a) contacting a sample comprising the polypeptide with a compound, and b) detecting antagonist activity in the sample.
  • Another embodiment provides a composition comprising an antagonist compound identified by the method and a pharmaceuticaUy acceptable excipient.
  • Yet another embodiment provides a method of treating a disease or condition associated with overexpression of functional IRAP, comprising administering to a patient in need of such treatment the composition.
  • Another embodiment provides a method of screening for a compound that specificaUy binds to a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, b) a polypeptide comprising a naturaUy occu ⁇ ing amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32.
  • the method comprises a) combining the polypeptide with at least one test compound under suitable conditions, and b) detecting binding of the polypeptide to the test compound, thereby identifying a compound that specifically binds to the polypeptide.
  • Yet another embodiment provides a method of screening for a compound that modulates the activity of a polypeptide selected from the group consisting of a) a polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, b) a polypeptide comprising a naturaUy occurring amino acid sequence at least 90% identical or at least about 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NO:l-32, c) a biologicaUy active fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32, and d) an immunogenic fragment of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1-32.
  • the method comprises a) combining the polypeptide with at least one test compound under conditions permissive for the activity ofthe polypeptide, b) assessing the activity of the polypeptide in the presence of the test compound, and c) comparing the activity ofthe polypeptide in the presence ofthe test compound with the activity of the polypeptide in the absence ofthe test compound, wherein a change in the activity ofthe polypeptide in the presence ofthe test compound is indicative of a compound that modulates the activity ofthe polypeptide.
  • StiU yet another embodiment provides a method for screening a compound for effectiveness in altering expression of a target polynucleotide, wherein said target polynucleotide comprises a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, the method comprising a) contacting a sample comprising the target polynucleotide with a compound, b) detecting altered expression of the target polynucleotide, and c) comparing the expression of the target polynucleotide in the presence of varying amounts of the compound and in the absence of the compound.
  • Another embodiment provides a method for assessing toxicity of a test compound, said method comprising a) treating a biological sample containing nucleic acids with the test compound; b) hybridizing the nucleic acids ofthe treated biological sample with a probe comprising at least 20 contiguous nucleotides of a polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, ii) a polynucleotide comprising a naturaUy occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, iii) a polynucleotide having a sequence complementary to i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent
  • Hybridization occurs under conditions whereby a specific hybridization complex is formed between said probe and a target polynucleotide in the biological sample, said target polynucleotide selected from the group consisting of i) a polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, ii) a polynucleotide comprising a naturally occurring polynucleotide sequence at least 90% identical or at least about 90% identical to a polynucleotide sequence selected from the group consisting of SEQ ID NO:33-64, iii) a polynucleotide complementary to the polynucleotide of i), iv) a polynucleotide complementary to the polynucleotide of ii), and v) an RNA equivalent of i)-iv).
  • the target polynucleotide can comprise a fragment of a polynucleotide selected from the group consisting of i)-v) above; c) quantifying the amount of hybridization complex; and d) comparing the amount of hybridization complex in the treated biological sample with the amount of hybridization complex in an untreated biological sample, wherein a difference in the amount of hybridization complex in the treated biological sample is indicative of toxicity of the test compound.
  • Table 1 summarizes the nomenclature for full length polynucleotide and polypeptide embodiments of the invention.
  • Table 2 shows the GenBank identification number and annotation of the nearest GenBank homolog, and the PROTEOME database identification numbers and annotations of PROTEOME database homologs, for polypeptide embodiments of the invention.
  • the probabihty scores for the matches between each polypeptide and its homolog(s) are also shown.
  • Table 3 shows structural features of polypeptide embodiments, including predicted motifs and domains, along with the methods, algorithms, and searchable databases used for analysis of the polypeptides.
  • Table 4 lists the cDNA and/or genomic DNA fragments which were used to assemble polynucleotide embodiments, along with selected fragments of the polynucleotides.
  • Table 5 shows representative cDNA libraries for polynucleotide embodiments.
  • Table 6 provides an appendix which describes the tissues and vectors used for construction of the cDNA libraries shown in Table 5.
  • Table 7 shows the tools, programs, and algorithms used to analyze polynucleotides and polypeptides, along with appUcable descriptions, references, and threshold parameters.
  • Table 8 shows single nucleotide polymorphisms found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • IRAP refers to the amino acid sequences of substantiaUy purified IRAP obtained from any species, particularly a mammahan species, including bovine, ovine, porcine, murine, equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of IRAP.
  • Agonists may include proteins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of IRAP either by directly interacting with IRAP or by acting on components of the biological pathway in which IRAP participates.
  • an "aUelic variant” is an alternative form of the gene encoding IRAP.
  • AUelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose stracture or function may or may not be altered.
  • a gene may have none, one, or many aUelic variants of its naturally occurring for Common mutational changes which give rise to aUelic variants are generaUy ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination with the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding IRAP include those sequences with deletions, insertions, or substitutions of different nucleotides, resulting in a polypeptide the same as IRAP or a polypeptide with at least one functional characteristic of IRAP. Included within this definition are polymorphisms which may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding IRAP, and improper or unexpected hybridization to aUelic variants, with a locus other than the normal chromosomal locus for the polynucleotide encoding IRAP.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a sUent change and result in a functionaUy equivalent IRAP.
  • Deliberate amino acid substitutions may be made on the basis of one or more sirnUarities in polarity, charge, solubility, hydrophobicity, hydrophUicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of IRAP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids with uncharged polar side chains having simUar hydrophUicity values may include: asparagine and glutamine; and serine and threonine.
  • Amino acids with uncharged side chains having simUar hydrophUicity values may include: leucine, isoleucine, and valine; glycine and alanine; and phenylalanine and tyrosine.
  • amino acid and amino acid sequence can refer to an oligopeptide, a peptide, a polypeptide, or a protein sequence, or a fragment of any of these, and to naturaUy occurring or synthetic molecules. Where “amino acid sequence” is recited to refer to a sequence of a naturaUy occurring protein molecule, “amino acid sequence” and like terms are not meant to limit the amino acid sequence to the complete native amino acid sequence associated with the recited protein molecule.
  • Amplification relates to the production of additional copies of a nucleic acid. Amplification may be carried out using polymerase chain reaction (PCR) technologies or other nucleic acid amplification technologies weU known in the art.
  • PCR polymerase chain reaction
  • antagonists refers to a molecule which inhibits or attenuates the biological activity of IRAP. Antagonists may include proteins such as antibodies, anticalins, nucleic acids, carbohydrates, smaU molecules, or any other compound or composition which modulates the activity of IRAP either by directly interacting with IRAP or by acting on components of the biological pathway in which IRAP participates.
  • antibody refers to intact immunoglobulin molecules as weU as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, which are capable of binding an epitopic determinant.
  • Antibodies that bind IRAP polypeptides can be prepared using intact polypeptides or using fragments containing smaU peptides of interest as the immunizing antigen.
  • the polypeptide or oligopeptide used to immunize an animal e.g., a mouse, a rat, or a rabbit
  • an animal e.g., a mouse, a rat, or a rabbit
  • chemicaUy Commonly used carriers that are chemicaUy coupled to peptides include bovine seram albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH). The coupled peptide is then used to immunize the animal.
  • antigenic determinant refers to that region of a molecule (i.e., an epitope) that makes contact with a particular antibody.
  • a protein or a fragment of a protein is used to immunize a host animal, numerous regions of the protein may induce the production of antibodies wliich bind specificaUy to antigenic determinants (particular regions or three-dimensional stractures on the protein).
  • An antigenic determinant may compete with the intact antigen (i.e., the immunogen used to elicit the immune response) for binding to an antibody.
  • aptamer refers to a nucleic acid or oligonucleotide molecule that binds to a specific molecular target.
  • Aptamers are derived from an in vitro evolutionary process (e.g., SELEX (Systematic Evolution of Ligands by Exponential Enrichment), described in U.S. Patent No. 5,270,163), which selects for target-specific aptamer sequences from large combinatorial libraries.
  • Aptamer compositions may be double-stranded or single-stranded, and may include deoxyribonucleotides, ribonucleotides, nucleotide derivatives, or other nucleotide-like molecules.
  • the nucleotide components of an aptamer may have modified sugar groups (e.g., the 2'-OH group of a ribonucleotide may be replaced by 2'-F or 2'-NH 2 ), which may improve a desired property, e.g., resistance to nucleases or longer lifetime in blood.
  • Aptamers may be conjugated to other molecules, e.g., a high molecular weight carrier to slow clearance ofthe aptamer from the circulatory system.
  • Aptamers may be specificaUy cross-linked to their cognate hgands, e.g., by photo-activation of a cross-linker (Brody, E.N. and L. Gold (2000) J. Biotechnol. 74:5-13).
  • introduction refers to an aptamer which is expressed in vivo.
  • a vaccinia virus-based RNA expression system has been used to express specific RNA aptamers at high levels in the cytoplasm of leukocytes (Blind, M. et al. (1999) Proc. Natl. Acad. Sci. USA 96:3606-3610).
  • spiegelmer refers to an aptamer which includes L-DNA, L-RNA, or other left- handed nucleotide derivatives or nucleotide-like molecules. Aptamers containing left-handed nucleotides are resistant to degradation by naturaUy occurring enzymes, which normaUy act on substrates containing right-handed nucleotides.
  • antisense refers to any composition capable of base-pairing with the "sense"
  • Antisense compositions may include DNA; RNA; peptide nucleic acid (PNA); ohgonucleotides having modified backbone linkages such as phosphorothioates, methylphosphonates, orbenzylphosphonates; ohgonucleotides having modified sugar groups such as 2'-methoxyethyl sugars or 2'-methoxyethoxy sugars; or ohgonucleotides having modified bases such as 5-methyl cytosine, 2'-deoxyura l, or 7-deaza-2'- deoxyguanosine.
  • Antisense molecules may be produced by any method including chemical synthesis or transcription.
  • the complementary antisense molecule base-pairs with a naturaUy occurring nucleic acid sequence produced by the ceU to form duplexes which block either transcription or translation.
  • the designation "negative” or “minus'' can refer to the antisense strand, and the designation “positive” or “plus” can refer to the sense strand of a reference DNA molecule.
  • biologicalcaUy active refers to a protein having structural, regulatory, or biochemical functions of a naturaUy occurring molecule.
  • immunologicalaUy active or “immunogenic” refers to the capability ofthe natural, recombinant, or synthetic IRAP, or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or ceUs and to bind with specific antibodies.
  • Complementary describes the relationship between two single-stranded nucleic acid sequences that anneal by base-pairing. For example, 5'-AGT-3' pairs with its complement, 3 * -TCA-5'.
  • composition comprising a given polynucleotide and a “composition comprising a given polypeptide” can refer to any composition containing the given polynucleotide or polypeptide.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotides encoding IRAP or fragments of IRAP may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl), detergents (e.g., sodium dodecyl sulfate; SDS), and other components (e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.).
  • salts e.g., NaCl
  • detergents e.g., sodium dodecyl sulfate; SDS
  • other components e.g., Denhardt's solution, dry milk, salmon sperm DNA, etc.
  • Consensus sequence refers to a nucleic acid sequence which has been subjected to repeated DNA sequence analysis to resolve uncalled bases, extended using the XL-PCR kit (Applied Biosystems, Foster City CA) in the 5' and/or the 3' direction, and resequenced, or which has been assembled from one or more overlapping cDNA, EST, or genomic DNA fragments using a computer program for fragment assembly, such as the GELVTEW fragment assembly system (Accelrys, Burlington MA) or Phrap (University of Washington, Seattle WA). Some sequences have been both extended and assembled to produce the consensus sequence.
  • Constant amino acid substitutions are those substitutions that are predicted to least interfere with the properties of the original protein, i.e., the structure and especiaUy the function of the protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generaUy maintain (a) the structure of the polypeptide backbone in the area ofthe substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to a chemically modified polynucleotide or polypeptide.
  • Chemical modifications of a polynucleotide can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide which retains at least one biological or immunological function of the natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegylation, or any simUar process that retains at least one biological or immunological function of the polypeptide from which it was derived.
  • a “detectable label” refers to a reporter molecule or enzyme that is capable of generating a measurable signal and is covalently or noncovalently joined to a polynucleotide or polypeptide.
  • “Differential expression” refers to increased or upregulated; or decreased, downregulated, or absent gene or protein expression, determined by comparing at least two different samples. Such comparisons may be carried out between, for example, a treated and an untreated sample, or a diseased and a normal sample.
  • Exon shuffling refers to the recombination of different coding regions (exons). Since an exon may represent a stractural or functional domain of the encoded protein, new proteins may be assembled through the novel reassortment of stable substructures, thus aUowing acceleration of the evolution of new protein functions .
  • a “fragment” is a unique portion of IRAP or a polynucleotide encoding IRAP which can be identical in sequence to, but shorter in length than, the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from about 5 to about 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes maybe at least 5, 10, 15, 16, 20, 25, 30, 40, 50, 60, 75, 100, 150, 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentiaUy selected from certain regions of a molecule.
  • a polypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50%) of a polypeptide as shown in a certain defined sequence.
  • these lengths are exemplary, and any length that is supported by the specification, including the Sequence Listing, tables, and figures, may be encompassed by the present embodiments.
  • a fragment of SEQ ID NO:33-64 can comprise a region of unique polynucleotide sequence that specificaUy identifies SEQ ID NO:33-64, for example, as distinct from any other sequence in the genome from which the fragment was obtained.
  • a fragment of SEQ ID NO:33-64 can be employed in one or more embodiments of methods of the invention, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO:33-64 from related polynucleotides.
  • the precise length of a fragment of SEQ ID NO:33-64 and the region of SEQ ID NO:33-64 to which the fragment corresponds are routinely determinable by one of ordinary skiU in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO:l-32 is encoded by a fragment of SEQ ID NO:33-64.
  • a fragment of SEQ ID NO: 1-32 can comprise a region of unique amino acid sequence that specificaUy identifies SEQ ID NO:l-32.
  • a fragment of SEQ ID NO:l-32 can be used as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO:l-32.
  • the precise length of a fragment of SEQ ID NO:l-32 and the region of SEQ ID NO:l-32 to which the fragment corresponds can be determined based on the intended purpose for the fragment using one or more analytical methods described herein or otherwise known in the art.
  • a “fuU length” polynucleotide is one containing at least a translation initiation codon (e.g., methionine) foUowed by an open reading frame and a translation termination codon.
  • a “full length” polynucleotide sequence encodes a "fuU length” polypeptide sequence.
  • “Homology” refers to sequence similarity or, alternatively, sequence identity, between two or more polynucleotide sequences or two or more polypeptide sequences.
  • percent identity and % identity refer to the percentage of identical nucleotide matches between at least two polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize ahgnment between two sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • Percent identity between polynucleotide sequences may be determined using one or more computer algorithms or programs known in the art or described herein. For example, percent identity can be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence ahgnment program. This program is part ofthe LASERGENE software package, a suite of molecular biological analysis programs (DNASTAR, Madison WI). CLUSTAL V is described in Higgins, D.G. and P.M. Sharp (1989; CABIOS 5:151-153) and in Higgins, D.G. et al. (1992; CABIOS 8:189-191).
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Ahgnment Search Tool
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Ahgnment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases.
  • BLAST 2 Sequences are commonly used with gap and other parameters set to default settings. For example, to compare two nucleotide sequences, one may use blastn with the "BLAST 2 Sequences” tool Version 2.0.12 (April-21-2000) set at default parameters. Such default parameters may be, for example: Matrix: BLOSUM62
  • Percent identity may be measured over the length of an entire defined sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20, at least 30, at least 40, at least 50, at least 70, at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • Nucleic acid sequences that do not show a high degree of identity may nevertheless encode simUar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantiaUy the same protein.
  • percent identity and % identity refer to the percentage of identical residue matches between at least two polypeptide sequences ahgned using a standardized algorithm.
  • Methods of polypeptide sequence ahgnment are weU-known. Some ahgnment methods take into account conservative amino acid substitutions. Such conservative substitutions, explained in more detaU above, generaUy preserve the charge and hydrophobicity at the site of substitution, thus preserving the stracture (and therefore function) of the polypeptide.
  • percent simUarity and % simUarity refer to the percentage of residue matches, including identical residue matches and conservative substitutions, between at least two polypeptide sequences ahgned using a standardized algorithm. In contrast, conservative substitutions are not included in the calculation of percent identity between polypeptide sequences. Percent identity between polypeptide sequences may be determined using the default parameters of the CLUSTAL V algorithm as incorporated into the MEGALIGN version 3.12e sequence ahgnment program (described and referenced above).
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences Version 2.0.12 (April-21-2000) with blastp set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15, at least 20, at least 30, at least 40, at least 50, at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size and which contain aU of the elements required for chromosome replication, segregation and maintenance.
  • humanized antibody refers to an antibody molecule in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of complementarity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency of the hybridization process, with more stringent conditions aUowing less non-specific binding, i.e. , binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skiU in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1% (w/v) SDS, and about 100 /ig/ml sheared, denatured salmon sperm DNA.
  • GeneraUy stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are typicaUy selected to be about 5°C to 20°C lower than the thermal melting point (T ⁇ for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • An equation for calculating T m and conditions for nucleic acid hybridization are well known and can be found in Sambrook, J. and D.W. RusseU (2001 ; Molecular Cloning: A Laboratory Manual, 3rd ed., vol. 1-3, Cold Spring Harbor Press, Cold Spring Harbor NY, ch. 9).
  • High stringency conditions for hybridization between polynucleotides of the present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS, for 1 hour. Alternatively, temperatures of about 65°C, 60°C, 55°C, or 42°C may be used. SSC concentration may be varied from about 0.1 to 2 x SSC, with SDS being present at about 0.1 %.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance, sheared and denatured salmon sperm DNA at about 100-200 ⁇ g/ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • Useful variations on these wash conditions wiU be readUy apparent to those of ordinary skiU in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary simUarity between the nucleotides. Such simUarity is strongly indicative of a simUar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acids by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R 0 t analysis) or formed between one nucleic acid present in solution and another nucleic acid immobilized on a sohd support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which ceUs or their nucleic acids have been fixed).
  • insertion and “addition” refer to changes in an amino acid or polynucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides, respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemo ines, and other signaling molecules, which may affect ceUular and systemic defense systems.
  • an "hnmunogenic fragment” is a polypeptide or ohgopeptide fragment of IRAP which is capable of eliciting an immune response when introduced into a living organism, for example, a mammal.
  • immunogenic fragment also includes any polypeptide or ohgopeptide fragment of IRAP which is useful in any of the antibody production methods disclosed herein or known in the art.
  • microa ⁇ ay refers to an anangement of a plurality of polynucleotides, polypeptides, antibodies, or other chemical compounds on a substrate.
  • element and “array element” refer to a polynucleotide, polypeptide, antibody, or other chemical compound having a unique and defined position on a microa ⁇ ay.
  • modulate refers to a change in the activity of IRAP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of IRAP.
  • nucleic acid and nucleic acid sequence refer to a nucleotide, oligonucleotide, polynucleotide, or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin which may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA), or to any DNA-like or RNA-like material.
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with a second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • Operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition.
  • PNAs preferentiaUy bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the ceU.
  • Post-translational modification of an IRAP may involve hpidation, glycosylation, phosphorylation, acetylation, racemization, proteolytic cleavage, and other modifications known in the art. These processes may occur syntheticaUy or biochemicaUy. Biochemical modifications w l vary by ceU type depending on the enzymatic milieu of IRAP.
  • Probe refers to nucleic acids encoding IRAP, their complements, or fragments thereof, which are used to detect identical, aUelic or related nucleic acids. Probes are isolated ohgonucleotides or polynucleotides attached to a detectable label or reporter molecule.
  • Typical labels include radioactive isotopes, ligands, chemiluminescent agents, and enzymes.
  • "Primers" are short nucleic acids, usuaUy DNA ohgonucleotides, which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid, e.g., by the polymerase chain reaction (PCR).
  • Probes and primers as used in the present invention typicaUy comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides of the disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used. Methods for preparing and using probes and primers are described in, for example, Sambrook, J. and D.W.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991, Whitehead Institute for Biomedical Research, Cambridge MA).
  • Ohgonucleotides for use as primers are selected using software known in the art for such purpose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of ohgonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kUobases.
  • SimUar primer selection programs have incorporated additional features for expanded capabilities.
  • the PrimOU primer selection program (avaUable to the public from the Genome Center at University of Texas South West Medical Center, DaUas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • Primer3 primer selection program (avaUable to the public from the Whitehead Institute/MIT Center for Genome Research, Cambridge MA) aUows the user to input a "nispriming library," in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of ohgonucleotides for microarrays.
  • the source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user's specific needs.
  • the PrimeGen program (avaUable to the pubhc from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby aUowing selection of primers that hybridize to either the most conserved or least conserved regions of ahgned nucleic acid sequences. Hence, this program is useful for identification of both unique and conserved ohgonucleotides and polynucleotide fragments.
  • oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fuUy or partiaUy complementary polynucleotides in a sample of nucleic acids. Methods of ohgonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a nucleic acid that is not naturaUy occu ⁇ ing or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook and RusseU (supra).
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a ceU.
  • such recombinant nucleic acids maybe part of a viral vector, e.g., based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a “regulatory element” refers to a nucleic acid sequence usuaUy derived from untranslated regions of a gene and includes enhancers, promoters, introns, and 5' and 3' untranslated regions (UTRs). Regulatory elements interact with host or viral proteins wliich control transcription, translation, or RNA stability.
  • Reporter molecules are chemical or biochemical moieties used for labeling a nucleic acid, amino acid, or antibody. Reporter molecules include radionuchdes; enzymes; fluorescent, chemUuminescent, or chromogenic agents; substrates; cof actors; inhibitors; magnetic particles; and other moieties known in the art.
  • RNA equivalent in reference to a DNA molecule, is composed of the same linear sequence of nucleotides as the reference DNA molecule with the exception that aU occu ⁇ ences of the nitrogenous base thymine are replaced with uracU, and the sugar backbone is composed of ribose instead of deoxyribose.
  • sample is used in its broadest sense.
  • a sample suspected of containing IRAP, nucleic acids encoding IRAP, or fragments thereof may comprise a bodUy fluid; an extract from a ceU, chromosome, organeUe, or membrane isolated from a ceU; a ceU; genomic DNA, RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print; etc.
  • binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a smaU molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular stracture of the protein, e.g., the antigenic determinant or epitope, recognized by the binding molecule. For example, if an antibody is specific for epitope "A,” the presence of a polypeptide comprising the epitope A, or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody wiU reduce the amount of labeled A that binds to the antibody.
  • substantiallyUy purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably at least about 75% free, and most preferably at least about 90% free from other components with which they are naturaUy associated.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, shdes, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as weUs, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • a “transcript image” or “expression profile” refers to the coUective pattern of gene expression by a particular ceU type or tissue under given conditions at a given time.
  • Transformation describes a process by which exogenous DNA is introduced into a recipient ceU. Transformation may occur under natural or artificial conditions according to various methods weU known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host ceU. The method for transformation is selected based on the type of host ceU being transformed and may include, but is not limited to, bacteriophage or viral infection, electroporation, heat shock, lipofection, and particle bombardment.
  • transformed ceUs includes stably transformed ceUs in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as weU as transiently transformed ceUs which express the inserted DNA or RNA for limited periods of time.
  • a "transgenic organism,” as used herein, is any organism, including but not limited to animals and plants, in which one or more of the cells of the organism contains heterologous nucleic acid introduced by way of human intervention, such as by transgenic techniques well known in the art.
  • the nucleic acid is introduced into the cell, directly or indirectly by introduction into a precursor of the cell, by way of deliberate genetic manipulation, such as by microinjection or by infection with a recombinant virus.
  • the nucleic acid can be introduced by infection with a recombinant viral vector, such as a lentiviral vector (Lois, C. et al. (2002) Science 295:868-872).
  • the term genetic manipulation does not include classical cross-breeding, or in vitro fertilization, but rather is directed to the introduction of a recombinant DNA molecule.
  • the transgenic organisms contemplated in accordance with the present invention include bacteria, cyanobacteria, fungi, plants and animals.
  • the isolated DNA of the present invention can be introduced into the host by methods known in the art, for example infection, transfection, transformation or transconjugation. Techniques for transferring the DNA ofthe present invention into such organisms are widely known and provided in references such as Sambrook and Russell (supra).
  • a "variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one ofthe nucleic acid sequences using blastn with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91 %, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity over a certain defined length.
  • a variant may be described as, for example, an "aUelic” (as defined above), "splice,” “species,” or “polymorphic” variant.
  • a splice variant may have significant identity to a reference molecule, but wiU generaUy have a greater or lesser number of polynucleotides due to alternate splicing during mRNA processing.
  • the co ⁇ esponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotides that vary from one species to another.
  • the resulting polypeptides wiU generaUy have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass "single nucleotide polymorphisms" (SNPs) in which the polynucleotide sequence varies by one nucleotide base. The presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • SNPs single nucleotide polymorphisms
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity or sequence simUarity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07-1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%, at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or greater sequence identity or sequence simUarity over a certain defined length of one of the polypeptides.
  • Various embodiments of the invention include new human immune response associated proteins (IRAP), the polynucleotides encoding IRAP, and the use of these compositions for the diagnosis, treatment, or prevention of immune system, neurological, developmental, muscle, and ceU prohferative disorders.
  • IRAP human immune response associated proteins
  • Table 1 summarizes the nomenclature for the full length polynucleotide and polypeptide embodiments of the invention. Each polynucleotide and its corresponding polypeptide are correlated to a single Incyte project identification number (Incyte Project ID). Each polypeptide sequence is denoted by both a polypeptide sequence identification number (Polypeptide SEQ ID NO:) and an Incyte polypeptide sequence number (Incyte Polypeptide ID) as shown.
  • Each polynucleotide sequence is denoted by both a polynucleotide sequence identification number (Polynucleotide SEQ ID NO:) and an Incyte polynucleotide consensus sequence number (Incyte Polynucleotide ID) as shown.
  • Column 6 shows the Incyte ID numbers of physical, full length clones corresponding to the polypeptide and polynucleotide sequences of the invention.
  • the fuU length clones encode polypeptides which have at least 95% sequence identity to the polypeptide sequences shown in column 3.
  • Table 2 shows sequences with homology to polypeptide embodiments of the invention as identified by BLAST analysis against the GenBank protein (genpept) database and the PROTEOME database.
  • Columns 1 and 2 show the polypeptide sequence identification number (Polypeptide SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for polypeptides of the invention.
  • Column 3 shows the GenBank identification number (GenBank ID NO:) of the nearest GenBank homolog and the PROTEOME database identification numbers (PROTEOME ID NO:) of the nearest PROTEOME database homologs.
  • Column 4 shows the probabihty scores for the matches between each polypeptide and its homolog(s).
  • Column 5 shows the annotation of the GenBank and PROTEOME database homolog(s) along with relevant citations where applicable, aU of which are expressly incorporated by reference herein.
  • Table 3 shows various structural features of the polypeptides ofthe invention.
  • Columns 1 and 2 show the polypeptide sequence identification number (SEQ ID NO:) and the corresponding Incyte polypeptide sequence number (Incyte Polypeptide ID) for each polypeptide of the invention.
  • Column 3 shows the number of amino acid residues in each polypeptide.
  • Column 4 shows amino acid residues comprising signature sequences, domains, motifs, potential phosphorylation sites, and potential glycosylation sites.
  • Column 5 shows analytical methods for protein structure/function analysis and in some cases, searchable databases to which the analytical methods were applied.
  • Tables 2 and 3 summarize the properties of polypeptides of the invention, and these properties estabhsh that the claimed polypeptides are immune response associated proteins.
  • SEQ ID NO:10 is 88% identical, from residue Ml to residue A181, to human interferon regulatory factor 7 (GenBank ID gl621457) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.)
  • the BLAST probabUity score is 1.6e-83, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ID NO:10 also has homology to a DNA binding protein that plays a critical role in the virus-mediated induction of type I interferon genes, is involved in virus gene repression and DNA damage responses through JNK signaling, as weU as in the innate immune response to viras infection and is interferon regulatory factor 7, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO: 10 also contains interferon regulatory factor and interferon regulatory factor transcription factor domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM/SMART databases of conserved protein famUies/domains. (See Table 3.) Data from BLIMPS, MOTIFS, and PROFILESCAN analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:10 is an interferon regulatory factor.
  • HMM hidden Markov model
  • SEQ ID NO:12 is 98% identical, fromresidue G26 to residue N100, to human smaU inducible cytokine A23 precursor (GenBank ID g3719362) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 3.5e-47, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance.
  • BLAST Basic Local Ahgnment Search Tool
  • SEQ ID NO: 12 also has homology to proteins that are locahzed extraceUularly, bind CC chemokine receptor 1 and induces calcium influx, inhibit proliferation of myeloid progenitor ceUs, induce chemotaxis in monocytes and resting T lymphocytes, and are smaU inducible cytokine A23, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO: 12 also contains a intercrine alpha family domain and small cytokine domains as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based SMART and PFAM databases of conserved protein families/domains.
  • HMM hidden Markov model
  • SEQ ID NO: 12 is a cytokine.
  • SEQ ID NO:24 is 87% identical, from residue Ml to residue
  • L161 to human tumor necrosis factor receptor superfamUy, member 9 (GenBank ID gl3623201) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 1.7e-79, which indicates the probabUity of obtaining the observed polypeptide sequence ahgnment by chance.
  • SEQ ID NO:24 also has homology to proteins that are localized to the plasma membrane, induce apoptosis, control cell proliferation, regulate T-ceU, peripheral monocyte, and B-ceU survival, and are tumor necrosis factor receptors, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:24 also contains a rumor necrosis factor receptor / nerve growth factor receptor repeat domain and a TNFR/NGFR cysteine-rich region domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based SMART and PFAM databases of conserved protein families/domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further co ⁇ oborative evidence that SEQ ID NO:24 is a tumor necrosis factor receptor.
  • HMM hidden Markov model
  • SEQ ID NO:28 is a splice variant of human IL12 receptor component (GenBank ID g507151) as determined by the Basic Local Ahgnment Search Tool
  • SEQ ID NO:28 also has homology to proteins that are locahzed to the plasma membrane, that mediate IL12-induced Thl cell responses to pathogens, that play a role in resisting mycobacteria and salmoneUa infections, and are signaling receptors, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:28 also contains a Fibronectin type 3 domain, as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein families/domains. (See Table 3.) Data from BLIMPS and MOTIFS analyses, and BLAST analyses against the PRODOM and DOMO databases, provide further co ⁇ oborative evidence that SEQ ID NO:28 is a component of the IL12 receptor.
  • SEQ ID NO:30 is 96% identical, fromresidue G10 to residue W213, to human CD1E antigen, isoform 1 (GenBank ID g8249469) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabUity score is 4.2e-l 14, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:30 also has homology to proteins that are locahzed to the golgi or endosome, are involved in nonpeptide antigen processing, and are members of the CD1 family of nonclassical MHC class I glycoproteins, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:30 also contains an immunoglobulin C-type domain and an Ig superfamily from SCOP domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)- based SMART and INCY databases of conserved protein famihes/domains. (See Table 3.) Data from MOTIFS analysis, and BLAST analysis against DOMO database, provide further corroborative evidence that SEQ ID NO:30 is a CD1 antigen.
  • HMM hidden Markov model
  • SEQ ID NO:32 is 97% identical, fromresidue Q18 to residue K98, to human T3 delta protein (GenBank ID g755754) as determined by the Basic Local Ahgnment Search Tool (BLAST). (See Table 2.) The BLAST probabihty score is 5.3e-46, which indicates the probabihty of obtaining the observed polypeptide sequence ahgnment by chance. SEQ ID NO:32 also has homology to protein CD3 antigen delta polypeptide, which is the delta subunit of the CD3 (T3) T-ceU receptor complex, and is involved in T ceU signaUng and activation, as determined by BLAST analysis using the PROTEOME database.
  • CD3 antigen delta polypeptide which is the delta subunit of the CD3 (T3) T-ceU receptor complex, and is involved in T ceU signaUng and activation, as determined by BLAST analysis using the PROTEOME database.
  • SEQ ID NO:32 also contains a immunoreceptor tyrosine-based activation motif domain as determined by searching for statisticaUy significant matches in the hidden Markov model (HMM)-based PFAM and SMART databases of conserved protein famihes/domains. (See Table 3.) Data from BLAST analyses against the PRODOM and DOMO databases, provide further corroborative evidence that SEQ ID NO:32 is a T3 delta protein.
  • SEQ ID NO:l-9, SEQ ID NO:ll, SEQ ID NO:13-23, SEQ ID NO:25-27, SEQ ID NO:29, and SEQ ID NO:31 were analyzed and annotated in a simUar manner. The algorithms and parameters for the analysis of SEQ ID NO:l-32 are described in Table 7.
  • the fuU length polynucleotide embodiments were assembled using cDNA sequences or coding (exon) sequences derived from genomic DNA, or any combination of these two types of sequences.
  • Column 1 lists the polynucleotide sequence identification number (Polynucleotide SEQ ID NO:), the corresponding Incyte polynucleotide consensus sequence number (Incyte ID) for each polynucleotide of the invention, and the length of each polynucleotide sequence in basepairs.
  • Column 2 shows the nucleotide start (5') and stop (3') positions of the cDNA and/or genomic sequences used to assemble the fuU length polynucleotide embodiments, and of fragments of the polynucleotides which are useful, for example, in hybridization or amplification technologies that identify SEQ ID NO.-33-64 or that distinguish between SEQ ID NO:33-64 and related polynucleotides.
  • the polynucleotide fragments described in Column 2 of Table 4 may refer specificaUy, for example, to Incyte cDNAs derived from tissue-specific cDNA libraries or from pooled cDNA libraries.
  • the polynucleotide fragments described in column 2 may refer to GenBank cDNAs or ESTs which contributed to the assembly of the full length polynucleotides.
  • the polynucleotide fragments described in column 2 may identify sequences derived from the ENSEMBL (The Sanger Centre, Cambridge, UK) database (Le., those sequences including the designation "ENST").
  • the polynucleotide fragments described in column 2 may be derived from the NCBI RefSeq Nucleotide Sequence Records Database (Le., those sequences including the designation "NM” or “NT”) or the NCBI RefSeq Protein Sequence Records (i.e. , those sequences including the designation "NP”).
  • the polynucleotide fragments described in column 2 may refer to assemblages of both cDNA and Genscan-predicted exons brought together by an "exon stitching" algorithm.
  • a polynucleotide sequence identified as FL_XXXXX_N,_N 2 _YYYY_N 3 _N 4 represents a "stitched" sequence in which XXXXX is the identification number of the cluster of sequences to which the algorithm was apphed, and YYYYY is the number of the prediction generated by the algorithm, and N l ⁇ 2 ⁇ 3 .. , if present, represent specific exons that may have been manuaUy edited during analysis (See Example V).
  • the polynucleotide fragments in column 2 may refer to assemblages of exons brought together by an "exon-stretching" algorithm.
  • a polynucleotide sequence identified as FLXXXXXX_gAAAAA_gBBBBB_l_N is a "stretched" sequence, with XXXXXbeing the Incyte project identification number, g ⁇ AAAA being the GenBank identification number of the human genomic sequence to which tlie "exon-stretching" algorithm was apphed, gBBBBB being the GenBank identification number or NCBI Ref eq identification number of the nearest GenBank protein homolog, and N referring to specific exons (See Example V).
  • a RefSeq identifier (denoted by "NM,” “NP,” or “NT”) may be used in place of the GenBank identifier (Le. , gBBBBB).
  • a prefix identifies component sequences that were hand-edited, predicted from genomic DNA sequences, or derived from a combination of sequence analysis methods.
  • the foUowing Table hsts examples of component sequence prefixes and co ⁇ esponding sequence analysis methods associated with the prefixes (see Example IV and Example V).
  • Incyte cDNA coverage redundant with the sequence coverage shown in Table 4 was obtained to confirm the final consensus polynucleotide sequence, but the relevant Incyte cDNA identification numbers are not shown.
  • Table 5 shows the representative cDNA hbraries for those full length polynucleotides which were assembled using Incyte cDNA sequences.
  • the representative cDNA library is the Incyte cDNA library which is most frequently represented by the Incyte cDNA sequences which were used to assemble and confirm the above polynucleotides.
  • the tissues and vectors which were used to construct the cDNA hbraries shown in Table 5 are described in Table 6.
  • Table 8 shows single nucleotide polymorphisms (SNPs) found in polynucleotide sequences of the invention, along with aUele frequencies in different human populations.
  • Columns 1 and 2 show the polynucleotide sequence identification number (SEQ ID NO:) and the co ⁇ esponding Incyte project identification number (PID) for polynucleotides of the invention.
  • Column 3 shows the Incyte identification number for tlie EST in which the SNP was detected (EST ID), and column 4 shows the identification number for the SNP (SNP ID).
  • Column 5 shows the position within the EST sequence at which the SNP is located (EST SNP), and column 6 shows the position ofthe SNP within the full- length polynucleotide sequence (CB 1 SNP).
  • Column 7 shows the allele found in the EST sequence.
  • Columns 8 and 9 show tlie two aheles found at the SNP site.
  • Column 10 shows the amino acid encoded by the codon including the SNP site, based upon the allele found in the EST.
  • Columns 11- 14 show the frequency of allele 1 in four different human populations. An entry of n/d (not detected) indicates that the frequency of aUele 1 in the population was too low to be detected, while n/a (not available) indicates that the allele frequency was not determined for the population.
  • the invention also encompasses IRAP variants.
  • IRAP variants can have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91 %, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% amino acid sequence identity to the IRAP amino acid sequence, and can contain at least one functional or stractural characteristic of IRAP.
  • Various embodiments also encompass polynucleotides which encode IRAP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO:33-64, which encodes IRAP.
  • the polynucleotide sequences of SEQ ID NO:33-64, as presented in the Sequence Listing, embrace the equivalent RNA sequences, wherein occurrences of the nitrogenous base thymine are replaced with uracU, and the sugar backbone is composed of ribose instead of deoxyribose.
  • the invention also encompasses variants of a polynucleotide encoding IRAP.
  • a variant polynucleotide wiU have at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polynucleotide sequence identity to a polynucleotide encoding IRAP.
  • a particular aspect of the invention encompasses a variant of a polynucleotide comprising a sequence selected from the group consisting of SEQ ID NO:33-64 which has at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO:33-64.
  • Any one of the polynucleotide variants described above can encode a polypeptide which contains at least one functional or structural characteristic of IRAP.
  • a polynucleotide variant of the invention is a splice variant of a polynucleotide encoding IRAP.
  • a splice variant may have portions which have significant sequence identity to a polynucleotide encoding IRAP, but wiU generaUy have a greater or lesser number of nucleotides due to additions or deletions of blocks of sequence arising from alternate splicing during mRNA processing.
  • a sphce variant may have less than about 70%, or alternatively less than about 60%, or alternatively less than about 50% polynucleotide sequence identity to a polynucleotide encoding IRAP over its entire length; however, portions of the sphce variant wUl have at least about 70%, or alternatively at least about 85%, or alternatively at least about 95%, or alternatively 100% polynucleotide sequence identity to portions of the polynucleotide encoding IRAP.
  • a polynucleotide comprising a sequence of SEQ ID NO:34, a polynucleotide comprising a sequence of SEQ ID NO:35, a polynucleotide comprising a sequence of SEQ ID NO:37, a polynucleotide comprising a sequence of SEQ ID NO:40, a polynucleotide comprising a sequence of SEQ ID NO:41, and a polynucleotide comprising a sequence of SEQ ID NO:55 are sphce variants of each other;
  • a polynucleotide comprising a sequence of SEQ ID NO:36, a polynucleotide comprising a sequence of SEQ ID NO:39, and a polynucleotide comprising a sequence of SEQ ID NO:62 are sphce variants of each other; a polynucleotide comprising a sequence of SEQ ID NO:42 and a polynucleot
  • polynucleotides which encode IRAP and its variants are generaUy capable of hybridizing to polynucleotides encoding naturaUy occurring IRAP under appropriately selected conditions of stringency, it may be advantageous to produce polynucleotides encoding IRAP or its derivatives possessing a substantiaUy different codon usage, e.g., inclusion of non-naturaUy occurring codons. Codons may be selected to increase the rate at which expression ofthe peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utihzed by the host.
  • RNA transcripts having more desirable properties such as a greater half-hfe, than transcripts produced from the naturaUy occurring sequence.
  • the invention also encompasses production of polynucleotides which encode IRAP and IRAP derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic polynucleotide may be inserted into any of the many avaUable expression vectors and ceU systems using reagents weU known in the art.
  • synthetic chemistry may be used to introduce mutations into a polynucleotide encoding IRAP or any fragment thereof.
  • Embodiments ofthe invention can also include polynucleotides that are capable of hybridizing to the claimed polynucleotides, and, in particular, to those having the sequences shown in SEQ ID NO:33-64 and fragments thereof, under various conditions of stringency (Wahl, G.M. and S.L. Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A.R. (1987) Methods Enzymol. 152:507-511). Hybridization conditions, including annealing and wash conditions, are described in "Definitions.” Methods for DNA sequencing are weU known in the art and may be used to practice any of the embodiments of the invention.
  • the methods may employ such enzymes as the Klenow fragment of DNA polymerase I, SEQUENASE (US Biochemical, Cleveland OH), Taq polymerase (Apphed Biosystems), thermostable T7 polymerase (Amersham Biosciences, Piscataway NJ), or combinations of polymerases and proofreading exonucleases such as those found in the ELONGASE amplification system (Invitrogen, Carlsbad CA).
  • sequence preparation is automated with machines such as the MICROLAB 2200 hquid transfer system (HamUton, Reno NV), PTC200 thermal cycler (MJ Research, Watertown MA) and ABI CATALYST 800 thermal cycler (Apphed Biosystems).
  • Sequencing is then ca ⁇ ied out using either the ABI 373 or 377 DNA sequencing system (Apphed Biosystems), the MEGABACE 1000 DNA sequencing system (Amersham Biosciences), or other systems known in the art.
  • the resulting sequences are analyzed using a variety of algorithms which are well known in the art (Ausubel et al., supra, ch. 7; Meyers, R.A. (1995) Molecular Biology and Biotechnology, Wiley VCH, New York NY, pp. 856-853).
  • the nucleic acids encoding IRAP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements.
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (Sarkar, G. (1993) PCR Methods Applic. 2:318-322).
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template.
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (Trigha, T. et al.
  • a third method involves PCR amplification of DNA fragments adjacent to known sequences in human and yeast artificial chromosome DNA (Lagerstrom, M. et al. (1991) PCR Methods Applic. 1:111-119).
  • multiple restriction enzyme digestions and ligations maybe used to insert an engineered double-stranded sequence into a region of unknown sequence before performing PCR.
  • Other methods which may be used to retrieve unknown sequences are known in the art (Parker, J.D. et al. (1991) Nucleic Acids Res. 19:3055-3060).
  • primers maybe designed using commerciaUy avaUable software, such as OLIGO 4.06 primer analysis software (National Biosciences, Plymouth MN) or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C.
  • hbraries When screening for full length cDNAs, it is preferable to use hbraries that have been size-selected to include larger cDNAs. In addition, random-primed hbraries, which often include sequences containing the 5' regions of genes, are preferable for situations in which an ohgo d(T) library does not yield a full-length cDNA. Genomic hbraries may be useful for extension of sequence into 5' non-transcribed regulatory regions.
  • CapiUary electrophoresis systems which are commerciaUy avaUable may be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products.
  • capUlary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific, laser-stimulated fluorescent dyes, and a charge coupled device camera for detection of the emitted wavelengths.
  • Output/light intensity may be converted to electrical signal using appropriate software (e.g., GENOTYPER and SEQUENCE NAVIGATOR, Apphed Biosystems), and the entire process from loading of samples to computer analysis and electronic data display maybe computer controUed.
  • CapUlary electrophoresis is especially preferable for sequencing smaU DNA fragments which may be present in limited amounts in a particular sample.
  • polynucleotides or fragments thereof which encode IRAP may be cloned in recombinant DNA molecules that direct expression of IRAP, or fragments or functional equivalents thereof, in appropriate host ceUs. Due to the inherent degeneracy of the genetic code, other polynucleotides which encode substantiaUy the same or a functionaUy equivalent polypeptides may be produced and used to express IRAP.
  • the polynucleotides of the invention can be engineered using methods generally known in the art in order to alter IRAP-encoding sequences for a variety of purposes including, but not limited to, modification of the cloning, processing, and/or expression of the gene product.
  • DNA shuffling by random fragmentation and PCR reassembly of gene fragments and synthetic oligonucleotides may be used to engineer the nucleotide sequences.
  • ohgonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosylation patterns, change codon preference, produce sphce variants, and so forth.
  • the nucleotides of the present invention may be subjected to DNA shuffling techniques such as MOLECULARBREEDING (Maxygen Inc., Santa Clara CA; described in U.S. Patent No.
  • DNA shuffling is a process by which a library of gene variants is produced using PCR-mediated recombination of gene fragments. The library is then subjected to selection or screening procedures that identify those gene variants with the desired properties.
  • prefe ⁇ ed variants may then be pooled and further subjected to recursive rounds of DNA shuffling and selection/screening.
  • genetic diversity is created through "artificial" breeding and rapid molecular evolution. For example, fragments of a single gene containing random point mutations may be recombined, screened, and then reshuffled until the desired properties are optimized. Alternatively, fragments of a given gene may be recombined with fragments of homologous genes in the same gene family, either from the same or different species, thereby maximizing the genetic diversity of multiple naturally occu ⁇ ing genes in a directed and controllable manner.
  • polynucleotides encoding IRAP may be synthesized, in whole or in part, using one or more chemical methods weU known in the art (Caruthers, M.H. et al. (1980) Nucleic Acids Symp. Ser. 7:215-223; Horn, T. et al. (1980) Nucleic Acids Symp. Ser. 7:225-232).
  • IRAP itself or a fragment thereof may be synthesized using chemical methods known in the art.
  • peptide synthesis can be performed using various solution-phase or solid-phase techniques (Creighton, T. (1984) Proteins, Structures and Molecular Properties, WH Freeman, New York NY, pp.
  • IRAP amino acid sequence of IRAP, or any part thereof, may be altered during direct synthesis and/or combined with sequences from other proteins, or any part thereof, to produce a variant polypeptide or a polypeptide having a sequence of a naturaUy occurring polypeptide.
  • the peptide may be substantiaUy purified by preparative high performance liquid chromatography (Chiez, R.M. and F.Z. Regnier (1990) Methods Enzymol. 182:392-421).
  • composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing (Creighton, supra, pp. 28-53).
  • the polynucleotides encoding IRAP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control ofthe inserted coding sequence in a suitable host.
  • These elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3 'untranslated regions in the vector and in polynucleotides encoding IRAP. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of polynucleotides encoding IRAP.
  • Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • no additional transcriptional or translational control signals may be needed.
  • exogenous translational control signals including an in- frame ATG initiation codon should be provided by the vector.
  • Exogenous translational elements and initiation codons may be of various origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of enhancers appropriate for the particular host ceU system used (Scharf, D. et al. (1994) Results Probl. CeU Differ. 20:125-162).
  • Methods wliich are weU known to those skilled in the art may be used to construct expression vectors containing polynucleotides encoding IRAP and appropriate transcriptional and translational control elements. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination (Sambrook and RusseU, supra, ch. 1-4, and 8; Ausubel et al., supra, ch. 1, 3, and 15).
  • a variety of expression vector/host systems may be utihzed to contain and express polynucleotides encoding IRAP.
  • microorganisms such as bacteria transformed with recombinant bacteriophage, plasmid, or cosmid DNA expression vectors; yeast transformed with yeast expression vectors; insect ceU systems infected with viral expression vectors (e.g., baculovirus); plant ceU systems transformed with viral expression vectors (e.g., cauliflower mosaic virus, CaMV, or tobacco mosaic viras, TMV) or with bacterial expression vectors (e.g., Ti or pBR322 plasmids); or animal cell systems (Sambrook and Russell, supra; Ausubel et al., supra; VanHeeke, G.
  • Expression vectors derived from retroviruses, adenoviruses, or herpes or vaccinia viruses, or from various bacterial plasmids may be used for delivery of polynucleotides to the targeted organ, tissue, or cell population (Di Nicola, M. et al. (1998) Cancer Gen. Ther. 5:350- 356; Yu, M. et al. (1993) Proc. Natl. Acad. Sci. USA 90:6340-6344; Buller, R.M. et al. (1985) Nature 317:813-815; McGregor, D.P. et al. (1994) Mol. Immunol. 31:219-226; Verma, I.M. and N. Somia (1997) Nature 389:239-242).
  • the invention is not limited by the host ceU employed.
  • a number of cloning and expression vectors may be selected depending upon the use intended for polynucleotides encoding IRAP.
  • routine cloning, subcloning, and propagation of polynucleotides encoding IRAP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene, La JoUa CA) or PSPORT1 plasmid (Invitrogen).
  • PBLUESCRIPT Stratagene, La JoUa CA
  • PSPORT1 plasmid Invitrogen.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence (Van Heeke, G. and S.M. Schuster (1989) J. Biol. Chem. 264:5503-5509).
  • vectors which direct high level expression of IRAP may be used.
  • vectors containing the strong, inducible SP6 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of IRAP.
  • a number of vectors containing constitutive or inducible promoters, such as alpha factor, alcohol oxidase, and PGH promoters, may be used in the yeast Saccharomyces cerevisiae or Pichia pastoris.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • such vectors direct either the secretion or intraceUular retention of expressed proteins and enable integration of foreign polynucleotide sequences into the host genome for stable propagation
  • Plant systems may also be used for expression of IRAP. Transcription of polynucleotides encoding IRAP may be driven by viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. (1987) EMBO J. 3:17-311). Alternatively, plant promoters such as the smaU subunit of RUBISCO or heat shock promoters may be used (Corazzi, G. et al. (1984) EMBO J. 3:1671-1680; Broghe, R. et al. (1984) Science 224:838-843; Winter, J. et al. (1991) Results Probl. Cell Differ. 17:85-105). These constructs can be introduced into plant cells by direct DNA transformation or pathogen-mediated transfection (The McGraw Hill Yearbook of Science and Technology (1992) McGraw HiU, New York NY, pp. 191-196).
  • a number of viral-based expression systems may be utUized.
  • polynucleotides encoding IRAP may be hgated into an adenovirus transcriptiontranslation complex consisting of the late promoter and tripartite leader sequence. Insertion in a non-essential El or E3 region of the viral genome may be used to obtain infective virus which expresses IRAP in host ceUs (Logan, J. and T. Shenk (1984) Proc. Natl. Acad. Sci. USA 81:3655-3659).
  • transcription enhancers such as the Rous sarcoma virus (RS V) enhancer
  • RS V Rous sarcoma virus
  • SV40 or EBV-based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes, polycationic amino polymers, or vesicles) for therapeutic purposes (Harrington, J.J. et al. (1997) Nat. Genet. 15:345- 355).
  • liposomes, polycationic amino polymers, or vesicles for therapeutic purposes.
  • stable expression of IRAP in ceU lines is preferred.
  • polynucleotides encoding IRAP can be transformed into ceU lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • ceUs may be aUowed to grow for about 1 to 2 days in enriched media before being switched to selective media.
  • the purpose of the selectable marker is to confer resistance to a selective agent, and its presence aUows growth and recovery of ceUs which successfuUy express the introduced sequences.
  • Resistant clones of stably transformed ceUs may be propagated using tissue culture techniques appropriate to the ceU type.
  • any number of selection systems may be used to recover transformed ceU lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tk ⁇ and apr ⁇ ceUs, respectively (Wigler, M. et al. (1977) CeU 11:223-232; Lowy, I. et al. (1980) CeU 22:817-823). Also, antimetabolite, antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-418
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively
  • trpB and hisD which alter ceUular requirements for metabohtes
  • Visible markers e.g., anthocyanins, green fluorescent proteins (GFP; BD Clontech), ⁇ -glucuronidase and its substrate ⁇ -glucuronide, or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants, but also to quantify the amount of transient or stable protein expression attributable to a specific vector system (Rhodes, CA. (1995) Methods Mol. Biol. 55:121-131).
  • marker gene expression suggests that the gene of interest is also present, the presence and expression of the gene may need to be confirmed.
  • sequence encoding IRAP is inserted within a marker gene sequence, transformed cells containing polynucleotides encoding IRAP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding IRAP under the control of a single promoter. Expression of the marker gene in response to induction or selection usually indicates expression of the tandem gene as weU.
  • host ceUs that contain the polynucleotide encoding IRAP and that express IRAP may be identified by a variety of procedures known to those of skiU in the art. These procedures include, but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of IRAP using either specific polyclonal or monoclonal antibodies are known in the art.
  • ELISAs enzyme-linked imrnunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated ceU sorting
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding IRAP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • polynucleotides encoding IRAP, or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • Such vectors are known in the art, are commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by addition of an appropriate RNA polymerase such as T7, T3, or SP6 and labeled nucleotides. These procedures may be conducted using a variety of commerciaUy available kits, such as those provided by Amersham Biosciences, Promega (Madison WI), and US Biochemical. Suitable reporter molecules or labels which may be used for ease of detection include radionuclides, enzymes, fluorescent, chemUuminescent, or chromogenic agents, as weU as substrates, cofactors, inhibitors, magnetic particles, and the like.
  • Host ceUs transformed with polynucleotides encoding IRAP may be cultured under conditions suitable for the expression and recovery of the protein from cell culture.
  • the protein produced by a transformed ceU may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • expression vectors containing polynucleotides which encode IRAP may be designed to contain signal sequences which direct secretion of IRAP through a prokaryotic or eukaryotic ceU membrane.
  • a host ceU strain may be chosen for its ability to modulate expression of the inserted polynucleotides or to process the expressed protein in the desired fashion.
  • Such modifications ofthe polypeptide include, but are not limited to, acetylation, carboxylation, glycosylation, phosphorylation, hpidation, and acylation.
  • Post-translational processing which cleaves a "prepro” or “pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host ceUs which have specific ceUular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa, MDCK, HEK293, and WI38) are avaUable from the American Type Culture CoUection (ATCC, Manassas VA) and maybe chosen to ensure the co ⁇ ect modification and processing of the foreign protein.
  • natural, modified, or recombinant polynucleotides encoding IRAP may be hgated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric IRAP protein containing a heterologous moiety that can be recognized by a commerciaUy avaUable antibody may facilitate the screening of peptide hbraries for inhibitors of IRAP activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commerciaUy avaUable affinity matrices.
  • Such moieties include, but are not limited to, glutathione S-transferase (GST), maltose binding protein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP), 6-His, FLAG, c-myc, and hemagglutinin (HA).
  • GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobihzed glutathione, maltose, phenylarsine oxide, calmodulin, and metal- chelate resins, respectively.
  • FLAG, c-myc, and hemagglutinin (HA) enable immunoaffinity purification of fusion proteins using commerciaUy avaUable monoclonal and polyclonal antibodies that specificaUy recognize these epitope tags.
  • a fusion protein may also be engineered to contain a proteolytic cleavage site located between the IRAP encoding sequence and the heterologous protein sequence, so that IRAP may be cleaved away from the heterologous moiety foUowing purification. Methods for fusion protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16).
  • a variety of commerciaUy avaUable kits may also be used to facUitate expression and purification of fusion proteins.
  • synthesis of radiolabeled IRAP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operably associated with the T7, T3, or SP6 promoters. Translation takes place in the presence of a radiolabeled amino acid precursor, for example, 35 S -methionine.
  • IRAP fragments of IRAP, or variants of IRAP may be used to screen for compounds that specifically bind to IRAP.
  • One or more test compounds may be screened for specific binding to IRAP.
  • 1, 2, 3, 4, 5, 10, 20, 50, 100, or 200 test compounds can be screened for specific binding to IRAP.
  • Examples of test compounds can include antibodies, anticalins, oligonucleotides, proteins (e.g., ligands or receptors), or small molecules.
  • variants of IRAP can be used to screen for binding of test compounds, such as antibodies, to IRAP, a variant of IRAP, or a combination of IRAP and/or one or more variants IRAP.
  • a variant of IRAP can be used to screen for compounds that bind to a variant of IRAP, but not to IRAP having the exact sequence of a sequence of SEQ ID NO: 1-32.
  • IRAP variants used to perform such screening can have a range of about 50% to about 99% sequence identity to IRAP, with various embodiments having 60%, 70%, 75%, 80%, 85%, 90%, and 95% sequence identity.
  • a compound identified in a screen for specific binding to IRAP can be closely related to the natural ligand of IRAP, e.g., a ligand or fragment thereof, a natural substrate, a stractural or functional mimetic, or a natural binding partner (Coligan, J.E. et al. (1991) Cu ⁇ ent Protocols in Immunology l(2):Chapter 5).
  • the compound thus identified can be a natural ligand of a receptor IRAP (Howard, A.D. et al. (2001) Trends Pharmacol. Sci.22: 132-140; Wise, A. et al. (2002) Drag Discovery Today 7:235-246).
  • a compound identified in a screen for specific binding to IRAP can be closely related to the natural receptor to which IRAP binds, at least a fragment ofthe receptor, or a fragment of the receptor including aU or a portion of the ligand binding site or binding pocket.
  • the compound may be a receptor for IRAP which is capable of propagating a signal, or a decoy receptor for IRAP which is not capable of propagating a signal (Ashkenazi, A. and V.M. Divit (1999) Cu ⁇ . Opin. Cell Biol. 11 :255-260; Mantovani, A. et al. (2001) Trends Immunol. 22:328- 336).
  • the compound can be rationally designed using known techniques.
  • Etanercept is an engineered p75 tumor necrosis factor (TNF) receptor dimer linked to the Fc portion of human IgGi (Taylor, P.C et al. (2001) Cu ⁇ . Opin. Immunol. 13:611-616).
  • TNF tumor necrosis factor
  • two or more antibodies having simUar or, alternatively, different specificities can be screened for specific binding to IRAP, fragments of IRAP, or variants of IRAP.
  • the binding specificity of the antibodies thus screened can thereby be selected to identify particular fragments or variants of IRAP.
  • an antibody can be selected such that its binding specificity allows for preferential identification of specific fragments or variants of IRAP.
  • an antibody can be selected such that its binding specificity allows for preferential diagnosis of a specific disease or condition having increased, decreased, or otherwise abnormal production of IRAP.
  • anticalins can be screened for specific binding to IRAP, fragments of IRAP, or variants of IRAP.
  • Anticalins are ligand-binding proteins that have been constracted based on a lipocahn scaffold (Weiss, G.A. and H.B. Lowman (2000) Chem. Biol. 7:R177-R184; Ske ⁇ a, A. (2001) J. Biotechnol. 74:257-275).
  • the protein architecture of lipocalins can include a beta-ba ⁇ el having eight antiparallel beta-strands, which supports four loops at its open end.
  • loops form the natural ligand-binding site of the lipocalins, a site which can be re-engineered in vitro by amino acid substitutions to impart novel binding specificities.
  • the amino acid substitutions can be made using methods known in the art or described herein, and can include conservative substitutions (e.g., substitutions that do not alter binding specificity) or substitutions that modestly, moderately, or significantly alter binding specificity.
  • screening for compounds which specifically bind to, stimulate, or inhibit IRAP involves producing appropriate cells which express IRAP, either as a secreted protein or on the ceU membrane.
  • Preferred cells can include cells from mammals, yeast, Drosophila, or E. coli. Cells expressing IRAP or ceU membrane fractions which contain IRAP are then contacted with a test compound and binding, stimulation, or inhibition of activity of either IRAP or the compound is analyzed.
  • An assay may simply test binding of a test compound to the polypeptide, wherein binding is detected by a fluorophore, radioisotope, enzyme conjugate, or other detectable label.
  • the assay may comprise the steps of combining at least one test compound with IRAP, either in solution or affixed to a solid support, and detecting the binding of IRAP to the compound.
  • the assay may detect or measure binding of a test compound in the presence of a labeled competitor.
  • the assay may be carried out using cell-free preparations, chemical libraries, or natural product mixtures, and the test compound(s) may be free in solution or affixed to a sohd support.
  • An assay can be used to assess the ability of a compound to bind to its natural ligand and/or to inhibit the binding of its natural ligand to its natural receptors.
  • assays include radio-labeling assays such as those described in U.S. Patent No. 5,914,236 and U.S. Patent No.
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a receptor) to improve or alter its ability to bind to its natural hgands (Matthews, D.J. and J.A. Wells. (1994) Chem. Biol. 1:25-30).
  • one or more amino acid substitutions can be introduced into a polypeptide compound (such as a ligand) to improve or alter its ability to bind to its natural receptors (Cunningham, B.C. and J.A. Wells (1991) Proc. Natl. Acad. Sci. USA 88:3407-3411; Lowman, H.B. et al. (1991) J. Biol. Chem. 266:10982-10988).
  • IRAP fragments of IRAP, or variants of IRAP may be used to screen for compounds that modulate the activity of IRAP.
  • Such compounds may include agonists, antagonists, or partial or inverse agonists.
  • an assay is performed under conditions permissive for IRAP activity, wherein IRAP is combined with at least one test compound, and the activity of IRAP in the presence of a test compound is compared with the activity of IRAP in the absence of the test compound. A change in the activity of IRAP in the presence of the test compound is indicative of a compound that modulates the activity of IRAP.
  • test compound is combined with an in vitro or ceU-free system comprising IRAP under conditions suitable for IRAP activity, and the assay is performed.
  • a test compound which modulates the activity of IRAP may do so indirectly and need not come in direct contact with the test compound. At least one and up to a plurality of test compounds may be screened.
  • polynucleotides encoding IRAP or their mammalian homologs may be "knocked out" in an animal model system using homologous recombination in embryonic stem (ES) ceUs.
  • ES embryonic stem
  • Such techniques are weU known in the art and are useful for the generation of animal models of human disease (see, e.g., U.S. Patent No. 5,175,383 and U.S. Patent No. 5,767,337).
  • mouse ES ceUs such as the mouse 129/SvJ ceU line, are derived from the early mouse embryo and grown in culture.
  • the ES ceUs are transformed with a vector containing the gene of interest disrupted by a marker gene, e.g., the neomycin phosphotransferase gene (neo; Capecchi, M.R. (1989) Science 244:1288-1292).
  • the vector integrates into the corresponding region of the host genome by homologous recombination.
  • homologous recombination takes place using the Cre-loxP system to knockout a gene of interest in a tissue- or developmental stage-specific manner (Marth, J.D. (1996) Clin. Invest. 97:1999-2002; Wagner, K.U. et al. (1997) Nucleic Acids Res. 25:4323-4330).
  • Transformed ES ceUs are identified and microinjected into mouse ceU blastocysts such as those from the C57BL/6 mouse strain.
  • the blastocysts are surgically transferred to pseudopregnant dams, and the resulting chimeric progeny are genotyped and bred to produce heterozygous or homozygous strains.
  • Transgenic animals thus generated may be tested with potential therapeutic or toxic agents.
  • Polynucleotides encoding IRAP may also be manipulated in vitro in ES ceUs derived from human blastocysts. Human ES ceUs have the potential to differentiate into at least eight separate ceU lineages including endoderm, mesoderm, and ectodermal ceU types.
  • ceU lineages differentiate into, for example, neural ceUs, hematopoietic lineages, and cardiomyocytes (Thomson, J.A. et al. (1998) Science 282:1145-1147).
  • Polynucleotides encoding IRAP can also be used to create "knockin" humanized animals
  • a region of a polynucleotide encoding IRAP is injected into animal ES ceUs, and the injected sequence integrates into the animal ceU genome.
  • Transformed cells are injected into blastulae, and the blastulae are implanted as described above.
  • Transgenic progeny or inbred lines are studied and treated with potential pharmaceutical agents to obtain information on treatment of a human disease.
  • a mammal inbred to overexpress IRAP e.g., by secreting IRAP in its milk, may also serve as a convenient source of that protein (Janne, J. et al. (1998) Biotechnol. Annu. Rev. 4:55-74). THERAPEUTICS
  • IRAP appears to play a role in immune system, neurological, developmental, muscle, and ceU prohferative disorders.
  • IRAP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IRAP.
  • disorders include, but are not limited to, an immune system disorder such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Braton, common variable immunodeficiency (CVl), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak- Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondylitis, amyloidosis, anemia, asthma,
  • an immune system disorder
  • a vector capable of expressing IRAP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IRAP including, but not limited to, those described above.
  • compositions comprising a substantiaUy purified IRAP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IRAP including, but not limited to, those provided above.
  • an agonist which modulates the activity of IRAP may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of IRAP including, but not limited to, those listed above.
  • an antagonist of IRAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IRAP.
  • disorders include, but are not limited to, those immune system, neurological, developmental, muscle, and ceU prohferative disorders described above.
  • an antibody which specificaUy binds IRAP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to ceUs or tissues wliich express IRAP.
  • a vector expressing the complement of the polynucleotide encoding IRAP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of IRAP including, but not limited to, those described above.
  • any protein, agonist, antagonist, antibody, complementary sequence, or vector embodiments may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skUl in the art, according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergisticaUy to effect the treatment or prevention of the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • An antagonist of IRAP may be produced using methods which are generally known in the art. In particular, purified IRAP may be used to produce antibodies or to screen hbraries of pharmaceutical agents to identify those wliich specificaUy bind IRAP.
  • Antibodies to IRAP may also be generated using methods that are weU known in the art. Such antibodies may include, but are not limited to, polyclonal, monoclonal, chimeric, and single chain antibodies, Fab fragments, and fragments produced by a Fab expression hbrary. In an embodiment, neutralizing antibodies (i.e., those which inhibit dimer formation) can be used therapeuticaUy.
  • Single chain antibodies e.g., from camels or llamas
  • various hosts including goats, rabbits, rats, mice, camels, dromedaries, Uamas, humans, and others may be immunized by injection with IRAP or with any fragment or ohgopeptide thereof which has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to, Freund's, mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oU emulsions, KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Cotynebacterium paivum are especiaUy preferable.
  • oligopeptides, peptides, or fragments used to induce antibodies are preferred. It is preferred that the oligopeptides, peptides, or fragments used to induce antibodies to
  • IRAP have an amino acid sequence consisting of at least about 5 amino acids, and generaUy wUl consist of at least about 10 amino acids. It is also preferable that these ohgopeptides, peptides, or fragments are substantiaUy identical to a portion ofthe amino acid sequence ofthe natural protein.
  • IRAP amino acids Short stretches of IRAP amino acids may be fused with those of another protein, such as KLH, and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to IRAP may be prepared using any technique which provides for the production of antibody molecules by continuous ceU lines in culture. These include, but are not limited to, the hybridoma technique, the human B-ceU hybridoma technique, and the EBV- hybridoma technique (Kohler, G. et al. (1975) Nature 256:495-497; Kozbor, D. et al. (1985) J.
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin hbraries or panels of highly specific binding reagents as disclosed in the literature (Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837;
  • Antibody fragments which contain specific binding sites for IRAP may also be generated.
  • such fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression hbraries may be constructed to aUow rapid and easy identification of monoclonal Fab fragments with the desired specificity (Huse, W.D. et al.
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with estabhshed specificities are weU known in the art.
  • Such immunoassays typicaUy involve the measurement of complex formation between IRAP and its specific antibody.
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering IRAP epitopes is generaUy used, but a competitive binding assay may also be employed (Pound, supra).
  • K a is defined as the molar concentration of IRAP-antibody complex divided by the molar concentrations of free antigen and free antibody under equihbrium conditions.
  • K a determined for a preparation of polyclonal antibodies, which are heterogeneous in their affinities for multiple IRAP epitopes, represents the average affinity, or avidity, of the antibodies for IRAP.
  • High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are prefe ⁇ ed for use in immunoassays in which the IRAP-antibody complex must withstand rigorous manipulations.
  • Low-affinity antibody preparations with K a ranging from about 10 6 to 10 7 L/mole are preferred for use in immunopurification and simUar procedures which ultimately require dissociation of IRAP, preferably in active form, from the antibody (Catty, D. (1988) Antibodies, Volume I: A Practical Approach, IRL Press, Washington DC; Liddell, J.E. and A. Cryer (1991) A Practical Guide to Monoclonal Antibodies, John WUey & Sons, New York NY).
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream apphcations.
  • a polyclonal antibody preparation containing at least 1-2 mg specific antibody/ml, preferably 5-10 mg specific antibody/ml is generaUy employed in procedures requiring precipitation of IRAP-antibody complexes.
  • Procedures for evaluating antibody specificity, titer, and avidity, and guidelines for antibody quality and usage in various apphcations, are generaUy avaUable (Catty, supra; Cohgan et al, supra).
  • polynucleotides encoding IRAP may be used for therapeutic purposes.
  • modifications of gene expression can be achieved by designing complementary sequences or antisense molecules (DNA, RNA, PNA, or modified ohgonucleotides) to the coding or regulatory regions of the gene encoding IRAP.
  • complementary sequences or antisense molecules DNA, RNA, PNA, or modified ohgonucleotides
  • antisense ohgonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding IRAP (Agrawal, S., ed. (1996) Antisense Therapeutics, Humana Press, Totawa NJ).
  • Antisense sequences can be delivered intracellularly in the form of an expression plasmid wliich, upon transcription, produces a sequence complementary to at least a portion of the cellular sequence encoding the target protein (Slater, J.E. et al. (1998) J. Allergy Clin. Immunol. 102:469-475; Scanlon, KJ. et al. (1995) FASEB J. 9:1288- 1296).
  • Antisense sequences can also be introduced intraceUularly through the use of viral vectors, such as retrovirus and adeno-associated viras vectors (Miller, A.D.
  • polynucleotides encoding IRAP may be used for somatic or germline gene therapy.
  • Gene therapy may be performed to (i) correct a genetic deficiency (e.g., in the cases of severe combined immunodeficiency (SCID)-X1 disease characterized by X- linked inheritance (Cavazzana-Calvo, M. et al. (2000) Science 288:669-672), severe combined immunodeficiency syndrome associated with an inherited adenosine deaminase (ADA) deficiency
  • SCID severe combined immunodeficiency
  • ADA adenosine deaminase
  • hepatitis B or C viras HBV, HCV
  • fungal parasites such as Candida albicans and Paracoccidioides brasiliensis
  • protozoan parasites such as Plasmodium falciparum and Ttypanosoma cruzi.
  • IRAP expression or regulation causes disease
  • the expression of IRAP from an appropriate population of transduced ceUs may aUeviate the clinical manifestations caused by the genetic deficiency.
  • IRAP are treated by constructing mamtnahan expression vectors encoding IRAP and introducing these vectors by mechanical means into IRAP-deficient cells.
  • Mechanical transfer technologies for use with ceUs in vivo or ex vitro include (i) direct DNA microinjection into individual cells, (ii) ballistic gold particle delivery, (ih) liposome-mediated transfection, (iv) receptor-mediated gene transfer, and (v) the use of DNA transposons (Morgan, R.A. and W.F. Anderson (1993) Annu. Rev. Biochem 62:191-217; Ivies, Z. (1997) CeU 91:501-510; Boulay, J.-L. and H. Recipon (1998) Curr. Opin. Biotechnol. 9:445-450).
  • Expression vectors that may be effective for the expression of IRAP include, but are not limited to, the PCDNA 3.1, EPITAG, PRCCMV2, PREP, PVAX, PCR2-TOPOTA vectors (Invitrogen, Carlsbad CA), PCMV-SCRIPT, PCMV-TAG, PEGSH/PERV (Stratagene, La JoUa CA), and PTET-OFF, PTET-ON, PTRE2, PTRE2-LUC, PTK-HYG (BD Clontech, Palo Alto CA).
  • IRAP maybe expressed using (i) a constitutively active promoter, (e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes), (ii) an inducible promoter (e.g., the tetracycline-regulated promoter (Gossen, M. and H. Bujard (1992) Proc. Natl. Acad. Sci. USA 89:5547-5551; Gossen, M. et al. (1995) Science 268:1766-1769; Rossi, F.M.V. and H.M. Blau (1998) Cu ⁇ . Opin. Biotechnol.
  • a constitutively active promoter e.g., from cytomegalovirus (CMV), Rous sarcoma virus (RSV), SV40 virus, thymidine kinase (TK), or ⁇ -actin genes
  • CommerciaUy available liposome transformation kits e.g., the PERFECT LIPID TRANSFECTION KIT, avaUable from Invitrogen
  • aUow one with ordinary skUl in the art to deliver polynucleotides to target cells in culture and require minimal effort to optimize experimental parameters.
  • transformation is performed using the calcium phosphate method (Graham, F.L. and A.J. Eb (1973) Virology 52:456-467), or by electroporation (Neumann, E. et al. (1982) EMBO J. 1 :841-845).
  • the introduction of DNA to primary ceUs requires modification of these standardized mammalian transfection protocols.
  • diseases or disorders caused by genetic defects with respect to IRAP expression are treated by constructing a retrovirus vector consisting of (i) the polynucleotide encoding IRAP under the control of an independent promoter or the retrovirus long terminal repeat (LTR) promoter, (U) appropriate RNA packaging signals, and (ih) a Rev-responsive element (RRE) along with additional retrovirus cis-acting RNA sequences and coding sequences required for efficient vector propagation.
  • Retrovirus vectors e.g., PFB and PFBNEO
  • Retrovirus vectors are commerciaUy avaUable (Stratagene) and are based on published data (Riviere, I. et al. (1995) Proc. Natl. Acad.
  • the vector is propagated in an appropriate vector producing cell line (VPCL) that expresses an envelope gene with a tropism for receptors on the target ceUs or a promiscuous envelope protein such as VSVg (Armentano, D. et al. (1987) J. Virol. 61:1647-1650; Bender, M.A. et al. (1987) J. Virol. 61:1639-1646; Adam, M.A. and A.D. MUler (1988) J. Virol. 62:3802-3806; DuU, T. et al. (1998) J. Virol. 72:8463-8471; Zufferey, R.
  • VPCL vector producing cell line
  • U.S. Patent No. 5,910,434 to Rigg discloses a method for obtaining retrovirus packaging ceU lines and is hereby incorporated by reference. Propagation of retrovirus vectors, transduction of a population of ceUs (e.g., CD4 + T- ceUs), and the return of transduced ceUs to a patient are procedures weU known to persons skilled in the art of gene therapy and have been weU documented (Ranga, U. et al. (1997) J. Virol. 71 :7020- 7029; Bauer, G.
  • an adenoviras-based gene therapy delivery system is used to dehver polynucleotides encoding IRAP to ceUs which have one or more genetic abnormahties with respect to the expression of IRAP.
  • the construction and packaging of adenoviras-based vectors are weU known to those with ordinary skUl in the art. Replication defective adenovirus vectors have proven to be versatUe for importing genes encoding immunoregulatory proteins into intact islets in the pancreas (Csete, M.E. et al. (1995) Transplantation 27:263-268). PotentiaUy useful adenoviral vectors are described in U.S. Patent No.
  • Adadenovirus vectors for gene therapy hereby incorporated by reference.
  • adenoviral vectors see also Antinozzi, P.A. et al. (1999; Annu. Rev. Nutr. 19:511-544) and Verma, I.M. and N. Somia (1997; Nature 18:389:239-242).
  • a herpes-based, gene therapy delivery system is used to dehver polynucleotides encoding IRAP to target ceUs which have one or more genetic abnormalities with respect to the expression of IRAP.
  • the use of herpes simplex virus (HS V)-based vectors may be especiaUy valuable for introducing IRAP to ceUs ofthe central nervous system, for which HSV has a tropism.
  • the construction and packaging of herpes-based vectors are well known to those with ordinary sk l in the art.
  • a rephcation-competent herpes simplex virus (HSV) type 1 -based vector has been used to dehver a reporter gene to the eyes of primates (Liu, X.
  • HSV-1 virus vector has also been disclosed in detaU in U.S. Patent No. 5,804,413 to DeLuca ("Herpes simplex viras strains for gene transfer"), which is hereby incorporated by reference.
  • U.S. Patent No. 5,804,413 teaches the use of recombinant HSV d92 which consists of a genome containing at least one exogenous gene to be transferred to a ceU under the control of the appropriate promoter for purposes including human gene therapy. Also taught by this patent are the constraction and use of recombinant HSV strains deleted for ICP4, ICP27 and ICP22.
  • HSV vectors see also Goins, W.F. et al. (1999; J. Virol. 73:519-532) and Xu, H. et al. (1994; Dev. Biol. 163:152-161).
  • the manipulation of cloned herpesviras sequences, the generation of recombinant virus following the transfection of multiple plasmids containing different segments ofthe large herpesviras genomes, the growth and propagation of herpesviras, and the infection of ceUs with herpesviras are techniques weU known to those of ordinary skUl in the art.
  • an alphaviras (positive, single-stranded RNA viras) vector is used to dehver polynucleotides encoding IRAP to target ceUs.
  • SFV Se liki Forest Viras
  • RNA replicates to higher levels than the full length genomic RNA, resulting in the overproduction of capsid proteins relative to the viral proteins with enzymatic activity (e.g., protease and polymerase).
  • enzymatic activity e.g., protease and polymerase.
  • WhUe alphaviras infection is typicaUy associated with ceU lysis within a few days, the ability to establish a persistent infection in hamster normal kidney ceUs (BHK-21) with a variant of Sindbis virus (SIN) indicates that the lytic rephcation of alphaviruses can be altered to suit the needs of the gene therapy apphcation (Dryga, S.A. et al. (1997) Virology 228:74-83).
  • the specific transduction of a subset of ceUs in a population may require the sorting of ceUs prior to transduction.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization of the ribozyme molecule to complementary target RNA, foUowed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specificaUy and efficiently catalyze endonucleolytic cleavage of RNA molecules encoding IRAP.
  • RNA sequences of between 15 and 20 ribonucleotides, co ⁇ esponding to the region of the target gene containing the cleavage site, may be evaluated for secondary stractural features which may render the oligonucleotide inoperable.
  • the suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary ohgonucleotides using ribonuclease protection assays.
  • RNA molecules may be generated by in vitro and in vivo transcription of DNA molecules encoding IRAP. Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6. Alternatively, these cDNA constructs that synthesize complementary RNA, constitutively or inducibly, can be mtroduced into ceU hnes, ceUs, or tissues.
  • RNA molecules may be modified to increase intraceUular stability and half-life. Possible modifications include, but are not limited to, the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' 0-methyl rather than phosphodiesterase linkages within the backbone of the molecule.
  • This concept is inherent in the production of PNAs and can be extended in aU of these molecules by the inclusion of nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and simUarly modified forms of adenine, cytosine, guanine, thymine, and uracU which are not as easUy recognized by endogenous endonucleases.
  • nontraditional bases such as inosine, queosine, and wybutosine, as weU as acetyl-, methyl-, thio-, and simUarly modified forms of adenine, cytosine, guanine, thymine, and uracU which are not as easUy recognized by endogenous endonucleases.
  • RNAi RNA interference
  • PTGS post-transcriptional gene sUencing
  • RNAi is a post-transcriptional mode of gene sUencing in which double-stranded RNA (dsRNA) introduced into a targeted ceU specificaUy suppresses the expression of the homologous gene (i.e., the gene bearing the sequence complementary to the dsRNA). This effectively knocks out or substantially reduces tlie expression of the targeted gene.
  • dsRNA double-stranded RNA
  • PTGS can also be accomplished by use of DNA or DNA fragments as weU. RNAi methods are described by Fire, A.
  • PTGS can also be initiated by introduction of a complementary segment of DNA into the selected tissue using gene delivery and/or viral vector delivery methods described herein or known in the art.
  • RNAi can be induced in mammalian ceUs by the use of smaU interfering RNA also known as siRNA.
  • siRNA are shorter segments of dsRNA (typicaUy about 21 to 23 nucleotides in length) that result in vivo from cleavage of introduced dsRNA by the action of an endogenous ribonuclease.
  • siRNA appear to be the mediators of the RNAi effect in mammals. The most effective siRNAs appear to be 21 nucleotide dsRNAs with 2 nucleotide 3 Overhangs.
  • the use of siRNA for inducing RNAi in mammahan ceUs is described by Elbashir, S.M. et al.
  • siRNA can be generated indirectly by introduction of dsRNA into the targeted ceU.
  • siRNA can be synthesized directly and introduced into a ceU by transfection methods and agents described herein or known in the art (such as hposome-mediated transfection, viral vector methods, or other polynucleotide dehvery/introductory methods).
  • Suitable siRNAs can be selected by examining a transcript of the target polynucleotide (e.g., mRNA) for nucleotide sequences downstream from the AUG start codon and recording the occu ⁇ ence of each nucleotide and the 3 ' adjacent 19 to 23 nucleotides as potential siRNA target sites, with sequences having a 21 nucleotide length being preferred.
  • Regions to be avoided for target siRNA sites include the 5' and 3' untranslated regions (UTRs) and regions near the start codon (within 75 bases), as these maybe richer in regulatory protein binding sites. UTR-binding proteins and/or translation initiation complexes may interfere with binding of the siRNP endonuclease complex.
  • the selected target sites for siRNA can then be compared to the appropriate genome database (e.g., human, etc.) using BLAST or other sequence comparison algorithms known in the art. Target sequences with significant homology to other coding sequences can be ehminated from consideration.
  • the selected siRNAs can be produced by chemical synthesis methods known in the art or by in vitro transcription using commerciaUy avaUable methods and kits such as the SILENCER siRNA constraction kit (Ambion, Austin TX).
  • long-term gene sUencing and or RNAi effects can be induced in selected tissue using expression vectors that continuously express siRNA.
  • This can be accomplished using expression vectors that are engineered to express hairpin RNAs (shRNAs) using methods known in the art (see, e.g., Brummelkamp, T.R. et al. (2002) Science 296:550-553; and Paddison, P.J. et al. (2002) Genes Dev. 16:948-958).
  • shRNAs can be delivered to target cells using expression vectors known in the art.
  • An example of a suitable expression vector for delivery of siRNA is the PSILENCER1.0-U6 (circular) plasmid (Ambion).
  • shRNAs are processed in vivo into siRNA-like molecules capable of carrying out gene-specific sUencing.
  • the expression levels of genes targeted by RNAi or PTGS methods can be determined by assays for mRNA and/or protein analysis.
  • Expression levels of tlie mRNA of a targeted gene can be determined, for example, by northern analysis methods using the NORTHERNMAX-GLY kit (Ambion); by microa ⁇ ay methods; by PCR methods; by real time PCR methods; and by other RNA/polynucleotide assays known in the art or described herein.
  • Expression levels of the protein encoded by the targeted gene can be determined, for example, by microarray methods; by polyacrylamide gel electrophoresis; and by Western analysis using standard techniques known in the art.
  • An additional embodiment of the invention encompasses a method for screening for a compound which is effective in altering expression of a polynucleotide encoding IRAP.
  • Compounds which may be effective in altering expression of a specific polynucleotide may include, but are not limited to, oligonucleotides, antisense ohgonucleotides, triple hehx-forming oligonucleotides, transcription factors and other polypeptide transcriptional regulators, and non-macromolecular chemical entities which are capable of interacting with specific polynucleotide sequences. Effective compounds may alter polynucleotide expression by acting as either inhibitors or promoters of polynucleotide expression.
  • a compound which specifically inhibits expression ofthe polynucleotide encoding IRAP may be therapeuticaUy useful, and in the treatment of disorders associated with decreased IRAP expression or activity, a compound which specifically promotes expression of the polynucleotide encoding IRAP may be therapeutically useful.
  • one or more test compounds may be screened for effectiveness in altering expression of a specific polynucleotide.
  • a test compound may be obtained by any method commonly known in the art, including chemical modification of a compound known to be effective in altering polynucleotide expression; selection from an existing, commercially- vailable or proprietary hbrary of naturally-occurring or non-natural chemical compounds; rational design of a compound based on chemical and/or structural properties of the target polynucleotide; and selection from a library of chemical compounds created combinatoriaUy or randomly.
  • a sample comprising a polynucleotide encoding IRAP is exposed to at least one test compound thus obtained.
  • the sample may comprise, for example, an intact or permeabihzed cell, or an in vitro cell-free or reconstituted biochemical system.
  • Alterations in the expression of a polynucleotide encoding IRAP are assayed by any method commonly known in the art. Typically, the expression of a specific nucleotide is detected by hybridization with a probe having a nucleotide sequence complementary to the sequence of the polynucleotide encoding IRAP. The amount of hybridization may be quantified, thus forming the basis for a comparison of the expression of the polynucleotide both with and without exposure to one or more test compounds. Detection of a change in the expression of a polynucleotide exposed to a test compound indicates that the test compound is effective in altering the expression of the polynucleotide.
  • a screen for a compound effective in altering expression of a specific polynucleotide can be carried out, for example, using a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Biophys. Res. Commun. 268:8-13).
  • a Schizosaccharomyces pombe gene expression system (Atkins, D. et al. (1999) U.S. Patent No. 5,932,435; Arndt, G.M. et al. (2000) Nucleic Acids Res. 28:E15) or a human cell line such as HeLa cell (Clarke, M.L. et al. (2000) Biochem. Bio
  • a particular embodiment of the present invention involves screening a combinatorial library of ohgonucleotides (such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides) for antisense activity against a specific polynucleotide sequence (Braice, T.W. et al. (1997) U.S. Patent No. 5,686,242; Bruice, T.W. et al. (2000) U.S. Patent No. 6,022,691). Many methods for introducing vectors into ceUs or tissues are avaUable and equaUy suitable for use in vivo, in vitro, and ex vivo.
  • ohgonucleotides such as deoxyribonucleotides, ribonucleotides, peptide nucleic acids, and modified ohgonucleotides
  • vectors may be introduced into stem ceUs taken from the patient and clonaUy propagated for autologous transplant back into that same patient. Dehvery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are weU known in the art (Goldman, CK. et al. (1997) Nat. Biotechnol. 15:462-466).
  • any of the therapeutic methods described above may be apphed to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys.
  • An additional embodiment of the invention relates to the administration of a composition which generaUy comprises an active ingredient formulated with a pharmaceuticaUy acceptable excipient.
  • Excipients may include, for example, sugars, starches, ceUuloses, gums, and proteins.
  • Various formulations are commonly known and are thoroughly discussed in the latest edition of Remington's Pharmaceutical Sciences (Maack Pubhshing, Easton PA).
  • Such compositions may consist of IRAP, antibodies to IRAP, and mimetics, agonists, antagonists, or inhibitors of IRAP.
  • compositions described herein may be administered by any number of routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • routes including, but not limited to, oral, intravenous, intramuscular, intra-arterial, intrameduUary, intrathecal, intraventricular, pulmonary, transdermal, subcutaneous, intraperitoneal, intranasal, enteral, topical, sublingual, or rectal means.
  • Compositions for pulmonary administration may be prepared in liquid or dry powder form. These compositions are generaUy aerosohzed immediately prior to inhalation by the patient. In the case of small molecules (e.g. traditional low molecular weight organic drugs), aerosol dehvery of fast-acting formulations is weU-known in the art. In the case of macromolecules (e.g.,
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended purpose.
  • the determination of an effective dose is weU within the capability of those skilled in the art.
  • Speciahzed forms of compositions may be prepared for direct intraceUular dehvery of macromolecules comprising IRAP or fragments thereof.
  • hposome preparations containing a ceU-impermeable macromolecule may promote ceU fusion and intraceUular dehvery of the macromolecule.
  • IRAP or a fragment thereof may be joined to a short cationic N- terminal portion from the HIV Tat-1 protein. Fusion proteins thus generated have been found to transduce into the ceUs of aU tissues, including the brain, in a mouse model system (Schwarze, S.R. et al. (1999) Science 285:1569-1572).
  • the therapeuticaUy effective dose can be estimated initiaUy either in ceU culture assays, e.g., of neoplastic ceUs, or in animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • ceU culture assays e.g., of neoplastic ceUs
  • animal models such as mice, rats, rabbits, dogs, monkeys, or pigs.
  • An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeuticaUy effective dose refers to that amount of active ingredient, for example IRAP or fragments thereof, antibodies of IRAP, and agonists, antagonists or inhibitors of IRAP, which amehorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeuticaUy effective in 50% of the population) or LD 50 (the dose lethal to 50% of the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD 50 /ED 50 ratio.
  • Compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from ceU culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration
  • the exact dosage wiU be determined by the practitioner, in light of factors related to the subject requiring treatment. Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity ofthe disease state, the general health ofthe subject, the age, weight, and gender of the subject, time and frequency of administration, drug combinations), reaction sensitivities, and response to therapy. Long-acting compositions maybe administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of dehvery is provided in the literature and generaUy avaUable to practitioners in the art. Those skUled in the art wiU employ different formulations for nucleotides than for proteins or their inhibitors. SimUarly, dehvery of polynucleotides or polypeptides wUl be specific to particular ceUs, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specificaUy bind IRAP may be used for the diagnosis of disorders characterized by expression of IRAP, or in assays to monitor patients being treated with IRAP or agonists, antagonists, or inhibitors of IRAP.
  • Antibodies useful for diagnostic purposes may be prepared in the same manner as described above for therapeutics. Diagnostic assays for IRAP include methods which utilize the antibody and a label to detect IRAP inhuman body fluids or in extracts of ceUs or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • IRAP A variety of protocols for measuring IRAP, including ELISAs, RIAs, and FACS, are known in the art and provide a basis for diagnosing altered or abnormal levels of IRAP expression.
  • Normal or standard values for IRAP expression are estabhshed by combining body fluids or ceU extracts taken from normal mammahan subjects, for example, human subjects, with antibodies to IRAP under conditions suitable for complex formation. The amount of standard complex formation may be quantitated by various methods, such as photometric means. Quantities of IRAP expressed in subject, control, and disease samples frombiopsied tissues are compared with the standard values. Deviation between standard and subject values estabhshes the parameters for diagnosing disease.
  • polynucleotides encoding IRAP may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotides, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of IRAP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of IRAP, and to monitor regulation of IRAP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotides, including genomic sequences, encoding IRAP or closely related molecules may be used to identify nucleic acid sequences which encode IRAP.
  • the specificity of tlie probe whether it is made from a highly specific region, e.g., tlie 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification wiU determine whether the probe identifies only naturaUy occurring sequences encoding IRAP, aUelic variants, or related sequences.
  • Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the IRAP encoding sequences.
  • the hybridization probes of the subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO:33-64 or from genomic sequences including promoters, enhancers, and introns of the IRAP gene.
  • Means for producing specific hybridization probes for polynucleotides encoding IRAP include the cloning of polynucleotides encoding IRAP or IRAP derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art, are commerciaUy avaUable, and may be used to synthesize RNA probes in vitro by means of the addition of the appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuchdes such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotides encoding IRAP may be used for the diagnosis of disorders associated with expression of IRAP.
  • disorders include, but are not limited to, an immune system disorder such as acquired immunodeficiency syndrome (AIDS), X-linked agammaglobinemia of Braton, common variable immunodeficiency (CVl), DiGeorge's syndrome (thymic hypoplasia), thymic dysplasia, isolated IgA deficiency, severe combined immunodeficiency disease (SCID), immunodeficiency with thrombocytopenia and eczema (Wiskott-Aldrich syndrome), Chediak- Higashi syndrome, chronic granulomatous diseases, hereditary angioneurotic edema, immunodeficiency associated with Cushing's disease, Addison's disease, adult respiratory distress syndrome, aUergies, ankylosing spondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis, autoimmune polyendocrinopathy- candidiasis-ectodermal dystrophy
  • Polynucleotides encoding IRAP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies; in PCR technologies; in dipstick, pin, and multiformat ELISA-like assays; and in microarrays utilizing fluids or tissues from patients to detect altered IRAP expression. Such qualitative or quantitative methods are weU known in the art.
  • polynucleotides encoding IRAP may be used in assays that detect the presence of associated disorders, particularly those mentioned above.
  • Polynucleotides complementary to sequences encoding IRAP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes. After a suitable incubation period, the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of polynucleotides encoding IRAP in the sample indicates the presence of the associated disorder.
  • Such assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profUe for expression is estabhshed. This may be accomphshed by combining body fluids or ceU extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding IRAP, under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantiaUy purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may aUow health professionals to employ preventative measures or aggressive treatment earher, thereby preventing the development or further progression of the cancer.
  • ohgonucleotides designed from the sequences encoding IRAP may involve the use of PCR. These ohgomers may be chemicaUy synthesized, generated enzymaticaUy, or produced in vitro.
  • Ohgomers wUl preferably contain a fragment of a polynucleotide encoding IRAP, or a fragment of a polynucleotide complementary to the polynucleotide encoding IRAP, and will be employed under optimized conditions for identification of a specific gene or condition.
  • Ohgomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • oligonucleotide primers derived from polynucleotides encoding IRAP may be used to detect single nucleotide polymorphisms (SNPs).
  • SNPs are substitutions, insertions and deletions that are a frequent cause of inherited or acquired genetic disease in humans.
  • Methods of SNP detection include, but are not limited to, single-stranded conformation polymorphism (SSCP) and fluorescent SSCP (fSSCP) methods.
  • SSCP single-stranded conformation polymorphism
  • fSSCP fluorescent SSCP
  • ohgonucleotide primers derived from polynucleotides encoding IRAP are used to amplify DNA using the polymerase chain reaction (PCR).
  • the DNA may be derived, for example, from diseased or normal tissue, biopsy samples, bodUy fluids, and the like.
  • SNPs in the DNA cause differences in the secondary and tertiary structures of PCR products in single-stranded form, and these differences are detectable using gel electrophoresis in non-denaturing gels.
  • the ohgonucleotide primers are fluorescently labeled, which aUows detection of the amplimers in high-throughput equipment such as DNA sequencing machines.
  • AdditionaUy sequence database analysis methods, termed in silico SNP (isSNP), are capable of identifying polymorphisms by comparing the sequence of individual overlapping DNA fragments which assemble into a common consensus sequence.
  • SNPs may be detected and characterized by mass spectrometry using, for example, the high throughput MASSARRAY system (Sequenom, Inc., San Diego CA).
  • SNPs may be used to study the genetic basis of human disease. For example, at least 16 common SNPs have been associated with non-insulin-dependent diabetes meUitus. SNPs are also useful for examining differences in disease outcomes in monogenic disorders, such as cystic fibrosis, sickle ceU anemia, or chronic granulomatous disease. For example, variants in the mannose-binding lectin, MBL2, have been shown to be correlated with deleterious pulmonary outcomes in cystic fibrosis. SNPs also have utility in pharmacogenomics, the identification of genetic variants that influence a patient's response to a drug, such as life-threatening toxicity.
  • N-acetyl transferase is associated with a high incidence of peripheral neuropathy in response to the anti-tuberculosis drug isoniazid, whUe a variation in the core promoter of the ALOX5 gene results in diminished clinical response to treatment with an anti-asthma drug that targets the 5-lipoxygenase pathway.
  • Analysis of the distribution of SNPs in different populations is useful for investigating genetic drift, mutation, recombination, and selection, as weU as for tracing the origins of populations and their migrations (Taylor, J.G. et al. (2001) Trends Mol. Med. 7:507-512; Kwok, P.-Y. and Z. Gu (1999) Mol. Med.
  • Methods which may also be used to quantify the expression of IRAP include radiolabeling or biotinylating nucleotides, coamplification of a control nucleic acid, and interpolating results from standard curves (Melby, P.C et al. (1993) J. Immunol. Methods 159:235-244; Duplaa, C. et al. (1993) Anal. Biochem. 212:229-236).
  • the speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the ohgomer or polynucleotide of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • ohgonucleotides or longer fragments derived from any of the polynucleotides described herein may be used as elements on a microa ⁇ ay.
  • the microa ⁇ ay can be used in transcript imaging techniques which monitor the relative expression levels of large numbers of genes simultaneously as described below.
  • the microa ⁇ ay may also be used to identify genetic variants, mutations, and polymorphisms.
  • This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, to monitor progression/regression of disease as a function of gene expression, and to develop and monitor the activities of therapeutic agents in the treatment of disease.
  • this information may be used to develop a pharmacogenomic profile of a patient in order to select the most appropriate and effective treatment regimen for that patient. For example, therapeutic agents which are highly effective and display the fewest side effects may be selected for a patient based on his/her pharmacogenomic profile.
  • IRAP fragments of IRAP, or antibodies specific for IRAP may be used as elements on a microarray.
  • the microarray may be used to monitor or measure protein- protein interactions, drag-target interactions, and gene expression profiles, as described above.
  • a particular embodiment relates to the use of the polynucleotides of the present invention to generate a transcript image of a tissue or ceU type.
  • a transcript image represents the global pattern of gene expression by a particular tissue or ceU type. Global gene expression patterns are analyzed by quantifying the number of expressed genes and their relative abundance under given conditions and at a given time (Seilhamer et al., "Comparative Gene Transcript Analysis," U.S. Patent No.
  • a transcript image may be generated by hybridizing the polynucleotides of the present invention or their complements to the totahty of transcripts or reverse transcripts of a particular tissue or ceU type.
  • the hybridization takes place in high-througliput format, wherein the polynucleotides of the present invention or their complements comprise a subset of a plurality of elements on a microarray.
  • the resultant transcript image would provide a profile of gene activity.
  • Transcript images may be generated using transcripts isolated from tissues, ceU lines, biopsies, or other biological samples.
  • the transcript image may thus reflect gene expression in vivo, as in the case of a tissue or biopsy sample, or in vitro, as in the case of a ceU line.
  • Transcript images which profile the expression of the polynucleotides of the present invention may also be used in conjunction with in vitro model systems and prechnical evaluation of pharmaceuticals, as weU as toxicological testing of industrial and naruraUy-occu ⁇ ing environmental compounds.
  • AU compounds induce characteristic gene expression patterns, frequently termed molecular fingerprints or toxicant signatures, which are indicative of mechanisms of action and toxicity (Nuwaysir, E.F. et al. (1999) Mol. Carcinog. 24:153-159; Steiner, S. and N.L. Anderson (2000) Toxicol. Lett. 112-113:467-471). If a test compound has a signature simUar to that of a compound with known toxicity, it is likely to share those toxic properties.
  • the toxicity of a test compound can be assessed by treating a biological sample containing nucleic acids with the test compound. Nucleic acids that are expressed in the treated biological sample are hybridized with one or more probes specific to the polynucleotides of the present invention, so that transcript levels co ⁇ esponding to the polynucleotides of the present invention may be quantified. The transcript levels in the treated biological sample are compared with levels in an untreated biological sample. Differences in the transcript levels between the two samples are indicative of a toxic response caused by the test compound in the treated sample.
  • proteome refers to the global pattern of protein expression in a particular tissue or cell type. Each protein component of a proteome can be subjected individuahy to further analysis. Proteome expression patterns, or profiles, are analyzed by quantifying the number of expressed proteins and their relative abundance under given conditions and at a given time. A profile of a ceU's proteome may thus be generated by separating and analyzing the polypeptides of a particular tissue or ceU type.
  • the separation is achieved using two-dimensional gel electrophoresis, in which proteins from a sample are separated by isoelectric focusing in the first dimension, and then according to molecular weight by sodium dodecyl sulfate slab gel electrophoresis in the second dimension (Steiner and Anderson, supra).
  • the proteins are visuahzed in the gel as discrete and uniquely positioned spots, typicaUy by staining the gel with an agent such as Coomassie Blue or silver or fluorescent stains.
  • the optical density of each protein spot is generaUy proportional to the level of the protein in the sample.
  • the optical densities of equivalently positioned protein spots from different samples are compared to identify any changes in protein spot density related to the treatment.
  • the proteins in the spots are partiaUy sequenced using, for example, standard methods employing chemical or enzymatic cleavage foUowed by mass spectrometry.
  • the identity of the protein in a spot may be determined by comparing its partial sequence, preferably of at least 5 contiguous amino acid residues, to the polypeptide sequences of interest. In some cases, further sequence data may be obtained for definitive protein identification.
  • a proteomic profile may also be generated using antibodies specific for IRAP to quantify the levels of IRAP expression.
  • the antibodies are used as elements on a microa ⁇ ay, and protein expression levels are quantified by contacting the microa ⁇ ay with the sample and detecting the levels of protein bound to each array element (Lueking, A. et al. (1999) Anal. Biochem. 270:103-111; Mendoze, L.G. et al. (1999) Biotechniques 27:778-788).
  • Detection may be performed by a variety of methods known in the art, for example, by reacting the proteins in the sample with a thiol- or amino-reactive fluorescent compound and detecting the amount of fluorescence bound at each a ⁇ ay element.
  • Toxicant signatures at the proteome level are also useful for toxicological screening, and should be analyzed in paraUel with toxicant signatures at the transcript level.
  • proteome toxicant signatures maybe useful in the analysis of compounds which do not significantly affect the transcript image, but which alter the proteomic profile.
  • the analysis of transcripts in body fluids is difficult, due to rapid degradation of mRNA, so proteomic profiling may be more rehable and informative in such cases.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins that are expressed in the treated biological sample are separated so that the amount of each protein can be quantified. The amount of each protein is compared to the amount of the corresponding protein in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample. Individual proteins are identified by sequencing the amino acid residues of the individual proteins and comparing these partial sequences to the polypeptides of the present invention.
  • the toxicity of a test compound is assessed by treating a biological sample containing proteins with the test compound. Proteins from the biological sample are incubated with antibodies specific to the polypeptides of the present invention. The amount of protein recognized by the antibodies is quantified. The amount of protein in the treated biological sample is compared with the amount in an untreated biological sample. A difference in the amount of protein between the two samples is indicative of a toxic response to the test compound in the treated sample.
  • Microarrays may be prepared, used, and analyzed using methods known in the art (Brennan, T.M. et al. (1995) U.S. Patent No. 5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci. USA 93:10614-10619; BaldeschweUer et al. (1995) PCT apphcation W095/25116; Shalon, D. et al. (1995) PCT application WO95/35505; HeUer, R.A. et al. (1997) Proc. Natl. Acad. Sci. USA 94:2150-2155; HeUer, M.J. et al. (1997) U.S.
  • nucleic acid sequences encoding IRAP may be used to generate hybridization probes useful in mapping the naturaUy occurring genomic sequence.
  • Either coding or noncoding sequences may be used, and in some instances, noncoding sequences may be preferable over coding sequences. For example, conservation of a coding sequence among members of a multi-gene family may potentiaUy cause undesired cross hybridization during chromosomal mapping.
  • sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA hbraries (Harrington, J.J. et al. (1997) Nat. Genet. 15:345-355; Price, CM. (1993) Blood Rev. 7:127-134; Trask, BJ. (1991) Trends Genet. 7:149-154).
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA hbraries
  • nucleic acid sequences may be used to develop genetic hnkage maps, for example, which co ⁇ elate the inheritance of a disease state with the inheritance of a particular chromosome region or restriction fragment length polymorphism (RFLP) (Lander, E.S. and D. Botstein (1986) Proc. Natl. Acad. Sci. USA 83:7353-7357).
  • RFLP restriction fragment length polymorphism
  • Fluorescent in situ hybridization may be correlated with other physical and genetic map data (Heinz-Uhich, et al. (1995) in Meyers, supra, pp. 965-968). Examples of genetic map data can be found in various scientific journals or at the Online Mendehan Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding IRAP on a physical map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder and thus may further positional cloning efforts.
  • OMIM Online Mendehan Inheritance in Man
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps.
  • physical mapping techniques such as linkage analysis using estabhshed chromosomal markers
  • linkage analysis using estabhshed chromosomal markers may be used for extending genetic maps.
  • the placement of a gene on the chromosome of another mammahan species, such as mouse may reveal associated markers even if the exact chromosomal locus is not known. This information is valuable to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation (Gatti, R.A. et al. (1988) Nature 336:577-580).
  • the nucleotide sequence of the instant invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • IRAP its catalytic or immunogenic fragments, or ohgopeptides thereof can be used for screening hbraries of compounds in any of a variety of drug screening techniques.
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a ceU surface, or located intraceUularly. The formation of binding complexes between IRAP and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest (Geysen, et al. (1984) PCT apphcation WO84/03564).
  • large numbers of different smaU test compounds are synthesized on a sohd substrate.
  • Tlie test compounds are reacted with IRAP, or fragments thereof, and washed.
  • Bound IRAP is then detected by methods weU known in the art.
  • Purified IRAP can also be coated directly onto plates for use in the aforementioned drag screening techniques.
  • non-neutralizing antibodies can be used to capture the peptide and immobUize it on a sohd support.
  • nucleotide sequences which encode IRAP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • Incyte cDNAs are derived from cDNA hbraries described in the LIFESEQ database (Incyte, Palo Alto CA). Some tissues are homogenized and lysed in guanidinium isothiocyanate, whUe others are homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Invitrogen), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates are centrifuged over CsCl cushions or extracted with chloroform. RNA is precipitated from the lysates with either isopropanol or sodium acetate and ethanol, or by other routine methods.
  • TRIZOL Invitrogen
  • RNA is treated with DNase.
  • poly(A)+ RNA is isolated using ohgo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA is isolated directly from tissue lysates using other RNA isolation kits, e.g., the POLY(A)PURE mRNA purification kit (Ambion, Austin TX).
  • Stratagene is provided with RNA and constructs the corresponding cDNA hbraries. Otherwise, cDNA is synthesized and cDNA hbraries are constracted with the UNIZAP vector system (Stratagene) or SUPERSCRIPT plasmid system (Invitrogen), using the recommended procedures or similar methods known in the art (Ausubel et al., supra, ch. 5). Reverse transcription is initiated using ohgo d(T) or random primers. Synthetic ohgonucleotide adapters are hgated to double stranded cDNA, and the cDNA is digested with the appropriate restriction enzyme or enzymes.
  • the cDNA is size-selected (300-1000 bp) using SEPHACRYL S 1000, SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Biosciences) or preparative agarose gel electrophoresis.
  • cDNAs are hgated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e.g., PBLUESCRIPT plasmid (Stratagene), PSPORT1 plasmid (Invitrogen, Carlsbad CA), PCDNA2.1 plasmid (Invitrogen), PBK-CMV plasmid (Stratagene), PCR2-TOPOTA plasmid (Invitrogen), PCMV-ICIS plasmid (Stratagene), pIGEN (Incyte, Palo Alto CA), pRARE (Incyte), or pINCY (Incyte), or derivatives thereof.
  • Recombinant plasmids are transformed into competent E. coli cells including XLl-Blue, XLl-BlueMRF, or SOLR from Stratagene or DH5 ⁇ , DH10B, or ElectroMAX DH10B from Invitrogen.
  • Plasmids obtained as described in Example I are recovered from host ceUs by in vivo excision using the UNIZAP vector system (Stratagene) or by ceU lysis. Plasmids are purified using at least one of the foUowing: a Magic or WIZARD Minipreps DNA purification system (Promega); an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD); and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid, QIAWELL 8 Ultra Plasmid purification systems or the R.E.A.L. PREP 96 plasmid purification kit from QIAGEN. FoUowing precipitation, plasmids are resuspended in 0.1 ml of distiUed water and stored, with or without lyophilization, at 4°C
  • plasmid DNA is amphfied from host ceU lysates using direct link PCR in a high-throughput format (Rao, V.B. (1994) Anal. Biochem. 216:1-14). Host ceU lysis and thermal cycling steps are ca ⁇ ied out in a single reaction mixture. Samples are processed and stored in 384- weU plates, and the concentration of amphfied plasmid DNA is quantified fluorometricaUy using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki, Finland). III. Sequencing and Analysis
  • Incyte cDNA recovered in plasmids as described in Example II are sequenced as follows. Sequencing reactions are processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Apphed Biosystems) thermal cycler or the PTC -200 thermal cycler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (HamUton) hquid transfer system. cDNA sequencing reactions are prepared using reagents provided by Amersham Biosciences or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Apphed Biosystems).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides are carried out using the MEG AB ACE 1000 DNA sequencing system (Amersham Biosciences); the ABI PRISM 373 or 377 sequencing system (Applied Biosystems) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences are identified using standard methods (Ausubel et al., supra, ch. 7). Some of the cDNA sequences are selected for extension using the techniques disclosed in Example VIII.
  • Polynucleotide sequences derived from Incyte cDNAs are vahdated by removing vector, linker, and poly(A) sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programming, and dinucleotide nearest neighbor analysis.
  • the Incyte cDNA sequences or translations thereof are then queried against a selection of public databases such as the GenBank primate, rodent, mammahan, vertebrate, and eukaryote databases, and BLOCKS, PRINTS, DOMO, PRODOM; PROTEOME databases with sequences from Homo sapiens, Rattus noi egicus, Mus musculus, Caenorhabditis elegans, Saccharomyces cerevisiae, Scliizosaccliaromyces pombe, and Candida albicans (Incyte, Palo Alto CA); hidden Markov model (HMM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM (Haft, D.H.
  • HMM hidden Markov model
  • HMM-based protein domain databases such as SMART (Schultz, J. et al. (1998) Proc. Natl. Acad. Sci. USA 95:5857-5864; Letunic, I. et al. (2002) Nucleic Acids Res. 30:242-244).
  • HMM is a probabilistic approach which analyzes consensus primary structures of gene families; see, for example, Eddy, S.R. (1996) Cu ⁇ . Opin. Struct. Biol. 6:361-365.
  • the queries are performed using programs based on BLAST, FASTA, BLIMPS, and HMMER.
  • the Incyte cDNA sequences are assembled to produce fuU length polynucleotide sequences.
  • GenBank cDNAs, GenBank ESTs, stitched sequences, stretched sequences, or Genscan-predicted coding sequences are used to extend Incyte cDNA assemblages to full length. Assembly is performed using programs based on Phred, Phrap, and Consed, and cDNA assemblages are screened for open reading frames using programs based on GeneMark, BLAST, and FASTA.
  • the full length polynucleotide sequences are translated to derive the co ⁇ esponding full length polypeptide sequences.
  • a polypeptide may begin at any of the methionine residues of the full length translated polypeptide.
  • FuU length polypeptide sequences are subsequently analyzed by querying against databases such as the GenBank protein databases (genpept), SwissProt, the PROTEOME databases, BLOCKS, PRINTS, DOMO, PRODOM, Prosite, bidden Markov model (HMM)-based protein famUy databases such as PFAM, INCY, and TIGRFAM; and HMM-based protein domain databases such as SMART.
  • Full length polynucleotide sequences are also analyzed using MACDNASIS PRO software (MiraiBio, Alameda CA) and LASERGENE software (DNASTAR).
  • Polynucleotide and polypeptide sequence ahgnments are generated using default parameters specified by the CLUSTAL algorithm as incorporated into the MEGALIGN multisequence ahgnment program (DNASTAR), which also calculates the percent identity between aligned sequences.
  • Table 7 summarizes tools, programs, and algorithms used for the analysis and assembly of Incyte cDNA and fuU length sequences and provides applicable descriptions, references, and threshold parameters.
  • the first column of Table 7 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, aU of which are incorporated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probabihty values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score or the lower the probability value, the greater the identity between two sequences).
  • Genscan is a general-purpose gene identification program which analyzes genomic DNA sequences from a variety of organisms (Burge, C and S. Karlin (1997) J. Mol. Biol. 268:78-94; Burge, C. and S. Karlin (1998) Cu ⁇ . Opin. Struct. Biol. 8:346-354).
  • the program concatenates predicted exons to form an assembled cDNA sequence extending from a methionine to a stop codon.
  • the output of Genscan is a FASTA database of polynucleotide and polypeptide sequences.
  • the maximum range of sequence for Genscan to analyze at once is set to 30 kb.
  • the encoded polypeptides are analyzed by querying against PFAM models for immune response associated proteins. Potential immune response associated proteins are also identified by homology to Incyte cDNA sequences that have been annotated as immune response associated proteins.
  • These selected Genscan-predicted sequences are then compared by BLAST analysis to the genpept and gbpri pubhc databases. Where necessary, the Genscan-predicted sequences are then edited by comparison to the top BLAST hit from genpept to correct e ⁇ ors in the sequence predicted by Genscan, such as extra or omitted exons.
  • BLAST analysis is also used to find any Incyte cDNA or pubhc cDNA coverage of the Genscan- predicted sequences, thus providing evidence for transcription.
  • Incyte cDNA coverage is avaUable, this information is used to correct or confirm the Genscan predicted sequence.
  • Full length polynucleotide sequences are obtained by assembling Genscan-predicted coding sequences with Incyte cDNA sequences and/or pubhc cDNA sequences using the assembly process described in Example III.
  • fuU length polynucleotide sequences are derived entirely from edited or unedited Genscan-predicted coding sequences.
  • Partial cDNA sequences are extended with exons predicted by the Genscan gene identification program described in Example IV. Partial cDNAs assembled as described in Example III are mapped to genomic DNA and parsed into clusters containing related cDNAs and Genscan exon predictions from one or more genomic sequences. Each cluster is analyzed using an algorithm based on graph theory and dynamic programming to integrate cDNA and genomic information, generating possible splice variants that are subsequently confirmed, edited, or extended to create a full length sequence. Sequence intervals in which the entire length of the interval is present on more than one sequence in the cluster are identified, and intervals thus identified are considered to be equivalent by transitivity.
  • Partial DNA sequences are extended to full length with an algorithmbased on BLAST analysis.
  • First, partial cDNAs assembled as described in Example III are queried against pubhc databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases using the BLAST program.
  • the nearest GenBank protein homolog is then compared by BLAST analysis to either Incyte cDNA sequences or GenScan exon predicted sequences described in Example IV.
  • a chimeric protein is generated by using the resultant high-scoring segment pahs (HSPs) to map the translated sequences onto the GenBank protein homolog. Insertions or deletions may occur in the chimeric protein with respect to the original GenBank protein homolog.
  • HSPs high-scoring segment pahs
  • GenBank protein homolog the chimeric protein, or both are used as probes to search for homologous genomic sequences from the pubhc human genome databases. Partial DNA sequences are therefore "stretched” or extended by the addition of homologous genomic sequences. The resultant stretched sequences are examined to determine whether they contain a complete gene. VI. Chromosomal Mapping of IRAP Encoding Polynucleotides
  • sequences used to assemble SEQ ID NO:33-64 are compared with sequences from the Incyte LIFESEQ database and public domain databases using BLAST and other implementations of the Smith- Waterman algorithm. Sequences from these databases that matched SEQ ID NO:33-64 are assembled into clusters of contiguous and overlapping sequences using assembly algorithms such as Phrap (Table 7). Radiation hybrid and genetic mapping data available from public resources such as the Stanford Human Genome Center (SHGC), Whitehead Institute for Genome Research (WIGR), and Genethon are used to determine if any of the clustered sequences have been previously mapped. Inclusion of a mapped sequence in a cluster results in the assignment of all sequences of that cluster, including its particular SEQ ID NO:, to that map location.
  • SHGC Stanford Human Genome Center
  • WIGR Whitehead Institute for Genome Research
  • Map locations are represented by ranges, or intervals, of human chromosomes.
  • the map position of an interval, in centiMorgans, is measured relative to the terminus ofthe chromosome's p- arm.
  • the centiMorgan (cM) is a unit of measurement based on recombination frequencies between chromosomal markers. On average, 1 cM is roughly equivalent to 1 megabase (Mb) of DNA in humans, although this can vary widely due to hot and cold spots of recombination.
  • the cM distances are based on genetic markers mapped by Genethon which provide boundaries for radiation hybrid markers whose sequences were included in each of the clusters. Human genome maps and other resources available to the public, such as the NCBI "GeneMap'99" World Wide Web site
  • ncbi.nlm.nih.gov/genemap/ can be employed to determine if previously identified disease genes map within or in proximity to the intervals indicated above.
  • 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 ceU type or tissue have been bound (Sambrook and RusseU, supra, ch. 7; Ausubel et al., supra, ch. 4).
  • Analogous computer techniques applying BLAST are used to search for identical or related molecules in databases such as GenBank or LIFESEQ (Incyte). This analysis is much faster than multiple membrane-based hybridizations.
  • the sensitivity of the computer search can be modified to determine whether any particular match is categorized as exact or simUar.
  • the basis of the search is the product score, which is defined as:
  • the product score takes into account both the degree of simUarity between two sequences and the length of the sequence match.
  • the product score is a normalized value between 0 and 100, and is calculated as foUows: the BLAST score is multiplied by the percent nucleotide identity and the product is divided by (5 times the length of the shorter of the two sequences).
  • the BLAST score is calculated by assigning a score of +5 for every base that matches in a high-scoring segment pair (HSP), and -4 for every mismatch. Two sequences may share more than one HSP (separated by gaps). If there is more than one HSP, then the pair with the highest BLAST score is used to calculate the product score.
  • the product score represents a balance between fractional overlap and quality in a BLAST ahgnment. For example, a product score of 100 is produced only for 100% identity over the entire length of the shorter of the two sequences being compared. A product score of 70 is produced either by 100% identity and 70% overlap at one end, or by 88% identity and 100% overlap at the other. A product score of 50 is produced either by 100% identity and 50% overlap at one end, or 79% identity and 100% overlap.
  • polynucleotides encoding IRAP are analyzed with respect to the tissue sources from which they are derived. For example, some full length sequences are assembled, at least in part, with overlapping Incyte cDNA sequences (see Example III).
  • Each cDNA sequence is derived from a cDNA library constracted from a human tissue.
  • Each human tissue is classified into one of the foUowing organ/tissue categories: cardiovascular system; connective tissue; digestive system; embryonic structures; endocrine system; exocrine glands; genitalia, female; genitalia, male; germ ceUs; hemic and immune system; hver; musculoskeletal system; nervous system; pancreas; respiratory system; sense organs; skin; stomatognathic system; unclassified/mixed; or urinary tract.
  • the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
  • each human tissue is classified into one of the foUowing disease/condition categories: cancer, ceU line, developmental, inflammation, neurological, trauma, cardiovascular, pooled, and other, and the number of hbraries in each category is counted and divided by the total number of hbraries across aU categories.
  • the resulting percentages reflect the tissue- and disease-specific expression of cDNA encoding IRAP.
  • cDNA sequences and cDNA library/tissue information are found in the LIFESEQ database (Incyte, Palo Alto CA). VIII. Extension of IRAP Encoding Polynucleotides
  • FuU length polynucleotides are produced by extension of an appropriate fragment of the fuU length molecule using ohgonucleotide primers designed from this fragment.
  • One primer is synthesized to initiate 5' extension of the known fragment, and the other primer is synthesized to initiate 3' extension of the known fragment.
  • the initial primers are designed using OLIGO 4.06 software (National Biosciences), or another appropriate program, to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the target sequence at temperatures of about 68 °C to about 72 °C. Any stretch of nucleotides wliich would result in hairpin stractures and primer-primer dimerizations is avoided.
  • Selected human cDNA hbraries are used to extend the sequence. If more than one extension is necessary or desired, additional or nested sets of primers are designed.
  • PCR is performed in 96-weU plates using the PTC-200 thermal cycler (MJ Research, Inc.).
  • the reaction mix contains DNA template, 200 nmol of each primer, reaction buffer containing Mg 2+ , (NH 4 ) 2 S0 4 , and 2-mercaptoethanol, Taq DNA polymerase (Amersham Biosciences), ELONGASE enzyme (Invitrogen), and Pfu DNA polymerase (Stratagene), with the following parameters for primer pair PCI A and PCI B: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 68°C, 2 min; Step 5: Steps 2, 3, and 4 repeated 20 times; Step 6: 68 °C, 5 min; Step 7: storage at 4°C In the alternative, the parameters for primer pair T7 and SK+ are as foUows: Step 1: 94°C, 3 min; Step 2
  • the plate is scanned in a Fluoroskan II (Labsystems Oy, Helsinki, Finland) to measure the fluorescence of the sample and to quantify the concentration of DNA.
  • a 5 ⁇ l to 10 ⁇ l ahquot of the reaction mixture is analyzed by electrophoresis on a 1 % agarose gel to determine which reactions are successful in extending the sequence.
  • Tlie extended nucleotides are desalted and concentrated, transfened to 384-well plates, digested with CviJI cholera virus endonuclease (Molecular Biology Research, Madison WI), and sonicated or sheared prior to rehgation into pUC 18 vector (Amersham Biosciences).
  • CviJI cholera virus endonuclease Molecular Biology Research, Madison WI
  • sonicated or sheared prior to rehgation into pUC 18 vector
  • the digested nucleotides are separated on low concentration (0.6 to 0.8%) agarose gels, fragments are excised, and agar digested with Agar ACE (Promega).
  • Extended clones were rehgated using T4 hgase (New England Biolabs, Beverly MA) into pUC 18 vector (Amersham Biosciences), treated with Pfu DNA polymerase (Stratagene) to fill-in restriction site overhangs, and transfected into competent E. coli ceUs. Transformed ceUs are selected on antibiotic-containing media, and individual colonies are picked and cultured overnight at 37° C in 384-weU plates in LB/2x carb hquid media.
  • the ceUs are lysed, and DNA is amplified by PCR using Taq DNA polymerase (Amersham Biosciences) and Pfu DNA polymerase (Stratagene) with the foUowing parameters: Step 1: 94°C, 3 min; Step 2: 94°C, 15 sec; Step 3: 60°C, 1 min; Step 4: 72°C, 2 min; Step 5: steps 2, 3, and 4 repeated 29 times; Step 6: 72°C, 5 min; Step 7: storage at 4°C.
  • DNA is quantified by PICOGREEN reagent (Molecular Probes) as described above. Samples with low DNA recoveries are reamphfied using the same conditions as described above.
  • Samples are dUuted with 20% dim ⁇ thysulfoxide (1:2, v/v), and sequenced using DYENAMIC energy transfer sequencing primers and the DYENAMIC DIRECT kit (Amersham Biosciences) or the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Applied Biosystems).
  • Preliminary filters remove the majority of basecall errors by requiring a minimum Phred quality score of 15, and remove sequence alignment e ⁇ ors and e ⁇ ors resulting from improper trimming of vector sequences, chimeras, and sphce variants.
  • An automated procedure of advanced chromosome analysis is apphed to the original chromatogram files in the vicinity of the putative SNP.
  • Clone enor filters use statisticaUy generated algorithms to identify e ⁇ ors introduced during laboratory processing, such as those caused by reverse transcriptase, polymerase, or somatic mutation.
  • Clustering enor fUters use statistically generated algorithms to identify e ⁇ ors resulting from clustering of close homologs or pseudogenes, or due to contamination by non-human sequences.
  • a final set of filters removes duplicates and SNPs found in immunoglobulins or T-cell receptors.
  • Certain SNPs are selected for further characterization by mass spectrometry using the high throughput MASSARRAY system (Sequenom, Inc.) to analyze allele frequencies at the SNP sites in four different human populations.
  • the Caucasian population comprises 92 individuals (46 male, 46 female), including 83 from Utah, four French, three Venezuelan, and two Amish individuals.
  • the African population comprises 194 individuals (97 male, 97 female), all African Americans.
  • the Hispanic population comprises 324 individuals (162 male, 162 female), all Mexican Hispanic.
  • the Asian population comprises 126 individuals (64 male, 62 female) with a reported parental breakdown of 43% Chinese, 31% Japanese, 13% Korean, 5% Vietnamese, and 8% other Asian. Allele frequencies are first analyzed in the Caucasian population; in some cases those SNPs which show no allelic variance in this population are not further tested in the other three populations. X. Labeling and Use of Individual Hybridization Probes
  • Hybridization probes derived from SEQ ID NO:33-64 are employed to screen cDNAs, genomic DNAs, or mRNAs. Although the labeling of ohgonucleotides, consisting of about 20 base pairs, is specifically described, essentiaUy the same procedure is used with larger nucleotide fragments.
  • Ohgonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each ohgomer, 250 ⁇ Ci of [ ⁇ - 32 P] adenosine triphosphate (Amersham Biosciences), and T4 polynucleotide kinase (DuPont NEN, Boston MA).
  • the labeled ohgonucleotides are substantiaUy purified using a SEPHADEX G- 25 superfine size exclusion dextran bead column (Amersham Biosciences). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane-based hybridization analysis of human genomic DNA digested with one of the foUowing endonucleases: Ase I, Bgl II, Eco RI, Pst I, Xba I, or Pvu II (DuPont NEN).
  • the linkage or synthesis of a ⁇ ay elements upon a microarray can be achieved utilizing photolithography, piezoelectric printing (ink-jet printing; see, e.g., Baldeschweiler et al., supra), mechanical microspotting technologies, and derivatives thereof.
  • the substrate in each of the aforementioned technologies should be uniform and sohd with a non-porous surface (Schena, M., ed. (1999) DNA Microa ⁇ ays: A Practical Approach, Oxford University Press, London). Suggested substrates include silicon, silica, glass shdes, glass chips, and sihcon wafers.
  • a procedure analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal, UV, chemical, or mechanical bonding procedures.
  • a typical array may be produced using avaUable methods and machines weU known to those of ordinary skill in the art and may contain any appropriate number of elements (Schena, M. et al. (1995) Science 270:467-470; Shalon, D. et al. (1996) Genome Res. 6:639-645; MarshaU, A. and J. Hodgson (1998) Nat. Biotechnol. 16:27-31).
  • FuU length cDNAs, Expressed Sequence Tags (ESTs), or fragments or ohgomers thereof may comprise the elements of the microa ⁇ ay. Fragments or ohgomers suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR).
  • the a ⁇ ay elements are hybridized with polynucleotides in a biological sample.
  • the polynucleotides in the biological sample are conjugated to a fluorescent label or other molecular tag for ease of detection.
  • a fluorescence scanner is used to detect hybridization at each array element.
  • laser desorbtion and mass spectrometry may be used for detection of hybridization.
  • microarray preparation and usage is described in detaU below.
  • Total RNA is isolated from tissue samples using the guanidinium thiocyanate method and poly(A) + RNA is purified using the oligo-(dT) cellulose method.
  • Each poly(A) + RNA sample is reverse transcribed using MMLV reverse-transcriptase, 0.05 pg/ ⁇ l oligo-(dT) primer (21mer), IX first strand buffer, 0.03 units/ ⁇ l RNase inhibitor, 500 ⁇ M dATP, 500 ⁇ M dGTP, 500 ⁇ M dTTP, 40 ⁇ M dCTP, 40 ⁇ M dCTP-Cy3 (BDS) or dCTP-Cy5 (Amersham Biosciences).
  • the reverse transcription reaction is performed in a 25 ml volume containing 200 ng poly(A) + RNA with GEMB RIGHT kits (Incyte).
  • Specific control poly(A) + RNAs are synthesized by in vitro transcription from non-coding yeast genomic DNA. After incubation at 37° C for 2 hr, each reaction sample (one with Cy3 and another with Cy5 labehng) is treated with 2.5 ml of 0.5M sodium hydroxide and incubated for 20 minutes at 85° C to the stop the reaction and degrade the RNA.
  • Samples are purified using two successive CHROMA SPIN 30 gel filtration spin columns (BD Clontech, Palo Alto CA) and after combining, both reaction samples are ethanol precipitated using 1 ml of glycogen (1 mg/ml), 60 ml sodium acetate, and 300 ml of 100% ethanol. The sample is then dried to completion using a SpeedVAC (Savant Instruments Inc., HolbrookNY) and resuspended in 14 ⁇ l 5X SSC/0.2% SDS.
  • SpeedVAC SpeedVAC
  • Sequences of the present invention are used to generate a ⁇ ay elements.
  • Each array element is amplified from bacterial cells containing vectors with cloned cDNA inserts.
  • PCR amplification uses primers complementary to the vector sequences flanking the cDNA insert.
  • Array elements are amplified in thirty cycles of PCR from an initial quantity of 1-2 ng to a final quantity greater than 5 ⁇ g.
  • Amphfied a ⁇ ay elements are then purified using SEPHACRYL-400 (Amersham Biosciences).
  • Purified a ⁇ ay elements are immobilized on polymer-coated glass slides.
  • Glass microscope shdes (Corning) are cleaned by ultrasound in 0.1 % SDS and acetone, with extensive distilled water washes between and after treatments.
  • Glass slides are etched in 4% hydrofluoric acid (VWR Scientific Products Corporation (VWR), West Chester PA), washed extensively in distilled water, and coated with 0.05% aminopropyl silane (Sigma- Aldrich, St. Louis MO) in 95% ethanol.
  • Coated shdes are cured in a 110°C oven.
  • a ⁇ ay elements are applied to the coated glass substrate using a procedure described in U.S.
  • Patent No. 5,807,522 incorporated herein by reference.
  • 1 ⁇ l ofthe anay element DNA, at an average concentration of 100 ng/ ⁇ l, is loaded into the open capUlary printing element by a high-speed robotic apparatus.
  • the apparatus then deposits about 5 nl of a ⁇ ay element sample per slide.
  • Microa ⁇ ays are UV-crosslinked using a STRATALINKER UV-crosslinker (Stratagene). Microa ⁇ ays are washed at room temperature once in 0.2% SDS and three times in distUled water. Non-specific binding sites are blocked by incubation of microa ⁇ ays in 0.2% casein in phosphate buffered saline (PBS) (Tropix, Inc., Bedford MA) for 30 minutes at 60° C followed by washes in 0.2% SDS and distilled water as before. Hybridization Hybridization reactions contain 9 ⁇ l of sample mixture consisting of 0.2 ⁇ g each of Cy3 and
  • the chamber containing the a ⁇ ays is incubated for about 6.5 hours at 60° C
  • the a ⁇ ays are washed for 10 min at 45° C in a first wash buffer (IX SSC, 0.1% SDS), three times for 10 minutes each at 45°C in a second wash buffer (0.1X SSC), and dried.
  • Detection Reporter-labeled hybridization complexes are detected with a microscope equipped with an
  • Innova 70 mixed gas 10 W laser (Coherent, Inc., Santa Clara CA) capable of generating spectral lines at 488 nm for excitation of Cy3 and at 632 nm for excitation of Cy5.
  • the excitation laser light is focused on the anay using a 20X microscope objective (Nikon, Inc., MelviUe NY).
  • the slide containing the a ⁇ ay is placed on a computer-controlled X-Y stage on the microscope and raster- scanned past the objective.
  • the 1.8 cm x 1.8 cm anay used in the present example is scanned with a resolution of 20 micrometers.
  • a mixed gas multiline laser excites the two fluorophores sequentiaUy. Emitted light is split, based on wavelength, into two photomultiplier tube detectors (PMT R1477, Hamamatsu Photonics Systems, Bridgewater NJ) co ⁇ esponding to the two fluorophores. Appropriate filters positioned between the array and the photomultiplier tubes are used to filter the signals.
  • the emission maxima of the fluorophores used are 565 nm for Cy3 and 650 nm for Cy5.
  • Each a ⁇ ay is typically scanned twice, one scan per fluorophore using the appropriate filters at the laser source, although the apparatus is capable of recording the spectra from both fluorophores simultaneously.
  • the sensitivity of the scans is typicaUy calibrated using the signal intensity generated by a cDNA control species added to the sample mixture at a known concentration.
  • a specific location on the a ⁇ ay contains a complementary DNA sequence, allowing the intensity of the signal at that location to be co ⁇ elated with a weight ratio of hybridizing species of 1:100,000.
  • the calibration is done by labeling samples ofthe calibrating cDNA with the two fluorophores and adding identical amounts of each to the hybridization mixture.
  • the output ofthe photomultiplier tube is digitized using a 12-bit RTI-835H analog-to-digital (A/D) conversion board (Analog Devices, Inc., Norwood MA) installed in an IBM-compatible PC computer.
  • the digitized data are displayed as an image where the signal intensity is mapped using a linear 20-color transformation to a pseudocolor scale ranging from blue (low signal) to red (high signal).
  • the data is also analyzed quantitatively. Where two different fluorophores are excited and measured simultaneously, the data are first co ⁇ ected for optical crosstalk (due to overlapping emission spectra) between the fluorophores using each fluorophore's emission spectrum.
  • a grid is superimposed over the fluorescence signal image such that the signal from each spot is centered in each element ofthe grid.
  • the fluorescence signal within each element is then integrated to obtain a numerical value conesponding to the average intensity of the signal.
  • the software used for signal analysis is the GEMTOOLS gene expression analysis program (Incyte). Array elements that exhibit at least about a two-fold change in expression, a signal-to-background ratio of at least about 2.5, and an element spot size of at least about 40%, are considered to be differentiaUy expressed.
  • PBMCs from 4 healthy volunteer donors were stimulated in vitro with LPS in the presence or absence of naproxen for 4, 24, and 72 hours.
  • PBMCs were incubated in the presence of naproxen alone for the same duration.
  • Treated PBMCs were compared to untreated PBMCs maintained in culture for 24 hours.
  • SEQ ID NO:43 was found to be upregulated by at least two fold in PBMCs treated with LPS alone for 4, 24, and 72 hours as weU as in ceUs treated with LPS + naproxen for 4 and 24 hours. Treatment with naproxen alone had no effect.
  • SEQ ID NO:43 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, U) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • PBMCs from 4 healthy volunteer donors were treated with LPS in the presence or absence of ibuprofen for 4, 24, and 72 hours.
  • PBMCs were treated with ibuprofen alone and with LPS + vehicle (ethanol) for the same duration.
  • Treated PBMCs were compared to PBMCs from the same donors maintained in culture for 24 hours.
  • SEQ ID NO:43 was found to be upregulated by at least two fold in PBMCs treated with LPS alone for 4, 24 , and 72 hours as weU as in ceUs treated with LPS + ibuprofen for 24 hours. Treatment with ibuprofen alone had no effect.
  • SEQ ID NO:43 can be used for one or more of the foUowing: i) monitoring treatment of immune disorders and related diseases and conditions, ii) diagnostic assays for immune disorders and related diseases and conditions, and hi) developing therapeutics and/or other treatments for immune disorders and related diseases and conditions.
  • SEQ ID NO:45 was up-regulated in lung tumor tissue versus normal lung tissue as determined by microa ⁇ ay analysis. Expression of SEQ ID NO:45 was increased at least two-fold in lung tumor tissue versus matched normal lung tissue from the same donor in one of ten donors tested. Therefore, in various embodiments, SEQ ID NO:45 can be used for one or more of the foUowing: i) monitoring treatment of lung cancer, ii) diagnostic assays for lung cancer, and ih) developing therapeutics and/or other treatments for lung cancer.
  • expression of SEQ ID NO:48 was up-regulated in colon tumor tissue versus normal colon tissue as determined by microarray analysis. Expression of SEQ ID NO:48 was increased at least two-fold in colon tumor tissue versus matched normal tumor tissue from the same donor in one of seven donors tested. Therefore, in various embodiments, SEQ ID NO:48 can be used for one or more of the foUowing: i) monitoring treatment of colon cancer, ii) diagnostic assays for colon cancer, and hi) developing therapeutics and/or other treatments for colon cancer. For example, SEQ ID NO:45 showed tissue-specific expression as determined by microarray analysis. RNA samples isolated from a variety of normal human tissues were compared to a common reference sample.
  • Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uteras (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individuaUy hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another.
  • the expression of SEQ ID NO:45 was increased by at least two-fold in thymus gland as compared to the reference sample. Therefore, SEQ ID NO:45 can be used as a tissue marker for thymus gland.
  • SEQ ID NO:63 showed tissue-specific expression as determined by microarray analysis.
  • RNA samples isolated from a variety of normal human tissues were compared to a common reference sample. Tissues contributing to the reference sample were selected for their ability to provide a complete distribution of RNA in the human body and include brain (4%), heart (7%), kidney (3%), lung (8%), placenta (46%), smaU intestine (9%), spleen (3%), stomach (6%), testis (9%), and uteras (5%).
  • the normal tissues assayed were obtained from at least three different donors. RNA from each donor was separately isolated and individually hybridized to the microarray. Since these hybridization experiments were conducted using a common reference sample, differential expression values are directly comparable from one tissue to another.
  • SEQ ID NO:63 was increased by at least two-fold in testis tissue from 4 out of 5 donors as compared to the reference sample. Therefore, SEQ ID NO:63 can be used as a tissue marker for testis.
  • Sequences complementary to the IRAP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturaUy occurring IRAP.
  • ohgonucleotides comprising from about 15 to 30 base pahs
  • essentiaUy the same procedure is used with smaUer or with larger sequence fragments.
  • Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of IRAP.
  • a complementary ohgonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence.
  • a complementary ohgonucleotide is designed to prevent ribosomal binding to the IRAP-encoding transcript.
  • IRAP IRAP expression and purification of IRAP is achieved using bacterial or virus-based expression systems.
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription.
  • promoters include, but are not limited to, the trp-lac (tac) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction with the lac operator regulatory element.
  • Recombinant vectors are transformed into suitable bacterial hosts, e.g., BL21(DE3).
  • Antibiotic resistant bacteria express IRAP upon induction with isopropyl beta-D- thiogalactopyranoside (IPTG).
  • IRAP in eukaryotic ceUs is achieved by infecting insect or mammahan ceU lines with recombinant Autographica californica nuclear polyhedrosis viras (AcMNPV), commonly known as baculovirus.
  • AcMNPV Autographica californica nuclear polyhedrosis viras
  • the nonessential polyhedrin gene of baculovirus is replaced with cDNA encoding IRAP by either homologous recombination or bacterial-mediated transposition involving transfer plasmid intermediates. Viral infectivity is maintained and the strong polyhedrin promoter drives high levels of cDNA transcription.
  • baculovirus Recombinant baculovirus is used to infect Spodoptera frugiperda (Sf9) insect ceUs in most cases, or human hepatocytes, in some cases. Infection of the latter requires additional genetic modifications to baculovirus (Engelhard, E.K. et al. (1994) Proc. Natl. Acad. Sci. USA 91:3224-3227; Sandig, V. et al. (1996) Hum Gene Ther. 7:1937-1945).
  • IRAP is synthesized as a fusion protein with, e.g., glutathione S- transferase (GST) or a peptide epitope tag, such as FLAG or 6-His, permitting rapid, single-step, affinity-based purification of recombinant fusion protein from crude cell lysates.
  • GST glutathione S- transferase
  • a peptide epitope tag such as FLAG or 6-His
  • GST a 26- kUodalton enzyme from Schistosoma japonicum, enables the purification of fusion proteins on immobilized glutathione under conditions that maintain protein activity and antigenicity (Amersham Biosciences).
  • the GST moiety can be proteolyticaUy cleaved from IRAP at specificaUy engineered sites.
  • FLAG an 8-amino acid peptide
  • 6-His a stretch of six consecutive histidine residues, enables purification on metal-chelate resins (QIAGEN).
  • Methods for protein expression and purification are discussed in Ausubel et al. (supra, ch. 10 and 16) .
  • Purified IRAP obtained by these methods can be used dhectiy in the assays shown in Examples XVII and XVIII, where apphcable.
  • IRAP function is assessed by expressing the sequences encoding IRAP at physiologicaUy elevated levels in mammalian ceU culture systems.
  • cDNA is subcloned into a mammahan expression vector containing a strong promoter that drives high levels of cDNA expression.
  • Vectors of choice include PCMV SPORT plasmid (Invitrogen, Carlsbad CA) and PCR3.1 plasmid (Invitrogen), both of which contain the cytomegalovirus promoter.
  • 5- 10 ⁇ g of recombinant vector are transiently transfected into a human ceU line, for example, an endothelial or hematopoietic ceU line, using either liposome formulations or electroporation.
  • 1-2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected.
  • Expression of a marker protein provides a means to distinguish transfected ceUs fromnontransfected ceUs and is a rehable predictor of cDNA expression from the recombinant vector.
  • Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP; BD Clontech), CD64, or a CD64-GFP fusion protein.
  • Flow cytometry (FCM) an automated, laser optics-based technique, is used to identify transfected ceUs expressing GFP or CD64-GFP and to evaluate the apoptotic state of the ceUs and other ceUular properties.
  • FCM detects and quantifies the uptake of fluorescent molecules that diagnose events preceding or coincident with ceU death. These events include changes in nuclear DNA content as measured by staining of DNA with propidium iodide; changes in ceU size and granularity as measured by forward light scatter and 90 degree side light scatter; down-regulation of DNA synthesis as measured by decrease in bromodeoxyuridine uptake; alterations in expression of ceU surface and intraceUular proteins as measured by reactivity with specific antibodies; and alterations in plasma membrane composition as measured by the binding of fluorescein-conjugated Annexin V protein to the ceU surface. Methods in flow cytometry are discussed in Ormerod, M.G. (1994; Flow Cytometry, Oxford, New York NY). The influence of IRAP on gene expression can be assessed using highly purified populations of cells transfected with sequences encoding IRAP and either CD64 or CD64-GFP. CD64 and
  • CD64-GFP are expressed on the surface of transfected ceUs and bind to conserved regions of human immunoglobulin G (IgG). Transfected ceUs are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL, Lake Success NY). mRNA can be purified from the ceUs using methods weU known by those of skill in the art. Expression of mRNA encoding IRAP and other genes of interest can be analyzed by northern analysis or microa ⁇ ay techniques. XV. Production of IRAP Specific Antibodies
  • the IRAP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a co ⁇ esponding ohgopeptide is synthesized and used to raise antibodies by means known to those of skUl in the art.
  • oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Apphed Biosystems) using FMOC chemistry and coupled to KLH (Sigma- Aldrich, St. Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (Ausubel et al., supra).
  • ABI 431 A peptide synthesizer Apphed Biosystems
  • KLH Sigma- Aldrich, St. Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized with the ohgopeptide-KLH complex in complete Freund's adjuvant. Resulting antisera are tested for antipeptide and anti-IRAP activity by, for example, binding the peptide or IRAP to a substrate, blocking with 1 % BSA, reacting with rabbit antisera, washing, and reacting with radio-iodinated goat anti-rabbit IgG.
  • IRAP is substantiaUy purified by immunoaffinity chromatography using antibodies specific for IRAP.
  • An immunoaffinity column is constructed by covalently coupling anti-IRAP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Biosciences). After the coupling, the resin is blocked and washed according to the manufacturer's instructions.
  • IRAP Media containing IRAP are passed over the immunoaffinity column, and the column is washed under conditions that aUow the preferential absorbance of IRAP (e.g., high ionic strength buffers in the presence of detergent).
  • the column is eluted under conditions that disrupt antibody/IRAP binding (e.g., a buffer of pH 2 to pH 3, or a high concentration of a chaotrope, such as urea or thiocyanate ion), and IRAP is coUected.
  • Candidate molecules previously arrayed in the wells of a multi-weU plate are incubated with the labeled IRAP, washed, and any weUs with labeled IRAP complex are assayed. Data obtained using different concentrations of IRAP are used to calculate values for the number, affinity, and association of IRAP with the candidate molecules.
  • IRAP molecules interacting with IRAP are analyzed using the yeast two-hybrid system as described in Fields, S. and O. Song (1989; Nature 340:245-246), or using commercially avaUable kits based on the two-hybrid system, such as the MATCHMAKER system (BD Clontech).
  • IRAP may also be used in the PATHCALLING process (CuraGen Corp., New Haven CT) wliich employs the yeast two-hybrid system in a high-throughput manner to determine aU interactions between the proteins encoded by two large hbraries of genes (Nandabalan, K. et al. (2000) U.S. Patent No. 6,057,101).
  • An assay for IRAP activity measures the ability of IRAP to recognize and precipitate antigens from serum. This activity can be measured by the quantitative precipit n reaction (Golub, E.S. et al. (1987) Immunology: A Synthesis, Sinauer Associates, Sunderland, MA, pages 113-115).
  • IRAP is isotopicaUy labeled using methods known in the art. Various seram concentrations are added to constant amounts of labeled IRAP. IRAP-antigen complexes precipitate out of solution and are coUected by centrifugation. The amount of precipitable IRAP-antigen complex is proportional to the amount of radioisotope detected in the precipitate. The amount of precipitable IRAP-antigen complex is plotted against the serum concentration.
  • the amount of precipitable IRAP-antigen complex initiaUy increases proportionately with increasing serum concentration, peaks at the equivalence point, and then decreases proportionately with further increases in seram concentration.
  • the amount of precipitable IRAP-antigen complex is a measure of IRAP activity which is characterized by sensitivity to both limiting and excess quantities of antigen.
  • an assay for IRAP activity measures the expression of IRAP on the ceU surface.
  • cDNA encoding IRAP is transfected into a non-leukocytic ceU line.
  • CeU surface proteins are labeled with biotin (de la Fuente, M.A. et al. (1997) Blood 90:2398-2405).
  • Immunoprecipitations are performed using IRAP-specific antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques. The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of IRAP expressed on the ceU surface.
  • an assay for IRAP activity measures the amount of ceU aggregation induced by overexpression of IRAP.
  • cultured cells such as NIH3T3 are transfected with cDNA encoding IRAP contained within a suitable mammahan expression vector under control of a strong promoter.
  • Cotransfection with cDNA encoding a fluorescent marker protein, such as Green Fluorescent Protein (CLONTECH) is useful for identifying stable transfectants.
  • the amount of ceU agglutination, or clumping, associated with transfected ceUs is compared with that associated with untransfected ceUs.
  • the amount of ceU agglutination is a direct measure of IRAP activity.

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Abstract

Dans divers modes de réalisation, l'invention concerne des protéines associées à une réponse immunitaire (IRAP) et des polynucléotides identifiant les IRAP et codant pour celles-ci. Dans certains modes de réalisation, l'invention concerne également des vecteurs d'expression, des cellules hôtes, des anticorps, des agonistes et des antagonistes. Dans d'autres modes de réalisation, l'invention se rapporte à des méthodes de diagnostic, de traitement ou de prévention de troubles associés à l'expression aberrante des IRAP.
PCT/US2004/007716 2003-03-12 2004-03-11 Proteines associees a une reponse immunitaire WO2004081197A2 (fr)

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US60/454,451 2003-03-12
US45869503P 2003-03-27 2003-03-27
US60/458,695 2003-03-27
US46053003P 2003-04-04 2003-04-04
US60/460,530 2003-04-04
US46923603P 2003-05-09 2003-05-09
US60/469,236 2003-05-09
US48329403P 2003-06-26 2003-06-26
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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007066094A1 (fr) * 2005-12-05 2007-06-14 Aberdeen University Identification d’epitopes immunologiquement actifs a la surface d’erythrocytes et leur utilisation pour induire une tolerance

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1006183A1 (fr) * 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
WO2001082774A2 (fr) * 2000-04-21 2001-11-08 Rutgers, The State University Of New Jersey Procedes et compositions destines au diagnostic de la schizophrenie

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1006183A1 (fr) * 1998-12-03 2000-06-07 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Récepteurs Fc recombinantes et solubles
WO2001082774A2 (fr) * 2000-04-21 2001-11-08 Rutgers, The State University Of New Jersey Procedes et compositions destines au diagnostic de la schizophrenie

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2007066094A1 (fr) * 2005-12-05 2007-06-14 Aberdeen University Identification d’epitopes immunologiquement actifs a la surface d’erythrocytes et leur utilisation pour induire une tolerance

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