WO2000001817A2 - Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes - Google Patents

Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes Download PDF

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Publication number
WO2000001817A2
WO2000001817A2 PCT/US1999/012366 US9912366W WO0001817A2 WO 2000001817 A2 WO2000001817 A2 WO 2000001817A2 US 9912366 W US9912366 W US 9912366W WO 0001817 A2 WO0001817 A2 WO 0001817A2
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WIPO (PCT)
Prior art keywords
polypeptide
seq
sequence
primate
amino acids
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PCT/US1999/012366
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English (en)
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WO2000001817A3 (fr
Inventor
Elizabeth Esther Mary Bates
Serge J. E. Lebecque
Erin E. Murphy
Jeanine D. Mattson
Daniel M. Gorman
Joseph A. Hedrick
Luquan Wang
Albert Zlotnik
Nicholas J. Murgolo
Jonathan R. Greene
James A. Johnston
Jose Fernando Bazan
Daniel Mahony
Emma M. Lees
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Schering Corporation
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Priority to AU48185/99A priority Critical patent/AU4818599A/en
Priority to JP2000558207A priority patent/JP2002519062A/ja
Priority to EP99931753A priority patent/EP1093516A2/fr
Priority to CA002332573A priority patent/CA2332573A1/fr
Publication of WO2000001817A2 publication Critical patent/WO2000001817A2/fr
Publication of WO2000001817A3 publication Critical patent/WO2000001817A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
    • C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
    • C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
    • C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
    • C12N9/6472—Cysteine endopeptidases (3.4.22)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4738—Cell cycle regulated proteins, e.g. cyclin, CDC, INK-CCR
    • 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
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/521—Chemokines
    • 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
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide

Definitions

  • the present invention pertains to compositions related to proteins which: function in cellular physiology, development, and differentiation of mammalian cells; exhibit sequence similarity to TNF receptors which function in controlling activation and expansion of mammalian cells, e.g., cells of a mammalian immune system; or function in controlling the cell cycle and growth.
  • genes, proteins, antibodies, and related reagents useful e.g., to separate or identify particular cell types, or to regulate activation, development, differentiation, and function of various cell types, including hematopoietic cells; which exhibit high structural similarity to proteins that exhibit the biological capacity to serve as a carrier mediated transporters of charged organic anions across cellular membranes, which typically can be used in prostaglandin and thromboxane physiology, e.g., transportation, influx, efflux, clearance, or degradation; which regulate or evidence development, differentiation, and function of various cell types, including hematopoietic cells; or to regulate cell division and proliferation of various cell types, including tumor cells.
  • Prostaglandin ⁇ (PGs) and thromboxanes (TXs) play widespread physiological, and therapeutic roles in health and disease such as glaucoma; pregnancy, labor, delivery, and abortion; gastric protection and peptic ulcer formation; intestinal fluid secretion; liver protection and damage; airway resistance and asthma; blood pressure control; and modulation of inflammatory cells.
  • PGs Prostaglandin ⁇
  • TXs thromboxanes
  • PGs are charged anions at physiological pH that diffuse poorly across biological membranes. This limited simple diffusion appears to be augmented by carrier mediated transport in many diverse tissues such as the lung, choroid plexus, liver, anterior chamber of the eye, vagina, uterus, and placenta. Understanding the role of prostaglandins in the development and functioning of the immune system is presently incomplete. Specifically, the influence of prostaglandins (PGs) on antigen presenting cells (APCs) of the immune system (e.g., dendritic cells) is, as yet, poorly understood.
  • APCs antigen presenting cells
  • DCs Dendritic cells
  • LPS bacterial lipopolysaccharides
  • TNF ⁇ tumor necrosis factor alpha
  • Maturational changes in DCs include, e.g., silencing of antigen uptake by endocytosis, upregulation of surface molecules related to T cell activation, and active production of a number of cytokines including TNF ⁇ and IL-12. Upon local accumulation of
  • TNF ⁇ DCs migrate to the T cell areas of secondary lymphoid organs to activate antigen specific T cells.
  • PGE2 has been shown to have an influence on DC maturity and the production of cytokines by DCs. Seem e.g., Kalinski, et al . (1997) J. Immunol. 159:28-35; Kuhn, et al . (1997)
  • PGs influence cells of the immune system. It seems likely that PGs, like cytokines, effect immune system development and activation.
  • the present invention contributes to satisfying that need and is directed generally to a novel mammalian gene encoding a prostaglandin-like transporter (PGT) .
  • PTT prostaglandin-like transporter
  • the activation of resting T cells is critical to most immune responses and allows these cells to exert their regulatory or effector capabilities.
  • APC antigen presenting cells
  • mAb immobilized monoclonal antibodies
  • T- cell activation and T cell expansion depends upon engagement of the T-cell receptor (TCR) and co-stimulatory signals provided by accessory cells.
  • TCR T-cell receptor
  • TNF Tumor Necrosis Factor
  • the discovery of new cell markers is always potentially useful. Moreover, the inability to modulate activation signals prevents control of inappropriate developmental or physiological responses in the immune system.
  • the present invention provides at least one alternative costimulatory molecule, which will be useful as a marker for cell types, and agonists and antagonists of which will be useful in modulating a plethora of immune conditions or responses .
  • the circulating component of the mammalian circulatory system comprises various cell types, including red and white blood cells of the erythroid and myeloid cell lineages. See, e.g., Rapaport (1987) Introduction to Hematology (2d ed. ) Lippincott,
  • Lymphokines apparently mediate cellular activities in a variety of ways . They have been shown to support the proliferation, growth, and differentiation of the pluripotential hematopoietic stem cells into vast numbers of progenitors comprising diverse cellular lineages making up a complex immune system. These interactions between the cellular components are necessary for a healthy immune response. These different cellular lineages often respond in a different manner when lymphokines are administered in conjunction with other agents.
  • the chemokines are a large and diverse superfamily of proteins. The superfamily is subdivided into two classical branches, based upon whether the first two cysteines in the chemokine motif are adjacent (termed the "C-C” branch) , or spaced by an intervening residue ("C-X-C"). A more recently identified branch of chemokines lacks two cysteines in the corresponding motif, and is represented by the chemokines known as lymphotactins . Another recently identified branch has three intervening residues between the two cysteines, e.g., CX3C chemokines. See, e.g., Schall and Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy & Immunol. 109:97-109.
  • Ubiquitin (Ub) is a highly conserved 76 amino acid polypeptide that plays an important role in the regulation of protein degradation, cell-cycle progression, gene transcription and signal transduction.
  • the ubiquitination pathway is fine tuned and controlled, in part, by deubiquitination enzymes, which remove ubiquitin from proteins. Misregulation of the ubiquitination pathway may contribute problems in the protein quantity regulation, which may be associated, e.g., with malignant transformation, and oncogenesis through oncogenic counterparts of normally processed ubiquitinated proteins. Other clinical problems will often result from excessive or insufficient protein levels. Therefore, understanding the ubiquitination roles, e.g., in immune function, will increase our understanding of cell biology, which should have relevance, e.g., to malignant transformation .
  • B lymphocytes normal resting B cells
  • the resting cells are activated to pass from the G Q to G ] _ phase of the cell cycle. See, e.g., Alberts, et al . (eds. 1989) Molecular Biology of the Cell Garland Publ . , NY; and Darnell, et al . (1990) Molecular Cell Biology Freeman, NY.
  • the activated cells are induced to proliferate. See, e.g., Paul, ed. (1989) Fundamental Immunology. 2nd ed. , Raven Press, NY; and the third edition.
  • T cells also referred to as "T lymphocytes" are also induced to proliferate by certain factors, which include phytohemagglutinin, anti-T cell receptor monoclonal antibodies, anti-CD3 monoclonal antibodies , and other agents .
  • B7 (CD80) and B70 (CD86) are the second "group" of molecules which strongly mediate B and T cell interaction. These molecules, on B cells, interact with their ligands CD28 and CTLA-4 on T cells. These interactions are major co-stimulatory signals for activation of both B and T cells .
  • Monoclonal antibodies against RP105 also induce strong proliferation of mouse B cells and protects mouse B cells from irradiation-induced apoptosis in a similar fashion to anti-CD40 antibody or CD40-ligand. See Miyake, et al . (1994) J. Exp. Med. 180:1217-1224.
  • the RP105 molecule and its ligand MD-1 may be an additional pair of molecules that play key roles in the activation of T cells and B cells. See Miyake, et al . (1998) J. Immunol. 161:1349-1353; and Chan, et al., (1998) J. Exp. Med. 188:93-101 However, the human sequence of MD-1, has remained undetermined. The present invention provides this and also provides a previously undescribed second human homolog of mouse MD-1, (i.e., MD-2).
  • Cancer can occur in many tissues of the body. It results from a change in certain cells that causes them to evade the normal growth limiting mechanisms, e.g., to escape the feedback controls that normally stop cellular growth and reproduction after a given number of such cells have developed. Cell division and transcription are highly coordinated processes that play important roles in this feedback control. See, e.g., Beeson, et al . (eds. 1979) Textbook of Medicine, 15th ed. , W.B. Saunders Co., Philadelphia, PA.; DeVita, et al . (eds. 1997) Cancer: Principles and Practice of Oncology. 5th ed. , Lippincott, Philadelphia, PA; Neal and Hoskin (1997) Clinical Oncology: Basic Principles and Practice Oxford University Press, NY; Kastan (1997) Checkpoint
  • the cell cycle can be divided into four phases: the presynthetic phases (Go and Gi) ; the phase of DNA synthesis (S) ; and the postsynthetic phase (G2).
  • Go and Gi the presynthetic phases
  • S DNA synthesis
  • G2 the postsynthetic phase
  • Effective chemotherapeutic agents are often those which target diseased cells in the S phase, e.g., choriocarcinoma, acute lymphocytic leukemia, lyphocytic lymphosarcoma, Burkitt's lymphoma, Hodgkin's disease, testicular neoplasms, Wilm's tumor, and Ewing 's sarcoma.
  • choriocarcinoma acute lymphocytic leukemia
  • lyphocytic lymphosarcoma Burkitt's lymphoma
  • Hodgkin's disease testicular neoplasms
  • Wilm's tumor Wilm's tumor
  • Ewing 's sarcoma Ewing 's sarcoma
  • the lack of knowledge regarding the control of the cell cycle has hampered the ability of medical science to specifically regulate cell division or immune responses.
  • the present invention provides compositions which will be important in the control of cell division and transcription.
  • the present invention is based, in part, upon the characterization of the genes and products relating to the DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5 chemokine, Dubll, Dubl2 , MD-1, MD- 2, and cyclin E2. It provides nucleic acids, polypeptides, antibodies, and methods for making and using such compositions.
  • the invention provides an isolated or recombinant antigenic polypeptide comprising: a plurality of distinct segments, wherein each segment has identity to at least 12 contiguous amino acids from the mature SEQ ID NO: 2; or at least 17 contiguous amino acids from the mature SEQ ID NO: 2.
  • the plurality of segments includes one of at least 19 contiguous amino acids; or two of at least 15 contiguous amino acids.
  • polypeptides include those wherein the polypeptide: comprises the mature SEQ ID NO: 2; binds with specificity to a polyclonal antibody which specifically binds to SEQ ID NO: 2; or the polypeptide: is a natural allelic variant of SEQ ID NO: 2; is at least 30 amino acids in length; exhibits at least two non-overlapping epitopes specific for SEQ ID NO: 2; is a synthetic polypeptide; is attached to a solid substrate; or is a 5-fold or less conservative substitution from SEQ ID NO: 2.
  • Fusion polypeptides are also provided, e.g., comprising first and second portions, the first portion comprising a sequence as described and the second portion comprising a detectable marker.
  • Pharmaceutical compositions are made available, e.g., comprising a sterile polypeptide, as described, in a pharmaceutically acceptable carrier.
  • Polynucleotide embodiments include an isolated or recombinant polynucleotide encoding a described polypeptide. Preferred forms will be such a polynucleotide which: comprises the mature polypeptide coding portion of SEQ ID NO: 1; or encodes the mature SEQ ID NO : 2. Preferred embodiments include wherein the polynucleotide is: a PCR product; a hybridization probe; a mutagenesis primer; or made by chemical synthesis. Alternatively, the polynucleotide is: detectably labeled; a deoxyribonucleic acid; or double stranded.
  • an expression vector comprising the described polynucleotide, including wherein the polypeptide specifically binds polyclonal antibodies generated against an immunogen of mature SEQ ID NO: 2; which selectively hybridizes under stringent hybridization conditions to a target polynucleotide sequence having at least 60 contiguous nucleotides from SEQ ID NO: 1; encodes a polypeptide having at least 50 contiguous amino acid residues from mature SEQ ID NO: 2; or is suitable for transfection into a prokaryote or eukaryote host cell.
  • the host cell is: a mammalian cell; a bacterial cell; an insect cell; a prokaryote; a eukaryote; or a COS cell.
  • a method is provided, e.g., of making a polypeptide comprising expressing the vector in the host cell.
  • polynucleotides include an isolated or recombinant polynucleotide which hybridizes to the coding portion of SEQ ID NO: 1 under stringent hybridization and wash conditions of at least 50° C, a salt concentration of less than 400 mM, and 50% formamide.
  • a nucleic acid may be an expression vector, which may hybridize to the coding portion of SEQ ID NO: 1 under stringent hybridization and wash conditions of at least 60° C, a salt concentration of less than 200 mM, and 50% formamide.
  • the vector encodes a polypeptide which specifically binds an antibody generated against a mature SEQ ID NO: 2.
  • polynucleotide which hybridizes to SEQ ID NO: 1, wherein the polynucleotide is: a PCR product; a hybridization probe; a mutagenesis primer; or made by chemical synthesis .
  • Methods are provided, e.g., of modulating the physiology or development of a cell, comprising contacting the cell with an agonist or antagonist of a described polypeptide; of detecting the presence of a complementary polynucleotide in a sample, comprising contacting a described polynucleotide that selectively hybridizes with the complementary polynucleotide in the sample to form a detectable duplex; thereby indicating the presence of the polynucleotide in the sample; or for identifying a compound that binds to a described polypeptide, comprising: incubating components comprising the compound and the polypeptide under conditions sufficient to allow the components to interact; and measuring the binding of the compound to the polypeptide.
  • the invention further provides an isolated or recombinant polynucleotide encoding an antigenic polypeptide comprising at least 17 contiguous amino acids from: the mature polypeptide from SEQ ID NO: 6; the mature polypeptide from SEQ ID NO: 8; the mature polypeptide from SEQ ID NO: 10; the mature polypeptide from SEQ ID NO: 12; the mature polypeptide from SEQ ID NO: 17; the mature polypeptide from SEQ ID NO: 19; the mature polypeptide from SEQ ID NO: 21; or the mature polypeptide from SEQ ID NO: 23.
  • such polynucleotide will encode all of the polypeptide of: signal processed SEQ ID NO: 6; signal processed SEQ ID NO: 8; signal processed SEQ ID NO: 10; signal processed SEQ ID NO: 12; signal processed SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO: 23.
  • polynucleotide which hybridizes at 55° C, less than 500 mM salt, and 50% formamide to the: mature protein coding portion of SEQ ID NO: 5; signal processed coding portion of SEQ ID NO: 7; signal processed coding portion of SEQ ID NO: 9; signal processed coding portion of SEQ ID NO: 11; mature protein coding portion of SEQ ID NO: 16; polypeptide coding portion of SEQ ID NO: 18; polypeptide coding portion of SEQ ID NO: 20; or polypeptide coding portion of SEQ ID NO: 22.
  • polynucleotides comprising at least 35 contiguous nucleotides of: mature protein coding portion of SEQ ID NO: 5; signal processed coding portion of SEQ ID NO: 7; signal processed coding portion of SEQ ID NO: 9; signal processed coding portion of SEQ ID NO: 11; mature protein coding portion of SEQ ID NO: 16; polypeptide coding portion of SEQ ID NO: 18; polypeptide coding portion of SEQ ID NO: 20; or polypeptide coding portion of SEQ ID NO: 22.
  • Various expression vectors are provided comprising such a polynucleotide.
  • the invention also provides a host cell containing the expression vector, including a eukaryotic cell.
  • Methods are provided, e.g., making an antigenic polypeptide comprising expressing a recombinant polynucleotide; for detecting a polynucleotide, comprising contacting the polynucleotide with a probe that hybridizes, under stringent conditions, to at least 25 contiguous nucleotides of the: mature protein coding portion of SEQ ID NO: 5; signal processed coding portion of SEQ ID NO: 7; signal processed coding portion of SEQ ID NO: 9; signal processed coding portion of SEQ ID NO: 11; mature protein coding portion of SEQ ID NO: 16; polypeptide coding portion of SEQ ID NO: 18; polypeptide coding portion of SEQ ID NO: 20; or polypeptide coding portion of SEQ ID NO: 22; to form a duplex, wherein detection of the duplex indicates the presence of the polynucleotide.
  • Kits are provided, e.g., for the detection of a described polynucleotide, comprising a compartment containing a probe that hybridizes, under stringent hybridization conditions, to at least 17 contiguous nucleotides of a described polynucleotide to form a duplex.
  • the probe is detectably labeled.
  • Binding compounds are provided, including antibodies, comprising an antibody binding site which specifically binds to a polypeptide comprising at least 17 contiguous amino acids from: signal processed SEQ ID NO 6; signal processed SEQ ID NO: 8; signal processed SEQ ID NO 10; signal processed SEQ ID NO: 12; signal processed SEQ ID NO 17; SEQ ID NO: 19 ; SEQ ID NO: 21; or SEQ ID NO: 23.
  • the antibody binding site is: selectively immunoreactive with the: signal processed SEQ ID NO: 6; signal processed SEQ ID NO: 8; signal processed SEQ ID NO: 10; signal processed SEQ ID NO: 12; signal processed SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO: 23; raised against a purified or recombinantly produced human HDTEA84 protein; raised against a purified or recombinantly produced human HSLJD37R protein; or in a monoclonal antibody, Fab, or F(ab)2; or the binding compound is: an antibody molecule; a polyclonal antiserum; detectably labeled; sterile; or in a buffered composition.
  • compositions allow various methods, including using the binding compound, comprising contacting the binding compound with a biological sample comprising an antigen, thereby forming a binding compound: antigen complex.
  • the biological sample is from a human, and the binding compound is an antibody.
  • Such also allow for production of a detection kit comprising the binding compound, and: instructional material for the use of the binding compound for the detection; or a compartment providing segregation of the binding compound.
  • Polypeptides are also made available, e.g., a substantially pure or isolated antigenic polypeptide, which binds to the described binding composition, and further comprises at least 17 contiguous amino acids from: signal processed SEQ ID NO: 6; signal processed SEQ ID NO: 8; signal processed SEQ ID NO: 10; signal processed SEQ ID NO: 12; signal processed SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO: 23.
  • Preferred polypeptides include those which: comprise at least a fragment of at least 25 contiguous amino acid residues from: a primate HDTEA84 protein; a primate HSLJD37R protein; or a rodent or primate RANKL protein; or are soluble polypeptides; are detectably labeled; are in a sterile composition; are in a buffered composition; bind to an sialic acid residue; are recombinantly produced; or have a naturally occurring polypeptide sequence.
  • the polypeptide comprises at least 17 contiguous amino acids from the: signal processed SEQ ID NO: 6; signal processed SEQ ID NO: 8; signal processed SEQ ID NO: 12; signal processed SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO: 23.
  • Methods including a method of modulating a precursor cell physiology or function comprising a step of contacting the cell with: a binding compound which binds to a described polypeptide; an HDTEA84 polypeptide; an HSLJD37R polypeptide; or a RANKL polypeptide.
  • the method may be one wherein the contacting is in combination with a TNF family ligand, or an antagonist of the TNF family ligand.
  • the present invention provides compositions related to other chemokine, Dub, or surface protein genes.
  • Polypeptide embodiments include: a substantially pure or recombinant HCC5 polypeptide exhibiting identity over a length of at least 12 amino acids to SEQ ID NO: 25; an isolated natural sequence HCC5 of mature SEQ ID NO: 25; a fusion protein comprising HCC5 sequence; a substantially pure or recombinant Dubll polypeptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 32 or 34; an isolated natural sequence Dubll of mature SEQ ID NO: 32 or 34; a fusion protein comprising Dubll sequence; a substantially pure or recombinant Dubl2 polypeptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 36 or 38; an isolated natural sequence Dubl2 of mature SEQ ID NO: 36 or 38; a fusion protein comprising Dubl2 sequence; a substantially pure or recombinant MD-1 poly
  • the HCC5 polypeptide is from a primate, including a human; comprises at least one polypeptide segment of SEQ ID NO: 25; exhibits a plurality of portions exhibiting the identity; is a natural allelic variant of HCC5; has a length at least about 30 amino acids; exhibits at least two non- overlapping epitopes which are specific for a primate HCC5 ; exhibits a sequence identity over a length of at least 35 amino acids to a HCC5; is glycosylated; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from natural sequence; or is a deletion or insertion variant from a natural sequence; or the Dubll polypeptide: is from a primate
  • compositions comprising such polypeptides are also provided, along with those comprising: the HCC5 polypeptide and: a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; another chemokine, including one selected from the group of HCC1, HCC2 , HCC3 , and HCC4; or an antibody antagonist for a chemokine, including one selected from the group of HCC1, HCC2, HCC3 , and HCC4; the Dubll polypeptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; the Dubl2 polypeptide and a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral,
  • Fusion proteins are provided, e.g., comprising: mature protein sequence of SEQ ID NO: 25; mature protein sequence of SEQ ID NO: 32, SEQ ID NO: 34, SEQ ID NO: 36 or SEQ ID NO: 38; mature protein sequence of SEQ ID NO: 42, SEQ ID NO: 44, SEQ ID NO: 46, SEQ ID NO: 48, or SEQ ID NO: 49; a detection or purification tag, including a FLAG, His6, or Ig sequence; or sequence of another chemokine protein with the chemokine polypeptide Kits are provided, e.g., comprising a described polypeptide and: a compartment comprising the polypeptide; and/or instructions for use or disposal of reagents in the kit.
  • Binding compounds comprising an antigen binding portion from an antibody, which specifically binds to a natural: HCC5 polypeptide, wherein the antibody: is raised against a peptide sequence of a mature HCC5 polypeptide sequence of SEQ ID NO: 25; is raised against a mature HCC5; is raised to a purified HCC5; is immunoselected; is a polyclonal antibody; binds to a denatured HCC5; or exhibits a Kd to HCC5 antigen of at least 30 ⁇ M; or Dubll polypeptide, wherein the antibody: is raised against a peptide sequence of a mature Dubll polypeptide sequence of SEQ ID NO: 32 or SEQ ID NO: 34; is raised against a mature Dubll; is raised to a purified Dubll; is immunoselected; is a polyclonal antibody; binds to a denatured
  • Dubll or exhibits a Kd to Dubll antigen of at least 30 ⁇ M; or
  • Dubl2 polypeptide wherein the antibody: is raised against a peptide sequence of a mature Dubl2 polypeptide sequence of SEQ ID NO: 36 or SEQ ID NO: 38; is raised against a mature Dubl2; is raised to a purified Dubl2; is immunoselected; is a polyclonal antibody; binds to a denatured Dubl2; or exhibits a Kd to Dubl2 antigen of at least 30 ⁇ M; or a primate MD-1 polypeptide, wherein the antibody: is raised against a peptide sequence of a mature polypeptide sequence of SEQ ID NO: 42; is raised against a mature MD-1; is raised to a purified MD-1; is immunoselected; is a polyclonal antibody; binds to a denatured MD-1; or exhibits a Kd to MD-1 antigen of at least 30 ⁇ M; or a primate MD-2 polypeptide, wherein the antibody: is raised against a peptide sequence of
  • the binding composition will be one wherein: the polypeptide is from a primate or rodent; the binding compound is an Fv, Fab, or Fab2 fragment; the binding compound is conjugated to another chemical moiety; is attached to a solid substrate, including a bead or plastic membrane; is in a sterile composition; or is detectably labeled, including a radioactive or fluorescent label.
  • Kits comprising the binding compound, and: a compartment comprising the binding compound; a compartment comprising purified antigen; and/or instructions for use or disposal of reagents in the kit.
  • Methods are provided for producing an antigen : antibody complex, comprising contacting an antibody and: a primate HCC5 polypeptide; a primate Dubll polypeptide; a primate Dubl2 polypeptide; a primate MD-1 polypeptide; a primate MD-2 polypeptide; or a rodent MD-2 polypeptide; thereby allowing the complex to form.
  • compositions are provided, e.g., the binding compound and: a carrier, wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; or an antibody antagonist for another chemokine, including one selected from the group of HCC1, HCC2 , HCC3 , and HCC4.
  • a carrier wherein the carrier is: an aqueous compound, including water, saline, and/or buffer; and/or formulated for oral, rectal, nasal, topical, or parenteral administration; or an antibody antagonist for another chemokine, including one selected from the group of HCC1, HCC2 , HCC3 , and HCC4.
  • Nucleic acid embodiments include, e.g., an isolated or recombinant nucleic acid encoding a polypeptide or fusion protein described, wherein: the HCC5 : polypeptide is from a primate, including a human; or nucleic acid: encodes an antigenic HCC5 peptide sequence of SEQ ID NO: 25 encodes a plurality of antigenic HCC5 peptide sequences of SEQ ID NO: 25; exhibits identity over at least 25 nucleotides to a natural cDNA encoding the HCC5 segment; is a hybridization probe for a gene encoding the HCC5 polypeptide; or further encodes another chemokine, including one selected from the group of HCC1, HCC2 , HCC3 , and HCC4; or the Dubll: polypeptide is from a primate, including a human; or nucleic acid: encodes a Dubll antigenic peptide sequence of SEQ ID NO: 32; or SEQ ID NO: 34;
  • nucleic acid embodiments include the described, which: is an expression vector; further comprises an origin of replication; is from a natural source; comprises a detectable label; comprises synthetic nucleotide sequence is less than 6 kb, preferably less than 3 kb; is from a primate, including a human; comprises a natural full length coding sequence; or is a PCR primer, PCR product, or mutagenesis primer.
  • Various cells are provided, including a cell or tissue comprising a described recombinant nucleic acid, including wherein the cell is: a prokaryotic cell; a eukaryotic cell; a bacterial cell; a yeast cell; an insect cell; a mammalian cell; a mouse cell; a primate cell; or a human cell.
  • Kits are provided, e.g., comprising a described nucleic acid, and: a compartment comprising the nucleic acid; a compartment comprising a nucleic acid encoding another chemokine, including
  • HCC1, HCC2, HCC3, and HCC4 instructions for use or disposal of reagents in the kit.
  • Alternative nucleic acids include those which: hybridize under wash conditions of 45° C and less than 2M salt to the polypeptide coding portion of SEQ ID NO: 24; hybridize under wash conditions of 45° C and less than 2M salt to the polypeptide coding portions of SEQ ID NO: 31 or 33; hybridize under wash conditions of 45° C and less than 2M salt to the coding portions of SEQ ID NO: 35 or 37; hybridize under wash conditions of 45° C and less than 2M salt to the coding portion of SEQ ID NO: 41; hybridize under wash conditions of 45° C and less than 2M salt to the coding portion of SEQ ID NO: 43 or 45. or hybridize under wash conditions of 45° C and less than 2M salt to the coding portion of SEQ ID NO: 47.
  • the wash conditions are at 55° C and/or 500 mM salt; or at 65° C and/or 150 mM salt.
  • methods are provided, e.g., of modulating physiology or development of a cell or tissue culture cells comprising exposing the cell to an agonist or antagonist of HCC5, primate MD-1, primate MD-2, or rodent MD-2.
  • Others include methods of detecting specific binding to a compound, comprising: contacting the compound to a composition selected from the group of: an antigen binding site which specifically binds to: an HCC5 chemokine; a Dubll; a Dubl2; a primate MD-1; a primate MD-2; a rodent MD-2; or an expression vector encoding: an HCC5 chemokine or fragment thereof; a Dubll or fragment thereof; a Dubl2 or fragment thereof; a primate MD-1 or fragment thereof; a primate MD-2 or fragment thereof; or a rodent MD-2 or fragment thereof; a substantially pure protein which is specifically recognized by the antigen binding site of the described antigen binding sites; a substantially pure HCC5 chemokine or peptide thereof
  • Particular polynucleotide embodiments include an isolated or recombinant polynucleotide which: encodes at least 17 contiguous amino acid residues of SEQ ID NO: 54; encodes at least two distinct segments of at least 10 contiguous amino acid residues of SEQ ID NO 54; or comprises one or more segments at least 21 contiguous nucleotides of SEQ ID NO: 53.
  • Such polynucleotides allow methods of making: a polypeptide comprising expressing a described expression vector, thereby producing the polypeptide; a duplex nucleic acid comprising contacting a polynucleotide with a complementary nucleic acid, thereby resulting in production of the duplex nucleic acid; a synthetic polynucleotide, comprising chemically polymerizing nucleotides to produce the polynucleotide; or a polynucleotide comprising using a PCR method.
  • Cyclin polypeptide embodiments include an isolated or recombinant antigenic polypeptide comprising at least: one segment comprising at least 17 contiguous amino acids from SEQ ID NO: 54; or at least two distinct segments of at least 11 contiguous amino acids from SEQ ID NO: 54.
  • Such polypeptide may: comprise at least one segment comprising at least 17 contiguous amino acids from SEQ ID NO: 54; and exhibit at least two non-overlapping epitopes which are selective for primate protein of SEQ ID NO: 54.
  • Other embodiments include those wherein the polypeptide: is a 5-fold or less substitution from a natural sequence; is a deletion or insertion variant from a natural sequence; or comprises at least two distinct segments of at least 11 contiguous amino acids from SEQ ID NO: 54.
  • the polypeptide is antigenic, and will typically comprise at least one sequence from (SEQ ID NO: 54) KESRYVHD (residues 120-127), DKHFEVLH (residues 127-134), HSDLEPQM (residues 134-141), QKDINKNM (residues 177-184), YAPKLQEF (residues 203-210), SEEDILRM (residues 219-226), LRMELIIL
  • the segments of at least 11 contiguous amino acids comprise one the segment with at least 14 contiguous amino acids from SEQ ID NO: 54.
  • Such polypeptides may further exhibit at least two non-overlapping epitopes which are selective for primate protein of SEQ ID NO: 54; and/or may: comprise a mature sequence of SEQ ID NO: 2; bind with selectivity to an antibody generated against an immunogen of SEQ ID NO: 54; comprise a plurality of polypeptide segments of 17 contiguous amino acids of SEQ ID NO: 54; or be a natural allelic variant of SEQ ID NO: 54.
  • the polypeptide may: be in a sterile composition; have a length at least 30 amino acids; be not glycosylated; be denatured; be a synthetic polypeptide; be attached to a solid substrate; or be a fusion protein with a detection or purification tag, including a FLAG, His6, or Ig sequence.
  • Other embodiments include those wherein the polypeptide: is a 5-fold or less substitution from a natural sequence; or is a deletion or insertion variant from a natural sequence.
  • kits which comprise such polypeptides and instructions for the use or disposal of the polypeptide or other reagents of the kit.
  • Methods are provided, e.g., to label the polypeptide, comprising labeling the polypeptide with a radioactive label; to separate the polypeptide from another polypeptide in a mixture, comprising running the mixture on a chromatography matrix, thereby separating the polypeptides; to identify a compound that binds selectively to the polypeptide, comprising incubating the compound with the polypeptide under appropriate conditions; thereby causing the component to bind to the polypeptide; to conjugate the polypeptide to a matrix, comprising derivatizing the polypeptide with a reactive reagent, and conjugating the polypeptide to the matrix; or inducing an antibody response to the polypeptide, comprising introducing the polypeptide as an antigen to an immune system, thereby inducing the response.
  • Binding compounds are provided, e.g., antibodies, comprising an antigen binding portion from an antibody which binds with selectivity to described polypeptides. Methods are made available for evaluating the selectivity of binding of a compound to cyclin E2, comprising contacting the compound to a recombinant cyclin E2 polypeptide and at least one other cyclin; and comparing binding of the compound to the cyclins.
  • proteins which: mediate uptake of substrates, e.g., prostaglandin- like molecules, modulate or mediate, e.g., induce or prevent trafficking, proliferation, or differentiation of, interacting cells, or are intracellular proteins which are important in various cellular processes, e.g., deubiquitination of proteins or cell cycle regulation.
  • substrates e.g., prostaglandin- like molecules
  • modulate or mediate e.g., induce or prevent trafficking, proliferation, or differentiation of, interacting cells
  • intracellular proteins which are important in various cellular processes, e.g., deubiquitination of proteins or cell cycle regulation.
  • the Prostaglandin-like Transporter (PGT) of the present invention is expressed particularly in antigen presenting cells of the immune system, e.g., dendritic cells.
  • the transporter is designated a dendritic cell prostaglandin-like transporter (DC-PGT) . Consequently, the DC-PGT of the present invention offers the means to establish fundamental understanding on the role of PG influence on immune function.
  • the present invention provides DNA sequence encoding a mammalian protein that exhibits structural features characteristic of functionally significant proteins, particularly which serve as organic anion transporters.
  • This family of organic anion transporters includes: the prostaglandin transporters of man (Lu, et al. (1996) J. Clin. Invest. 98:1142-1149) and rat; organic anion transporters in man and rat; brain digoxyin transporters and Matrin F/G of rat (Kanai, et al . (1995) Science 268:866-869).
  • Transporters of this family typically are 12 transmembrane proteins of approximately 650 amino acids in length. Characteristic of this group of proteins is a cysteine rich region located in one of the extracellular loops, which resembles a zinc finger motif. It is not entirely certain whether these polypeptides mediate primarily the influx or efflux of prostaglandins and organic anions, and they may, under different circumstances produce influx or efflux depending, e.g., on the intracellular concentration of the organic anions concerned.
  • the DC PGT protein of the present invention is closest in homology to the prostaglandin transporters and it is probable that a prostaglandin is the major anion transported.
  • DC- PGT DC- PGT
  • primate e.g., human (GenBank: locus HSU70867, accession U70867)
  • rodent e.g., rat (prostaglandin transporter - rat, GenBank Ace. No. 1083766; Kanai, et al . (1995) Science 268:866-869).
  • the antigens HDTEA84, HSLJD37R, and RANKL, and fragments, or antagonists will be useful in physiological modulation of cells expressing receptors for, e.g., ligands of the TNF family.
  • Some of these antigens appear to lack a membrane spanning segment, suggesting that they are soluble forms of receptor. This suggests that the soluble proteins can serve as antagonists of the TNF-like ligands.
  • membrane spanning forms exist, which serve as signaling receptors mediating cellular response to the ligands.
  • the HDTEA84 gene has been detected in cDNA libraries derived form Hodgkin ' s lymphoma, endothelial cells, keratinocytes, prostrate, and cerebellum. It exhibits significant: sequence similarity to the osteoprotegerin ligand receptor reported by Lacey, et al . (1998) Cell 93:165-176.
  • the HDTEA84 will likely modulate proliferation or development by antagonizing its respective ligand.
  • Membrane associated forms should exist, likely alternatively spliced transcription products .
  • the HSLJD37R exhibits like similarity to receptors for TNF. While the first embodiment is an incomplete sequence, the available portion currently lacks an identified transmembrane segment. Additional efforts provide a full length sequence, and an alternative splice variant.
  • the rodent 427152#4 Rank-like (RANKL) was detected in a rodent cDNA library panel probed with Mouse 427152#4 (204 bp) .
  • Positive signals were detected in CH12 (B cell line) ; rag-1 thymus; rag-1 heart; rag-1 brain (best signal) ; rag-1 testes; rag- 1 liver; normal lung; rag-1 lung; asthmatic lung; tolerized and challenged lung; Nippo-infected lung; Nippo IL-4 K.O. lung; Nippo anti-IL-5 treated lung; influenza lung; guinea pig allergic lung; w.t. stomach; and w.t. colon on a 3 day exposure at -80° C with an inten ⁇ ifier screen.
  • the primate e.g., human, Rank-like (RANKL) homologs of rodent 427152#4 were detected in a human cDNA library panel probed with mouse 427152#4 (204 bp) . Signals were detected in monkey asthma lung 4 h (1.6-2.0 kb) and adult placenta (2.5-3.0 kb) on a 3 day exposure at -80° C with screen.
  • RNKL Rank-like
  • CDla+ 95% DC activated CHA kidney epithelial carcinoma cell line
  • monkey lung normal normal
  • psoriasis skin fetal lung
  • fetal ovary fetal testes
  • fetal spleen fetal spleen
  • Each of these proteins will also be useful as antigens, e.g., immunogens, for raising antibodies to various epitopes on the protein, linear and/or conformational epitopes.
  • the molecules may be useful in defining various cell subsets, either by the molecules produced by, or by expression of membrane forms of the receptors. Such cells should be responsive to the respective ligands . Soluble forms of the receptors should serve as antagonists of the ligand, binding to the ligand and preventing interaction with membrane forms, which would mediate signaling. Both genes express proteins which exhibit structural motifs characteristic of a member of the TNF receptor family.
  • HDTEA84 interesting features include: signal sequence from about 1-11; TNF receptor Cys rich domains I (about 32-72) , II (about 73-113), III (about 114-150), and IV (about 151-193); and unique region from about 194-300.
  • HSLJD37R SEQ ID NO: 10 form
  • signal sequence from about 1-41; TNF receptor Cys rich domains I (about 42-90), II (about 91-131), III (about 132-168), and IV (about 169- 211); transmembrane segment from about 354-370. Similar alignment of the other variants will identify similar features . Segments including combinations or excluding such segments may be desired.
  • HDTEA84, HSLJD37R, and RANKL to members of the TNF receptor family suggests related function of these molecules. See, e.g., Lacey, et al . (1998) Cell 93:165-176.
  • the sequences however, mostly lack a transmembrane segment, suggesting that the proteins are soluble receptor forms . They may well also have membrane bound forms resulting, e.g., from alternatively spliced transcript variants.
  • the soluble forms are likely to be antagonists of the ligand, e.g., blocking the binding of ligand to a membrane bound form of signaling receptor.
  • these molecules may be useful in the treatment of abnormal immune or developmental disorders .
  • the natural antigens should be capable of modulating various biochemical responses which lead to biological or physiological responses in target cells.
  • the embodiments characterized herein are from primate, e.g., human, but other species variants almost surely exist, e.g., rodents, etc. See below.
  • the descriptions below are directed, for exemplary purposes, to primate HDTEA84, HSLJD37R, or RANKL, but are likewise applicable to related embodiments from other species .
  • the HDTEA84, HSLJD37R, and RANKL clones were assembled through the careful analysis of ESTs present in various databases, e.g., Merck-WashU public database. These genes exhibit structural motifs characteristic of a member of the TNF receptor family.
  • Table 3 discloses the nucleic acid and predicted amino acid sequences for primate, e.g., human, HDTEA84.
  • the ESTs were identified from several different libraries .
  • the ESTs were identified from several different libraries derived from: smooth muscle, pancreas tumor, adipocytes, HUVEC cells, adult pulmonary, endothelial cells, prostate cell line PC3 , microvascular endothelial cells, fetal heart, and dendritic cells. Other sequences were detected in libraries from: multiple sclerosis lesions, breast, kidney, and germinal center B cells .
  • SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22 provide the sequences of various mammalian genes designated RANKL.
  • rodent RANKL interesting features include: signal sequence from about 1-29; TNF receptor Cys rich domain I (about 33-74), II (about 75-114), and III (about 115-135).
  • interesting features of the primate RANKL include: TNF receptor Cys rich domain I (about 1-43), II (about 44-83), and III (about 84-104); transmembrane segment from about 139-155. Alignment with other TNF receptors will identify additional interesting corresponding features. Segments with boundaries at these positions may be especially interesting.
  • Hybridization signals with RANKL were detected with rodent, e.g., mouse sequence, in CH12 (B cell line), rag-1 thymus, rag-1 heart, rag-1 brain (strongest signal), rag-1 testes, rag-1 liver, normal lung, rag-1 lung, asthmatic lung, tolerized and challenged lung, Nippo-infected lung, Nippo IL-4 K.O. lung, Nippo anti-IL-5 lung, influenza lung, guinea pig allergic lung, w.t. stomach, and w.t. colon on a 3 day exposure at -80° C with a screen. On a 2 week exposure at -80° C with screen, signals were also detected in the following libraries: Mel 14+ naive, Mell4+ Thl, Mell4+ Th2,
  • the invention provides a chemokine.
  • chemokine for a review of the chemokine family, see, e.g., Lodi, et al. (1994) Science 263:1762-1767; Gronenborn and Clore (1991) Protein Engineering 4:263-269; Miller and Kranger (1992) Proc. Nat ' 1 Acad. Sci. USA 89:2950-2954; Matsushima and Oppenheim (1989) Cvtokine 1:2-13; Stoeckle and Baker (1990) New Biol. 2:313-323; Oppenheim, et al. (1991) Ann. Rev. Immunol. 9:617-648; Schall (1991) Cvtokine 3:165-183; and Thomson (ed. 1994) The Cvtokine Handbook 2d ed. Academic Press, NY.
  • HCC5 which is a CC chemokine. See SEQ ID NO: 24 and SEQ ID NO: 25.
  • the descriptions are directed, for exemplary purposes, to the human HCC5 natural allele described, but are likewise applicable to allelic and/or polymorphic variants, e.g., from other individuals, as well as splicing variants, e.g., natural forms.
  • HCC5 belongs to the CC chemokine family. See, e.g., stem cell mobilizing chemokine (CKbeta-1) from Kreider, et al .
  • Patent WO 9715594 SEQ ID NO: 26
  • GenBank Accession number 97P-W17659 macrophage inflammatory protein-1-gamma (MIP-1) from Adams, et al . (1995) Patent WO 9517092 (SEQ ID NO: 27) and GenBank Accession number 95P-R76128; human MIP-4, a chemoattractant for leukocytes from Adams, et al . (1997) Patent WO 9634891 (SEQ ID NO: 28) and GenBank Accession number 96P-W07203; pituitary expressed chemokine (PGEC) from Bandman, et al . , Patent WO 9616979 (SEQ ID NO: 29) and GenBank Accession number 96P-R95691; and human chemokine HCC-1 from
  • the HCC5 chemokine was discovered through searches and careful analysis of database sequences.
  • the HCC5 sequence was discovered in a cDNA library from pooled bulk breast tumor tissue. Absence of overlapping sequences from other sources suggests extremely specific tissue expression, or highly regulated expression. Amino acid homology analysis suggests that the HCC5 gene encodes a member of a group of related family of chemokines.
  • the primate e.g., human, HCC5 chemokine is most closely related in sequence to the chemokines , human chemokine HCC1 ; human pituitary expressed chemokine (PGEC) ; human MIP-4 (a chemoattractant for leukocytes) ; human macrophage inflammatory protein-1-gamma (MlP-l ⁇ ) ; and human stem cell mobilizing chemokine (CKbeta-1) .
  • PGEC pituitary expressed chemokine
  • MIP-4 a chemoattractant for leukocytes
  • MlP-l ⁇ macrophage inflammatory protein-1-gamma
  • CKbeta-1 human stem cell mobilizing chemokine
  • the HCC5 chemokine is seemingly specifically expressed, since its sequence has not appeared from many sources .
  • the structural similarity to other chemokines suggests that signals important in inflammation, cell differentiation, and development are mediated by it.
  • the HCC5 may actually be an antagonist of one, some, or all, of many related chemokines.
  • combination compositions may be desired.
  • a combined group of functional agonists and antagonists for specific receptors may be called for, e.g., a combination of chemokines and antibody antagonists of others.
  • HCC5 may be useful to block HIV or HTLV infection, which viruses may use the respective receptors for infection.
  • the HCC5 chemokine exhibits limited similarity to portions of known chemokines. See, e.g., Matsushima and Oppenheim (1989) Cvtokine 1:2-13; Oppenheim, et al . (1991) Ann. Rev. Immunol. 9:617-648; Schall (1991) Cvtokine 3:165-183; and Gronenborn and Clore (1991) Protein Engineering 4:263-269. Other features of comparison are apparent between the HCC5 chemokine and chemokine families. See, e.g., Lodi, et al . (1994) Science 263:1762-1766.
  • ⁇ -sheet and ⁇ -helix residues can be determined using, e.g., RASMOL program, see Sayle and Milner-White (1995)
  • Antagonists might be created by N-terminal modification, e.g., either truncation of addition of an N-terminal methionine. Since HCC5 is structurally related to the HCC chemokines, it may well exhibit similar behaviors and functions.
  • HCC5 chemokines especially in dendritic cells, or in Thl T cells, B cells, and macrophages, suggest roles in immune functions, e.g., it will likely attract T cells and monocytes .. Thus, the HCC5 chemokine is likely to recruit these cell types in vivo, thereby enhancing the immune response mediated by these cell types.
  • the expression patterns appear consistent with a functional importance of the ligands in a TH1/TH2 regulation and/or response, including, e.g., in a cancer therapy.
  • ligands and homologs are identified as possible immune adjuvants, e.g., for cellular responses, but also as possible adjuvants to modulate soluble antigen responses, e.g., vaccines .
  • the invention further provides mammalian, e.g., primate, DNA sequences encoding proteins which exhibit structural properties of likely intracellular deubiquitmating protein enzymes. These proteins are designated deubiqutmating 11 (Dubll) and deubiqutinating 12 (Dubl2) .
  • deubiqutmating 11 Dubll
  • Dubl2 deubiqutinating 12
  • deubiquitinating enzymes have also been reported to have additional functions besides deubiquitination. See, e.g., Hochstrasser (1996) Cell 84:813-815; Hicke and Riezman (1996) Cell 84:277-287; and Chen, et al . (1996) Cell 84:853-862.
  • the descriptions typically are directed, for exemplary- purposes, to the human Dubll and human Dubl2 natural alleles described, but are likewise applicable to allelic and/or polymorphic variants, e.g., from other individuals, as well as splicing variants, e.g., natural forms, and species variants from other primates or other species. These genes will allow isolation of other primate genes encoding proteins related to this, further extending the family beyond the specific embodiments described.
  • Dubll or Dubl2 proteins may be useful in the treatment of conditions associated with abnormal physiology or development, such as, e.g., uterine carcinoma associated with p53 dysregulation associated with human papilloma virus or mental retardation of Angelman syndrome (AS) due to disruption of the 5' end of the UBE3A (E6-AP) gene which codes for a disubiquitination protein.
  • Pharmacological intervention which alters the half-lives of cellular proteins associated with these diseases may have wide therapeutic potential.
  • T cell growth factor interleukin-2 regulates lymphocyte proliferation by inducing the expression of growth promoting genes.
  • HTLV-1 transformed cell lines derived from Adult T-cell Leukemia can exhibit constitutive activation of the IL-2-induced JAK/STAT pathway. See Migone, et al . (1998) Proc. Nat'l Acad. Sci. USA 95:3845-3850. ATL cell lines were examined for expression of IL-2 induced genes.
  • Dubl2 is an oncogene that, when constitutively expressed, can induce cytokine-independent growth in lymphocytes and may be implicated in leukemogenesis . It is likely that Dub2 controls cell growth by regulating the ubiquitin- dependent proteolysis or the ubiquitin-dependent state of a critical intracellular substrate. Functional similarity of the Dubll and Dubl2 would be expected. Thus, the biological role of Dub2 suggests similar important roles for the other Dub family members .
  • Screening for inhibitors of the DUB enzymes can also be easily accomplished using the known assays for activity.
  • Such assays can be developed into high throughput screening efforts, testing, particularly, compounds known to affect protein turnover, or similar enzymatic sites .
  • Small molecule antagonists of the enzymes, which will be membrane permeable, would be particularly desirable therapeutically.
  • mammalian, e.g., primate, and rodent, e.g., mouse DNA sequences are provided encoding proteins which exhibit structural properties of ligands for proteins exhibiting a leucine-rich protein motif (LRR) that is important, e.g., in immune function.
  • LRR leucine-rich protein motif
  • human MD-1 is a primate homolog of the previously described rodent MD-1, see, e.g., Miyake, et al . (1998) J. Immunol. 161:1348-1353, while human MD-2 and mouse MD-2 are newly discovered MD-1 homolog.
  • LRR proteins see, e.g., Kobe and
  • the descriptions below are directed, for exemplary- purposes, to the primate, e.g., human, MD-1 and MD-2, and rodent, e.g., mouse, MD-2 natural alleles described, but are likewise applicable to allelic and/or polymorphic variants, e.g., from other individuals, as well as splicing variants, e.g., natural forms, and species variants.
  • the MD-1 or MD-2 proteins naturally occurring or recombinant
  • fragments thereof, and antibodies thereto should be useful in the treatment of conditions associated with abnormal physiology or development, such as, e.g., the recognition of specific pathogenic molecules and the activation of B cell physiology.
  • MD-1 and MD-2 exhibit structural motifs characteristic of ligands for the RP105 or BAS-1 surface receptors.
  • soluble forms, antibodies, or small molecule drugs which disrupt intercellular signaling mediated by these receptors, will find use in modulating cellular response. These responses will be important in normal or abnormal clinical situations .
  • the matching of the MD and RP105 may also be easily tested. Identification of the counter receptor for the MD-2 may include testing both the RP105 and BAS-1 genes, along with other screening methods, as described.
  • the likely counter receptor structure for the MDs are RP105, BAS-1, and related genes. Associated proteins which bind to these, including the DUB proteins, may be identified using these techniques, among others.
  • Another aspect of the invention provides members of the cyclin proteins .
  • the cyclins and their partner catalytic subunits, the cyclin-dependent kinases (Cdks) play key roles in the regulation of eukaryotic cell cycle events. See, e.g., Draetta (1994) Curr. Qpin. Cell Biol. 6:842-846; Sherr (1994) Cell 79:551-555; and Ohtsubo, et al . (1995) Mol. Cell. Biol. 15:2612- 2624. Cyclins were first identified in marine invertebrates on the basis of their dramatic cell cycle periodic expression during meiotic and mitotic divisions .
  • cyclins A-H A large family of cyclins, designated cyclins A-H, bind and activate different Cdks which are serine/threonine kinases essential for cell cycle progression.
  • the timing of the expression of the various cyclins is key in determining at which phase of the cell cycle (S, Go, Gi, or G2 ) their associated Cdk is active.
  • D-type cyclins are synthesized early in Gi and bind and activate CDK4 and CDK6.
  • Cyclin E-Cdk2 and Cyclin A-Cdk2 complexes form later in Gi as cells prepare to begin DNA synthesis.
  • Cyclin B-cdc2 is active during G2 and mitosis. See, e.g., Lees (1995) Curr. Qpin. Cell Biol. 7:773-780.
  • Cyclin-Cdk complex associated proteins are critical for modulation of cyclin activity. Proteins that co- immunoprecipitated with cyclin E were visualized by SDS-PAGE.
  • Cdks can also exert control on cell division and proliferation by phosphorylating specific intracellular target proteins. This phosphorylation event can induce the cellular transition from the Gi to the S phase of the cell cycle. See, e.g., Strahler, et al . (1992) Biochem. Biophys . Res. Comm. 185:197-203; Brattsand, et al . (1994) Eur. J. Biochem. 220:359- 368; and Li, et al . (1996) Cell 85:319-329. Regulation of the cell cycle machinery is important in development and control of cellular proliferation. Misregulation may lead to proliferative disorders, e.g., neoplastic conditions and cancer.
  • cyclin E2 The novel cyclin gene, designated cyclin E2 , exhibits about 49% structural identity to the known human cyclin E. See, e.g., Lew, et al. (1991) Cell 66:1197-1206; and NCBI Entrez accession number M74093.
  • the new variant cyclin E2 sequences are provided in SEQ ID NO: 52 and SEQ ID NO: 53. Notable features on the E2 sequence include the cyclin box running from about residue 120- 254; and a putative phosphorylation site at thr392. The phosphorylation site is trigger in cyclin E for ubiquitin dependent degradation. See Clurman, et al.
  • Particularly interesting segments include, e.g., from about 93-100; 98-106; 104-113; 107-121; 120- 128; 124-134; 131-137; 172-177; 176-185; 189-193; 196-202; 200- 210; 218-223; 228-232; 236-242; 240-245; and 248-252.
  • TNF receptor family antagonists may act as a co- stimulatory molecule for regulation of T cell mediated cell activation, and may in fact, cause a shift of T helper cell types, e.g., between Thl and Th2 ; chemokines have recognized functional properties; intracellular Dubs have been described and the role in oncogenesis established; membrane associated or soluble forms of signaling proteins such as the MDs are well known; and the role of cyclins in cell cycle regulation are recognized.
  • the ligands or binding structures for the cell surface antigens may serve to regulate cell proliferation or development.
  • TNF and FAS can kill cells expressing their respective receptors, including fibroblasts, liver cells, and lymphocytes.
  • Some members of this class of ligands exhibit effects on cellular proliferation of cells expressing their respective receptors, e.g., B cells expressing CD40. These effects on proliferation may also effect subsequent differentiation steps, and may lead, directly or indirectly, to changes in cytokine expression profiles.
  • the members of the TNF ligand family also exhibit costimulation effects, which may also regulate cellular differentiation or apoptosis.
  • Receptor expressing cells may be protected from activation induced cell death (AICD) or apoptosis.
  • AICD activation induced cell death
  • CD40 ligand can have effects on T and B lymphocytes.
  • the embodiments characterized herein are mostly from human, but additional sequences for proteins in other mammalian species, e.g., primates and rodents, will also be available. See below.
  • reverse translation e.g., using the genetic code, software is available, which will indicate what nucleic acid sequences could encode them. See, e.g., MacVector, Oxford Molecular Group Software.
  • artificial genes, or redundant oligonucleotides may be selected to isolate natural variants or species counterparts.
  • Purified Protein Primate, e.g., human, DC-PGT polypeptide sequence is shown in SEQ ID NO: 2; primate, e.g., human, HDTEA84 polypeptide sequence is shown in SEQ ID NO: 6; primate, e.g., human, HSLJD37R polypeptide sequences are shown in SEQ ID NO: 8, 10, and 12; rodent, e.g., murine, RANKL polypeptide sequence is shown in SEQ ID NO: 17; primate forms of RANKL polypeptide sequence are shown in SEQ ID NO: 19, 21, and 23; primate, e.g., human, HCC5 chemokine polypeptide sequence is shown in SEQ ID NO: 25; primate, e.g., human, Dubll polypeptide sequences are shown in SEQ ID NO: 32 and 34; primate, e.g., human, Dubl2 polypeptide sequences are shown in SEQ ID NO: 36 and 38; primate, e.g.
  • amino acid sequences provided amino to carboxy, are important in providing sequence information in the antigen allowing for distinguishing the protein from other proteins and exemplifying numerous variants.
  • the peptide sequences allow preparation of peptides to generate antibodies to recognize such segments, and nucleotide sequences allow preparation of oligonucleotide probes, both of which are strategies for detection or isolation, e.g., cloning, of genes or cDNAs encoding such sequences .
  • the purified protein, or proteins will typically comprise a plurality of distinct, e.g., nonoverlapping, segments of the specified length. Typically, the plurality will be at least two, more usually at least three, and preferably 5, 7, or even more. While the length minima are provided, longer lengths, of various sizes, may be appropriate, e.g., one of length 7, and two of length 12.
  • Such peptides are useful for generating antibodies by standard methods, as described herein.
  • Synthetic peptides or purified protein can be presented to an immune system to generate a specific binding composition, e.g., monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (Current ed.) Antibodies : A Laboratory Manual Cold Spring Harbor Press.
  • the specific binding composition could be used for screening of an expression library made from a cell line which expresses a clone encoding, e.g., a prostaglandin transporter.
  • the screening can be standard staining of surface expressed protein, or by panning. Screening of intracellular expression can also be performed by various staining or immunofluorescence procedures.
  • the binding compositions could be used to affinity purify or sort out cells expressing the protein.
  • the binding compositions may also be useful in determining qualitative and quantitative expression levels of the proteins in various biological samples, including, e.g., cell types or tissues.
  • the term e.g., "human DC-PGT” shall encompass, when used in a protein context, a protein having amino acid sequence shown in SEQ ID NO: 2. Significant polypeptide fragments of such a protein should preserve some of the properties, biological or physical, of the full length protein. Other essentially identical or equivalent proteins may be found in other primates or related species.
  • binding components e.g., antibodies
  • binding components typically bind to, e.g., a DC-PGT, with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM.
  • Homologous proteins would be found in mammalian species other than human, e.g., primates or rodents. Non-mammalian species should also possess structurally or functionally related genes and proteins, e.g., birds or amphibians. Similar meanings apply in reference to HDTEA84, HSLJD37R, RANKL, HCC5, Dubll, Dubl2 , MD-1, MD-2, and cyclin E2.
  • polypeptide includes a significant fragment or segment, and encompasses a stretch of amino acid residues of at least about 8 amino acids, generally at least 10 amino acids, more generally at least 12 amino acids, often at least 14 amino acids, more often at least 16 amino acids, typically at least 18 amino acids, more typically at least 20 amino acids, usually at least 22 amino acids, more usually at least 24 amino acids, preferably at least 26 amino acids, more preferably at least 28 amino acids, and, in particularly preferred embodiments, at least about 30 or more amino acids.
  • the segments may have lengths of at least 37, 45, 53, 61, 70, 80, 90, etc., and often will encompass a plurality of such matching sequences. The specific ends of such a segment will be at any combinations within the protein.
  • the plurality will be at least two, more usually at least three, and preferably 5, 7, or even more. While the length minima are provided, longer lengths, of various sizes, may be appropriate, e.g., one of length 7, and two of length 12.
  • binding composition refers to molecules that bind with specificity to the respective protein or polypeptide, e.g., DC-PGT, e.g., in a cell adhesion pairing type fashion, or an antibody-antigen interaction.
  • Other compounds include, e.g., proteins, which specifically associate with DC-PGT, including in a natural physiologically relevant protein-protein interaction, either covalent or non-covalent.
  • the molecule may be a polymer, or chemical reagent.
  • a functional analog may be an antigen with structural modifications, or it may be a molecule which has a molecular shape which interacts with the appropriate binding determinants.
  • the compounds may serve as agonists or antagonists of the binding interaction, see, e.g., Goodman, et al . (eds. 1990) Goodman & Gilman's: The Pharmacological Bases of Therapeutics (8th ed. ) Pergamon Press.
  • Substantially pure in the polypeptide context, typically means that the protein is free from other contaminating proteins, nucleic acids, and other biologicals derived from the original source organism or cell. Purity may be assayed by standard methods, and will ordinarily be at least about 40% pure, more ordinarily at least about 50% pure, generally at least about 60% pure, more generally at least about 70% pure, often at least about 75% pure, more often at least about 80% pure, typically at least about 85% pure, more typically at least about 90% pure, preferably at least about 95% pure, more preferably at least about 98% pure, and in most preferred embodiments, at least 99% pure. The analysis may be weight or molar percentages, evaluated, e.g., by gel staining, spectrophotometry, or terminus labeling. Carriers or excipients will often be subsequently added.
  • Solubility of a polypeptide or fragment depends upon the environment and the polypeptide. Many parameters affect polypeptide solubility, including temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and nature of the solvent. Typically, the temperature at which the polypeptide is used ranges from about 4° C to about 65° C. Usually the temperature at use is greater than about 18° C. For diagnostic purposes, the temperature will usually be about room temperature or warmer, but less than the denaturation temperature of components in the assay. For therapeutic purposes, the temperature will usually be body temperature, typically about 37° C for humans and mice, though under certain situations the temperature may be raised or lowered in situ or in vitro.
  • the size and structure of the polypeptide should generally be in a substantially stable state, and usually not in a denatured state.
  • the polypeptide may be associated with other polypeptides in a quaternary structure, e.g., to confer solubility, or associated with lipids or detergents in a manner which approximates natural lipid bilayer interactions.
  • the solvent and electrolytes will usually be a biologically compatible buffer, of a type used for preservation of biological activities, and will usually approximate a physiological aqueous solvent.
  • the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7.5.
  • one or more detergents will be added, typically a mild non-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS (3- [3-cholamidopropyl)dimethylammonio] -1-propane sulfonate), or a low enough concentration as to avoid significant disruption of structural or physiological properties of the protein.
  • Solubility is reflected by sedimentation measured in Svedberg units, which are a measure of the sedimentation velocity of a molecule under particular conditions .
  • the determination of the sedimentation velocity was classically performed in an analytical ultracentrifuge, but is typically now performed in a standard ultracentrifuge. See, Freifelder (1982) Physical Biochemistry (2d ed.), W.H. Freeman; and Cantor and Schimmel (1980) Biophysical
  • a soluble particle or polypeptide will typically be less than about 3OS, more typically less than about 15S, usually less than about 10S, more usually less than about 6S, and, in particular embodiments, preferably less than about 4S, and more preferably less than about 3S.
  • the human complimentary DNA and deduced amino acid sequence provided here for DC-PGT contains sequences corresponding to twelve putative transmembrane (TM) segments, based upon a hydropathicity and structural analysis of DC-PGT.
  • TM transmembrane
  • the 12 transmembrane segments correspond to hydrophobic stretches which run approximately from amino acids 47-68 (TMl) ; 88-107 (TM2); 117-136 (TM3); 188-208 (TM4); 225-244 (TM5); 279-294 (TM6) 367-386 (TM7); 412-431 (TM8) ; 450-474 (TM9); 561-578 (TM10) ; 597-616 (TMll) ; and 651-675 (TM12).
  • Charged amino residues located within the transmembrane domains are: glutamine at amino residues 59, 62, 196, 207, 380, 469, 602, 655, and 675; glutamic acid at residue 95; and arginine at residues 607 and 674.
  • Extracellular loops correspond approximately to amino acid residues 69-87, 137-187, 295-366, 432- 449, 579-596, and 617-650. Putative N-glycosylation sites in the exposed, extracellular face of the molecule are located in the second and fifth extracellular loops at Asn-X-Ser/Thr motifs (e.g., 146-148; 176-178; and 538-540) . Intracellular portions correspond approximately to amino acid residues 1-46, 108-116, 209-224, 295-366, 432-449, 579-596, and 676-709. These boundaries will often be the boundaries of segments of interest, which be include multiple described segments.
  • Transporters of this family are typically 12 transmembrane proteins of approximately 650 amino acids in length and include the organic anion transporters in man and rat, prostaglandin transporters of man (Lu, et al. (1996) J. Clin. Invest. 98:1142- 1149) and rat; brain digoxyin transporters and Matrin F/G of rat (Kanai, et al . (1995) Science 268:866-869).
  • Characteristic of this family of organic anion transporter proteins is a cysteine rich region located in one of the extracellular loops, which resembles a zinc finger motif.
  • the DC-PGT cysteine rich region is located in extracellular loop 5 with cysteines approximately at positions 489, 493, 495, 504, 516, 520, 539, 541, 554, and 557.
  • TNF receptors TNF receptors
  • Dubs TNF receptors
  • MDs cyclin E
  • cyclin E segments of comparison with other proteins or genes .
  • This invention also encompasses proteins or peptides having substantial amino acid sequence homology with the amino acid sequences of the described proteins .
  • the variants include species and polymorphic variants, e.g., naturally occurring forms. Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches, if necessary, by introducing gaps as required. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453; Sankoff, et al . (1983) Chapter One in Time Warps . String Edits, and Macromolecules : The Theory and
  • Sequence identity changes when considering conservative substitutions as matches.
  • Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine.
  • homologous amino acid sequences are typically intended to include natural polymorphic or allelic and interspecies variations in each respective protein sequence.
  • Typical homologous proteins or peptides will have from 25-100% identity (if gaps can be introduced) , to 50-100% identity (if conservative substitutions are included) with the amino acid sequence of the HDTEA84.
  • Identity measures will be at least about 35%, generally at least about 40%, often at least about 50%, typically at least about 60%, usually at least about 70%, preferably at least about 80%, and more preferably at least about 90%.
  • sequence comparison typically one sequence acts as a reference sequence, to which test sequences are compared.
  • test and reference sequences are input into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated.
  • sequence comparison algorithm then calculates the percent sequence identity for the test sequence (s) relative to the reference sequence, based on the designated program parameters .
  • Optical alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman (1981) Adv. Appl . Math. 2:482, by the homology alignment algorithm of Needleman and Wunsch (1970) J. Mol. Biol. 48:443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat ' 1 Acad. Sci. USA 85:2444, by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics Software Package, Genetics Computer Group, 575 Science Dr., Madison, WI) , or by visual inspection (see generally Ausubel et al., supra).
  • PILEUP creates a multiple sequence alignment from a group of related sequences using progressive, pairwise alignments to show relationship and percent sequence identity. It also plots a tree or dendrogram showing the clustering relationships used to create the alignment. PILEUP uses a simplification of the progressive alignment method of Feng and Doolittle (1987) J. Mol. Evol . 35:351-360. The method used is similar to the method described by Higgins and Sharp (1989) CABIOS 5:151-153. The program can align up to 300 sequences, each of a maximum length of 5,000 nucleotides or amino acids . The multiple alignment procedure begins with the pairwise alignment of the two most similar sequences, producing a cluster of two aligned sequences.
  • This cluster is then aligned to the next most related sequence or cluster of aligned sequences.
  • Two clusters of sequences are aligned by a simple extension of the pairwise alignment of two individual sequences .
  • the final alignment is achieved by a series of progressive, pairwise alignments.
  • the program is run by designating specific sequences and their amino acid or nucleotide coordinates for regions of sequence comparison and by designating the program parameters. For example, a reference sequence can be compared to other test sequences to determine the percent sequence identity relationship using the following parameters: default gap weight (3.00), default gap length weight (0.10), and weighted end gaps.
  • HSPs high scoring sequence pairs
  • initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them.
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLAST program uses as defaults a wordlength (W) of 11, the BLOSUM62 scoring matrix (see Henikoff and Henikoff (1989) Proc. Nat'l Acad. Sci.
  • the BLAST algorithm also performs a statistical analysis of the similarity between two sequences (see, e.g., Karlin and Altschul (1993) Proc. Nat'l Acad. Sci. USA 90:5873-5787).
  • One measure of similarity provided by the BLAST algorithm is the smallest sum probability (P(N)), which provides an indication of the probability by which a match between two nucleotide or amino acid sequences would occur by chance.
  • a nucleic acid is considered similar to a reference sequence if the smallest sum probability in a comparison of the test nucleic acid to the reference nucleic acid is less than about 0.1, more preferably less than about 0.01, and most preferably less than about 0.001.
  • a further indication that two nucleic acid sequences of polypeptides are substantially identical is that the polypeptide encoded by the first nucleic acid is immunologically cross reactive with the polypeptide encoded by the second nucleic acid, as described below.
  • a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions.
  • nucleic acid sequences are substantially identical is that the two molecules hybridize to each other under stringent conditions, as described below.
  • the isolated DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5, Dub, MD- 1, MD-2, or cyclin E2 DNAs can be readily modified by nucleotide substitutions, nucleotide deletions, nucleotide insertions, and inversions of nucleotide stretches. These modifications result in novel DNA sequences which encode these antigens, their derivatives, or proteins having similar physiological, immunogenic, antigenic, or other functional activity. These modified sequences can be used to produce mutant antigens or to enhance expression.
  • Enhanced expression may involve gene amplification, increased transcription, increased translation, and other mechanisms.
  • “Mutant HDTEA84” encompasses a polypeptide otherwise falling within the sequence identity definition of the HDTEA84 as set forth above, but having an amino acid sequence which differs from that of HDTEA84 as normally found in nature, whether by way of deletion, substitution, or insertion.
  • This generally includes proteins having significant identity with a protein having sequence of SEQ ID NO: 6, and as sharing various biological activities, e.g., antigenic or immunogenic, with those sequences, and in preferred embodiments contain most of the full length disclosed sequences. Full length sequences will typically be preferred, though truncated versions, e.g., soluble constructs and intact domains, will also be useful, likewise, genes or proteins found from natural sources are typically most desired.
  • HDTEA84 proteins particularly those found in various warm blooded animals, e.g., mammals and birds, or fish. These descriptions are generally meant to encompass all HDTEA84 proteins, not limited to the particular human embodiment specifically discussed. Similar concepts apply to the other polypeptides provided.
  • DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5, Dub, MD-1, MD-2, or cyclin E2 mutagenesis can also be conducted by making amino acid insertions or deletions. Although site specific mutation sites are predetermined, mutants need not be site specific. Protein mutagenesis can be conducted by making amino acid insertions or deletions, or combinations may be generated to arrive at a final construct. Insertions include amino- or carboxy- terminal fusions. Random mutagenesis can be conducted at a target codon and the expressed mutants can then be screened for the desired activity.
  • the mutations in the DNA normally should not place coding sequences out of reading frames and preferably will not create complementary regions that could hybridize to produce secondary mRNA structure such as loops or hairpins.
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins.
  • a heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner.
  • the fusion product of an immunoglobulin with a polypeptide is a continuous protein molecule having sequences fused in a typical peptide linkage, typically made as a single translation product and exhibiting properties derived from each source peptide.
  • a similar concept applies to heterologous nucleic acid sequences .
  • the present invention also provides recombinant proteins, e.g., heterologous fusion proteins using segments from these proteins.
  • a heterologous fusion protein is a fusion of proteins or segments which are naturally not normally fused in the same manner.
  • Fusion proteins will be useful as sources for cleaving, separating, and purifying portions thereof.
  • new constructs may be made from combining similar functional domains from other proteins.
  • target-binding or other segments may be "swapped" between different new fusion polypeptides or fragments. See, e.g., Cunningham, et al . (1989) Science 243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.
  • a double stranded fragment will often be obtained either by synthesizing the complementary strand and annealing the strand together under appropriate conditions or by adding the complementary strand using DNA polymerase with an appropriate primer sequence, e.g., PCR techniques.
  • the blocking of physiological response with, e.g., HDTEA84, HSLJD37R, RANKL, HCC5 chemokine, MD-1, or MD-2 may result from the inhibition of binding of the respective ligand to signaling form of receptor or binding counterstructure, e.g., through competitive inhibition.
  • binding affinity to substrate may be modifiable or competed with, e.g., DC-PGT, Dubs, or cyclin E2.
  • in vitro assays of the present invention will often use isolated protein, soluble fragments comprising ligand or substrate binding segments of these proteins, or forms attached to solid phase substrates. These assays will also allow for the diagnostic determination of the effects of either binding segment mutations and modifications, or antigen mutations and modifications, e.g., HDTEA84, HSLJD37R, RANKL, MD-1, or MD-2 analogs.
  • This invention also contemplates the use of competitive drug screening assays, e.g., where neutralizing antibodies to antigen or binding fragments compete with a test compound for binding to the protein, e.g., of natural protein sequence.
  • a test compound for binding to the protein, e.g., of natural protein sequence.
  • substrate binding e.g., competitive inhibitors
  • receptor interaction e.g., competitive inhibitors
  • covalent or aggregate conjugates with other chemical moieties e.g., by standard means.
  • glycosylation alterations are included, e.g., made by modifying the glycosylation patterns of a polypeptide during its synthesis and processing, or in further processing steps. See, e.g., Elbein (1987) Ann. Rev. Biochem. 56:497-534. Also embraced are versions of the peptides with the same primary amino acid sequence which have other minor modifications, including phosphorylated amino acid residues, e.g., phosphotyrosine, phosphoserine, or phosphothreonine .
  • Fusion polypeptides between these proteins and other homologous or heterologous proteins are also provided.
  • Many cytokine receptors or other surface proteins are multimeric, e.g., homodimeric entities, and a repeat construct may have various advantages, including lessened susceptibility to proteolytic cleavage.
  • Typical examples are fusions of a reporter polypeptide, e.g., luciferase, with a segment or domain of a protein, e.g., a receptor-binding segment, so that the presence or location of the fused ligand may be easily determined. See, e.g., Dull, et al . , U.S. Patent No. 4,859,609.
  • gene fusion partners include bacterial ⁇ -galactosidase, trpE, Protein A, ⁇ -lactamase, alpha amylase, alcohol dehydrogenase, yeast alpha mating factor, and detection or purification tags such as a FLAG sequence of His6 sequence. See, e.g., Godowski, et al . (1988) Science 241:812-816. Of particular interest are fusion constructs of receptor with a membrane attachment domain.
  • Fusion peptides will typically be made by either recombinant nucleic acid methods or by synthetic polypeptide methods .
  • Techniques for nucleic acid manipulation and expression are described generally, e.g., in Sambrook, et al . (1989) Molecular Cloning: A Laboratory Manual (2d ed. ) , vols. 1-3, Cold Spring Harbor Laboratory; and Ausubel, et al . (eds. 1993) Current Protocols in Molecular Biology, Greene and Wiley, NY. Techniques for synthesis of polypeptides are described, e.g., in Merrifield
  • Covalent or aggregative derivatives will be useful as immunogens , as reagents in immunoassays, or in purification methods such as for affinity purification of binding partners, e.g., other antigens.
  • the desired proteins can be immobilized by covalent bonding to a solid support such as cyanogen bromide-activated SEPHAROSE, by methods which are well known in the art, or adsorbed onto polyolefin surfaces, with or without glutaraldehyde cross-linking, for use in the assay or purification of antibodies or an alternative binding composition.
  • the protein can also be labeled with a detectable group, e.g., for use in diagnostic assays. Purification may be effected by an immobilized antibody or complementary binding partner.
  • a solubilized protein or fragment of this invention can be used as an immunogen for the production of antisera or antibodies specific for binding to the antigen or fragments thereof.
  • Purified antigen can be used to screen monoclonal antibodies or antigen-binding fragments, encompassing antigen binding fragments of natural antibodies, e.g., Fab, Fab', F(ab)2, etc.
  • Purified protein can also be used as a reagent to detect antibodies generated in response to the presence of elevated levels of the antigen or cell fragments containing the antigen, both of which may be diagnostic of an abnormal or specific physiological or disease condition.
  • This invention contemplates antibodies raised against amino acid sequences encoded by nucleotide sequences described, or fragments of proteins containing it.
  • this invention contemplates antibodies having binding affinity to or being raised against specific fragments, e.g., which are predicted to lie outside of the lipid bilayer, both extracellular or intracellular.
  • the present invention contemplates the isolation of additional closely related species variants .
  • Southern and Northern blot analysis should establish that similar genetic entities exist in other mammals. It is likely that these proteins are widespread in species variants, e.g., rodents, lagomorphs, carnivores, artiodactyla, perissodactyla, and primates.
  • the invention also provides means to isolate a group of related antigens displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of the molecules will be greatly accelerated by the isolation and characterization of additional distinct species variants of them.
  • the present invention provides useful probes for identifying additional homologous genetic entities in different species.
  • the isolated genes will allow transformation of cells lacking expression of a corresponding protein, e.g., either species types or cells which lack corresponding antigens and exhibit negative background activity. This should allow analysis of the function of genes in comparison to untransformed control cells. Dissection of critical structural elements which effect the various activation or differentiation functions mediated through these antigens is possible using standard techniques of modern molecular biology, particularly in comparing members of the related class. See, e.g., the homolog-scanning mutagenesis technique described in Cunningham, et al . (1989) Science 243:1339- 1336; and approaches used in O'Dowd, et al . (1988) J. Biol. Chem.
  • the invention also provides, in the context of the DC-PGT, means to isolate a group of related organic anion transporters, e.g., other vertebrate prostaglandin transporters, displaying both distinctness and similarities in structure, expression, and function. Elucidation of many of the physiological effects of the antigens will be greatly accelerated by the isolation and characterization of distinct species variants.
  • the present invention provides useful probes for identifying additional homologous genetic entities in different species. The results described above indicate that sufficiently homologous genes exist in other species that cross-species hybridization is likely to allow successful cloning.
  • the isolated genes will allow transformation of cells lacking expression of a described gene, e.g., prostaglandin transporter.
  • Various species types or cells which lack corresponding proteins can be isolated, and should exhibit negative background activity.
  • Expression of transformed genes will allow isolation of antigenically pure cell lines, with defined or single specie variants. This approach will allow for more sensitive detection and discrimination of the physiological effects of the gene, e.g., prostaglandin transporters.
  • Subcellular fragments e.g., cytoplasts or membrane fragments, can be isolated and used.
  • the DC-PGT genes may also be useful to increase the rate of transport of desired prostaglandins into transformed cells.
  • the transporter may be transformed into cells for targeting of incorporation of desired substrates or analogs.
  • it may be useful to incorporate specific modified prostaglandins into those cells, which may become more susceptible to other treatments, or directly affected.
  • specific dendritic cell subsets may be transformed to become more sensitive to prostaglandins or specific substrates.
  • such cells may be useful screening targets to identify entities which can block transport, thereby preventing uptake of substrate.
  • prostaglandins and analogs thereof may be screened for either transporter binding affinity or transporter transfer.
  • the transporter may require a direct energy- source, e.g., ATP or other nucleotide triphosphate , or may depend upon an ion gradient, as described above.
  • intracellular functions would probably involve segments of the antigen which are normally accessible to the cytosol, as would segments of the receptors.
  • protein internalization may occur under certain circumstances, and interaction between intracellular components and "extracellular" components may occur.
  • the specific segments of interaction of protein with other intracellular components may be identified by mutagenesis or direct biochemical means, e.g., cross-linking or affinity methods. Structural analysis by crystallographic or other physical methods will also be applicable. Further investigation of the mechanism of signal transduction will include study of associated components which may be isolatable by affinity methods or by genetic means, e.g., complementation analysis of mutants. Further study of the expression and control of the proteins will be pursued.
  • the controlling elements associated with the antigens should exhibit differential physiological, developmental, tissue specific, or other expression patterns. Upstream or downstream genetic regions, e.g., control elements, are of interest. In particular, physiological or developmental variants, e.g., multiple alternatively processed forms of the antigen might be found. Thus, differential splicing of message may lead to an assortment of membrane bound forms, soluble forms, and modified versions of antigen.
  • Antibodies can be raised to the various described polypeptides, including species, polymorphic, or allelic variants, and fragments thereof, both in their naturally occurring forms and in their recombinant forms. Additionally, antibodies can be raised to the proteins in either their active forms or in their inactive forms, including native or denatured versions. Anti- idiotypic antibodies are also contemplated.
  • Antibodies including binding fragments and single chain versions, against predetermined fragments of the antigens can be raised by immunization of animals with conjugates of the fragments with immunogenic proteins.
  • Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to normal or defective polypeptide, or screened for agonistic or antagonistic activity.
  • Antibodies may be agonistic or antagonistic, e.g., by sterically blocking partner or substrate binding. These monoclonal antibodies will usually bind with at least a Krj of about 1 mM, more usually at least about
  • 300 ⁇ M typically at least about 100 ⁇ M, more typically at least about 30 ⁇ M, preferably at least about 10 ⁇ M, and more preferably at least about 3 ⁇ M or better. More preferred embodiments may have even higher affinities, e.g., at least 300 nM, 30 nM, 3 nM, or perhaps even picomolar affinity.
  • binding composition refers to molecules that bind with affinity and selectivity to, e.g., the DC-PGT, e.g., in an antibody-antigen interaction.
  • other compounds e.g., accessory proteins, may also specifically and/or selectively associate with the antigen to the exclusion of other molecules.
  • the association will be in a natural physiologically relevant protein-protein interaction, either covalent or non- covalent, and may include members of a multiprotein complex, including carrier compounds or dimerization partners .
  • the molecule may be a polymer, or chemical reagent. No implication as to whether an antigen is necessarily a convex shaped molecule, e.g.
  • the ligand or the receptor of a ligand-receptor interaction is necessarily represented, other than whether the interaction exhibits similar specificity, e.g., specific or selective affinity.
  • a functional analog may be a polypeptide with structural modifications, e.g., a mutein, or may be a wholly unrelated molecule, e.g., which has a molecular shape which interacts with the appropriate ligand binding determinants .
  • the ligands may serve as agonists or antagonists of the receptor, see, e.g., Goodman, et al . Goodman & Gilman's: The Pharmacological Bases of Therapeutics (current edition) Pergamon Press, Tarrytown,
  • binding agent : antigen complex refers to a complex of a binding agent and antigen, e.g., a DC-PGT protein, that is formed by specific binding of the binding agent to antigen.
  • Specific or selective binding of the binding agent means that the binding agent has a specific binding site, e.g., antigen binding site, that recognizes a site on the antigen.
  • antibodies raised to a DC-PGT protein and recognizing an epitope on the protein are capable of forming a binding agent :DC- PGT protein complex by specific selective binding.
  • the formation of a binding agent .-DC-PGT protein complex allows the qualitative or quantitative measurement of DC-PGT protein in a mixture of other proteins and biologies.
  • antibody DC- PGT protein complex
  • the term "antibody: DC- PGT protein complex” refers to an embodiment in which the binding agent, e.g., is the antigen binding portion from an antibody.
  • the antibody may be monoclonal, polyclonal, or a binding fragment of an antibody, e.g., an Fab or F(ab)2 fragment.
  • the antibody will preferably be a polyclonal antibody for cross-reactivity testing purposes .
  • “specifically immunoreactive with”, when referring to a protein or peptide, refers to a binding reaction which is determinative of the presence of the protein in the presence of a heterogeneous population of other proteins and other biological components .
  • the specified antibodies bind to a particular protein and do not significantly bind other proteins present in the sample.
  • Specific binding to an antibody under such conditions may require an antibody that is selected for its specificity or selectivity for a particular protein.
  • the serum can be immunoselected or immunodepleted, to minimize crossreactivity with a specific target protein.
  • a DC-PGT polypeptide that specifically binds to, or that is specifically immunoreactive with, an antibody, e.g., such as a polyclonal antibody, generated against a defined immunogen, e.g., such as an immunogen consisting of an amino acid sequence of SEQ ID NO: 2, or fragments thereof, or a polypeptide generated from the nucleic acid of SEQ ID NO: 1 is typically determined in an immunoassay. Included within the metes and bounds of the present invention are those nucleic acid sequences described herein, including functional variants, that encode polypeptides that selectively bind to polyclonal antibodies generated against the prototypical DC-PGT polypeptide as structurally and functionally defined herein.
  • the immunoassay typically uses a polyclonal antiserum which was raised, e.g., to a protein of SEQ ID NO: 2. This antiserum is selected to have low crossreactivity against appropriate other PGT family members, preferably from the same species, and any such crossreactivity is removed by immunoabsorption prior to use in the immunoassay. Appropriate selective serum preparations can be isolated, and characterized.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described above. Synthetic peptides or purified protein can be presented to an immune system to generate monoclonal or polyclonal antibodies.
  • HDTEA84 can be used as a specific binding reagent, and advantage can be taken of its specificity of binding, much like an antibody would be used.
  • the specific binding composition could be used for screening of an expression library made from a cell line which expresses an HDTEA84, HSLJD37R, or RANKL.
  • the screening can be standard staining of surface expressed antigen constructs, or by panning. Screening of intracellular expression can also be performed by various staining or immunofluorescence procedures.
  • the binding compositions could be used to affinity purify or sort out cells expressing the protein.
  • the protein e.g., of SEQ ID NO: 2
  • recombinant protein may be produced in a mammalian cell line.
  • An appropriate host e.g., an inbred strain of mice such as Balb/c
  • a standard adjuvant such as Freund's adjuvant
  • a standard mouse immunization protocol see Harlow and Lane
  • a substantially full length synthetic peptide derived from the sequences disclosed herein can be used as an immunogen.
  • Polyclonal sera are collected and titered against the immunogen protein in an immunoassay, e.g., a solid phase immunoassay with the immunogen immobilized on a solid support.
  • Polyclonal antisera with a titer of 10 ⁇ or greater are selected and tested for their cross reactivity against other PGT family members, e.g., human or rat PGT, using a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.
  • PGT family members e.g., human or rat PGT
  • a competitive binding immunoassay such as the one described in Harlow and Lane, supra, at pages 570-573.
  • PGT family members are used in this determination in conjunction with the target.
  • These PGT family members can be produced as recombinant proteins and isolated using standard molecular biology and protein chemistry techniques as described herein.
  • antibody preparations can be identified or produced having desired selectivity or specificity for subsets of PGT family members.
  • Immunoassays in the competitive binding format can be used for the crossreactivity determinations.
  • the protein of SEQ ID NO: 2 can be immobilized to a solid support. Proteins added to the assay compete with the binding of the antisera to the immobilized antigen. The ability of the above proteins to compete with the binding of the antisera to the immobilized protein is compared to the protein of SEQ ID NO : 2. The percent crossreactivity for the above proteins is calculated, using standard calculations. Those antisera with less than 10% crossreactivity with each of the proteins listed above are selected and pooled. The cross-reacting antibodies are then removed from the pooled antisera by immunoabsorption or immunodepletion with the above-listed proteins.
  • the immunoabsorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare a second protein to the immunogen protein.
  • the two proteins are each assayed at a wide range of concentrations and the amount of each protein required to inhibit 50% of the binding of the antisera to the immobilized protein is determined. If the amount of the second protein required is less than twice the amount of the protein of, e.g., SEQ ID NO: 2 that is required, then the second protein is said to specifically bind to an antibody generated to the immunogen.
  • the antibodies of this invention can also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for ability to bind to the antigens without inhibiting binding by a partner.
  • neutralizing antibodies they can be useful in competitive binding assays. They will also be useful in detecting or quantifying a described protein or its binding partners. See, e.g., Chan (ed. 1987) Immunology: A Practical Guide, Academic Press, Orlando, FL; Price and Newman (eds. 1991) Principles and Practice of Immunoassay, Stockton Press, N.Y. ; and Ngo (ed. 1988) Nonisotopic Immunoassay, Plenum Press, N.Y. Cross absorptions or depletions and other tests will identify antibodies which exhibit various spectra of specificities, e.g., unique or shared species specificities.
  • the antibodies, including antigen binding fragments, of this invention can be potent antagonists that bind to the antigen and inhibit functional binding or inhibit the ability of a binding partner to elicit a biological response. They also can be useful as non-neutralizing antibodies and can be coupled to toxins or radionuclides so that when the an'cibody binds to antigen, a cell expressing it, e.g., on its surface, is killed. Further, these antibodies can be conjugated to drugs or other therapeutic agents, either directly or indirectly by means of a linker, and may effect drug targeting. They may be labeled for histology- evaluation.
  • Antigen fragments may be joined to other materials, particularly polypeptides, as fused or covalently joined polypeptides to be used as immunogens .
  • An antigen and its fragments may be fused or covalently linked to a variety of immunogens, such as keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc. See Microbiology, Hoeber Medical Division, Harper and Row, 1969; Landsteiner (1962) Specificity of Serological Reactions, Dover Publications, New York; Williams, et al. (1967) Methods in Immunology and Immunochemistry, vol. 1,
  • monoclonal antibodies from various mammalian hosts, such as mice, rodents, primates, humans, etc. Description of techniques for preparing such monoclonal antibodies may be found in, e.g., Stites, et al . (eds.) Basic and Clinical Immunology (4th ed. ) , Lange Medical Publications, Los Altos, CA, and references cited therein; Harlow and Lane (1988) Antibodies : A Laboratory Manual , CSH Press;
  • polypeptides and antibodies of the present invention may be used with or without modification, including chimeric or humanized antibodies. Frequently, the polypeptides and antibodies will be labeled by joining, either covalently or non-covalently, a substance which provides for a detectable signal.
  • labels and conjugation techniques are known and are reported extensively in both the scientific and patent literature. Suitable labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent moieties, chemiluminescent moieties, magnetic particles, and the like. Patents, teaching the use of such labels include U.S. Patent Nos.
  • the antibodies of this invention can also be used for affinity chromatography in isolating the protein.
  • Columns can be prepared where the antibodies are linked to a solid support, e.g., particles, such as agarose, Sephadex, or the like, where a cell lysate may be passed through the column, the column washed, followed by increasing concentrations of a mild denaturant, whereby the purified protein will be released. See, e.g., Wilchek et al. (1984) Meth. Enzymol. 104:3-55.
  • Antibodies raised against each protein will also be useful to raise anti-idiotypic antibodies. These will be useful in detecting or diagnosing various immunological conditions related to expression of the respective antigens.
  • nucleic Acids The described peptide sequences and the related reagents are useful in detecting, isolating, or identifying a DNA clone encoding, e.g., the DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5, Dubll, Dubl2 , MD-1, MD-2, or cyclin E2 polypeptides, e.g., from a natural source.
  • the nucleic acids, particularly natural genes will be useful in isolating a gene from mammal, and similar procedures will be applied to isolate genes from other species, e.g., warm blooded animals, such as birds and mammals.
  • prostaglandin transporter shall encompass, when used in a protein context, a protein having an amino acid sequence shown in Table 1, or a significant fragment of such a protein. It also refers to a vertebrate, e.g., mammal, including human, derived polypeptide which exhibits similar biological function, e.g., antigenic, or interacts with prostaglandin transporter specific binding components, e.g., specific antibodies.
  • binding components typically bind to a prostaglandin transporter with high affinity, e.g., at least about 100 nM, usually better than about 30 nM, preferably better than about 10 nM, and more preferably at better than about 3 nM. Still higher affinities are possible, e.g., 100 pM, 30 pM, 100 fM, etc.
  • This invention contemplates use of isolated DNA or fragments of the present invention to encode a structurally related, e.g., antigenically related, or biologically active protein, e.g., substrate binding or transporting, prostaglandin transporter, TNF receptor-like proteins, chemokine, Dubs, surface receptors, or cell cycle regulatory proteins, or polypeptide fragments thereof.
  • a structurally related, e.g., antigenically related, or biologically active protein e.g., substrate binding or transporting, prostaglandin transporter, TNF receptor-like proteins, chemokine, Dubs, surface receptors, or cell cycle regulatory proteins, or polypeptide fragments thereof.
  • this invention covers isolated or recombinant DNA which encodes a structurally related or biologically active protein or polypeptide and that is capable of hybridizing under appropriate conditions with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact antigen, or fragment, and have an amino acid sequence as disclosed in Tables 1
  • this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which are homologous to the respective genes or which were isolated using cDNA encoding the proteins as a probe.
  • homologous genes or proteins will be natural forms isolated from other vertebrates , e.g., warm blooded animals , including mammals, such as primates.
  • the isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others.
  • nucleic acid is a nucleic acid, e.g., an RNA, DNA, or a mixed polymer, which is substantially separated from other components which naturally accompany a native sequence, e.g., ribosomes, polymerases, and flanking genomic sequences from the originating species.
  • the term embraces a nucleic acid sequence which has been removed from its naturally occurring intracellular environment, and includes recombinant or cloned DNA isolates and chemically synthesized analogs or analogs biologically synthesized by heterologous systems.
  • a substantially pure molecule includes once or currently isolated forms of the molecule. Alternatively, a purified species may be separated from host components from a recombinant expression system.
  • the nucleic acid will be in a vector or fragment less than about 50 kb, usually less than about 30 kb, typically less than about 10 kb, and preferably less than about 6 kb.
  • An isolated nucleic acid will generally be a homogeneous composition of molecules, but will, in some embodiments, contain minor heterogeneity. This heterogeneity is typically found at the polymer ends or portions not critical to a desired biological function or activity.
  • the peptide segments can also be used to predict appropriate oligonucleotides to screen a library.
  • the genetic code e.g., reverse translation, can be used to select appropriate oligonucleotides useful as probes for screening. See, e.g., SEQ ID NO: 1
  • This invention contemplates use of isolated DNA or fragments to encode a biologically active corresponding polypeptide.
  • this invention covers isolated or recombinant DNA which encodes a biologically active protein or polypeptide which is capable of hybridizing under appropriate conditions with the DNA sequences described herein.
  • Said biologically active protein or polypeptide can be an intact antigen, or fragment, and have an amino acid sequence disclosed in, e.g., SEQ ID NO: 2, 6, 8, 10,
  • this invention covers the use of isolated or recombinant DNA, or fragments thereof, which encode proteins which are homologous to a described protein or which was isolated using cDNA encoding such protein as a probe.
  • the isolated DNA can have the respective regulatory sequences in the 5' and 3' flanks, e.g., promoters, enhancers, poly-A addition signals, and others.
  • a "recombinant" nucleic acid is defined either by its method of production or its structure. In reference to its method of production, e.g., a product made by a process, the process is use of recombinant nucleic acid techniques, e.g., involving human intervention in the nucleotide sequence, typically selection or production. Alternatively, it can be a nucleic acid made by generating a sequence comprising fusion of two fragments which are not naturally contiguous to each other, but is meant to exclude products of nature, e.g., naturally occurring mutants.
  • products made by transforming cells with any unnaturally occurring vector is encompassed, as are nucleic acids comprising sequence derived using any synthetic oligonucleotide process. Such is often done to replace a codon with a redundant codon encoding the same or a conservative amino acid, while typically introducing or removing a sequence recognition site.
  • nucleic acid segments of desired functions are joined together to generate a single genetic entity comprising a desired combination of functions not found in the commonly available natural forms .
  • Restriction enzyme recognition sites are often the target of such artificial manipulations, but other site specific targets, e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • site specific targets e.g., promoters, DNA replication sites, regulation sequences, control sequences, or other useful features may be incorporated by design.
  • a similar concept is intended for a recombinant, e.g., fusion, polypeptide.
  • synthetic nucleic acids which, by genetic code redundancy, encode polypeptides similar to fragments of these antigens, and fusions of sequences from various different species variants .
  • a significant "fragment" in a nucleic acid context is a contiguous segment of at least about 17 nucleotides, generally at least about 22 nucleotides, ordinarily at least about 29 nucleotides, more often at least about 35 nucleotides, typically at least about 41 nucleotides, usually at least about 47 nucleotides, preferably at least about 55 nucleotides, and in particularly preferred embodiments will be at least about 60 or more nucleotides.
  • a DNA which codes for a DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5, Dub, MD-1, MD-2, or cyclin E2 protein will be particularly useful to identify genes, mRNA, and cDNA species which code for related or homologous proteins, as well as DNAs which code for homologous proteins from different species. There are likely homologs in other species, including primates, rodents, birds, and fish. Various such proteins should be homologous and are encompassed herein. However, even genes encoding proteins that have a more distant evolutionary relationship to the antigen can readily be isolated under appropriate conditions using these sequences if they are sufficiently homologous. Primate proteins are of particular interest.
  • Recombinant clones derived from the genomic sequences, e.g., containing introns, will be useful for transgenic studies, including, e.g., transgenic cells and organisms, and for gene therapy. See, e.g., Goodnow (1992) "Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology, Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al . (1991) Science 254:707-710; Capecchi (1989) Science 244:1288;
  • Substantial homology in the nucleic acid sequence comparison context means either that the segments, or their complementary strands, when compared, are identical when optimally aligned, with appropriate nucleotide insertions or deletions, in at least about 50% of the nucleotides, generally at least about 58%, ordinarily at least about 65%, often at least about 71%, typically at least about 77%, usually at least about 85%, preferably at least about 95 to 98% or more, and in particular embodiments, as high as about 99% or more of the nucleotides.
  • substantial homology exists when the segments will hybridize under selective hybridization conditions, to a strand, or its complement, typically using a sequence of DC-PGT, e.g., in SEQ ID NO: 1.
  • selective hybridization will occur when there is at least about 55% homology over a stretch of at least about 30 nucleotides, preferably at least about 75% over a stretch of about 25 nucleotides, and most preferably at least about 90% over about 20 nucleotides.
  • the length of homology comparison, as described, may be over longer stretches, and in certain embodiments will be over a stretch of at least about 17 nucleotides, usually at least about 28 nucleotides, typically at least about 40 nucleotides, and preferably at least about 75 to 100 or more nucleotides.
  • Stringent conditions in referring to homology in the hybridization context, will be stringent combined conditions of salt, temperature, organic solvents, and other parameters, typically those controlled in hybridization reactions.
  • Stringent temperature conditions will usually include temperatures in excess of about 30° C, usually in excess of about 37° C, typically in excess of about 55° C, preferably in excess of about 70° C.
  • Stringent salt conditions will ordinarily be less than about 1000 mM, usually less than about 400 mM, typically less than about 250 mM, preferably less than about 150 mM. However, the combination of parameters is much more important than the measure of any single parameter. See, e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370.
  • Hybridization under stringent conditions should give a background of at least 2-fold over background, preferably at least 3-5 or more.
  • DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5 , Dubll, Dubl2 , MD-1, MD-2, or cyclin E2 from other mammalian species can be cloned and isolated by cross-species hybridization of closely related species. Homology may be relatively low between distantly related species, and thus hybridization of relatively closely related species is advisable. Alternatively, preparation of an antibody preparation which exhibits less species specificity may be useful in expression cloning approaches.
  • Nucleic acids which encodes the described proteins, or fragments thereof can be obtained by chemical synthesis, screening cDNA libraries, or screening genomic libraries prepared from a wide variety of cell lines or tissue samples. See, e.g., Okayama and Berg (1982) Mol. Cell. Biol. 2:161-170; Gubler and Hoffman (1983) Gene 25:263-269; and Glover (ed. 1984) DNA Cloning: A Practical Approach, IRL Press, Oxford.
  • the sequences provided herein provide useful PCR primers or allow synthetic or other preparation of suitable genes encoding a receptor; including, naturally occurring embodiments.
  • DNA can be expressed in a wide variety of host cells for the synthesis of a full-length protein, or fragments, which can in turn, e.g., be used to generate polyclonal or monoclonal antibodies; for binding studies; for construction and expression of modified molecules; for structure/function studies; and for controls in detection assays.
  • Each antigen or its fragments can be expressed in host cells that are transformed or transfected with appropriate expression vectors. These molecules can be substantially purified to be free of protein or cellular contaminants, other than those derived from the recombinant host, and therefore are particularly useful in pharmaceutical compositions when combined with a pharmaceutically acceptable carrier and/or diluent.
  • the antigen, or portions thereof, may be expressed as fusions with other proteins .
  • Vectors as used herein, comprise plasmids, viruses, bacteriophage, integratable DNA fragments, and other vehicles which enable the integration of DNA fragments into the genome of the host. See, e.g., Pouwels, et al. (1985 and Supplements) Cloning Vectors : A Laboratory Manual , Elsevier, N.Y. ; and
  • Expression vectors are typically self-replicating DNA or RNA constructs containing the desired antigen gene or its fragments, usually operably linked to suitable genetic control elements that are recognized in a suitable host cell. These control elements are capable of effecting expression within a suitable host. The specific type of control elements necessary to effect expression will depend upon the eventual host cell used.
  • the genetic control elements can include a prokaryotic promoter system or a eukaryotic promoter expression control system, and typically include a transcriptional promoter, an optional operator to control the onset of transcription, transcription enhancers to elevate the level of mRNA expression, a sequence that encodes a suitable ribosome binding site, and sequences that terminate transcription and translation.
  • Expression vectors also usually contain an origin of replication that allows the vector to replicate independently of the host cell.
  • DNA sequences are operably linked when they are functionally related to each other.
  • DNA for a presequence or secretory leader is operably linked to a polypeptide if it is expressed as a preprotein or participates in directing the polypeptide to the cell membrane or in secretion of the polypeptide.
  • a promoter is operably linked to a coding sequence if it controls the transcription of the polypeptide;
  • a ribosome binding site is operably linked to a coding sequence if it is positioned to permit translation.
  • operably linked means contiguous and in reading frame, however, certain genetic elements such as represser genes are not contiguously linked but still bind to operator sequences that in turn control expression.
  • Suitable expression vectors include pCDNAl; pCD, see Okayama, et al . (1985) Mol. Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas, et al . (1987) Cell
  • expression vectors are designed for stable replication in their host cells or for amplification to greatly increase the total number of copies of the desirable gene per cell. It is not always necessary to require that an expression vector replicate in a host cell, e.g., it is possible to effect transient expression of the antigen or its fragments in various hosts using vectors that do not contain a replication origin that is recognized by the host cell. It is also possible to use vectors that cause integration of a gene or its fragments into the host DNA by recombination, or to integrate a promoter which controls expression of an endogenous gene.
  • Adenovirus techniques are available for expression of the genes in various cells and organs. See, e.g., Hitt, et al . (1997) Adv. Pharmaco1. 40:137-195; and literature from Quantum Biotechnologies, Montreal, Canada. Animals may be useful to determine the effects of the gene on various developmental or physiologically functional animal systems.
  • Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes.
  • Prokaryotes include both gram negative and gram positive organisms, e.g., E. coli and B. subtilis.
  • Lower eukaryotes include yeasts, e.g., S. cerevisiae and Pichia, and species of the genus Dictyostelium.
  • Higher eukaryotes include established tissue culture cell lines from animal cells, both of non-mammalian origin, e.g., insect cells, and birds, and of mammalian origin, e.g., human, primates, and rodents.
  • Prokaryotic host-vector systems include a wide variety of vectors for many different species. As used herein, E. coli and its vectors will be used generically to include equivalent vectors used in other prokaryotes. A representative vector for amplifying DNA is pBR322 or many of its derivatives.
  • Vectors that can be used to express the prostaglandin transporter or its fragments include, but are not limited to, such vectors as those containing the lac promoter (pUC-series) ; trp promoter (pBR322-trp) ; Ipp promoter (the pIN-series) ; lambda-pP or pR promoters (pOTS) ; or hybrid promoters such as ptac (pDR540) . See Brosius et al .
  • Lower eukaryotes e.g., yeasts and Dictyostelium, may be transformed with vectors encoding vertebrate prostaglandin transporters.
  • the most common lower eukaryotic host is the baker's yeast, Saccharomyces cerevisiae. It will be used to generically represent lower eukaryotes although a number of other strains and species are also available.
  • Yeast vectors typically consist of a replication origin (unless of the integrating type) , a selection gene, a promoter, DNA encoding the desired protein or its fragments, and sequences for translation termination, polyadenylation, and transcription termination.
  • Suitable expression vectors for yeast include such constitutive promoters as 3-phosphoglycerate kinase and various other glycolytic enzyme gene promoters or such inducible promoters as the alcohol dehydrogenase 2 promoter or metallothionine promoter.
  • Suitable vectors include derivatives of the following types: self-replicating low copy number (such as the YRp-series) , self-replicating high copy number (such as the YEp- series) ; integrating types (such as the Yip-series) , or mini- chromosomes (such as the YCp-series) .
  • Higher eukaryotic tissue culture cells are the preferred host cells for expression of the functionally active prostaglandin transporter.
  • eukaryotic tissue culture cell lines are workable, e.g., insect baculovirus expression systems, whether from an invertebrate or vertebrate source.
  • mammalian cells are preferred, in that the processing, both cotranslationally and posttranslationally is more likely to simulate natural forms. Transformation or transfection and propagation of such cells has become a routine procedure.
  • useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insect cell lines, bird cell lines, and monkey (COS) cell lines.
  • Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, RNA splice sites (if genomic DNA is used) , a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or amplification gene. Suitable expression vectors may be plasmids, viruses, or retroviruses carrying promoters derived, e.g., from such sources as from adenovirus, SV40, parvoviruses , vaccinia virus, or cytomegalovirus . Representative examples of suitable expression vectors include pCDNAl; pCD, see Okayama et al . (1985) Mol . Cell Biol. 5:1136-1142; pMClneo Poly-A, see Thomas et al . (1987) Cell 51:503-512; and a baculovirus vector such as pAC 373 or pAC 610.
  • DC-PGT DC-PGT
  • HDTEA84 HSLJD37R
  • RANKL HCC5
  • Dubll Dubl2
  • MD-1 MD-2
  • cyclin E2 polypeptide a system which provides a specific or defined glycosylation pattern. See, e.g., Luckow and Summers (1988)
  • prokaryotic forms lack eukaryotic glycosylation patterns.
  • the pattern will be modifiable by exposing the polypeptide, e.g., an unglycosylated form, to appropriate glycosylating proteins introduced into a heterologous expression system.
  • the desired gene may be cotransformed with one or more genes encoding mammalian or other glycosylating enzymes. Using this approach, certain mammalian glycosylation patterns will be achievable or approximated in prokaryote or other cells.
  • the DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5, Dubll, Dubl2 , MD- 1, MD-2, or cyclin E2 , or a fragment thereof may be engineered to be phosphatidyl inositol (PI) linked to a cell membrane, but can be removed from membranes by treatment with a phosphatidyl inositol cleaving enzyme, e.g., phosphatidyl inositol phospholipase-C.
  • a phosphatidyl inositol cleaving enzyme e.g., phosphatidyl inositol phospholipase-C.
  • Transformed cells include cells, preferably mammalian, that have been transformed or transfected with vectors containing a prostaglandin transporter gene, typically constructed using recombinant DNA techniques.
  • Transformed host cells usually express the antigen or its fragments, but for purposes of cloning, amplifying, and manipulating its DNA, do not need to express the protein.
  • This invention further contemplates culturing transformed cells in a nutrient medium, thus permitting the protein, or soluble fragments, to accumulate in the culture. Soluble protein can be recovered, either from the culture or from the culture medium, and membrane associated proteins may be prepared from suitable cell subtractions .
  • an azide process for example, an acid chloride process, an acid anhydride process, a mixed anhydride process, an active ester process (for example, p- nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester) , a carbodiimidazole process, an oxidative-reductive process, or a dicyclohexylcarbodiimide (DCCD) /additive process can be used.
  • Solid phase and solution phase syntheses are both applicable to the foregoing processes.
  • the proteins, fragments, or derivatives are suitably prepared in accordance with the above processes as typically employed in peptide synthesis, generally either by a so-called stepwise process which comprises condensing an amino acid to the terminal amino acid, one by one in sequence, or by coupling peptide fragments to the terminal amino acid.
  • Amino groups that are not being used in the coupling reaction are typically protected to prevent coupling at an incorrect location.
  • the C-terminal amino acid is bound to an insoluble carrier or support through its carboxyl group.
  • the insoluble carrier is not particularly limited as long as it has a binding capability to a reactive carboxyl group.
  • examples of such insoluble carriers include halomethyl resins, such as chloromethyl resin or bromomethyl resin, hydroxymethyl resins, phenol resins, tert-alkyloxycarbonyl- hydrazidated resins, and the like.
  • An amino group-protected amino acid is bound in sequence through condensation of its activated carboxyl group and the reactive amino group of the previously formed peptide or chain, to synthesize the peptide step by step. After synthesizing the complete sequence, the peptide is split off from the insoluble carrier to produce the peptide.
  • This solid-phase approach is generally described by Merrifield et al . (1963) in J. Am. Chem. Soc. 85:2149-2156, which is incorporated herein by reference.
  • the prepared protein and fragments thereof can be isolated and purified from the reaction mixture by means of peptide separation, e.g., by extraction, precipitation, electrophoresis and various forms of chromatography, and the like.
  • the proteins of this invention can be obtained in varying degrees of purity depending upon its desired use. Purification can be accomplished by use of the protein purification techniques disclosed herein or by the use of the antibodies herein described in immunoabsorbent affinity chromatography.
  • This immunoabsorbent affinity chromatography is carried out by first linking the antibodies to a solid support and then contacting the linked antibodies with solubilized lysates of appropriate source cells, lysates of other cells expressing the protein, or lysates or supernatants of cells producing the desired protein as a result of DNA techniques, see below.
  • Detergents may be necessary to include in the methods to maintain protein solubility.
  • the present invention provides reagents which will find use in diagnostic applications as described elsewhere herein, e.g., in the general description for cell mediated conditions, or below in the description of kits for diagnosis.
  • the genes will be useful in forensic analyses, e.g., to identify species, or to diagnose different cell subsets or types.
  • DC-PGT is used to clear prostaglandins (PGs) and other metabolically active organic anions from the body (in the liver, fetal liver, lung and placenta) it is easy to suppose that an alteration in the capacity of this mechanism could augment the allergic response.
  • Prostaglandin PGF2 ⁇ an( GD2 , and PGG2 and thrombaxane A2 can cause airway obstruction, particularly in the peripheral lung, while PGE2 and PGI2 are bronchodilators .
  • Use of the transporter of the invention could help transport or remove these prostaglandins to modulate airway obstruction.
  • prostaglandins play an important role in secondary immunosuppression seen following surgical stress. Alexander (1990) J. Trauma 30:S70; Faist, et al . (1987) J. Trauma 27:837; Ninneman, et al . (1984) J. Trauma 24:201; Wood, et al. (1987) Arch. Surg. 122:179; Polk, et al . in Eremin and Sewell (eds. 1992) The Immunological Basis of Surgical Science and Practice, Oxford U. Press.
  • PGE2 inhibits lymphocyte proliferation, decreases IL-2 release, decreases response to IL-2, inhibits natural killer cells, and activates supressor cells.
  • Prostaglandin E2 through locally produced vasodilatory effects, may play a role in rheumatoid arthritis by promoting the entry of inflammatory cells into the joint.
  • polymorphonuclear leukocytes can ingest immune complexes, which, in turn, cause neutrophils to produce reactive oxygen metabolites and other inflammatory mediators to further enchance an inflammatory cascade. Henson, et al . (1987) J. Clin. Invest. 79:699.
  • the present invention to modulate prostaglandins in a subject suffering from trauma, injury, disease or in post-surgical treatments.
  • Immune system cells may be synthesizing PGs and thus using DC-PGT in an efflux role for removing PGs from the intracellular space may be useful.
  • DC-PGT might transport a specific organic anion.
  • Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using the compositions provided herein.
  • a disease or disorder associated with abnormal function of a prostaglandin transporter should be a likely target for a substrate or blocking substrate.
  • the transporter may be a useful means for supplying important metabolites or metabolite blockers to the respective cells.
  • transformation with the transporter may increase availability of the substrate to the cell.
  • a prostaglandin analog might be advantageously supplied to the cell.
  • the prostaglandin analog might confer high susceptibility to further treatment, e.g., radiation sensitivity or otherwise, or may directly affect normal metabolism, e.g., nucleic acid related enzymes.
  • the transporter may be useful to screen for antagonists or inhibitors, which might be effective in blocking the normal availability to the cell of the natural substrate. Screening methods for such prostaglandin analogs are provided.
  • prostaglandin transporter for binding metabolites or compounds having binding affinity to the transporter can be performed, including isolation of associated components . Subsequent biological assays can then be utilized to determine if the compound has intrinsic biological activity and is therefore an agonist or antagonist in that it blocks an activity of the transporter.
  • prostaglandin analogs may be useful in blocking binding of the natural target or otherwise blocking transporter activity.
  • various other analogs may be useful in blocking an ion transporter, or organic anion source.
  • This invention further contemplates the therapeutic use of antibodies to prostaglandin transporter as antagonists . This approach should be particularly useful with other prostaglandin transporter species variants and other members of the family.
  • Antagonists of the transporter activity may be useful in various medical conditions. These would include immune, inflammatory or allergic abnormalities, all of which are important where transfer of organic anions take place. Certain congenital diseases of prostaglandin physiology will be susceptible to such a therapeutic approach.
  • the HDTEA84, HSLJD37R, RANKL, HCC5, MD-1, or MD-2 (naturally occurring or recombinant) , fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to them, should be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions.
  • modulation of development of lymphoid cells will be achieved by appropriate therapeutic treatment using the compositions provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaling by a ligand or receptor should be a likely target for an agonist or antagonist of the antigen.
  • the antigen plays a role in regulation or development of hematopoietic cells, e.g., lymphoid cells, which affect immunological responses, e.g., autoimmune disorders .
  • the antigen may provide a costimulatory signal to cell activation, or be involved in regulation of cell proliferation or differentiation.
  • the HDTEA84, HSLJD37R, RANKL, HCC5, MD-1, or MD-2 will likely modulate cells which possess a receptor therefor, e.g., T cell mediated interactions with other cell types . These interactions would lead, in particular contexts, to modulation of cell growth, cytokine synthesis by those or other cells, or development of particular effector cells.
  • the HDTEA84, HSLJD37R, RANKL, HCC5, MD-1, or MD-2 or antagonists could redirect T cell responses, e.g., between Thl and Th2 polarization, or with ThO cells, or may affect B cells or other lymphoid cell subsets .
  • these agonists should be various antibodies which recognize the appropriate epitopes, e.g., which mimic binding of ligand or receptor to its partner. Alternatively, they may bind to epitopes which sterically can block receptor binding. Bone morphogenesis may be regulated by these receptor segments .
  • the ligands or receptors may provide a selective and powerful way to modulate immune responses in abnormal situations, e.g., autoimmune disorders, including rheumatoid arthritis, systemic lupus erythematosis (SLE) , Hashimoto's autoimmune thyroiditis, as well as acute and chronic inflammatory responses in which T cell activation, expansion, and/or immunological T cell memory play an important role.
  • autoimmune disorders including rheumatoid arthritis, systemic lupus erythematosis (SLE) , Hashimoto's autoimmune thyroiditis, as well as acute and chronic inflammatory responses in which T cell activation, expansion, and/or immunological T cell memory play an important role.
  • SLE systemic lupus erythematosis
  • Hashimoto's autoimmune thyroiditis as well as acute and chronic inflammatory responses in which T cell activation, expansion, and/or immunological T cell memory play an important role.
  • compositions with other modulators of signaling would be useful, especially with the TNF receptor-like genes.
  • Such other signaling molecules might include, e.g., TCR reagents, CD40, CD40L, CTLA-8, CD28, SLAM, FAS, osteoprotegerin, and their respective antagonists, including antibodies .
  • Cyclin E2 nucleotides e.g., human cyclin E2 DNA or RNA
  • the nucleotide sequences provided may be labeled using, e.g., 32p or biotin and used to probe standard restriction fragment polymorphism blots, providing a measurable character to aid in distinguishing between individuals. Such probes may be used in well-known forensic techniques such as genetic fingerprinting.
  • nucleotide probes made from cyclin E2 sequences may be used in in situ assays to detect chromosomal abnormalities.
  • cyclin E2 gene rearrangements in the human chromosome encoding a cyclin E2 gene may be detected via well-known in situ techniques, using cyclin E2 probes in conjunction with other known chromosome markers .
  • the cyclin E2 gene may have useful prognostic utility in various cancers, e.g., breast, etc.
  • Antibodies and other binding agents directed towards cyclin E2 proteins or nucleic acids may be used to purify the corresponding cyclin E2 molecule. As described in the Examples below, antibody purification of cyclin E2 protein components is both possible and practicable. Antibodies and other binding agents may also be used in a diagnostic fashion to determine whether cyclin E2 protein components are present in a tissue sample or cell population using well-known techniques described herein.
  • cyclin E2 protein expression is limited to specific tissue types. By directing a probe, such as an antibody or nucleic acid to a cyclin E2 protein it is possible to use the probe to distinguish tissue and cell types in situ or in vitro.
  • This invention also provides reagents with significant therapeutic value.
  • the cyclin E2 protein (naturally occurring or recombinant) , fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity to a cyclin E2 protein, can be useful in the treatment of conditions associated with abnormal physiology or development, including abnormal proliferation, e.g., cancerous conditions, or degenerative conditions. Abnormal proliferation, regeneration, degeneration, and atrophy may be modulated by appropriate therapeutic treatment using the compositions provided herein.
  • a disease or disorder associated with abnormal expression or abnormal signaling by a cyclin E2 protein is a target for an agonist or antagonist of the protein.
  • the proteins likely play a role in regulation or development of neuronal or hematopoietic cells, e.g., lymphoid cells, which affect immunological responses.
  • Various abnormal conditions are known in each of the cell types shown to possess, e.g., HDTEA84, mRNA by Northern blot analysis. See Berkow (ed.) The Merck Manual of Diagnosis and Therapy, Merck & Co., Rahway, NJ; Thorn, et al . Harrison' s Principles of Internal Medicine, McGraw-Hill, NY; and Weatherall, et al. (eds.) Oxford Textbook of Medicine, Oxford University Press, Oxford.
  • T cells or are T cell mediated may be responsive to treatment by an agonist or antagonist provided herein.
  • an agonist or antagonist provided herein. See, e.g., Stites and Terr (eds; 1991) Basic and Clinical Immunology Appleton and Lange, Norwalk, CT; and Samter, et al. (eds) Immunological Diseases Little, Brown and Co.
  • These problems should be susceptible to prevention or treatment using compositions provided herein.
  • Specific, or selective, antibodies can be purified and then administered to a patient, veterinary or human.
  • reagents can be combined for therapeutic use with additional active or inert ingredients, e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers, excipients, or preservatives .
  • additional active or inert ingredients e.g., in conventional pharmaceutically acceptable carriers or diluents, e.g., immunogenic adjuvants, along with physiologically innocuous stabilizers, excipients, or preservatives .
  • diluents e.g., immunogenic adjuvants
  • physiologically innocuous stabilizers e.g., excipients, or preservatives
  • These combinations can be sterile filtered and placed into dosage forms as by lyophilization in dosage vials or storage in stabilized aqueous preparations.
  • This invention also contemplates use of antibodies or binding fragments thereof, including forms which are not complement binding.
  • Drug screening using proteins or fragments thereof can be performed to identify compounds having binding affinity to or other relevant biological effects on antigen functions, including isolation of associated components. Subsequent biological assays can then be utilized to determine if the compound has intrinsic stimulating activity or is a blocker or antagonist in that it blocks the activity of the antigen, e.g., mutein antagonists. Likewise, a compound having intrinsic stimulating activity can activate the signal pathway and is thus an agonist in that it overcomes any blocking activity of these soluble forms of receptors. This invention further contemplates the therapeutic use of blocking antibodies to ligands or receptors as agonists or antagonists and of stimulatory molecules, e.g., muteins, as agonists. This approach should be particularly useful with other soluble receptor species variants .
  • Another therapeutic approach included within the invention involves direct administration of reagents or compositions by any conventional administration techniques (e.g., but not restricted to local injection, inhalation, or administered systemically) , to the subject with an immune, allergic, or trauma disorder.
  • the reagents, formulations, or compositions included within the bounds and metes of the invention may also be targeted to specific cells or transporters by methods described herein.
  • the actual dosage of reagent, formulation, or composition that modulates an immune, allergic, or trauma disorder depends on many factors, including the size and health of an organism, however one of ordinary skill in the art can use the following teachings describing the methods and techniques for determining clinical dosages. See, e.g., Spilker (1984) Guide to Clinical Studies and Developing Protocols.
  • the dose will be in the range of about between 0.5 fg/ml and 500 ⁇ g/ml, inclusive, final concentration administered per day to an adult in a pharmaceutically acceptable carrier.
  • reagents necessary for effective therapy will depend upon many different factors, including means of administration, target site, physiological state of the patient, and other medicants administered. Thus, treatment dosages should be titrated to optimize safety and efficacy. Typically, dosages used in vitro may provide useful guidance in the amounts useful for in situ administration of these reagents. Animal testing of effective doses for treatment of particular disorders will provide further predictive indication of human dosage. Various considerations are described, e.g., in Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics, 8th Ed., Pergamon Press; and Remington's Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Perm.
  • Pharmaceutically acceptable carriers will include water, saline, buffers, and other compounds described, e.g., in the Merck Index. Merck & Co., Rahway, New Jersey. Dosage ranges would ordinarily be expected to be in amounts lower than 1 mM concentrations, typically less than about 10 ⁇ M concentrations, usually less than about 100 nM, preferably less than about 10 pM (picomolar) , and most preferably less than about 1 fM (femtomolar) , with an appropriate carrier.
  • Slow release formulations, or a slow release apparatus will often be utilized for continuous or long term administration. See, e.g., Langer (1990) Science 249:1527-1533.
  • Ligands, receptors, enzymes, fragments thereof, and antibodies to it or its fragments, antagonists, and agonists may be administered directly to the host to be treated or, depending on the size of the compounds, it may be desirable to conjugate them to carrier proteins such as ovalbumin or serum albumin prior to their administration.
  • Therapeutic formulations may be administered in many conventional dosage formulations. While it is possible for the active ingredient to be administered alone, it is preferable to present it as a pharmaceutical formulation.
  • Formulations typically comprise at least one active ingredient, as defined above, together with one or more acceptable carriers thereof. Each carrier should be both pharmaceutically and physiologically acceptable in the sense of being compatible with the other ingredients and not injurious to the patient.
  • Formulations include those suitable for oral, rectal, nasal, topical, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration.
  • the formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. See, e.g., Gilman, et al. (eds. 1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics , 8th Ed., Pergamon Press; and Remington ' s Pharmaceutical Sciences. 17th ed. (1990), Mack Publishing Co., Easton, Perm.; Avis, et al . (eds. 1993) Pharmaceutical Dosage Forms : Parenteral Medications .
  • the therapy of this invention may be combined with or used in association with other agents, e.g., other modulators of cell activation, e.g., CD40, CD40 ligand, CD28, CTLA-4, B7, B70, SLAM, T cell receptor signaling entities, or their respective antagonists.
  • other modulators of cell activation e.g., CD40, CD40 ligand, CD28, CTLA-4, B7, B70, SLAM, T cell receptor signaling entities, or their respective antagonists.
  • kits and assay methods which are capable of screening compounds for binding activity to the proteins.
  • automating assays have been developed in recent years so as to permit screening of tens of thousands of compounds in a short period. See, e.g., Fodor, et al . (1991) Science 251:767-773, which describes means for testing of binding affinity by a plurality of defined polymers synthesized on a solid substrate.
  • suitable assays can be greatly facilitated by the availability of large amounts of purified, soluble proteins or nucleic acids as provided by this invention.
  • Other methods can be used to determine the critical residues in the substrate, ligand, or receptor binding interactions.
  • Mutational analysis can be performed, e.g., see Somoza, et al. (1993) J . EXP . Med. 178:549-558, to determine specific residues critical in the interaction and/or signaling. This will allow study of both extracellular domains, involved in the soluble ligand interaction, or intracellular domain of a transmembrane form, which provides interactions important in intracellular signaling.
  • antagonists can normally be found once the antigen has been structurally defined, e.g., by tertiary structure data. Testing of potential interacting analogs is now possible upon the development of highly automated assay methods using a purified protein. In particular, new agonists and antagonists will be discovered by using screening techniques described herein. Of particular importance are compounds found to have a combined binding affinity for a spectrum of protein molecules, e.g., compounds which can serve as antagonists for species variants of the antigens .
  • One method of drug screening utilizes eukaryotic or prokaryotic host cells which are stably transformed with recombinant DNA molecules expressing desired protein.
  • Cells may be isolated which express a selected protein in isolation from other molecules.
  • Such cells either in viable or fixed form, can be used for standard binding partner binding assays. See also, Parce, et al . (1989) Science 246:243-247; and Owicki, et al . (1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitive methods to detect cellular responses.
  • Another technique for drug screening involves an approach which provides high throughput screening for compounds having suitable binding affinity to a desired target protein, and is described in detail in Geysen, European Patent Application
  • kits for detecting, e.g., the presence of protein or binding partner.
  • the kit will have a compartment containing either a described polypeptide or gene segment or a reagent which recognizes one or the other, e.g., fragments or antibodies.
  • kits may be nucleic acid based.
  • a kit for determining the binding affinity of a test compound to, e.g., an HDTEA84 would typically comprise a test compound; a labeled compound, for example a binding partner or antibody having known binding affinity for HDTEA84; a source of HDTEA84 (naturally occurring or recombinant) ; and a means for separating bound from free labeled compound, such as a solid phase for immobilizing the molecule.
  • a test compound for example a binding partner or antibody having known binding affinity for HDTEA84
  • a source of HDTEA84 naturally occurring or recombinant
  • a means for separating bound from free labeled compound such as a solid phase for immobilizing the molecule.
  • a preferred kit for determining the concentration of, e.g., an HDTEA84 in a sample would typically comprise a labeled compound, e.g., binding partner or antibody, having known binding affinity for the antigen, a source of antigen (naturally occurring or recombinant) and a means for separating the bound from free labeled compound, e.g., a solid phase for immobilizing the HDTEA84. Compartments containing reagents, and instructions, will normally be provided.
  • Antibodies including antigen binding fragments, specific for, e.g., the HDTEA84 or fragments, are useful in diagnostic applications to detect the presence of elevated levels of HDTEA84 and/or its fragments.
  • diagnostic assays can employ lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and further can involve the detection of antigens related to the antigen in serum, or the like. Diagnostic assays may be homogeneous (without a separation step between free reagent and antigen-binding partner complex) or heterogeneous (with a separation step) .
  • RIA radioimmunoassay
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • EMIT enzyme-multiplied immunoassay technique
  • SFIA substrate-labeled fluorescent immunoassay
  • Anti-idiotypic antibodies may have similar use to diagnose presence of antibodies against a described protein, as such may be diagnostic of various abnormal states.
  • Overproduction of prostaglandin transporter may reflect various medical conditions, which may be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal differentiation.
  • leukemias and lymphomas may exhibit altered transporter expression, which may reflect their altered physiology and may provide means to selectively target.
  • overproduction of HDTEA84, HSLJD37R, RANKL, HCC5, MD-1, or MD-2 may result in production of various immunological reactions which may be diagnostic of abnormal physiological states, particularly in proliferative cell conditions such as cancer or abnormal activation or differentiation.
  • Expression levels of DC-PGT, Dubs, or cyclin E2 may likewise be diagnostic of specific therapeutic conditions, advantageous or disadvantageous .
  • the reagents for diagnostic assays are supplied in kits, so as to optimize the sensitivity of the assay.
  • the protocol, and the label either labeled or unlabeled antibody or binding partner, or labeled HDTEA84 is provided. This is usually in conjunction with other additives, such as buffers, stabilizers, materials necessary for signal production such as substrates for enzymes, and the like.
  • the kit will also contain instructions for proper use and disposal of the contents after use.
  • the kit has compartments for each useful reagent.
  • the reagents are provided as a dry lyophilized powder, where the reagents may be reconstituted in an aqueous medium providing appropriate concentrations of reagents for performing the assay.
  • labeling may be achieved by covalently or non-covalently joining a moiety which directly or indirectly provides a detectable signal.
  • the binding partner, test compound, HDTEA84, or antibodies thereto can be labeled either directly or indirectly.
  • Possibilities for direct labeling include label groups: radiolabels such as 125j_ f enzymes (U.S. Pat. No. 3,645,090) such as peroxidase and alkaline phosphatase, and fluorescent labels (U.S. Pat. No. 3,940,475) capable of monitoring the change in fluorescence intensity, wavelength shift, or fluorescence polarization.
  • Possibilities for indirect labeling include biotinylation of one constituent followed by binding to avidin coupled to one of the above label groups.
  • Suitable matrices include plastic such as an ELISA plate, filters, and beads. See, e.g., Coligan, et al . (eds. 1993) Current Protocols in Immunology. Vol. 1, Chapter 2, Greene and Wiley, NY.
  • suitable separation techniques include, without limitation, the fluorescein antibody magnetizable particle method described in Rattle, et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magnetic particle separation as described in U.S. Pat. No. 4,659,678.
  • Another diagnostic aspect of this invention involves use of oligonucleotide or polynucleotide sequences taken from the sequence of a described protein. These sequences can be used as probes for detecting levels of the message in samples from patients suspected of having an abnormal condition, e.g., cancer or developmental problem. Since the antigen is a marker for activation, it may be useful to determine the numbers of activated T cells to determine, e.g., when additional suppression may be called for.
  • the preparation of both RNA and DNA nucleotide sequences, the labeling of the sequences, and the preferred size of the sequences has received ample description and discussion in the literature. See, e.g., Langer-Safer, et al . (1982) Proc.
  • antibodies may be employed which can recognize specific duplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes, or DNA-protein duplexes.
  • the antibodies in turn may be labeled and the assay carried out where the duplex is bound to a surface, so that upon the formation of duplex on the surface, the presence of antibody bound to the duplex can be detected.
  • probes to the novel anti-sense RNA may be carried out in any conventional techniques such as nucleic acid hybridization, plus and minus screening, recombinational probing, hybrid released translation (HRT) , and hybrid arrested translation (HART) . This also includes amplification techniques such as polymerase chain reaction (PCR) .
  • PCR polymerase chain reaction
  • kits which also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on the combination of multiple indications used as markers. Thus, kits may test for combinations of markers. See, e.g., Viallet, et al . (1989) Progress in Growth Factor Res . 1:89-97. Other kits may be used to evaluate T cell subsets . X. Methods for Isolating Substrates/Specific Partners
  • the DC-PGT should interact with its substrate target.
  • the substrate will be similar to the organic molecules which are subject to transport.
  • the Dubs and cyclin E2 will also be screened for substrate identification.
  • the HDTEA84, HSLJD37R, and RANKL protein should interact with a TNF ligand, based, e.g., upon its similarity in structure and function to other cell surface antigens exhibiting similar structure and cell type specificity of expression.
  • the MD-1 and MD-2 antigens are related to known proteins, which interact with B cell antigens. Methods to isolate a ligand are made available by the ability to make purified protein for screening programs. Similar techniques will be applicable to the HCC5 chemokine, and the MD-1 and MD-2 surface receptors. Sequences provided herein will allow for screening or isolation of specific ligands. Many methods exist for expression cloning, panning, affinity isolation, or other means to identify a ligand. A two-hybrid selection system may also be applied making appropriate constructs with the available sequences, as appropriate. See, e.g., Fields and Song (1989) Nature 340:245- 246.
  • Methods for protein purification include such methods as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, crystallization, and others. See, e.g., Ausubel, et al. (1987 and periodic supplements); Deutscher (1990) "Guide to Protein Purification” in Methods in Enzymol . vol. 182, and other volumes in this series; and manufacturer's literature on use of protein purification products, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond, CA. Combination with recombinant techniques allow fusion to appropriate segments, e.g., to a FLAG sequence or an equivalent which can be fused via a protease-removable sequence.
  • the membrane topologies of proteins e.g., DC-PGT, and a cohort of putative homologues were analyzed by a variety of methods that sought to determine the consensus number of domains, e.g., hydrophobic membrane-spanning helices and the likely cytoplasmic or extracellular exposure of the hydrophilic connecting loops.
  • the ALOM and MTOP Karlin, et al . (1985) Biochim. Bioohvs . Acta 815:468-476; and
  • Peripheral or cord blood cells are cultured in the presence of Stem Cell Factor (SCF) , GM-CSF, and TNF- ⁇ in endotoxin free RPMI 1640 medium (GIBCO, Grand Island, NY) supplemented with 10% (v/v) heat-inactivated fetal bovine serum (FBS; Flow Laboratories, Irvine, CA) , 10 mM HEPES, 2 M L- glutamine, 5 X 10 ⁇ 5 M 2-mercaptoethanol, penicillin (100 ⁇ g/ml) .
  • SCF Stem Cell Factor
  • GM-CSF GM-CSF
  • TNF- ⁇ in endotoxin free RPMI 1640 medium
  • FBS heat-inactivated fetal bovine serum
  • penicillin 100 ⁇ g/ml
  • CD34+ cells are seeded for expansion in 25 to 75 cm ⁇ flasks
  • CDla+ cells are routinely collected after 12 days of culture, eventually adherent cells are recovered using a 5 mM EDTA solution.
  • the CDla+ cells are activated by resuspension in complete medium at 5 x 10 ⁇ cells/ml and activated for the appropriate time (e.g., 1 or 6 h) with 1 ⁇ g/ml phorbol 12-myristate 13-acetate (PMA, Sigma) and 100 ng/ml ionomycin (Calbiochem, La Jolla, CA) .
  • PMA phorbol 12-myristate 13-acetate
  • ionomycin Calbiochem, La Jolla, CA
  • RNA Isolation and Library Construction Total RNA is isolated using, e.g., the guanidine thiocyanate/CsCl gradient procedure as described by Chirgwin, et al. (1978) Biochem. 18:5294-5299.
  • poly (A) + RNA is isolated using the OLIGOTEX mRNA isolation kit (QIAGEN) .
  • Double stranded cDNA are generated using, e.g., the SUPERSCRIPT plasmid system (Gibco BRL, Gaithersburg, MD) for cDNA synthesis and plasmid cloning.
  • the resulting double stranded cDNA is unidirectionally cloned, e.g., into pSportl and transfected by electroporation into ELECTROMAX DH10BTM Cells (Gibco BRL, Gaithersburg, MD) .
  • DNA isolated from randomly picked clones, or after subtractive hybridization using inactivated cells, are subjected to nucleotide sequence analysis using standard techniques.
  • selected isolated clones can be selected.
  • a Taq DiDeoxy Terminator cycle sequencing kit (Applied Biosystems, Foster City, CA) can be used.
  • the labeled DNA fragments are separated using a DNA sequencing gel of an appropriate automated sequencer.
  • the isolated clone is sequenced as described, e.g., in Maniatis, et al. (Current ed.) Molecular Cloning. A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor Press; Sambrook, et al . (Current ed. ) Molecular Cloning: A Laboratory Manual, (2d ed.), vols. 1-3, CSH Press, NY;
  • Poly(A) + RNA is isolated from appropriate cell populations, e.g., using the FastTrack mRNA kit (Invitrogen, San Diego, CA) . Samples are electrophoresed, e.g., in a 1% agarose gel containing formaldehyde and transferred to a GeneScreen membrane (NEN).
  • Hybridization is performed, e.g., at 65° C in 0.5 M NaHP0 pH 7.2, 7% SDS, 1 mM EDTA, and 1% BSA
  • filters are washed three times at 50° C in 0.2X SSC, 0.1% SDS, e.g., for 30 min, and exposed to film for 24 h.
  • a positive signal will typically be 2X over background, preferably 5-25X.
  • the recombinant gene construct may be used to generate a probe for detecting the message.
  • the insert may be excised and used in the detection methods described above.
  • Various standard methods for cross species hybridization and washes are well known in the art. See, e.g., Sambrook, et al . and Ausubel.
  • HDTEA84 Gene Cloning The HDTEA84 was assembled by careful analysis of ESTs found in various databases. These ESTs were from cDNA libraries derived from Hodgkin's lymphoma, endothelial cells, keratinocytes, prostate, and cerebellum. PCR primers are designed and synthesized and a PCR product is obtained from any of these libraries. This product is used as a hybridization clone to screen these libraries for a full length clone, which may include a transmembrane segment.
  • HSLJD37R was identified from sequences derived from cDNA libraries from: smooth muscle, pancreas tumor, adipocytes, HUVEC cells, adult pulmonary, endothelial cells, prostate cell line PC3 , microvascular endothelial cells, fetal heart, and dendritic cells.
  • a GenBank report by Pan, et al. has been submitted. See GenBank Accession 3549263.
  • Other sequences were detected in libraries from: multiple sclerosis lesions, breast, kidney, and germinal center B cells.
  • RT-PCT showed signal in B cells, PBL, granulocytes, T cells, monocytes, dendritic cell subpopulations including PMA/ionomycin treated, U937 cells, JY cells, MRC5 cells, CHA, Jurkat, and YC1 cells. This suggests that the transcript is widely expressed.
  • RANKL was also identified in cDNA libraries from specific tissues, as described. Likewise, the HCC5 chemokine sequence was identified.
  • the Dubll and Dubl2 genes were identified, in part, from their similarity to known Dubl and Dub2 genes.
  • the MD-1 and MD-2 were identified, in part, from their similarity to the ligand for the RP105 gene.
  • the cyclin E2 was identified based upon its similarity to cyclin E.
  • Plasmid DNA containing differential display PCR products are prepared using the QIAGEN Plasmid Maxi Kit (QIAGEN) following the manufacturer's instructions. Plasmid DNA is cut with EcoRI (Boehringer Mannheim) or BstXI (NE Biolabs, Mass.), gel extracted with the QIAEX gel extraction kit (QIAGEN) and random primed with [ 32 P]dCTP (Amersham) using the Prime-It II kit (Stratagene, La Jolla, CA) , all in accordance with manufacturer ' s instructions . Various primers may be used to quantitate expression of message. Means to block DNA hybridization signal, or RNA isolation, will be applicable to quantitate roughly the amount of expression of appropriate RNAs .
  • DC-PGT is highly expressed in both activated and non-activated dendritic cells (DC) , activated monocytes, activated granulocytes and adult lung. No expression was found in T or PBL cells (either activated or non-activated) . Minor expression was detected in B cell (both activated and non- activated) and limited expression was detected in the brain.
  • the results of the northern analysis suggests an expression in macrophages, rather than monocytes (Kuppfer cells in the liver, microglial cells in the brain, alveolar macrophages in the lung) particularly as there is no expression in PBL.
  • Southern expression analysis carried out using common techniques confirmed the expression pattern revealed in the Northern analysis.
  • the DC-PGT tissue distribution seems to have highest mRNA levels in kidney, placenta, liver, bone marrow, thymus, spleen, lung, and some in testis.
  • This distribution corresponds to organs with especially important ion exchange features, e.g., Na, K, or Ca, or in hematopoietic organs.
  • important ion exchange features e.g., Na, K, or Ca
  • the expression is higher in fibroblast and hematopoietic cells compared to neuronal cells.
  • a probe specific for cDNA encoding the HDTEA84, HSLJD37R, or RANKL is used to determine tissue distribution of message encoding the antigen.
  • Standard hybridization probes may be used to do a Northern analysis of RNA from appropriate sources, either cells, e.g., stimulated, or in various physiological states, in various tissues, e.g., spleen, liver, thymus, lung, etc., or in various species. Southern analysis of cDNA libraries may also provide valuable distribution information. Standard tissue blots or species blots are commercially available. Similar techniques will be useful for evaluating diagnostic or medical conditions which may correlate with expression in various cell types. PCR analysis using appropriate primers may also be used.
  • Antibody analysis including immunohistochemistry or FACS, may be used to determin cellular or tissue distribution.
  • Samples for mouse mRNA distribution may include, e.g., : resting mouse fibroblastic L cell line (C200) ; Braf:ER (Braf fusion to estrogen receptor) transfected cells, control (C201) ; T cells, THl polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IFN- ⁇ and anti IL-4; T200) ; T cells, TH2 polarized (Mell4 bright, CD4+ cells from spleen, polarized for 7 days with IL-4 and anti-IFN- ⁇ ; T201) ; T cells, highly THl polarized (see Openshaw, et al . (1995) J. EXP. Med.
  • T cells highly TH2 polarized (see Openshaw, et al . (1995) J. EXP. Med. 182:1357-1367; activated with anti-CD3 for 2, 6, 16 h pooled; T203); CD44- CD25+ pre T cells, sorted from thymus (T204) ; THl T cell clone Dl.l, resting for 3 weeks after last stimulation with antigen (T205) ; THl T cell clone Dl.l, 10 ⁇ g/ml ConA stimulated 15 h (T206); TH2 T cell clone CDC35, resting for 3 weeks after last stimulation with antigen (T207) ; TH2 T cell clone CDC35, 10 ⁇ g/ml ConA stimulated 15 h (T208) ; Mel 14+ naive T cells from spleen, resting (
  • Mell4+ T cells polarized to Thl with IFN- ⁇ /IL-12/anti-IL-4 for 6, 12, 24 h pooled (T210) ; Mel 14+ T cells, polarized to Th2 with IL- 4/anti-IFN- ⁇ for 6, 13, 24 h pooled (T211) ; unstimulated mature B cell leukemia cell line A20 (B200) ; unstimulated B cell line CH12 (B201) ; unstimulated large B cells from spleen (B202); B cells from total spleen, LPS activated (B203); metrizamide enriched dendritic cells from spleen, resting (D200); dendritic cells from bone marrow, resting (D201) ; monocyte cell line RAW 264.7 activated with LPS 4 h (M200) ; bone-marrow macrophages derived with GM and M-CSF (M201) ; macrophage cell line J774, resting
  • M202 macrophage cell line J774 + LPS + anti-IL-10 at 0.5, 1, 3, 6, 12 h pooled
  • M204 macrophage cell line J774 + LPS + IL-10 at 0.5, 1, 3, 5, 12 h pooled
  • aerosol challenged mouse lung tissue, Th2 primers, aerosol OVA challenge 7, 14, 23 h pooled see Garlisi, et al . (1995) Clinical Immunology and Immunopathology 75:75-83; X206) ; Nippostrongulus-infected lung tissue (see Coffman, et al.
  • Various cells, tissues, and developmental stages are stained with labeled antibodies.
  • the detection may be immuno- histochemical for solid tissue, by FACS in disperse cells,- and by other appropriate methods for other sample types.
  • Antibodies specific for the various forms may be used to distinguish between membrane associated and soluble fragments.
  • Various amplification means may be coupled to increase sensitivity.
  • Specific neutralizing antibodies should provide means to specifically block the biological activity of the prostaglandin transporter. Activities related to prostaglandin binding, or to prostaglandin transport may be measured by sensitive means based upon knowledge of the normal biological function of the various forms.
  • tissue types e.g., hematopoietic progenitors, or of other cell or tissue types will be useful to further determine distribution and likely function.
  • Other tissue types, at defined developmental stages, and pathology samples may be screened to determine whether pathological states or stages may be advantageously correlated with the biological activity of the transporter .
  • Protein Expression PCR is used to make a construct comprising the open reading frame, preferably in operable association with proper promoter, selection, and regulatory sequences.
  • the resulting expression plasmid is transformed into an appropriate cell type, e.g., the Topp5, E. coli strain (Stratagene, La Jolla, CA) .
  • Ampicillin resistant (50 ⁇ g/ml) transformants are grown in Luria Broth (Gibco) at 37° C until the optical density at 550 ran is 0.7.
  • Recombinant protein is induced with 0.4 mM isopropyl- ⁇ D-thiogalacto-pyranoside (Sigma, St. Louis, MO) and incubation of the cells continued at 20° C for a further 18 hours.
  • Cells from a 1 liter culture are harvested by centrifugation and resuspended, e.g., in 200 ml of ice cold 30% sucrose, 50 mM Tris HCI pH 8.0, 1 mM ethylenediaminetetraacetic acid. After 10 min on ice, ice cold water is added to a total volume of 2 liters. After 10 min on ice, ice cold water is added to a total volume of 2 liters. After
  • the recombinant protein is purified via standard purification methods, e.g., various ion exchange chromatography methods.
  • Immunoaffinity methods using antibodies described below can also be used. Affinity methods may be used where an epitope tag is engineered into an expression construct. Similar methods are used to prepare expression constructs and cells in eukaryotic cells. Eukaryotic promoters and expression vectors may be produced, as described above.
  • controlling elements associated with the antigens may exhibit differential developmental, tissue specific, or other expression patterns. Upstream or downstream genetic regions, e.g., control elements, are of interest.
  • transfected cell lines are screened for one which expresses the antigen, membrane bound, or soluble forms, at a high level compared with other cells.
  • Various cell lines are screened and selected for their favorable properties in handling.
  • Natural protein can be isolated from natural sources, or by expression from a transformed cell using an appropriate expression vector.
  • Purification of the expressed protein is achieved by standard procedures, or may be combined with engineered means for effective purification at high efficiency from cell lysates or supernatants .
  • the prostaglandin transporter is isolated by a combination of affinity chromatography using the prostaglandin transporter specific binding compositions, e.g., antibody, as a specific binding reagent in combination with protein purification techniques allowing separation from other proteins and contaminants. Various detergent combinations may be tested to determine what combinations will retain biological activity while solubilizing contaminants.
  • the purification may follow biological activity, e.g., prostaglandin binding or transport into membranes, or by ELISA or other structural binding reagents .
  • the described genes can be used as a hybridization probe to screen a library from a desired source, e.g., a primate cell cDNA library.
  • a desired source e.g., a primate cell cDNA library.
  • Many different species can be screened both for stringency necessary for easy hybridization, and for presence using a probe.
  • Appropriate hybridization conditions will be used to select for clones exhibiting specificity of cross hybridization .
  • cDNA libraries from the desired species are collected, from appropriate cell types. Screening by hybridization or PCR using degenerate probes based upon the peptide sequences will also allow isolation of appropriate clones. Alternatively, use of appropriate primers for PCR screening will yield enrichment of appropriate nucleic acid clones.
  • Species variants are isolated using cross-species hybridization techniques based upon isolation of a full length isolate or fragment from one species as a probe.
  • antibodies raised against proteins will be used to screen for cells which express cross-reactive proteins from an appropriate, e.g., cDNA library.
  • the purified protein or defined peptides are useful for generating antibodies by standard methods, as described above. Synthetic peptides or purified protein are presented to an immune system to generate monoclonal or polyclonal antibodies. See, e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene; and Harlow and Lane (1989) Antibodies : A Laboratory Manual Cold Spring Harbor Press .
  • the resulting antibodies are used, e.g., for screening, panning, or sorting.
  • DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transfer and hybridization are performed according to standard techniques. See Jenkins, et al . (1982) J. Virol. 43:26-36. Blots may be prepared with Hybond-N nylon membrane (Amersham) . The probe is labeled with 32 _ ⁇ (-j r p. washing is done to a final stringency, e.g., of 0. IX SSC, 0.1%
  • BIOS Laboratories New Haven, CT
  • PCR methods See Fan, et al . (1996) Immunogenetics 44:97-103.
  • Chromosome spreads are prepared. In situ hybridization is performed on chromosome preparations obtained from phytohemagglutinin-stimulated human lymphocytes cultured for 72 h.
  • 5-bromodeoxyuridine is added for the final seven hours of culture (60 ⁇ g/ml of medium) , to ensure a posthybridization chromosomal banding of good quality.
  • a PCR fragment amplified with the help of primers, is cloned into an appropriate vector.
  • the vector is labeled by nick- translation with 3 H.
  • the radiolabeled probe is hybridized to metaphase spreads at final concentration of 200 ng/ml of hybridization solution as described in Mattei, et al . (1985) Hum. Genet . 69:327-331.
  • chromosome spreads are first stained with buffered Giemsa solution and metaphase photographed. R-banding is then performed by the fluorochrome-photolysis-Giemsa (FPG) method and metaphases rephotographed before analysis.
  • FPG fluorochrome-photolysis-Giemsa
  • the DC-PGT gene was mapped to marker SHGC-3911 on chromosome llql3 with a resulting lod score of 1000.0.
  • Other markers in the SHGC-3911 region at chromosome llql3 include the Fc ⁇ RI receptor which is alleged to be associated with allergic conditions.
  • the ubiquitously expressed human PGT homologue of Lu et al . (described above) is localized to chromosome 7.
  • DC-PGT Constructs for the expression of, e.g., DC-PGT are made with a tag (FLAG) sequence (Hopp, et al . (1988) Biotechnology (NY) 6:1205-1210) introduced in the protein.
  • FLAG tag
  • the open reading frame of the DC-PGT cDNA of SEQ ID NO: 1 is amplified by appropriate PCR primers using standard methods to introduce the FLAG peptide sequence (IBI, New Haven, CT) at the C-terminus of the protein.
  • a PFU enzyme (Stratagene) with 12 cycles PCR: 94° C 30 sec; 55° C 1 min; 72 ° C 4 min.
  • PCR constructs are cloned into a PME18X vector (DNAX) using Xhol and Xbal sites incorporated into the 5' and 3' primers, respectively.
  • COS-7 cells are maintained in DMEM, 10% FCS, 4 mM L-glutamine
  • Plasmid DNA is transfected by electroporation (BIORAD, Hercules, CA) (20 ⁇ g / 1 x 10 7 cells) and plated into tissue culture dishes. The medium is replaced after 24 hours and cell lysates and media are collected three days after transfection. Lysis buffer (25 mM Hepes pH 7.5, 2 mM EDTA, 1.0% NP-40, 150 mM NaCl, 0.01% Aprotinin (Sigma, St. Louis, MO), 0.01% Leupeptin (Sigma)) is added to the plates. Plates are kept on ice for 45 minutes.
  • Lysates are centrifuged for 15 minutes to eliminate cell debris.
  • Supernatants of centrifuged cell lysates and sterile-filtered media from cultured cells are incubated with anti-FLAG M2 Affinity Gel (IBI) at 4 ° C overnight and washed four times with PBS.
  • Immunoprecipitates are eluted in a Econocolumn (BIORAD) with 2.5 M Glycine, pH 2.5. Eluates are neutralized with Hepes, pH 7.4 (JRH Biosciences) and concentrated by precipitation with 24% TCA and 2% deoxycholic sodium salt (Sigma) .
  • Pellets are eluted in 2 x Sample Buffer (NOVEX, San Diego, CA) , electrophoresed on 4-20% tris-glycine gels (Novex) and transferred to PVDF membranes (Immobilon-P, Millipore Corporation, Bedford,
  • Membranes are exposed to 3% non-fat milk for 1 h at 37° C.
  • Anti-FLAG M2 antibody is used as recommended (IBI) .
  • Anti-mouse Ig horseradish peroxidase conjugate (Amersham) is used at 1:2,000 dilution and the peroxidase detection is performed with ECL detection reagents (Amersham) .
  • fusion proteins can be produced, e.g., a recombinant prostaglandin transporter construct is prepared, e.g., as a fusion product with a useful affinity reagent, e.g., FLAG peptide.
  • This peptide segment may be useful for purifying the expression product of the construct. See, e.g., Crowe, et al . (1992) OIAexpress : The High Level Expression & Protein Purification System QUIAGEN, Inc. Chatsworth, CA; and Hopp, et al . (1988) Bio/Technology 6:1204- 1210.
  • Membranes comprising the transporter are assayed to determine the natural prostaglandin substrate.
  • the prostaglandin will be a uracil related prostaglandin, but may also include, at various levels of efficiency of binding or transport, pyrimidine or purine analogs. See, e.g., Goodman and Gilman (Current ed.), The Pharmacological Basis of Therapeutics ; Lukovics and Zablocka Nucleoside Synthesis: Organosilicon Methods Ellis Horwood, N.Y. ; Townsend, Chemistry of Nucleosides and Nucleotides. vols.
  • Expression cloning of cells transformed with an appropriate cDNA library may be sorted by FACS using antibody reagents described above. The sorted cells are isolated and expanded, and subjected to multiple selection cycles, leading to a high proportion of cells expressing the desired DNA.
  • the antibodies to, e.g., DC-PGT are used for screening of a library made from a cell line which expresses the polypeptide.
  • Standard staining techniques are used to detect or sort intracellular or surface expressed ligand, or surface expressing transformed cells are screened by panning. Screening of intracellular expression is performed by various staining or immunofluorescence procedures. See also McMahan, et al . (1991) EMBO J. 10:2821-2832.
  • HBSS Hank's Buffered Saline Solution
  • PFA paraformaldehyde
  • the antibodies to a selected protein are used to affinity purify or sort out cells expressing the antigen. See, e.g., Sambrook et al . or Ausubel et al, which are incorporated herein by reference.
  • the antigen is typically expressed on the cell surface.
  • Hybridization approaches may also be utilized to find closely related variants of the antigen based upon nucleic acid hybridization.
  • DC-PGT cDNAs can be expressed in HeLa cell monolayers or in Xenopus oocytes to determine the ability of DC-PGT to uptake various tracer labeled substrates e.g., prostaglandins such as PGE1, PGE2, PGE2a, PGD2 , thrombaxanes such as TxB2 or non- prostaglandin anionic substrates such as glutathione, p-amino hippurate, taurochoalate, urate, unconjugated and conjugated bilirubin, and estradiol glucouronide .
  • PGE1, PGE2, PGE2a thrombaxanes
  • TxB2 thrombaxanes
  • non- prostaglandin anionic substrates such as glutathione, p-amino hippurate, taurochoalate, urate, unconjugated and conjugated bilirubin, and estradiol glucouronide .
  • RNA complementary RNA
  • Uptake studies are performed 2 to 3 days after injection by washing of oocytes three times in Waymouth's solution, incubating for various periods at 27°C with radioactive substrates (approx. 0.25 ⁇ ci/ml; total concentration, approx. 1 nM) , washing three times with ice- cold Waymouth's solution, and lysing in 0.5 ml of 10% SDS.
  • Oocyte-associated radioactivity is determined by liquid scintillation spectroscopy.
  • vaccinia virus an the DNA-lipofectin complex are removed, and the cells are maintained overnight in Dulbecco ' s modified Eagle's medium supplemented with 5% fetal bovine serum. Uptake studies are performed 19 hours after transfection. Monolayers are washed three times with culture medium without serum and incubated for various times at 27° C with radioactive substrate (0.5 ⁇ Ci/ml per dish; total concentration, approx. 0.2 nM) . Uptake is stopped by washing cells once with ice-cold Waymouth's solution containing 5% bovine serum albumin and then four times with Waymouth's solution alone. Cells are scrapped and the associated radioactivity is measured by liquid scintillation spectroscopy.
  • compositions inhibiting DC-PGT uptake can also be measured.
  • uptakes at the end of the fatty acid chain can also be measured. For example to measure the inhibition of tracer PGE2 , uptakes at the end of the fatty acid chain.
  • DC-PGT of the invention can be used to screen for inhibitors or agonists of DC-PGT mediated tracer transport .
  • the efficacy of potential antagonists can be compared with known PG transport inhibitors such as furosemide, probenecid, or indomethacin.
  • Potential agonist or antagonist compositions are incubated, using a system as described above, for a time sufficient to allow binding of the test composition and the DC-PGT transporter. Enhancement or decrement in measures of tracer uptake can be correlated to the specific composition being tested.
  • a construct for expression of the product can be used as a specific binding reagent to identify its binding partner, e.g., ligand, by taking advantage of its specificity of binding, much like an antibody would be used.
  • a receptor reagent is either labeled as described above, e.g., fluorescence or otherwise, or immobilized to a substrate for panning methods . See also Anderson, et al. (1997) Nature 390:175-179, which is incorporated herein by reference.
  • the binding composition is used to screen an expression library made from a cell line which expresses a binding partner, e.g., TNF family ligand. Standard staining techniques are used to detect or sort intracellular or surface expressed receptor, or surface expressing transformed cells are screened by panning. Screening of intracellular expression is performed by various staining or immunofluorescence procedures. See also McMahan, et al. (1991) EMBO J. 10:2821-2832.
  • receptor reagents are used to affinity purify or sort out cells expressing a receptor. See, e.g., Sambrook, et al. or Ausubel, et al .
  • Another strategy is to screen for a membrane bound ligand by panning.
  • the cDNA containing ligand cDNA is constructed as described above.
  • the ligand can be immobilized and used to immobilize expressing cells. Immobilization may be achieved by use of appropriate antibodies which recognize, e.g., a FLAG sequence or a receptor fusion construct, or by use of antibodies raised against the first antibodies. Recursive cycles of selection and amplification lead to enrichment of appropriate clones and eventual isolation of ligand expressing clones.
  • Phage expression libraries can be screened by receptor. Appropriate label techniques, e.g., anti-FLAG antibodies, will allow specific labeling of appropriate clones.
  • Chemokine proteins are produced, e.g., in COS cells transfected with a plasmid carrying the chemokine cDNA by electroporation. See, Hara, et al . (1992) EMBO J. 10:1875-1884. Physical analytical methods may be applied, e.g., CD analysis, to compare tertiary structure to other chemokines to evaluate whether the protein has likely folded into an active conformation. After transfection, a culture supernatant is collected and subjected to bioassays. A mock control, e.g., a plasmid carrying the luciferase cDNA, is used. See, de Wet, et al . (1987) Mol. Cell. Biol . 7:725-757. A positive control, e.g., recombinant murine
  • MlP-l ⁇ from R&D Systems (Minneapolis, MN) , is typically used.
  • antibodies may be used to block the biological activities, e.g., as a control.
  • Lymphocyte migration assays are performed as previously described, e.g., in Bacon, et al . (1988) Br. J. Pharmacol. 95:966- 974. Other trafficking assays are also available. See, e.g., Quidling-Jarbrink, et al . (1995) Eur. J. Immunol. 25:322-327; Koch, et al. (1994) J. Clinical Investigation 93:921-928; and Antony, et al. (1993) J . Immunol . 151:7216-7223. Murine Th2 T cell clones, CDC-25 (see Tony, et al. (1985) J. EXP. Med.
  • Ca2+ flux upon chemokine stimulation is measured according to the published procedure described in Bacon, et al . (1995) J. Immunol . 154:3654-3666.
  • a robust and sensitive assay is selected as described above, e.g., on immune cells, neuronal cells, or stem cells.
  • Chemokine is added to the assay in increasing doses to see if a dose response is detected.
  • a proliferation assay cells are plated out in plates. Appropriate culture medium is provided, and chemokine is added to the cells in varying amounts. Growth is monitored over a period of time which will detect either a direct effect on the cells, or an indirect effect of the chemokine .
  • an activation assay or attraction assay is used.
  • An appropriate cell type is selected, e.g., hematopoietic cells, myeloid (macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (T cell, B cell, or NK cells), neural cells
  • stem cells e.g., progenitor cells which differentiate to other cell types, e.g., gut crypt cells and undifferentiated cell types.
  • Retroviral infection assays have also been described using, e.g., the CCR1, CCR3 , and CCR5 receptors. These receptors, which bind the RANTES and MIP-1 related chemokines, are likely also to be receptors for the HCC5 . Recent description of these chemokine receptors in retroviral infection processes, and the effects by the related RANTES and MIP-1 chemokines, suggest similar effects may exist with the HCC5 . See, e.g., Baiter (1996) Science 272:1740 (describing evidence for chemokine receptors as coreceptors for HIV); and Deng, et al . (1996) Nature 381:661-666.
  • Chemokines may also be assayed for activity in hemopoietic assays as described, e.g., by H. Broxmeyer. See Bellido, et al . (1995) J. Clinical Investigation 95:2886-2895; and Jilka, et al . (1995) Exot ' 1 Hematology 23:500-506. They may be assayed for angiogenic activities as described, e.g., by Streiter, et al . (1992) Am. J. Pathol . 141:1279-1284. Or for a role in inflammation. See, e.g., Wakefield, et al . (1996) J. Surgical Res. 64:26-31.
  • the DUB genes will be screened for the deubiquitinating activities, as described. See, e.g., Hochstrasser (1995) Curr. Qpin. Cell Biol. 7:215-223; Wilkinson, et al . (1995) Biochemistry 34:14535-14546; Baker, et al . (1992) J. Biol. Chem. 267:23364- 23375; Baek et al . (1998) J. Biol. Chem. 272:25560-25565; and Papa and Hochstrasser (1993) Nature 366:313-319.
  • the reaction is terminated by adding 50 ⁇ l of 40% (w/v) trichloroacetic acid and 50 ⁇ l of 1.2% (w/v) bovine serum albumin.
  • the samples are centrifuged, and the resulting supernatants are counted for their radioactivities using a counter.
  • the enzyme activity is expressed as a percentage of l ⁇ i-iabeiec! ub-PESTc hydrolyzed to acid-soluble products.
  • Dubll or Dubl2 can be expressed as a GST fusion protein according to the method of Zhu, et al. (1997) J. Biol. Chem.
  • the MD gene products will be screened for cell signaling activities. See, e.g., Miyake, et al . (1998) J. Immunol. 161:1348-1353; Kobe and Deisenhofer (1994) Trends Biochem. Sci. 19:412.
  • the inhibition of cell cycle progression is especially important for the control of abnormally proliferative diseases, e.g., cancer.
  • Several methods are available to accomplish this control.
  • the ability of cyclin binding is inhibited by the use, e.g., of antibodies raised against the cyclin binding proteins.
  • Other elements include, e.g., peptidomimetics which are peptides designed to mimic the binding site of cyclin associated proteins and disrupt the interaction of these proteins with cyclin.
  • the most effective method to block cell cycle progression is the use of small molecules, e.g., to block the interaction of the associated proteins with cyclin, or to block downstream activity of the associated proteins, as described, e.g., in Hung, et al. (1996) Chemistry and Biology 3:623-639. Exposure of a cell to these permeable small molecules should cause a conditional loss of function of the target protein.
  • Also included in this category is the use of gene therapy to block the expression of the cyclin associated protein or gene transcription factors. Methods of using gene therapy are described, e.g., in Goodnow (1992) "Transgenic Animals” in Roitt (ed.) Encyclopedia of Immunology, Academic Press, San Diego, pp. 1502-1504; Travis (1992) Science 256:1392-1394; Kuhn, et al. (1991) Science 254:707-710; Capecchi (1989) Science 244:1288; Robertson (1987) (ed.) Teratocarcinomas and Embryonic Stem Cells : A Practical Approach, IRL Press, Oxford; and Rosenberg (1992) J. Clinical Oncology 10:180-199. Also included is the use of antisense RNA in gene therapy to block expression of the target gene, or proper splicing of gene transcripts.

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Abstract

L'invention concerne des gènes purifiés, provenant d'un mammifère, des réactifs apparentés à ces gènes, notamment des protéines purifiées, des anticorps spécifiques et des acides nucléiques codant ces polypeptides. L'invention concerne encore des procédés d'utilisation de ces réactifs, ainsi que des nécessaires de diagnostic. Elle caractérise enfin des gènes et des produits associés à DC-PGT (transporteur du type prostaglandine de cellules dendritiques), HDTEA84, HSLJD37R et RANKL (associés à la famille des récepteurs de TNF), chimiokine HCC5, Dub 11 et Dub 12 (protéines de désubiquitination 11 et 12), MD-1 et MD-2 (protéines possédant des propriétés de ligands de protéines présentant un motif de protéine riche en leucines (LRR)) et cycline E2.
PCT/US1999/012366 1998-07-06 1999-07-06 Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes WO2000001817A2 (fr)

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Application Number Priority Date Filing Date Title
AU48185/99A AU4818599A (en) 1998-07-06 1999-07-06 Mammalian genes; dendritic cell prostaglandin-like transponder (dc-pgt), hdtea84, hsljd37r and rankl, hcc5 chemokine, deubiquitinating 11 and 12 (dub11, dub12),md-1, md2 and cyclin e2, related reagents and methods
JP2000558207A JP2002519062A (ja) 1998-07-06 1999-07-06 哺乳動物遺伝子；樹状細胞プロスタグランジン様トランスポーター（ｄｃ−ｐｇｔ）、ｈｄｔｅａ，ｈｓｌｊｄ３７ｒおよびｒａｎｋｌ、ｈｃｃ５ケモカイン、脱ユビキチン化１１および１２（ｄｕｂ１１、ｄｕｂ１２）、ｍｄ−１、ｍｄ−２およびサイクリンｅ２、関連試薬および方法
EP99931753A EP1093516A2 (fr) 1998-07-06 1999-07-06 Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes
CA002332573A CA2332573A1 (fr) 1998-07-06 1999-07-06 Genes mammiferes ; transporteur du type prostaglandine de cellules dendritiques (dc-pgt), hdtea84, hsljd37r et rankl, chimiokine hcc5, proteines de desubiquitination 11 et 12 (dub11, dub12), md-1, md-2 et cycline e2, reactifs apparentes et procedes associes

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US9389798P 1998-07-23 1998-07-23
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