JP2002519062A - Mammalian genes; dendritic cell prostaglandin-like transporter (DC-PGT), HDTEA, HSLJD37R and RANKL, HCC5 chemokine, deubiquitination 11 and 12 (DUB11, DUB12), MD-1, MD-2 and cyclin E2 , Related reagents and methods - Google Patents

Abstract

  (57) [Summary] Provided are genes purified from mammals and associated reagents, including purified proteins, specific antibodies and nucleic acids encoding their polypeptides. Methods and diagnostic kits using the reagents are also provided. Characterization of genes and products related to: DC-PGT (dendritic cell prostaglandin-like transporter), HDTEA84, HSLJD37R and RANKL (associated with the TNF receptor family), HCC5 chemokines, Dub11 and Dub12 (deubiquitination) 11 and 12), MD-1 and MD-2 (proteins that characterize ligands for proteins that display leucine-rich protein motifs (LRRs)) and cyclin E2.

Description

DETAILED DESCRIPTION OF THE INVENTION

FIELD OF THE INVENTION The present invention relates to proteins that function in the cell physiology, development, and differentiation of mammalian cells; to regulate the activation and expansion of mammalian cells, eg, cells of the mammalian immune system. A protein that exhibits sequence similarity to a functioning TNF receptor; or a composition related to a protein that functions in controlling the cell cycle and growth. In particular, it provides purified genes, proteins, antibodies, and related agents, such as: to isolate or identify specific cell types, or to activate, develop, and activate various cell types, including hematopoietic cells. Useful for regulating differentiation and function; exhibits a high degree of structural similarity to proteins that exhibit biological capacity to act as carrier-mediated transporters of charged organic anions across cell membranes;
Typically, the physiological functions of prostaglandins and thromboxanes, such as transport,
Can be used in influx, efflux, efflux, or degradation; modulates or demonstrates the development, differentiation and function of various cell types, including hematopoietic cells; or regulates cell division and proliferation of various cell types, including tumor cells Useful to

BACKGROUND OF THE INVENTION [0002] Prostaglandins (PG) and thromboxanes (TX) are glaucoma; pregnancy, labor, labor, and abortion; protection of the stomach and formation of peptic ulcers; secretion of intestinal fluid;
It plays a wide range of physiological and therapeutic roles in health or disease, such as liver protection and damage; airway resistance and asthma; regulation of blood pressure; and regulation of inflammatory cells.

[0003] PG is a charged anion at physiological pH and diffuses only slightly through biological membranes. This limited simple diffusion can cause the lungs, choroid plexus, liver, anterior chamber, vagina,
In many diverse tissues, such as the uterus and placenta, it appears to be increased by carrier-mediated transport.

[0004] The understanding of the role of prostaglandins in the development and functionalization of the immune system is currently incomplete. In particular, the effects of prostaglandins (PGs) on antigen-presenting cells (APCs) (eg, dendritic cells) of the immune system are still poorly understood.

[0005] Dendritic cells (DCs) are the most powerful antigen presenting cells. For example, Paul (eds., 1993) Fundamental Immunology 3rd edition, Rav
See en Press, NY. DC is derived from bacterial lipopolysaccharide (
It is highly responsive to inflammatory stimuli, such as cytokines such as LPS) and tumor necrosis factor alpha (TNFα). The presence of cytokines and LPS can induce a range of phenotypic and functional changes in DC, collectively called maturation. For example, Banchereau and Schmitt Dendri
tic Cells in Fundamental and Clinica
l See Immunology Plenum Press, NY.

[0006] Maturational changes in DC include, for example, termination of antigen uptake by endocytosis, upregulation of surface molecules associated with T cell activation, and TN
Includes active production of many cytokines, including Fα and IL-12. TNFα
Due to the local accumulation of, DCs migrate to the T cell area of secondary lymphoid organs and activate antigen-specific T cells.

[0007] Recent data indicate that DCs secrete PG. For example, Corm
(1986) Ann. Inst. Pasteur 137: 369-3
See 82. Furthermore, PGE ₂ has been shown to influence DC maturation and DC production of cytokines. For example, Kalinski et al. (19)
97) J. Immunol. 159: 28-35; Kuhn et al. (1997) Eu.
r. J. Immunol. 27: 3135-3142; and Rieser et al. (
1997) J. Amer. Exp. Med. 186: 1603-1608.

[0008] Currently, there is a need to understand how PGs affect cells of the immune system.
PGs, like cytokines, appear to result in the development and activation of the immune system. The present invention contributes to meeting that need, and generally relates to novel mammalian genes encoding a prostaglandin-like transporter (PGT).

In another aspect, activation of resting T cells is critical for most immune responses, and allows these cells to exert their regulatory or effector capabilities. For example, Paul (ed., 1993) Fundamental Immun
See Logic, 3rd Edition, Raven Press, NY. Increased adhesion between T cells and antigen presenting cells (APCs), or other forms of primary stimulus, such as immobilized monoclonal antibodies (mAbs), can enhance T cell receptor signals. T cell activation and T cell expansion depend on the involvement of costimulatory signals provided by T cell receptors (TCRs) and accessory cells. For example,
Jenkins and Johnson (1993) Curr. Opin. Imm
unol. 5: 361-367; Bierer and Hahn (1993) Se.
min. Immunol. 5: 249-261; June et al. (1990) Imm.
unol. Today 11: 211-216; and Jenkins (199
4) See Immunity 1: 443-446. The primary and well-studied costimulatory interaction of T cells is that CD28 or CTLA-
4 together with either B7 or B70 (Jenkins (
1994) Immunity 1: 443-446). CD28 deficient mouse (S
hahinian et al. (1993) Science 261: 609-612; G
Reen et al. (1994) Immunity 1: 501-508), and CT
Transgenic mice expressing LA-4 immunoglobulin (Ronchese et al. (
1994) J. Am. Exp. Med. 179: 809-817) indicate that these mice have normal primary immune and CTL responses to lymphocytic choriomeningitis virus and vesicular stomatitis virus, Several T cell response defects were revealed. As a result, both of these studies conclude that other costimulatory molecules must support T cell function. However, identification of these molecules that mediate another costimulatory signal has been difficult.

[0010] Tumor necrosis factor (TNF) is a fundamental member of the nascent cytokine family that functions as a prominent mediator of immunomodulatory and inflammatory responses. These ligands are typically Type II membrane proteins with homology at the carboxyl terminus. Soluble proteins processed by proteolysis are often produced. For example, Smith et al. (1994) Cell 76-959.
-962; Armitage (1994) Current Opinion i
n Immunology 6: 407-413; Gruss and Dower
(1995) Blood 85: 3378-3404; Wiley et al. (1995)
) Immunity 3: 673-682; and Baker and Reddy.
(1996) Oncogene 12: 1-9. The critical role of these family members has been demonstrated by a number of studies, and they are involved in apoptosis, peripheral tolerance, Ig maturation and isotype switching, and the regulation of general B and T cell function. . For example, Thomson (ed., 1994) The Cytokine Handbook Academic
Press, San Diego, CA; Naismith and Splan
g (1998) Trends Biochem. Sci. 23: 74-79; L
ucas et al. (1997) J. Am. Leukoc. Biol. 61: 551-558;
Reddi (1997) Cell 89: 159-161; Van Deven.
ter (1997) Gut 40: 443-448; Jablonska (19
97) Postepy. Hig. Med. Dosw. 51: 567-575; H
ill and Lunec (1996) Mol. Aspects Med. 17:
455-509; Aderka (1996) Cytokine Growth.
Factor Rev. 7: 231-240; Lotz et al. (1996) J. Mol. Le
ukoc. Biol. 60: 1-7; and Gruss and Dower (19
95) Cytokines Mol. Ther. 1: 75-105. These include a fundamental role in the immune and developmental networks associated with human therapeutic needs. The identification of ligands and cell surface receptors allows for pair determination, which is useful in modulating such signaling.

[0011] The discovery of new cell markers is always potentially useful. In addition, the inability to modulate activation signals prevents inappropriate development and control of physiological responses in the immune system. The present invention provides at least one alternative costimulatory molecule. It is useful as a cell type marker, and its agonists and antagonists are useful in modulating many immune states or responses.

[0012] The circulating components of the mammalian circulatory system include a variety of cell types, including red and white blood cells of the erythroid and myeloid cell lines. For example, Rapport (1987) In
reduction to Hematology (2nd edition) Lippinc
ott, Philadelphia, PA; Jandl (1987) Blood.
: Textbook of Hematology, Little, Brown
and Co. And Boston, MA; and Paul (eds., 1993) Fu.
mental Immunology (3rd edition) Raven Press
, N .; Y. checking ...

[0013] For some time, it has been known that the mammalian immune response is based on a complex series of cellular interactions called the "immune network." Recent studies have provided new insights into the inner workings of this network. While it remains clear that many responses actually circulate around network-like interactions of lymphocytes, macrophages, granulocytes, and other cells, immunologists now generally consider lymphokines,
There is the opinion that soluble proteins known as cytokines, or monokines, play a crucial role in controlling these cell interactions. Thus, there is considerable interest in the isolation, characterization, and mechanism of action of cell regulatory factors. Its understanding should lead to important advances in the diagnosis and treatment of many medical disorders, such as the immune system and other disorders.

[0014] Lymphokines apparently mediate the activity of cells in a variety of ways. They have been shown to support the division, proliferation, and differentiation of pluripotent hematopoietic stem cells into many ancestors, including the diverse cell lineages that make up the complex immune system. These interactions between cellular components are necessary for a healthy immune response. These different cell lines often respond in different ways when lymphokines are administered with other drugs.

[0015] Chemokines are a large and diverse superfamily of proteins.
The superfamily determines whether the first two cysteines in the chemokine motif are adjacent (referred to as the "CC" split) or separated by intervening residues ("C-X-C"). Is divided into two classical sects based on A more recently identified branch of chemokines lacks two cysteines in the corresponding motifs and is represented by the chemokine known as lymphotoactin. Another recently identified sect, such as the CX3C chemokine, has three intervening residues between two cysteines. For example, Schall
And Bacon (1994) Current Opinion in Imm
unology 6: 865-873; and Bacon and Schall (
1996) Int. Arch. Allergy & Immunol. 109:
See 97-109.

Because the physiology mediated by these soluble molecules is so important, the discovery of new chemokines is important in both diagnostic and therapeutic settings.

In addition, while the general importance of regulating protein synthesis has been universally accepted, the general importance of proteolysis has not been fully recognized. One mechanism of proteolysis is through ubiquitination signals and degradation pathways. Ubiquitin (Ub) is a highly conserved 76 amino acid polypeptide that plays an important role in regulating protein degradation, cell cycle progression, gene transcription and signal transduction. The ubiquitination pathway is well regulated and controlled by deubiquitinase, which partially removes ubiquitin from proteins. Misregulation of the ubiquitination pathway can contribute to problems in regulating protein levels. It can be associated with, for example, malignant transformation and tumorigenesis by tumorigenic counterparts of normally processed ubiquitinated proteins. Other clinical problems often arise from excessive or insufficient protein levels. Thus, understanding the role of ubiquitination, eg, in immune function, increases our understanding of cell biology, which should have relevance, for example, in malignant transformation.

In addition, the expansion of normally resting B cells (also called “B lymphocytes”) involves two distinct steps. First, resting cells pass is activated from G ₀ of the cell cycle into G ₁ phase. For example, Alberts et al. (Eds., 1989) Molecular.
Biology of the Cell Garland Publ. , NY
And Darnell et al. (1990) Molecular Cell Bio.
See logic Freeman, NY. The activated cells are then induced to proliferate. For example, Paul et al. (1989) Fundamental Im
Munology, 2nd edition, Raven Press, NY; and 3rd edition. Interleukin-1 (IL-1), IL-2, IL-4, IL-1
A number of factors have been identified that induce B cell proliferation, including 0, and IL-13. In addition, antibodies to certain B cell surface molecules have been shown to promote B cell proliferation. T cells (also called "T lymphocytes") are also induced to proliferate by certain factors. It is phytohemagglutinin,
Includes anti-T cell receptor monoclonal antibodies, anti-CD3 monoclonal antibodies, and other agents.

B7 (CD80) and B70 (CD86) are a second “group” of molecules that strongly mediate B and T cell interactions. These molecules on B cells are
Interacts with its ligands CD28 and CTLA-4 on cells. These interactions are the major costimulatory signals of activation of both B and T cells.

During the last 15 years, it has been shown that B7 (CD80) and B70 (CD86) play a fundamental role in T-cell and B-cell activation. Numerous in vitro and in vivo experiments have shown that these two pairs of molecules are important targets for immunosuppression. For example, Banchereau et al. (1994)
Ann. Rev .. Immunol. 12: 881-922; van Koote
n et al. (1996) Adv. Immunol. 61: 1-77; Linsley and Ledbetter (1993) Ann. Rev .. Immunol. 11:
See 191-212.

In 1995, another molecule called RP105 was cloned from mouse spleen cells. Miyake et al. (1995) J. Am. Immunol. 154: 33
See 33-3340. Monoclonal antibodies to RP105 also induce potent proliferation of mouse B cells and, in a similar manner to anti-CD40 antibodies or CD40 ligand, protect mouse B cells from irradiation-induced apoptosis.
Miyake et al. (1994) J. Am. Exp. Med. 180: 1217-1224
checking ...

The RP105 molecule and its ligand MD-1 may be an additional pair of molecules that play an important role in T and B cell activation. Miyake et al. (
1998) J. Immunol. 161: 1349-1353; and Chan
(1998) J. Am. Exp. Med. 188: 93-101. However, the human sequence of MD-1 remained undetermined. The present invention provides this, and also provides a previously undescribed second human homolog of mouse MD-1 (ie, MD-2).

A number of factors have been identified that affect the differentiation process of precursor cells or that modulate the physiology or migratory properties of particular cell types. These observations indicate that there are other factors whose function in immune function was not previously recognized. These factors provide a biological activity whose range of effects may differ from known differentiation or activators. Lack of knowledge about the structural, biological, and physiological properties of regulators that regulate cell physiology in vivo prevents the regulation of the effects of such factors. Thus, medical conditions that require regulation of the development or physiology of the cells involved remain out of control.

Thus, significant therapeutic needs exist in the areas of cytokine regulation of physiology, proteolysis, and B cell signaling. The present invention provides important insights and developments in these areas.

[0025] Cancer can occur in many tissues of the body. It occurs by changes in certain cells that allow them to escape normal growth-limiting mechanisms, for example, after a given number of such cells have developed, usually escape feedback control that stops cell growth and replication. Cell division and transcription are highly coordinated processes that play important roles in this feedback control. For example, Beeso
(eds., 1979) Textbook of Medicine, 15th edition,
W. B. Saunders Co. , Philadelphia, PA; DeV
(ita, 1997) Cancer: Principles and Pr
actice of Oncology, 5th edition, Lippincott, Ph
iladelphia, PA; Neal and Hoskin (1997) Cli.
natural Oncology: Basic Principles and
Practice Oxford University Press, NY;
Kastan (1997) Checkpoint Controls and
Cancer CSH Press, NY; and Thomas (eds, 1996)
) Apoptosis and Cell Cycle Control in
Cancer: Basic Mechanisms and Implica
Tions for Treating Maligant Disease
See BIOS Scientific, Oxford.

[0026] Molecules that function to regulate cell division play an important role in controlling the growth of various types of cells. Deviations in these controls can lead to various disease states, such as tumorigenesis, inappropriate wound healing, developmental abnormalities, and metabolic problems.

The cell cycle can be divided into four phases: a pre-synthesis phase (G ₀ and G ₁ ); a DNA synthesis phase (S); and a post-synthesis phase (G ₂ ). For example, Guyton (ed., 1976)
Textbook of Medical Physiology, 5th edition, W
. B. Saunders Co. , Philadelphia, PA; Albe
rts et al. (eds., 1994) Molecular Biology of the
Cell, Third Edition, Garland Publishing, New York
k, NY; and Darnell et al. (eds., 1990) Molecular Ce.
II Biology, 2nd edition, W.W. H. Freeman, New York,
See NY. Effective chemotherapeutic agents often target pathological cells in the S phase, such as choriocarcinoma, acute lymphocytic leukemia, lymphatic lymphosarcoma, Burkitt's lymphoma, Hodgkin's disease, testicular neoplasm, Wilms tumor, and Ewing sarcoma It is assumed that. Unfortunately, tumorigenic cells that are not actively dividing are less sensitive to these drugs.

[0028] Lack of knowledge of cell cycle regulation hinders the medical science's ability to specifically regulate cell division or the immune response. The present invention provides compositions that are important in regulating cell division and transcription.

SUMMARY OF THE INVENTION The present invention provides, in part, DC-PGT, HDTEA84, HSLJD37R,
RANKL, HCC5 chemokine, Dub11, Dub12, MD-1, MD-
2, and characterization of genes and products related to cyclin E2. The invention provides nucleic acids, polypeptides, antibodies, and methods of making and using such compositions.

In a DC-PGT embodiment, the present invention provides an isolated or recombinant antigenic polypeptide. The peptide comprises a plurality of discrete portions, wherein each segment is at least 12 contiguous amino acids from mature SEQ ID NO: 2;
Or has identity to at least 17 contiguous amino acids from mature SEQ ID NO: 2. In some embodiments, the plurality of segments comprises one of at least 19 contiguous amino acids; or two of at least 15 contiguous amino acids. Other polypeptides include the mature SEQ ID NO: 2; polypeptides that specifically bind to a polyclonal antibody that specifically binds to SEQ ID NO: 2; or polypeptides that are natural allelic variants of SEQ ID NO: 2 A polypeptide that is at least 30 amino acids in length; a polypeptide that exhibits at least two non-overlapping epitopes specific to SEQ ID NO: 2; a polypeptide that is a synthetic polypeptide; a polypeptide that binds to a solid substrate; Polypeptides that are 5 or less conservative substitutions. Also provided is a fusion protein comprising, for example, a first and second portion. First
The second part contains a sequence as described, and the second part contains a detectable marker. For example, pharmaceutical compositions comprising a sterile polypeptide as described in a pharmaceutically acceptable carrier are available.

[0031] Embodiments of the polynucleotide include isolated or recombinant polynucleotides that encode the desired polypeptide. A preferred format is a polynucleotide comprising a portion encoding the mature polypeptide of SEQ ID NO: 1; or a polynucleotide such as that encoding mature SEQ ID NO: 2. Preferred embodiments include a polynucleotide that is a PCR product; a polynucleotide that is a hybridization probe; a polynucleotide that is a mutagenic primer; or a polynucleotide made by chemical synthesis. Alternatively, the polynucleotide is detectably labeled; is a deoxyribonucleic acid; or is double-stranded. Also provided are expression vectors comprising the described polynucleotides, including those in which the polypeptide specifically binds to a polyclonal antibody generated against the mature SEQ ID NO: 2 immunogen. It specifically hybridizes under stringent hybridization conditions to a target polynucleotide sequence having at least 60 contiguous nucleotides from SEQ ID NO: 1; at least 50 contiguous amino acid residues from mature SEQ ID NO: 2 Or suitable for transfection into prokaryotic or eukaryotic host cells. Preferably, the host cell is a mammalian cell; a bacterial cell; an insect cell; a prokaryote; a eukaryote;
S cells. For example, there is provided a method of producing a polypeptide comprising expressing a vector in a host cell.

The other polynucleotide is at least 50 ° C., a salt concentration of less than 400 mM,
And an isolated or recombinant polynucleotide that hybridizes to the coding portion of SEQ ID NO: 1 under stringent hybridization and washing conditions of 50% formamide. Such a nucleic acid that can 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 can be an expression vector. Preferably, the vector encodes a polypeptide that specifically binds an antibody raised against mature SEQ ID NO: 2. Another embodiment is a polynucleotide that hybridizes to SEQ ID NO: 1. Here, the polynucleotide is a PCR product; a hybridization probe; a mutagenic primer; or produced by chemical synthesis.

For example, a method is provided for regulating the physiology or development of a cell. The method comprises the steps of contacting a cell with an agonist or antagonist of a described polypeptide; detecting the presence of a complementary polynucleotide in the sample, which selectively binds to the complementary polynucleotide in the sample. Contacting the specifically hybridized described polynucleotide to form a detectable duplex; thereby indicating the presence of the polynucleotide in the sample; or binds to the described polypeptide. Incubating a component comprising the compound and the polypeptide under conditions sufficient to allow the component to interact; and measuring the binding of the compound to the polypeptide to identify the compound; The step of performing

[0034] In a TNF receptor-like embodiment, the present invention further provides an isolated or recombinant polynucleotide encoding an antigenic polypeptide comprising at least 17 contiguous amino acids, derived from: A mature polypeptide from SEQ ID NO: 8; a mature polypeptide from SEQ ID NO: 10;
A mature polypeptide from SEQ ID NO: 12; a mature polypeptide from SEQ ID NO: 17;
A mature polypeptide from SEQ ID NO: 19; a mature polypeptide from SEQ ID NO: 21;
Or a mature polypeptide derived from SEQ ID NO: 23. In a preferred embodiment, such a polynucleotide is one in which the signal is processed (signal pro
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: 2
1; or encodes all of the polypeptides of SEQ ID NO: 23. Other embodiments include polynucleotides that hybridize with less than 500 mM salt and 50% formamide at 55 ° C. for: a mature protein-encoding portion of SEQ ID NO: 5; the signal of SEQ ID NO: 7 has been processed Coding part; signal part of the processed signal of SEQ ID NO: 9; coding part of the processed signal of SEQ ID NO: 11; mature protein coding part of SEQ ID NO: 16; SEQ ID NO: 18
The polypeptide coding portion of SEQ ID NO: 20; or the polypeptide coding portion of SEQ ID NO: 22. Other formats include a polynucleotide comprising at least 35 contiguous nucleotides of the following: the mature protein coding portion of SEQ ID NO: 5; the coding portion where the signal of SEQ ID NO: 7 is processed; the signal portion of SEQ ID NO: 9 is processed Coding part; signal processed part of SEQ ID NO: 11; mature protein coding part of SEQ ID NO: 16; polypeptide coding part of SEQ ID NO: 18; polypeptide coding part of SEQ ID NO: 20;
Or the polypeptide coding portion of SEQ ID NO: 22. Various expression vectors containing such polynucleotides are provided. The invention also provides a host cell comprising the expression vector, including a eukaryotic cell.

For example, provided is a method of making an antigenic polypeptide comprising expressing a recombinant polypeptide; for the step of detecting a polynucleotide, the polynucleotide is prepared under stringent conditions. Below, the following: the mature protein coding portion of SEQ ID NO: 5; the signal processed portion of SEQ ID NO: 7; the signal processed portion of SEQ ID NO: 9; the processed portion of the signal of SEQ ID NO: 11; A probe that hybridizes to at least 25 contiguous nucleotides of the mature protein coding portion of SEQ ID NO: 16; the polypeptide coding portion of SEQ ID NO: 18; the polypeptide coding portion of SEQ ID NO: 20; or the polypeptide coding portion of SEQ ID NO: 22. Contacting to form a duplex Include. Here, detection of this duplex indicates the presence of the polynucleotide. For example, for detection of the described polynucleotides, include a probe that hybridizes under stringent hybridization conditions to at least 17 contiguous nucleotides of the described polynucleotides to form a duplex. A kit comprising a compartment is provided. Preferably, the probe is detectably labeled.

[0036] Binding compounds including antibodies are provided. The binding compound comprises an antibody binding site that 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 Is processed; SEQ ID NO: 10; signal is processed; SEQ ID NO: 12; signal is processed, SEQ ID NO: 17;
SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO: 23. Preferably, the antibody binding site comprises the following: 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: 1
7; SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO: 23; selectively immunoreactive; purified or raised against recombinantly produced human HDTEA84 protein; purified Or recombinantly produced human HS
Raised against the LJD37R protein; or in a monoclonal antibody, Fab, or F (ab) 2; or, the binding compound is an antibody molecule; a polyclonal antiserum; Is labeled;
Sterile; or is present in a buffered composition.

Such compositions allow for a variety of methods, including using the binding compounds described above. The method includes contacting the binding compound with a biological sample containing the antigen, thereby forming a binding compound: antigen complex. Preferably, the biological sample is from a human, and the binding compound is an antibody. It also allows for the generation of a binding kit and instructions for use of the binding compound for detection; or a detection kit comprising a compartment that provides for separation of the binding compound.

[0038] Polypeptides, such as substantially pure or isolated antigenic polypeptides, will also be available. The polypeptide binds to the binding composition described,
And further comprising at least 17 contiguous amino acids from the following: signal processed SEQ ID NO: 6; signal processed SEQ ID NO: 8
SEQ ID NO: 10 in which the signal was processed; SEQ ID NO: 12 in which the signal was processed; SEQ ID NO: 17 in which the signal was processed; SEQ ID NO: 19; SEQ ID NO: 21; A preferred polypeptide is the primate HDTEA
84 comprising at least a fragment of at least 25 contiguous amino acid residues from the protein; 84 comprising at least a fragment of at least 25 contiguous amino acid residues from the primate HSLJD37R protein; or a rodent or primate Comprising at least a fragment of at least 25 contiguous amino acid residues from a RANKL-like protein; or a soluble polypeptide; detectably labeled; present in a sterile composition; That bind to sialic acid residues; that are recombinantly produced; or that have a naturally occurring polypeptide sequence. In another embodiment, the polypeptide comprises at least 17 contiguous amino acids from the following: signal processed SEQ ID NO: 6; signal processed SEQ ID NO: 8; signal processed SEQ ID NO: 12; SEQ ID NO: 17; SEQ ID NO: 19; SEQ ID NO: 21; or SEQ ID NO: 23 where the signal was processed.

Methods are provided that include methods of modulating the physiology or function of precursor cells. The method comprises the steps of: binding a cell and a binding compound to the described polypeptide; HDTEA84
Contacting the polypeptide; an HSLJD37R polypeptide; or a RANKL polypeptide. In this method, the contacting step may be in combination with a TNF family ligand, or an antagonist of the TNF family ligand.

In another embodiment, the present invention provides a composition associated with another chemokine, Dub, or surface protein gene. Embodiments of the polypeptide include: a substantially pure or recombinant HCC5 polypeptide that exhibits identity over a length of at least 12 amino acids to SEQ ID NO: 25; Native sequence HCC5; fusion protein comprising HCC5 sequence; SEQ ID NO: 32
Or a substantially pure or recombinant Dub11 polypeptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 32 or 34;
An isolated native sequence of Dub11; a fusion protein comprising the Dub11 sequence; a substantially pure or recombinant Dub12 polypeptide exhibiting identity to SEQ ID NO: 36 or 38 over a length of at least about 12 amino acids; An isolated native sequence Dub12 of mature SEQ ID NO: 36 or 38; a fusion protein comprising the Dub12 sequence; substantially pure or recombinant, exhibiting identity to SEQ ID NO: 42 over a length of at least about 12 amino acids MD-1 polypeptide; isolated native sequence MD-1 of mature SEQ ID NO: 42; fusion protein comprising primate MD-1 sequence; identity to SEQ ID NO: 44 or 46 over a length of at least about 12 amino acids A substantially pure or recombinant MD-2 polypeptide; mature SEQ ID NO: 44 or 4 An isolated native sequence MD-2; a fusion protein comprising a primate MD-2 sequence; substantially pure or showing identity to SEQ ID NO: 48 or 49 over a length of at least about 12 amino acids. Recombinant MD-2 polypeptide; isolated native sequence MD-2 of mature SEQ ID NO: 48; or mouse MD-
A fusion protein comprising two sequences. Preferred embodiments are over a length of at least 17 amino acids; more preferably over a length of at least 21 amino acids;
Substantially pure or isolated polypeptides that match this sequence over 5, 30, 35, 50, 75 or more. In other preferred embodiments, the HCC5 polypeptide is: derived from a primate, including a human; comprises at least one polypeptide segment of SEQ ID NO: 25; exhibits a plurality of portions exhibiting identity; A natural allelic variant of at least about 30
Has an amino acid length; exhibits at least two non-overlapping epitopes specific for primate HCC5; exhibits sequence identity to HCC5 over a length of at least 35 amino acids; is glycosylated; A synthetic polypeptide; attached to a solid substrate; conjugated to another chemical moiety; no more than 5-fold substitution from the native sequence; or a deletion or insertion variant of the native sequence. Yes; or the Dub11 polypeptide is: derived from a primate, including a human; comprises at least one polypeptide segment of SEQ ID NO: 32 or 34; exhibits multiple portions exhibiting identity; Is a naturally occurring allelic variant; has a length of at least about 30 amino acids; Also exhibit two non-overlapping epitopes; show sequence identity to Dub11 over a length of at least about 35 amino acids; 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 the native sequence;
Or a deletion or insertion variant of the native sequence; alternatively, the Dub12 polypeptide is derived from a primate, including a human; comprises at least one polypeptide segment of SEQ ID NO: 36 or 38; Indicate multiple parts; D
is a natural allelic variant of ub12; has at least about 30 amino acids in length; exhibits at least two non-overlapping epitopes specific to primate Dub12; Displays sequence identity over length; is glycosylated; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; A substitution; or a deletion or insertion variant of the native sequence; or the primate MD-1 polypeptide is: human-derived; comprises at least one polypeptide segment of SEQ ID NO: 42; Exhibiting multiple portions of identity; being a natural allelic variant of primate MD-1; being at least about 30 amino acids long Or having, at least 2 that is specific for primate MD-1
Show three non-overlapping epitopes; at least about 35 for primate MD-1
Displays sequence identity over the length of the amino acid; is glycosylated; is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; Or a deletion or insertion variant of the native sequence; alternatively, the primate MD-2 polypeptide is: human-derived; at least one polymorph of SEQ ID NO: 44 or 46 Comprises peptide segments; exhibits multiple parts of identity; is a natural allelic variant of primate MD-2; has at least about 30 amino acids in length;
Exhibits at least two non-overlapping epitopes that are specific for primate MD-2; exhibits sequence identity to primate MD-2 over a length of at least about 35 amino acids; is glycosylated; A polypeptide; attached to a solid substrate; conjugated to another chemical moiety; 5-fold or less substitution from the native sequence; or a deletion or insertion variant of the native sequence. Alternatively, the rodent MD-2 polypeptide is: derived from a mouse; SEQ ID NO: 48;
Or comprising at least one polypeptide segment of 49; displaying multiple portions of identity; being a natural allelic variant of rodent MD-2; having a length of at least about 30 amino acids Exhibits at least two non-overlapping epitopes that are specific for rodent MD-2; exhibits sequence identity to rodent MD-2 over a length of at least about 35 amino acids; Is a synthetic polypeptide; is attached to a solid substrate; is conjugated to another chemical moiety; is a 5-fold or less substitution from the native sequence; or is a deletion or insertion of the native sequence It is a variant. Sterile compositions comprising such polypeptides are also provided with those containing: HCC5 polypeptide and the following: a carrier, wherein the carrier is: water, saline and / or Aqueous compounds including buffers; and / or carriers, formulated for oral, rectal, nasal, topical or parenteral administration; HCC1, HCC2
Another chemokine comprising a chemokine selected from the group of HCC3 and HCC4; or an antibody antagonist to a chemokine comprising a chemokine selected from the group of HCC1, HCC2, HCC3 and HCC4;
1 polypeptide and a carrier, wherein the carrier is: water,
Aqueous compounds including saline and / or buffers; and / or orally,
A carrier, formulated for rectal, nasal, topical or parenteral administration; the Dub12 polypeptide and carrier, wherein the carrier is water, saline and / or buffer. An aqueous compound comprising: and / or a carrier formulated for oral, rectal, nasal, topical or parenteral administration; the MD-1 polypeptide and carrier, wherein the carrier comprises: The following: aqueous compounds including water, saline and / or buffers; and / or formulated for oral, rectal, nasal, topical or parenteral administration;
A carrier; said MD-2 polypeptide and a carrier, wherein said carrier is an aqueous compound comprising water, saline and / or buffer; and / or oral, rectal, nasal, topical or A carrier that is formulated for parenteral administration.

For example, the following: 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; 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 comprising a FLAG, His6, or Ig sequence;
Or a sequence of another chemokine protein having a chemokine polypeptide is provided. For example, the described polypeptides and:
A kit comprising a compartment containing the polypeptide; and / or instructions for using and disposing of the reagents in the kit is provided.

For example, a binding compound comprising an antibody is provided that comprises an antigen-binding portion derived from the antibody. The binding compound is the following: an HCC5 polypeptide, wherein the antibody is raised against the peptide sequence of the mature HCC5 polypeptide sequence of SEQ ID NO: 25; Raised against purified HCC5; immunoselected; polyclonal antibody; binds to denatured HCC5; or exhibits a Kd for the HCC5 antigen of at least 30 μM; Wherein the antibody is raised against the peptide sequence of the mature Dub11 polypeptide sequence of SEQ ID NO: 32 or SEQ ID NO: 34; whether it is raised against the mature Dub11; Elicited; immunoselected; polyclonal antibody ; Binds to denatured Dub11; or at least 30
the Kd for the Dub11 antigen in μM; or a Dub12 polypeptide, wherein said antibody is the mature Du of SEQ ID NO: 36 or SEQ
has been raised against the peptide sequence of the b12 polypeptide sequence; mature Dub
12; raised against purified Dub12; immunoselected; polyclonal antibody; binds to denatured Dub12; Or a primate MD-1 polypeptide, wherein said antibody is: SEQ ID NO: 42
Has been raised against the peptide sequence of the mature polypeptide sequence of the mature MD-
1, raised against purified MD-1; immunoselective; polyclonal antibody; bound to denatured MD-1; or at least 30 μM of MD-1 Indicates the Kd for the antigen; or MD
-2 polypeptide, wherein the antibody is: SEQ ID NO: 44 or SEQ ID NO: 4
6 against the peptide sequence of the mature MD-2 polypeptide sequence;
Raised against mature MD-2; raised against purified MD-2; immunoselected; polyclonal antibody; binds to denatured MD-2; or at least 30 μM MD The Kd against the -2 antigen; or a rodent MD-2 polypeptide, wherein said antibody is: SEQ ID NO: 4
8 or the mature MD-2 polypeptide sequence of SEQ ID NO: 49; raised against a mature rodent MD-2; raised against a purified rodent MD-2. Has been selected; is immunoselected; is a polyclonal antibody; binds to denatured rodent MD-2;
It specifically binds to its natural counterpart, showing a Kd for the M antigen. In certain embodiments, the binding composition comprises: whether the polypeptide is from a primate or rodent; whether the binding compound is an Fv, Fab, or Fab2 fragment;
Whether the binding compound is conjugated to another chemical moiety; is attached to a solid substrate, including beads or plastic membrane; is in a sterile composition;
Or a binding composition that is detectably labeled, including a radioactive or fluorescent label.

Provided are a binding compound, and a kit comprising: a compartment containing the binding compound; a compartment containing the purified antigen; and / or instructions for using or discarding the reagents in the kit. Provided is a method of generating an antigen: antibody complex, comprising: an antibody;
The following: primate HCC5 polypeptide; primate Dub11 polypeptide; primate D
contacting a ub12 polypeptide; a primate MD-1 polypeptide; a primate MD-2 polypeptide; or a rodent MD-2 polypeptide, thereby allowing formation of a complex. Other compositions, such as a binding compound and a carrier, wherein the carrier is an aqueous compound comprising water, saline, and / or a buffer; and / or oral, rectal, intranasal, topical, Or a carrier formulated for parenteral administration; or HCC1, HCC2, H
An antibody antagonist to another chemokine is provided, including a chemokine selected from the group of CC3, and HCC4.

Embodiments of the nucleic acids include, for example, isolated or recombinant nucleic acids encoding the described polypeptides or fusion proteins. Wherein the HCC5 is: the polypeptide is from a primate, including a human; or the nucleic acid encodes the following: an antigenic HCC5 peptide sequence of SEQ ID NO: 25;
Encoding a C5 peptide sequence; native cDN encoding an HCC5 segment
A; at least 25 nucleotides identical to A; a hybridization probe to a gene encoding an HCC5 polypeptide; or another chemokine, including a chemokine further selected from the group of HCC1, HCC2, HCC3, and HCC4. Encodes; or Dub11: the polypeptide is from a primate, including a human; or the nucleic acid is: SEQ ID NO: 3
Encodes the Dub11 antigenic peptide sequence of SEQ ID NO: 2 or SEQ ID NO: 34; encodes the multiple antigenic peptide sequences of SEQ ID NO: 32 or SEQ ID NO: 34; Dub
Exhibits identity over at least 25 nucleotides to the native cDNA encoding the 11 segment; or is it a hybridization probe to the gene encoding the Dub11 polypeptide; and if Dub12 is: the polypeptide is derived from a primate, including humans; Or the nucleic acid encodes the following: an antigenic Dub12 peptide sequence of SEQ ID NO: 36 or SEQ ID NO: 38; a plurality of antigenic peptide sequences of SEQ ID NO: 36 or SEQ ID NO: 38; a natural cDNA encoding a Dub12 segment A hybridizing probe to a gene encoding a Dub12 polypeptide; or a primate MD-1: wherein the polypeptide is from a primate, including a human. Or the nucleic acid encodes the following: an antigenic MD-1 peptide sequence of SEQ ID NO: 42; a plurality of antigenic peptide sequences of SEQ ID NO: 42; a native cDNA encoding an MD-1 segment and at least 25 Whether it shows identity over nucleotides; is a hybridization probe to a gene encoding an MD-1 polypeptide; or is primate MD-2: is the polypeptide derived from a human; or is a nucleic acid has the following sequence: Encoding the antigenic MD-2 peptide sequence of SEQ ID NO: 44 or SEQ ID NO: 46; encoding multiple antigenic peptide sequences of SEQ ID NO: 44 or SEQ ID NO: 46; Show identity over at least 25 nucleotides; primate MD-2 polypeptide Whether a hybridization probe for a gene encoding; or rodent MD-2 is: or polypeptide is derived from mice; or nucleic acid, the following: SEQ ID NO: 48 or SEQ ID NO: 4
9 encodes an antigenic MD-2 peptide sequence; 9 encodes a plurality of antigenic peptide sequences of SEQ ID NO: 48 or SEQ ID NO: 49; has identity over at least 25 nucleotides with a native cDNA encoding an MD-2 segment. Shown; or a hybridization probe to a gene encoding a rodent MD-2 polypeptide. Other nucleic acid embodiments are expression vectors; further comprising an origin of replication; derived from natural sources; containing a detectable label; containing a synthetic nucleotide sequence; less than 6 kb, preferably 3 kb Less than; from a primate, including a human; including a native full-length coding sequence; or comprising a described nucleic acid that is a PCR primer, PCR product, or mutagenic primer.

Various cells are provided, including cells or tissues containing the described recombinant nucleic acids. Here, the cells include cells that are: prokaryotic cells; eukaryotic cells; bacterial cells; yeast cells; insect cells; mammalian cells; mouse cells; primate cells;

For example, the described nucleic acids, and the following: compartments containing the nucleic acids; HCC1, HCC2,
A compartment containing nucleic acids encoding HCC3 and another chemokine, including HCC4;
Or instructions for using or disposing of the reagents in the kit.

The alternative nucleic acid hybridizes to the polypeptide-encoding portion of SEQ ID NO: 24 under wash conditions at 45 ° C. and less than 2M salt; Hybridizes to the coding portion of the polypeptide of SEQ ID NO: 33 or under a washing condition of less than 2M salt; hybridizes to the coding portion of SEQ ID NO: 35 or 37; Hybridizes to the coding part of SEQ ID NO: 41; under hybridizing conditions at 45 ° C. and less than 2M salt; or hybridizes to the coding part of SEQ ID NO. 43 or 45; , Including a nucleic acid that hybridizes to the coding portion of SEQ ID NO: 47. Preferably, the washing conditions are 55 ° C and / or 50 ° C.
0 mM salt concentration, or 65 ° C. and / or 150 mM salt concentration.

Further, for example, the physiology or development of cells or tissue culture cells comprising exposing the cells to an agonist or antagonist of HCC5, primate MD-1, primate MD-2, or rodent MD-2. A method for adjusting is provided. Other methods include contacting the compound with a composition selected from the group consisting of: then detecting specific binding to the compound, comprising detecting the binding of the composition to the compound. Detection methods include: an antigen-binding site that specifically binds to HCC5 chemokine; an antigen-binding site that specifically binds to Dub11; Dub12.
Binding site that specifically binds to primate MD-1; antigen binding site that specifically binds to primate MD-2; specifically binding to rodent MD-2 An antigen binding site to bind; or an expression vector encoding HCC5 chemokine or a fragment thereof; an expression vector encoding Dub11 or a fragment thereof; an expression vector encoding Dub12 or a fragment thereof; an expression encoding primate MD-1 or a fragment thereof An expression vector encoding a primate MD-2 or a fragment thereof; or an expression vector encoding a rodent MD-2 or a fragment thereof; a substance specifically recognized by the antigen-binding site of the antigen-binding site described. Pure protein; substantially pure H C5 chemokine or a fusion protein comprising the 30 amino acid sequence portion of the peptide, or HCC5 chemokine sequences; substantially pure D
ub11 or a peptide thereof, or a fusion protein comprising a 30 amino acid sequence portion of the Dub11 sequence; a substantially pure Dub12 or a peptide thereof, or a fusion protein comprising a 30 amino acid sequence portion of the Dub12 sequence; a substantially pure primate MD- A fusion protein comprising one or a peptide thereof, or a 30 amino acid sequence portion of the primate MD-1 sequence; comprising substantially pure primate MD-2 or a peptide thereof, or a 30 amino acid sequence portion of the primate MD-2 sequence. A fusion protein; substantially pure rodent MD-2 or a peptide thereof, or rodent M
A fusion protein comprising a 30 amino acid sequence portion of the D-2 sequence;

[0049] Certain polynucleotide embodiments include the following isolated or recombinant polynucleotides: a polynucleotide encoding at least 17 contiguous amino acid residues of SEQ ID NO: 54; A polynucleotide encoding at least two different segments of at least 10 contiguous amino acid residues of SEQ ID NO: 54; or a polynucleotide comprising one or more segments of at least 21 contiguous nucleotides of SEQ ID NO: 53. Such polynucleotides allow for a method of producing: a polypeptide (including the steps of expressing the described expression vector to produce the polypeptide); a double-stranded nucleic acid (e.g., Contacting with a complementary nucleic acid to produce the double-stranded nucleic acid); synthetic polynucleotides (including chemically polymerizing nucleotides to produce the polynucleotide); Or a polynucleotide (including a step using a PCR method).

Embodiments of a cyclin polypeptide include an isolated or recombinant antigenic polypeptide comprising at least the following: one segment comprising at least 17 contiguous amino acids from SEQ ID NO: 54 Or SEQ ID NO: 5
At least two different segments of at least 11 contiguous amino acids from 4. Such a polypeptide may include at least one segment comprising at least 17 contiguous amino acids from SEQ ID NO: 54; and at least two non-overlapping epitopes that are selective for the primate protein of SEQ ID NO: 54 Can be indicated. Other embodiments include the following polypeptides: a polypeptide that is a 5-fold or less substitution from the native sequence; a polypeptide that is a deletion or insertion variant from the native sequence; Comprising at least two different segments of at least 11 contiguous amino acids from
Polypeptide. Preferably, the polypeptide is antigenic, and typically from SEQ ID NO: 54: KERYVHD (residues 120-127), DK
HFEVLH (residues 127-134), HSDLEPQM (residues 134-141)
), QKDINKNM (residues 177-184), YAPKLQEF (residues 203
-210), SEEDILRM (residues 219-226), LRMELIIL (residues 224-231), ELCPVTII (residues 237-244), and LFL
SEQ ID NO: 54 comprises at least one sequence from QVDAL (residues 249-256);
It comprises one such segment having at least 14 contiguous amino acids from the same. Such a polypeptide may exhibit at least two non-overlapping epitopes that are selective for the primate protein of SEQ ID NO: 54; and / or may include the mature sequence of SEQ ID NO: 2; Can selectively bind to an antibody raised against the original; can comprise a multiple polypeptide segment of 17 contiguous amino acids of SEQ ID NO: 54; or a natural allelic variant of SEQ ID NO: 54 Can be The polypeptide may be present in a sterile composition; may have a length of at least 30 amino acids; may be unglycosylated; can be modified; can be a synthetic polypeptide; Or a fusion protein with a detection or purification tag comprising the sequence of FLAG, His6 or Ig. Other embodiments include the following: an embodiment in which the polypeptide is a 5-fold or less substitution from the native sequence, or a polypeptide is a deletion or insertion variant from the native sequence. Embodiment.

A variety of kits are provided, including, for example, such polypeptides and instructions for use or disposal of the polypeptides or other reagents of the kit.

A method for labeling the polypeptide, including, for example, labeling the polypeptide with a radioactive label; flowing the mixture through a chromatography matrix, thereby separating the polypeptide. A method for separating said polypeptide from another polypeptide in said compound; incubating said compound under appropriate conditions with said polypeptide, thereby binding said component to said polypeptide A method for identifying a compound that selectively binds to the polypeptide, comprising the steps of: derivatizing the polypeptide with a reactive reagent, and binding a matrix to the polypeptide. The polypeptide is conjugated to the matrix. Or a method of inducing an antibody response to the polypeptide, comprising introducing the polypeptide as an antigen into the immune system, thereby inducing an antibody response.

For example, provided are binding compounds, including antibodies, that include an antigen-binding portion derived from an antibody that selectively binds to the described polypeptide. A method for assessing the selectivity of binding of a compound to cyclin E2 has been made available,
The method includes contacting the compound with a recombinant cyclin E2 polypeptide and at least one other cyclin; and comparing the binding of the compound to the plurality of cyclins.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS All references cited herein are set forth as if each individual publication or patent application was specifically and individually incorporated by reference. To the same extent, it is incorporated herein by reference.

I. Overview It should be understood that this invention is not limited to the particular compositions, methods, and techniques described herein, since such compositions, Methods and techniques can vary. The terms used in the specification are for the purpose of describing particular embodiments and are not intended to limit the scope of the invention, which is defined by the appended claims. It should be understood that it should be restricted.

As used herein, including the appended claims, “a”, “an”
Words such as "a" and "the" include their corresponding pluralities unless the context clearly dictates otherwise. Thus, for example, reference to "a polynucleotide" includes one or more different polynucleotides,
Reference to "composition" includes one or more such compositions, and reference to "method" includes reference to equivalent steps and methods known to those skilled in the art.
And so on.

Unless defined otherwise, technical and scientific terms used herein are:
It has the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references discussed above are provided for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the invention is not entitled to antedate any such disclosure by its prior art.

The invention also provides amino acid and DNA sequences that encode various mammalian proteins, for example, polypeptides produced by selected cells. Among these proteins are the following: proteins that mediate the uptake of substrates (eg, prostaglandin-like molecules), eg, regulate or mediate the transport, proliferation, or differentiation of interacting cells A protein that is a protein (eg, induces or prevents) or an intracellular protein that is important in various cellular processes (eg, protein deubiquitination or cell cycle regulation).

[0059] The prostaglandin-like transporter (PGT) of the present invention is specifically expressed in antigen-presenting cells (eg, dendritic cells) of the immune system. Thus, this transporter is a dendritic cell prostaglandin-like transporter (DC-P
GT). As a result, the DC-PGT of the present invention has a PG
Provide a means to establish a basic understanding of the role of impacts

The present invention provides DNA sequences encoding mammalian proteins that exhibit characteristic structures unique to functionally important proteins, particularly as organic anion transporters. Organic anion transporters of this family include: human (Lu et al. (1996) J. Clin. Invest. 98: 1.
142-1149) and rat prostaglandin transporters; human and rat organic anion transporters; rat brain digoxin transporter and Martin F / G (Kanai et al., (1995) Science).
268: 866-869).

The transporters of this family are typically 12 transmembrane proteins about 650 amino acids in length. Unique to this group of proteins are cysteine-rich regions located in one of the extracellular loops. This region is similar to the zinc finger motif. These polypeptides primarily mediate the influx or efflux of prostaglandins and organic anions, and they enter or exit under different circumstances, for example, depending on the intracellular concentration of the organic anion involved. Is not completely certain.

[0062] The DC-PGT proteins of the present invention have the highest homology to the prostaglandin transporter, and prostaglandin may be the major anion transported. The human gene embodiments described herein (designated DC-
PGT or clone 240) contains an open reading frame encoding a putative protein of about 709 amino acids. This protein represents an intracellular, transmembrane, and extracellular protein segment and is known for mammalian development (eg, the development of dendritic cells of the immune system)
Reveal new aspects of organic anion transport that may be involved during

The introduction of evolutionary information in the form of sequence homologs significantly simplifies structural analysis for related molecules that share a common structural backbone, despite considerable sequence diversity (eg, Chothia and Lesk ( 1986) EMBO
J. 5: 823-826). While this concept can be effectively extended to strong prediction of TM regions across aligned protein families, any single sequence can provide an uncertain topology. Persson and Argos (1
994). Mol. Biol. 237: 182-192; and Rost et al. (1995) Protein Sci. 4: 521-533. DC
For -PGT, many sequence homologs were first assembled by comparative matching against protein and translated nucleotide databases (Alt
Schul et al. (1994) Nature Genet. 6: 119-129; K
oonin et al. (1994) EMBO J. 13: 493-503). DC-PG
These related to T include ubiquitously expressed PGT from primates (eg, humans) (GenBank: locus HSU70867, accession number U70867)
), And PGT from rodents (e.g., rats) (prostaglandin transporter-rat, GenBank accession number 1083766; Kanai et al. (
1995) Science 268: 866-869). These sequences provide the first Kyte as a means to predict the topology of integral membrane proteins.
And Doolittle (1982) were subjected to a parallel analysis by a comprehensive software program of a computer program which was greatly improved on the hydrophobic hydrophilicity profile. Four algorithms (ALOM, MTOP, MEMSAT and Top)
PredII) (Klein et al., (1985) Biochim. Biophys).
. Acta 815: 468-476; Hartmann et al. (1989) Pro.
c. Nat'l. Acad. Sci. USA 86: 5786-5790; Jo.
nes et al. (1994) Biochem. 33: 3038-3049; and C
laros and von Heijne (1994) Comp. Applic.
Biosci. 10: 685-686) were used to predict TM stretch and direction individually; these predictions were pooled and ClustalW and MAC
AW (Thompson et al. (1984) Nucl. Acids Res. 22:
4673-4680; and Schuler et al. (1991) Proteins.
9: 180-190).
In addition, these multiple aligned sequence files were used as inputs to PHD and TMAP for family prediction of shared TM regions (Ros
(1995) Protein Sci. 4: 521-533; Persso
n and Argos (1994) J. Am. Mol. Biol. 237: 182-19
2). The remaining structural feature in this two-step analysis appears to be a shared topological trait present in all members of the organic anion transporter family.

Also provided are HDTEA84, HSLJD37R and RANKL genes and proteins, which are involved in the TNF signaling pathway. Antigen HDT
EA84, HSLJD37R and RANKL, and fragments or antagonists thereof, are useful, for example, in the physiological regulation of cells that express receptors for the TNF family of ligands. Some of these antigens appear to lack segments spanning the membrane, suggesting that they are soluble forms of the receptor. This suggests that soluble proteins may serve as antagonists of TNF-like ligands. In addition, there appears to be a transmembrane form that acts as a signaling receptor that mediates the cellular response to the ligand.

The HDTEA84 gene was detected in cDNA libraries from Hodgkin lymphoma, endothelial cells, keratinocytes, prostrate, and cerebellum. This is described in Racey et al. (1998) Cell 93: 165-17.
Osteoprotegerin reported by No. 6
Shows significant sequence similarity to the ligand receptor. HDTEA84 appears to regulate growth or development by antagonizing its respective ligand. There should be a membrane-bound form that is probably an alternatively spliced transcript.

HSLJD37R shows similar similarities to the receptor for TNF. The first embodiment is an incomplete sequence, but the available portion is currently lacking the identified transmembrane segment. Further efforts will provide full length sequences, and alternative splice variants.

The rodent 427152 # 4 Rank-like (RANKL)
# 4 (204 bp) was detected in a panel of rodent cDNA libraries probed. Positive signal is an intensifying screen
exposure to −80 ° C. for 3 days with CH12 (
Rag-1 thymus; rag-1 heart; rag-1 brain (maximal signal); rag-1 testis; rag-1 liver; normal lung; rag-1 lung; asthmatic lung; And challenged lungs; Nippo infected lungs; Nippo IL-4K
. O. Lung; Nippo anti-IL-5 treated lung; influenza lung; guinea pig allergic lung; t. Stomach; and w. t. Detected in the colon. Exposure to −80 ° C. using a screen for 2 weeks also detected signals in the following libraries: Mel14 + unstimulated; Mel14 + Th1; Mel14 + T
h2; Th1 3 weeks Bl / 6; large B cells; bEnd3 + TNFα + IL−
10; guinea pig normal lung; and Rag Hh-colon.

Primates (eg, humans) Rank-like (RANKL) of rodent 427152 # 4
The homolog is human cDN probed with mouse 427152 # 4 (204 bp).
Detected in panel A library. Exposure to −80 ° C. for 3 days using a screen detected signals in monkey asthmatic lung 4h (1.6-2.0 kb) and adult placenta (2.5-3.0 kb). Using a screen
Two weeks of exposure at 80 ° C. also detected signals in the following libraries: CD1a + 95% DC-activated CHA (renal epithelial cell carcinoma cell line)
Monkey normal lung; psoriatic skin; fetal lung; fetal ovary; fetal testis; and fetal spleen.

Each of these proteins is also useful as an antigen (eg, an immunogen) for raising antibodies to various epitopes on the protein, linear and / or conformational epitopes. The molecule may be useful in defining various cell subsets, either by the molecule produced, or by the expression of a membrane form of the receptor. Such cells should respond to the respective ligand. The soluble form of the receptor should act as an antagonist of the ligand, thereby binding the ligand and preventing interaction with the membrane form that mediates signal transduction.

Both genes express proteins that display structural motifs characteristic of members of the TNF receptor family. SEQ ID NO: 5 and SEQ ID NO: 6, respectively,
The nucleic acid sequence and the deduced amino acid sequence of a primate (eg, human) HDTEA84 are provided. SEQ ID NO: 7 and SEQ ID NO: 8 are each primate (eg, human) H
3 provides the nucleic acid sequence and deduced amino acid sequence of SLJD37R.

Interesting features of HDTEA84 include: a signal sequence from about 1-11; Cys-rich domain I of the TNF receptor (about 32-7
2), II (about 73-113), III (about 114-150), and IV (about 151-193); and unique regions from about 194-300. Features of HSLJD37R (SEQ ID NO: 10 form) based in part on alignment with HDTEA84: signal sequence from about 1-41; Cys-rich domain I (about 42-90), II (about 91-131) of TNF receptor ), III (about 132-168)
), And IV (about 169-211); transmembrane segments from about 354-370. Similar alignments of other variants identify similar features. Segments that include combinations of such segments or segments that exclude such segments may be desirable.

HDTEA84, a HSLJD against members of the TNF receptor family
The structural homology of 37R and RANKL suggests a related function of these molecules. See, for example, Racey et al. (1998) Cell 93: 165-176. However, these sequences largely lack transmembrane segments, suggesting that the protein is in soluble receptor form. They can also have a membrane-bound form resulting, for example, from alternatively spliced transcript variants. The soluble form appears to be, for example, an antagonist of the ligand that prevents binding of the ligand to the membrane-bound form of the signaling receptor. Thus, these molecules may be useful in treating abnormal immune or developmental disorders.

The natural antigen should be able to modulate various biochemical responses leading to a biological or physiological response in the target cell. The embodiments characterized herein are from primates (eg, humans), but other species variants are almost certainly present (eg, rodents, etc.). See below. The following description is for illustrative purposes only.
It relates to the primates HDTEA84, HSLJD37R or RANKL, but is equally applicable to related embodiments from other species.

The clones of HDTEA84, HSLJD37R and RANKL have been identified in various databases (eg, the Merck-WashU public database).
Assembled by careful analysis of ST. These genes exhibit structural motifs characteristic of members of the TNF receptor family. See, for example, TNF receptor, NGF receptor, and FAS receptor. Table 3 shows
Disclose the nucleic acid sequence and deduced amino acid sequence of primate (eg, human) HDTEA84. ESTs have been identified from several different libraries.

SEQ ID NO: 7 and SEQ ID NO: 8 are primate (eg, human) HSLJs, respectively.
Disclosed are the partial nucleic acid sequence and the deduced amino acid sequence of D37R. This EST was identified from several different libraries derived from: smooth muscle, pancreatic tumor, adipocytes, HUVEC cells, adult lung, endothelial cells, prostate cell line PC3, microvascular endothelial cells, fetal heart, And dendritic cells. Other sequences were detected in libraries derived from: multiple sclerosis lesions, breast, kidney, and germinal center B
Cells (germinal center B cells).

SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20 and SEQ ID NO: 22
The sequences of various mammalian genes designated L are provided.

Interesting features of rodent RANKL include: a signal sequence derived from about 1-29; a Cys-rich domain I of the TNF receptor (about 33-
74), II (about 75-114), and III (about 115-135). Interesting features of the primate RANKL include: C of the TNF receptor
ys-rich domains I (about 1-43), II (about 44-83), and III (
About 84-104); a transmembrane segment derived from about 139-155. Other TNF
Alignment with the receptor identifies further interesting, corresponding features. Segments with boundaries at these locations can be of particular interest.

The hybridization signal with RANKL was measured using a screen -8.
Three days of exposure at 0 ° C., using rodent (eg, mouse) sequences, were detected in: CH12 (B cell line); rag-1 thymus, rag-1 heart, rag-1 brain. (Strongest signal), rag-1 testis, rag-1 liver, normal lung, rag-1 lung, asthmatic lung, tolerized and challenged lung, Nippo
Infected lung, Nippo IL-4K. O. Lung, Nippo anti-IL-5 lung, influenza lung, guinea pig allergic lung, w. t. Stomach, and w. t. colon. Exposure to the screen at −80 ° C. for 2 weeks also detected signals in the following libraries: Mel14 + unstimulated; Mel14 + Th1; Me
114 + Th2; Th1 3 weeks Bl / 6; large B cells; bEnd3 + TNF
α + IL-10; guinea pig normal lung; and Rag Hh-colon. Probes of the human library with rodent sequences were screened for 3 h of monkey asthmatic lung (1.6-2.0 kb) and adult placenta (2.5 k
b-3.0 kb) provided a detectable signal. Exposure to −80 ° C. for 2 weeks using a screen also detected signals in the following libraries: CD1a + 95% DC-activated CHA (renal epithelial cell carcinoma cell line); monkey normal lung; psoriatic skin; Fetal lung; fetal ovary; fetal testis; and fetal spleen.

In another embodiment, the present invention provides chemokines. For a review of the chemokine family, see, eg, Lodi et al. (1994) Science 26.
3: 1762-1767; Greenborn and Clore (1991).
Protein Engineering 4: 263-269; Miller
And Kranger (1992) Proc. Nat'l. Acad. Sci.
USA 89: 2950-2954; Matsushima and Oppenh.
eim (1989) Cytokine 1: 2-13; Stoeckle and Baker (1990) New Biol. 2: 313-323; Openh
eim et al. (1991) Ann. Rev .. Immunol. 9: 617-648;
Schall (1991) Cytokine 3: 165-183; and T
homson (1994) The Cytokine Handbook 2
Edition, Academic Press, NY. checking ...

[0080] The new chemokine described herein is the named HCC5, a CC chemokine. See SEQ ID NO: 24 and SEQ ID NO: 25. The description relates to the described human HCC5 natural allele for illustrative purposes, but likewise allelic and / or polymorphic variants (eg, from other individuals) and splicing variants (eg, natural Form). Based on sequence analysis of the chemokine protein sequences described below, it is clear that HCC5 belongs to the CC chemokine family: for example, Kreider et al. (1997).
Patent WO 9715594 (SEQ ID NO: 26) and GenBank accession number 97
Stem cell mobilization from P-W17659 (stem cell mobilizin)
g) Chemokine (Ckβ-1); Adams et al. (1995) Patent WO95170
Macrophage inflammatory protein-1-γ (MIP-1) from N.92 (SEQ ID NO: 27) and GenBank accession number 95P-R76128; Adams et al.
997) Leukocyte chemoattractant human MIP-4 from patent WO 9634891 (SEQ ID NO: 28) and GenBank accession number 96P-W07203; Ba
ndman et al., Patent WO 9616997 (SEQ ID NO: 29) and GenBank
pituitary-expressed chemokine (PGEC) from k accession number 96P-R95691; and Forsmann et al. (1998) patent WO9741230 (SEQ ID NO: 3).
0) and human chemokine H from GenBank accession number 97P-38171
CC-1.

The HCC5 chemokine was discovered through database sequence searches and careful analysis. The HCC5 sequence was derived from cDN from pooled bulk breast tumor tissue.
Found in the A library. The absence of overlapping sequences from other sources suggests highly specific tissue expression, or highly regulated expression. Amino acid homology analysis suggests that the HCC5 gene encodes a large group of related families of chemokines. Primate (eg, human) HCC5 chemokines are most closely related to sequences for the following chemokines: human chemokine HCC
1. Human pituitary-expressed chemokine (PGEC); human MIP-4 (a leukocyte chemoattractant); human macrophage inflammatory protein-1-γ (MIP-1γ); and human stem cell mobilized chemokine (CKβ-1).

The HCC5 chemokine is apparently specifically expressed. Because the sequence was not found in many sources. Structural similarity to other chemokines suggests that this mediates the importance of signals in inflammation, cell differentiation and development.

HCC5 may in fact be one, some or all antagonists of many related chemokines. In such cases, a combination composition may be desired. For example, a combination group of functional agonists and antagonists for a particular receptor may be referred to, for example, as a combination of other chemokines and antibody antagonists. In addition, HCC5 may be useful in blocking HIV or HTLV infection, which viruses can use their respective receptors for infection.

This HCC5 chemokine shows limited similarity to known parts of the chemokine. For example, Matsushima and Openheim (1989).
Cytokine 1: 2-13; Openheim et al. (1991) Ann.
Rev .. Immunol. 9: 617-648; Schall (1991) Cy.
token 3: 165-183; and Gronnborn and C
lore (1991) Protein Engineering 4: 263-
269. Other comparative features are evident between the HCC5 chemokine and the chemokine family. For example, Lodi et al. (1994) Science
263: 1762-1766. In particular, β-sheet and α-helical residues can be determined, for example, using the RASMOL program (Sayle and Milner-White (1995) TIBS 20: 374-376;
Or Greenenberg et al. (1991) Protein Engineer.
ing 4: 263-269; and other structural features can be found in Lodi
(1994) Science 263: 1762-1767. These secondary and tertiary structural features, along with the appropriate cysteine residue spacing, help to further define the structural features of C, CC, CXC and CX3C.

An antagonist may be generated by any truncation of the N-terminal modification, eg, the addition of an N-terminal methionine. Since HCC5 is structurally related to HCC chemokines, it may well exhibit similar behavior and function.

In particular, the distribution of HCC5 chemokines in dendritic cells or in Th1 T cells, B cells, and macrophages suggests a role in immune function (eg, it seems to attract T cells and monocytes) . Thus, HCC5 chemokines appear to recruit these cell types in vivo and thereby enhance the immune response mediated by these cell types. The expression pattern is
Appears in TH1 / TH2 regulation and / or response consistent with the functional importance of the ligand (including, for example, in cancer therapy). Thus, ligands and homologs are not only identified as potential immune adjuvants, eg, for cellular responses, but also as potential adjuvants for modulating soluble antigen responses (eg, vaccines). Is done.

The present invention further provides mammalian (eg, primate) DNA sequences that encode proteins that exhibit structural features, such as protein enzymes that deubiquitinate in cells. These proteins are named deubiquitinated 11 (Dub11) and deubiquitinated 12 (Dub12). For a review of the superfamily of deubiquitinase enzymes, see, for example, Hochstrasser (1995).
) Curr. Opin. Cell Biol. 7: 215-223; Wilki
nson et al. (1995) Biochemistry 34: 14535-145.
46; Baker et al. (1992) J. Mol. Biol. Chem. 267: 23364
~ 23375; and Papa and Hochstrasser (1993) N
ature 366: 313-319. However, deubiquitinase has also been reported to have additional functions in addition to deubiquitination. For example, Hochstrasser (1996) Cell 84: 813-8.
15; Hicke and Riezman (1996) Cell 84: 277-
287; and Chen et al. (1996) Cell 84: 853-862.

The description typically relates to the natural alleles of human Dub11 and human Dub12 described for illustrative purposes, but also, for example, alleles and / or polymorphisms from other individuals As well as splice variants (eg, natural forms) and species variants from other primates or other species. These genes allow for the isolation of genes in other primates that encode proteins in this regard, and further extend the family beyond the specific examples described.

The Dub11 or Dub12 protein (naturally occurring or recombinant), fragments thereof, and antibodies thereto (Dub11 or Dub1)
UBE3A (E6) encoding aberrant physiology or development (eg, uterine cancer associated with p53 dysregulation associated with human papilloma virus, ubiquinone disubiquitination protein), with compounds identified as having binding affinity for -AP) Prader-Willi syndrome (Angelman syndrome (AS)) due to disruption of the 5 'end of the gene
Mental retardation). Pharmacological interventions that alter the half-life of cellular proteins involved in these diseases can have broad therapeutic potential. Specifically, human papillomavirus positive tumors and possibly wild-type p5
P53 in all tumors that carry 3 but lack retinoblastoma gene function
Interference with ubiquitination (and subsequent degradation) can lead to programmed cell death. Furthermore, specific inhibitors of p27 and cyclin B ubiquitination are expected to be potent antiproliferative factors. Inhibitors of IKB ubiquitination should inhibit NFKB activation and may have utility in various autoimmune and inflammatory conditions. Finally, deubiquitinase may be a new, potential drug target. Because they also seem to regulate cell proliferation. These conditions or disease states can be modulated by appropriate therapeutic treatment using the deubiquitinated compositions provided herein.

Conversely, methods for blocking enzyme activity should have the opposite effect.
Small molecule drug screens to block the enzymatic activity of the protein can be performed to identify substances that block deubiquitination and, as appropriate, affect the proteolytic pathway.

The T-cell growth factor interleukin-2 (IL-2) regulates lymphocyte proliferation by inducing the expression of growth-promoting genes. HTLV-1 transformed cell lines (derived from adult T-cell leukemia (ATL)) are IL-2 induced JA
It may show constitutive activation of the K / STAT pathway. Migone et al. (1998) P
rc. Nat'l Acad. Sci. USA 95: 3845-3850. ATL cell lines are tested for IL-2 inducible gene expression. The deubiquitinase Dub2 was found to be constitutively expressed. Zh
u et al. (1997) J. Am. Biol. Chem. 272: 51-57.
In addition, Dub2 expression conferred cytokine-independent growth on the interleukin-3-dependent mouse Ba / F3 hematopoietic cell line. SCID mice (n = 18) injected subcutaneously with Dub2-expressing Ba / F3 cells developed tumors with a 6-week incubation period (but not with inactivated mutants of Dub2 CS). Cells from these tumors show constitutive tyrosine phosphorylation of STAT5 and also exhibit ATL down-regulation of protein tyrosine phosphatase SHP-1.
The cell line was mimicked. These findings indicate that when Dub12 is constitutively expressed,
Strongly indicates that it is an oncogene, which can induce cytokine-independent proliferation in lymphocytes and may be involved in leukemia induction. Dub2 appears to regulate cell proliferation by modulating ubiquitin-dependent proteolysis or ubiquitin-dependent state of key intracellular substrates. Functional similarity of Dub11 and Dub12 is expected. Thus, the biological role of Dub2 suggests a similar important role for other Dub family members.

[0092] Screening for inhibitors of the DUB enzyme can also be easily accomplished by using known assays for activity. Such assays can be developed into high-throughput screening approaches, particularly testing compounds or similar enzyme sites known to affect protein turnover. Antagonists of small molecules of the enzyme, which are membrane permeable, are particularly desirable therapeutically.

In an MD embodiment of the present invention, for example, a mammal encoding a protein that exhibits structural features of a ligand for a protein that exhibits a leucine-enriched protein motif (LRR) that is important in immune function, eg, a mammal, eg, Primate and rodent (eg, mouse) DNA sequences are provided. These proteins are designated herein as human MD-1, human MD-2 and mouse MD-2. Human MD-1 is a previously described rodent MD-1 (eg, Miyak).
e, et al. (1998) J. Am. Immunol. 161: 1348-1353). On the other hand, human MD-2 and mouse MD-2 are newly discovered MD-1 homologs. For a general review of LRR proteins, see, eg, Kobe and Deisenhofer (1994) Trends.
Biochem. Sci. 19: 412. For the role of LRR in specific immune defense, see, for example, Jones et al. (1994) Science.
266: 789; Dixon et al. (1996) Cell 84: 451; and Baker et al. (1997) Science 276: 726.

[0094] Similar sequences for proteins in other species should also be available. The following description, by way of example, describes the primate (eg, human) M
D-1 and MD-2, as well as native alleles of rodent (e.g., mouse) MD-2, as well as allelic and / or polymorphic variants (e.g., from other individuals). ) And splice variants (eg, natural forms)
And species variants.

The MD-1 or MD-2 protein (naturally occurring or recombinant) fragments thereof and antibodies thereto are known as MD-1 or MD-2
Along with compounds identified as having a binding affinity for, should be useful in treating abnormal physiological phenomena or conditions associated with development (eg, recognition of certain pathogenic molecules and activation of B cell physiology). is there. As described above, MD-1
And MD-2 characterize the structural motif of the ligand for the RP105 or BAS-1 surface receptor. Thus, soluble forms, antibodies, or small molecule drugs that disrupt intracellular signaling mediated by these receptors find use in modulating cellular responses. These responses are important in normal or abnormal clinical conditions.

The conformity of MD and RP105 can also be easily tested. Identification of a counter receptor for MD-2, as described above, together with other screening methods,
Testing both the RP105 gene and the BAS-1 gene may be included. M
Possible counter-receptor structures for D are RP105, BAS-1 and related genes. Related proteins that bind to them, including DUB proteins, can be identified, inter alia, using these techniques.

[0097] Another aspect of the present invention provides members of the cyclin proteins. Cyclin-dependent kinase, a catalytic subunit of cyclin and its partners (
Cdk) plays a major role in regulating eukaryotic cell cycle events. See, for example, Draetta (1994) Curr. Opin. Cell Biol. 6:
842-846; Sherr (1994) Cell 79: 551-555; and Ohtsubo et al. (1995) Mol. Cell. Biol. 15: 261
See 2-2624. Cyclin was first identified in marine invertebrates during meiosis and mitosis based on their dramatic cell cycle expression.

A large family of cyclins (named cyclins AH) bind and activate different Cdks, serine / threonine kinases that are essential for cell cycle progression. The timing of the expression of the various cyclins is important in determining in which phase of the cell cycle (S, G0, G1 or G2) their associated Cdks are active. D-type cyclins are synthesized early in G1 and bind to and activate CDK4 and CDK6. Cyclin E-
Cdk2 and the cyclin A-Cdk2 complex form late in G1 when cells prepare to initiate DNA synthesis. Cyclin B-cdc2 is active during G2 and mitosis. See, for example, Lees (1995) Curr. Opin. Ce
11 Biol. 7: 773-780.

[0099] Other cyclin-Cdk complex-related proteins are important in regulating cyclin activity. Proteins co-immunoprecipitated with cyclin E were analyzed by SDS-PAGE.
Visualized by However, the viability of the cyclin E “knockout” mice indicated the presence of redundancy. In addition, studies in other species have also suggested that homologs may exist in humans.

Cdks can also affect cell division and proliferation control by phosphorylating specific intracellular target proteins. This phosphorylation event is associated with cell cycle G1
Cell transition from phase to S phase. For example, Strahler et al. (1992)
) Biochem. Biophys. Res. Comm. 185: 197-20
3: 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 for the development and control of cell proliferation.
Failure to regulate can lead to proliferative disorders such as neoplastic conditions and cancer. See, for example, Sherr (1998) Science 274: 1672-1677.

The novel cyclin gene (referred to as cyclin E2) shows about 49% structural identity to known human cyclin E. For example, Lew et al. (1991)
Cell 66: 1197-1206; and NCBI Entrez Accession No. M74093. The new variant cyclin E2 sequence is SEQ ID NO: 52
And SEQ ID NO: 53. Salient features on the E2 sequence include a cyclin box spanning approximately amino acids 120-254; and a putative phosphorylation site at thr392. The phosphorylation site is a trigger in cyclin E for ubiquitin-dependent degradation. Clurman et al. (1996) Genes a.
nd Development 10: 1979-1990. Particularly interesting segments include, for example, about 93-100; 98-106;
To 113; 107 to 121; 120 to 128; 124 to 134; 131 to 137.
172 to 177; 176 to 185; 189 to 193; 196 to 202; 200
-210; 218-223; 228-232; 236-242; 240-245.
And 248-252.

[0102] The structural homology of these genes to the identified families suggests a related function of these molecules. For example, PGT homologs should function in transport across cell membranes; antagonists or agonists of the TNF receptor family can act as costimulatory molecules for modulation of T cell-mediated cell activation, and indeed A shift in the type of helper T cells can occur (eg, between Th1 and Th2); chemokines have recognized functional properties; intracellular Dubs have been described and roles in the oncogene have been established; MD like,
Membrane-associated or soluble forms of signaling proteins are well known; and the role of cyclins in cell cycle regulation is recognized.
Alternatively, the ligand or binding structure of a cell surface antigen serves to regulate cell growth or development.

For TNF ligand molecules, they typically regulate cell proliferation, viability and differentiation. For example, TNF and FAS can kill cells that express their respective receptors. This includes fibroblasts, hepatocytes and lymphocytes. Some members of this class of ligands have an effect on the cellular proliferation of cells expressing their respective receptors (eg, B cells expressing CD40). These effects on proliferation can also affect subsequent stages of differentiation and can directly or indirectly lead to changes in cytokine expression profiles.

[0104] Members of the TNF ligand family also show costimulatory effects, which can also regulate cell differentiation or apoptosis. Receptor-expressing cells can be protected from activation-induced cell death (AICD) or apoptosis. For example, CD
Forty ligand may have an effect on T and B lymphocytes.

The embodiments characterized herein are mostly of human origin,
Additional sequences for proteins in other mammalian species (eg, primates and rodents) are also available. See below. In particular, reverse translation (eg, using the genetic code) software is available with the provided polypeptide sequences. This indicates what nucleic acid sequence can encode it. For example, MacVector, Oxoford Molecular Group
See Software. Thus, artificial genes or redundant oligonucleotides can be selected to isolate their counterparts in natural variants or species.

II. Purified Protein The primate (eg, human) DC-PGT polypeptide sequence is shown in SEQ ID NO: 2; the primate (eg, human) HDTEA84 polypeptide sequence is SEQ ID NO: The primate (eg, human) HSLJD37R polypeptide sequence is shown in SEQ ID NOs: 8, 10, and 12; the rodent (eg, mouse) RANKL polypeptide sequence is shown in SEQ ID NO: 17. Primates of the RANKL polypeptide sequence are shown in SEQ ID NOs: 19, 21 and 23;
For example, the human) HCC5 chemokine polypeptide sequence is shown in SEQ ID NO: 25; the primate (eg, human) Dub11 polypeptide sequence is SEQ ID NO: 32.
And the primate (eg, human) Dub12 polypeptide sequence is shown in SEQ ID NO: 36 and SEQ ID NO: 38; the primate (eg, human) MD-1 polypeptide sequence has the sequence: The primate (eg, human) MD-2 polypeptide sequence is shown in SEQ ID NO: 44 and SEQ ID NO: 46; the rodent (eg, mouse) MD-2 polypeptide sequence is: SEQ ID NO: 48 and SEQ ID NO: 49; and a primate (eg, human) cyclin E2 is set forth in SEQ ID NO: 54.

[0107] These amino acid sequences (provided from amino to carboxy) allow identification of the protein from other proteins and are important in providing sequence information for antigens that exemplify many variants. . In addition, the peptide sequence allows for the preparation of peptides to generate antibodies that recognize such segments, and the nucleotide sequence allows for the preparation of oligonucleotide probes. Both of these are strategies for detection or isolation (eg, cloning of a gene or cDNA encoding such a sequence).

The purified protein (s) typically include a plurality of different (eg, non-overlapping) segments of a particular length. Typically, a plurality may be at least two, more usually at least three, and preferably even five, seven or more. A minimum length is provided, but longer lengths of various sizes (eg, 7
1 length, and 12 lengths 2) may be appropriate. Such peptides are useful for generating antibodies by standard methods, as described herein. Synthetic peptides or purified proteins can be presented to the immune system to generate specific binding compositions, such as monoclonal or polyclonal antibodies. For example, Coligan (1991) Current Protocols
in Immunology Wiley / Green; and Harlo
w and Lane (Edited by Current) Antibodies: A Labor
attory Manual Cold Spring Harbor Pres
See s.

For example, a specific binding composition can be used for screening of an expression library consisting of, for example, a cell line expressing a clone encoding a prostaglandin transporter. This screening can be standard staining of the surface expressed protein, or panning. Screening for intracellular expression can also be performed by various staining or immunofluorescence procedures. The binding composition can be used to affinity purify or sort cells that express the protein. The binding compositions can also be useful in determining qualitative and quantitative levels of protein expression in various biological samples, including, for example, cell types or tissues.

As used herein, the term, eg, “human DC-PGT”,
When used in the context of a protein, it includes a protein having the amino acid sequence set forth in SEQ ID NO: 2. Important polypeptide fragments of such proteins should preserve some characteristics (biological or physical characteristics) of the full-length protein. Other essentially identical or equivalent proteins can be found in other primates or related species. In addition, binding components (eg, antibodies) typically have high affinity (eg, at least about 100 nM, usually less than about 30 nM, preferably less than about 10 nM, and most preferably less than about 3 nm), eg, D
Binds to C-PGT. Homologous proteins can be found in non-human mammalian species, such as primates or rodents. Non-mammalian species (eg, birds or amphibians) should also have structurally or functionally related genes and proteins. Similar meanings are HDTEA84, HSLJD37R, RA
Applies to NKL, HCC5, Dub11, Dub12, MD-1, MD-2 and cyclin E2.

As used herein, the term polynucleotide includes significant fragments or segments and includes at least about 8 amino acids, generally at least 10 amino acids, and more usually at least 12 amino acids. 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 It comprises a stretch of amino acid residues of at least 26 amino acids, more preferably at least 28 amino acids, and in a particularly preferred embodiment at least about 30 or more amino acids. The segments are at least 37, 45, 53, 61, 70, 8
It may have a length of 0, 90, etc., and often includes a plurality of such alignments. The particular termini of such segments can be any combination within the protein. In certain embodiments, there are multiple different (eg, non-overlapping) segments of a particular length. Typically, the plurality is at least 2, more usually at least 3, and preferably 5, 7, or more. Although a minimum length is provided, longer lengths of various sizes (eg, one length 7 and two lengths 12) are suitable.

The term “binding composition” refers to a molecule (eg, DC-PGT) that binds specifically to a respective protein or polypeptide, eg, in a cell-adhesion pairing mode or in an antibody-antigen interaction. . Other compounds include, for example, DC-P
Proteins that specifically associate with GT (including covalent or non-covalent interactions between naturally occurring physiologically relevant proteins). This molecule is
It can be a polymer or a chemical reagent. A functional analog can be an antigen whose structure has been modified, or can be a molecule having the shape of a molecule that interacts with appropriate binding determinants. The compounds may act as agonists or antagonists of the binding interaction (eg, Goodman et al. (Eds., 1990) Goodman
And Gilman: The Pharmacological Bases
of Therapeutics (8th edition) Pergamon Press).

[0113] Substantially pure, in the context of polypeptides, typically means that the protein is free of other contaminating proteins, nucleic acids, and other biological material from the original source organism or cell. Means no. Purity can be assayed by standard methods, and is usually at least about 40% pure, more usually at least about 50% pure, generally at least about 60% pure, and more usually 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 95% pure. 98% pure, and in the most preferred embodiment, at least 99% pure. Assays can be evaluated in% by weight or mol% by, for example, gel staining, spectrophotometry, or end labeling. A carrier or excipient is often added subsequently.

The solubility of a polypeptide or fragment depends on its environment and on the polypeptide. Many parameters affect polypeptide solubility. The parameters include temperature, electrolyte environment, size and molecular characteristics of the polypeptide, and the nature of the solvent. Typically, the temperature at which the polypeptide is used will range from about 4 ° C to about 65 ° C. Typically, the temperature during use is about 18
Higher than ° C. For diagnostic purposes, this temperature is usually about room temperature or warmer, but lower than the denaturation temperature of the components in the assay. For therapeutic purposes, this temperature is usually body temperature, typically about 3 for humans and mice.
7 ° C. Depending on the particular situation, the temperature may be increased or decreased in situ or in vitro.

The size and structure of the polypeptide should generally be substantially stable, and usually not denatured. Polypeptides can be associated with other polypeptides in a quaternary structure, eg, to confer solubility, or with lipids or detergents in a manner that approximates the interaction of natural lipid bilayers.

Solvents and electrolytes are typically biocompatible buffers of the type used for preserving biological activity, and usually approximate physiological aqueous solvents. Usually, the solvent will have a neutral pH, typically between about 5 and 10, and preferably about 7
. 5 In some situations, typically a mild non-denaturing surfactant (eg, CHS (cholesteryl hemisuccinate) or CHAPS (3- [3-cholamidopropyl) dimethyl ammonium)]-
1-propanesulfonate)) or one or more detergents at a sufficiently low concentration to avoid significant disruption of the structure or physiological properties of the protein.

The solubility is reflected by the settling measured in Svedberg units.
The Svedberg unit is a measure of the rate of sedimentation of a molecule under certain conditions.
Determination of sedimentation velocity was performed classically in analytical ultracentrifugation, but is nowadays typically performed in standard ultracentrifugation. Freifelder (1982) Ph
physical Biochemistry (second edition), W.C. H. Freeman
And Cantor and Schimmel (1980) Biophysic;
al Chemistry, parts 1-3, W.C. H. Freeman & Co. ,
See San Francisco, each of which is incorporated herein by reference. As a crude determination, the sample containing the putative soluble polypeptide is spun in a standard full-size ultracentrifuge at about 50 Krpm for about 10 minutes, and the soluble molecules remain in the supernatant. Soluble particles or polypeptides are typically less than about 30S, more typically less than about 15S, usually less than about 10S, more usually less than about 6S, and in certain embodiments, preferably less than about 4S, And more preferably about 3S
Is less than.

The human complementary DNA sequences and deduced amino acid sequences provided herein for DC-PGT include sequences corresponding to twelve putative transmembrane (TM) segments, which indicate the hydropathy of DC-PGT. And structural analysis. DC-PGT
The sequence TopPredII (Claros and von Heijne (199)
4) Comp. Applic. (Biosci, 10: 685-686) The peaks indicated by the profile reach above the "estimated" or "specific" baseline. 1
The two transmembrane segments comprise approximately amino acids 47-68 (TM1);
7 (TM2); 117-136 (TM3); 188-208 (TM4); 225
-244 (TM5); 279-294 (TM6) 367-386 (TM7);
12-431 (TM8); 450-474 (TM9); 561-578 (TM1
0); 597-616 (TM11); and correspond to hydrophobic stretches ranging from 651-675 (TM12). Charged amino residues located within the transmembrane domain are amino residues 59, 62, 196, 207, 380, 469, 602, 655
Glutamine at, and 675; glutamic acid at residue 95; and residue 60
Arginine at 7 and 674. The extracellular loop is composed of amino acid residues 69-8
7, 137-187, 295-366, 432-449, 579-596, and 617-650. The putative N-glycosylation site on the exposed extracellular surface of the molecule is identified by the Asn-X-Ser / Thr motif (eg,
6-148; 176-178; and 538-540) are located in the second and fifth extracellular loops. The intracellular portion is composed of amino acid residues 1-46, 108-116
, 209-224, 295-366, 432-449, 579-596 and 6
76-709. These boundaries are often the boundaries of the segment of interest, which includes multiple described segments.

The transporters of this family are typically about 650 amino acids in length.
2 transmembrane proteins and an organic anionic transporter in humans and rats, the human prostaglandin transporter (Lu et al. (1996)
J.). Clin. Invest. 98: 1142-1149) and rat prostaglandin transporter; rat brain digoxin transporter and Matrin F / G (Kanai et al. (1995) Science 268).
: 866 to 869). Characterizing this family of organic anionic 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 approximately at positions 489, 493, 495, 504, 51
Located in extracellular loop 5 with cysteines at positions 6, 520, 539, 541, 554, and 557.

Other particularly interesting TNF receptor, Dub, MD, and cyclin E segments are indicated. These may also be segments in which the invention is comparable to other proteins or genes.

III. Physical Variants The invention also includes proteins or peptides that have substantial amino acid sequence homology to the amino acid sequence of the described proteins. The variants include species and polymorphic variants (eg, naturally occurring forms).

[0122] Amino acid sequence homology, or sequence identity, is determined by optimizing residue matches and, if necessary, by introducing the desired gap.
Needleham et al. (1970) J. Mol. Mol. Biol. 48: 443-45
3; Sankoff et al. (1983) Time Warps, String Ed.
it's and macromolecules: The Theory an
d Practice of Sequence Comparison
Chapter, Addison-Wesley, Reading, MA; and Intel
software packages from liGenetics, Mountain View, CA; and the University of Wisconsin Gen.
See etics Computer Group, Madison, WI. If conservative substitutions are considered a match, the sequence identity will change. 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 naturally occurring polymorphic or allelic variants and interspecies variants in each respective protein sequence. Representative homologous proteins or peptides range from 25-100% identity (if gaps can be introduced) to 50-100% identity (if conservative substitutions are included) to the amino acid sequence of HDTEA84. Having. Identity values are 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%.

For sequence comparisons, typically one sequence acts as a reference sequence, to which test sequences are compared. When using a sequence comparison algorithm, the test and reference sequences are entered into a computer, the coordinates of the subsequences are designed if necessary, and the parameters of the sequence algorithm program are designed. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence to the reference sequence, based on the parameters of the designed program.

[0124] Visual alignment of sequences for comparison can be performed, for example, by Smith and Wa.
terman (1981) Adv. Appl. Math. 2: 482, by the local homology algorithm or by Needleman and Wunsch (1970).
J. Mol. Biol. 48: 443, by the search for similarity method of Pearson and Lipman (1988) Proc. Nat'l
Acad. Sci. USA 85: 2444, or by computerized implementation of these algorithms (Wisconsin Gene).
GAP, BESTFIT, F in tics Software Package
ASTA and TFASTA, Genetics Computer Group
p, 575 Science Dr. , Madison, WI) or by visual inspection (see generally Ausubel et al., Supra).

One example of a useful algorithm is PILEUP. PILEUP creates multiple sequence alignments from groups of related sequences using progressive pairwise alignments to show relationships and percent sequence identity. It also plots in a pedigree or dendrogram showing the clustering relationships used to create the alignment. PILEUP is described in Feng and Doolittle (1987) J. Am. Mol. Evol. 35: 351-360
Use a simplified version of the progressive alignment method of The method used is described in Higgins and Sharp (1989) CABIOS 5: 151-
153 is similar to the method described by This program has a maximum length of 5,0
Up to 300 sequences of each of the 00 nucleotides or amino acids can be aligned. Multiple alignment procedures begin with a pairing alignment of the two most similar sequences, creating two clusters of aligned sequences. This cluster is then aligned to the next most related or cluster of aligned sequences. Two clusters of sequences are aligned by a simple extension of the pairing alignment of the two individual sequences. The final alignment is achieved by a series of progressive pairing alignments. The program is executed by setting specific sequences and their amino acid or nucleotide coordinates in the region of sequence comparison and by setting the parameters of the program. For example, a reference sequence can be compared to other test sequences and the percent sequence identity determined using the following parameters: default gap weight (3.00), default gap length weight (0.10), and Weight end gap.

Another example of an algorithm suitable for determining percent sequence identity and sequence similarity is the BLAST algorithm, which is described in Altschul et al. (1990) J. Am.
Mol. Biol. 215: 403-410). BLAST
The software for performing the analysis is available at National Center fo
r Biotechnology Information (http: www
. ncbi. nlm. nih. Gov /) is publicly available. The algorithm involves first identifying a high scoring sequence pair (HSP) by identifying short words of length W in the query sequence. The query sequence either matches or satisfies some positive value threshold score T when aligned with words of the same length in the database sequence. T is considered as the score threshold for adjacent words (Altschul et al., Supra). These first adjacent 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, as long as the cumulative alignment score can be increased. Word hit growth in each direction is stopped if: the cumulative alignment score drops by an amount X from its maximum attainment; due to the accumulation of one or more negative scoring residue alignments. If the cumulative score goes below 0; or if the end of any sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLAST program defaults to a word length (W) of 11 and a BLOSUM62 scoring matrix of 50 (Henikoff and Henikoff (1989) Proc. Na).
t'l Acad. Sci. USA 89: 10915) Alignment (B)
An expected value (E) of 10, M = 5, N = 4, and a comparison of both strands is used.

In addition to calculating percent sequence identity, the BLAST algorithm also performs statistical analysis of the similarity between two sequences (eg, Karlin and Alt)
Schul (1993) Proc. Nat'l Acad. Sci. USA 9
0: 5873-5787). One measure of similarity provided by the BLAST algorithm is the minimum sum probability (P (N)). This provides an indication of the probability that a match between two nucleotide or amino acid sequences will occur by chance. For example, the minimum sum probability in the 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, the nucleic acid is considered similar to the reference sequence.

A further indication that the two nucleic acid sequences of a polypeptide are substantially identical is
The polypeptide encoded by the first nucleic acid is immunologically cross-reactive with the polypeptide encoded by the second nucleic acid, shown below. Thus, a polypeptide is typically substantially identical to a second polypeptide, for example, where the two peptides differ only by conservative substitutions. Another indication that two 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 DNA can be readily modified by nucleotide substitution, nucleotide deletion, nucleotide insertion, and nucleotide stretch inversion. These modifications result in novel DNA sequences which encode these antigens, their derivatives, or proteins having similar physiological, immunological, antigenic or other functional activities. I do. These modified sequences can be used to produce or enhance mutant antigen. The enhanced expression is associated with gene amplification, increased transcription, increased translation,
And other mechanisms. For example, a "mutant HDTEA84" is included within the definition of sequence identity for HDTEA84, as shown above, but differs from the amino acid sequence of HDTEA84 normally found in nature by either deletion, substitution or insertion. Includes other polypeptides with different amino acid sequences. Generally, this refers to proteins having significant identity to proteins having the sequence of SEQ ID NO: 6 and sharing various biological activities (eg, antigenic or immunogenic activities) with those sequences. And, in preferred embodiments, comprises the majority of the full length of the disclosed sequences. Typically, full-length sequences are preferred, but truncated forms (eg, soluble constructs and intact domains) are also useful, as are genes or proteins found from natural sources, typically , Most desired. Similar concepts apply to different HDTEA84 proteins, especially those found in various warm-blooded animals (eg, mammals and birds, or fish). These descriptions generally include all HDTEA84 proteins and are not limited to the specific human embodiments specifically discussed. Similar concepts apply to other provided polypeptides.

DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5,
Mutagenesis of Dub, MD-1, MD-2, or cyclin E2 may also be performed by creating amino acid insertions or deletions. Although the site of the site-specific mutation is predetermined, the mutant need not be site-specific. Protein mutagenesis may be performed by generating amino acid insertions or deletions, or a combination may be generated to achieve a final construct. Insertions include amino-terminal or carboxy-terminal fusions. Random mutagenesis is performed at the target codon, and then the expressed mutants can be screened for the desired activity. Methods for generating substitution mutations at predetermined sites in DNA having a known sequence include:
Well known in the art (eg, by M13 primer mutagenesis or polymerase chain reaction (PCR) techniques). For example, see Sambrook et al.
9); Ausubel et al. (1987 and addendum); and Kunkel et al.
87) Methods in Enzymol. 154: 367-382.

[0131] Mutations in the DNA should not be located in the coding sequence outside of the reading frame, and preferably create complementary regions that can hybridize to create a secondary mRNA structure (eg, a loop or hairpin). should not do.

The present invention also provides recombinant proteins (eg, heterologous fusion proteins using segments derived from these proteins). A heterologous fusion protein is a fusion of proteins or segments that are not normally fused in nature in the same manner.
Thus, a fusion product of an immunoglobulin with a polypeptide will have the sequence fused with a typical peptide bond, will typically be produced as a single translation product, and will have properties from each source peptide. A continuous protein molecule to present. Similar concepts apply to heterologous nucleic acid sequences.

The present invention also provides recombinant proteins (eg, heterologous fusion proteins using segments derived from these proteins). A heterologous fusion protein is a fusion of proteins or segments that are not normally fused in nature in the same manner.
Similar concepts apply to heterologous nucleic acid sequences. Fusion proteins are useful as sources for cleavage, separation, and purification of some of them.

In addition, new constructs can be made from combinations of similar functional domains from other proteins. For example, a target binding segment or other segment may "swap" between different novel fusion polypeptides or fragments.
d) ". For example, Cunningham et al. (1989) Science.
243: 1330-1336; and O'Dowd et al. (1988) J. Am. Bio
l. Chem. 263: 15985-15992.

See Beaucage and Carruthers (1981) Tetra. Le
tts. 22: 1859-1862 produces the appropriate synthetic DNA fragment. Double-stranded fragments often add the appropriate primer sequence to their complement, either by synthesizing the complementary strand and annealing the strands together under appropriate conditions, or using DNA polylase (eg, , PCR techniques).

IV. Functional Variants Blocking the physiological response with, for example, HDTEA84, HSLJD37R, RANKL, HCC5, chemokines, MD-1 or MD-2 may be, for example, a receptor in the form of a signal transduction via competitive inhibition Or the corresponding structure (counterst
structure). In others, the binding affinity for a substrate can be modified or, for example, competed with DC-PGT, Dub, or cyclin E2. Thus, in vitro assays of the invention often employ isolated proteins, soluble fragments containing ligands or substrate binding segment segments of these proteins, or forms attached to solid substrates. These assays also include mutations and modifications of the binding segment or antigens (eg, HDTEA84, HSLJD37R, PANK).
L, MD-1, or MD-2 analog).

The present invention also contemplates the use of competitive drug screening assays, for example, where a neutralizing antibody against an antigen or binding fragment competes with a test compound for binding to a protein (eg, a native protein sequence). . This is applicable for substrate binding (eg, competitive inhibitors), and in receptor interactions, the protein has a binding facing structure.

“Derivatives” of antigens, eg, receptors, include amino acid sequence variants, glycosylation variants, and covalent or aggregate conjugates with other chemical moieties from naturally occurring forms. Covalent derivatives can be prepared, for example, by functional attachment to the amino acid side chain of the receptor, or to a group found at the N- or C-terminus, by standard means. For example, Lundblad and Noyes (1
988) Chemical Reagents for Protein Mo
modification, vol. 1-2, CRC Press, Inc. , Boca
Raton, FL; Hugli (eds., 1989) Technologies in.
Protein Chemistry, Academic Press, Sa
n Diego, CA; and Wong (1991) Chemistry of
Protein Conjugation and Cross Linki
ng, CRC Press, Boca Raton, FL.

In particular, glycosylation alterations are included, eg, made by altering the glycosylation pattern of a polypeptide during its synthesis and processing or in a further processing step. For example, Elbein (1987) An
n. Rev .. Biochem. 56: 497-534. Versions of peptides having the same primary amino acid sequence with other minor modifications, including phosphorylated amino acid residues (eg, phosphotyrosine, phosphoserine, or phosphothreonine) are also included.

[0140] Fusion polypeptides between these proteins and other homologous or other heterologous proteins are also provided. Many cytokine receptors or other surface proteins are multimeric (eg, homodimeric entities)
And repeat constructs can have various advantages, including reduced sensitivity to proteolytic cleavage. A representative example is the fusion of a segment or domain of a protein (eg, a receptor binding segment) with a reporter polypeptide (eg, luciferase), such that the presence or localization of the fused ligand is easily determined. Can be done. See, e.g., Dull et al., U.S. Patent No. 4,859,609.
See issue. Other gene fusion partners include bacterial β-galactosidase,
Detection or purification tags such as trpE, protein A, β-lactamase, α-amylase, alcohol dehydrogenase, yeast α-mating factor, and the FLAG sequence of the His6 sequence are included. See, for example, Godowski et al., (1988).
Science 241: 812-816. Of particular interest are:
It is a fusion construct of a membrane binding domain and a receptor.

[0141] Fusion peptides are typically made by either recombinant nucleic acid methods or synthetic polypeptide methods. Techniques for nucleic acid manipulation and expression generally include, for example,
Sambrook et al. (1989) Molecular Cloning: AL.
laboratory Manual (2nd edition), Volumes 1 to 3, Cold S
(1993) Current Protocols in Molecular, pp. harbor Laboratory; and Ausubel et al.
Biology, Greene and Wiley, NY. Techniques for the synthesis of polypeptides are described, for example, in Merrifield (1963).
J. Amer. Chem. Soc. 85: 2149-2156; Merrifi
eld (1986) Science 232: 341-347; Atherto
(1989) Solid Phase Peptide Syntheses.
is: A Practical Approach, IRL Press, Ox
ford; and Grant (1992) Synthetic Peptide
s: A User's Guide, W.S. H. Freeman, NY.

The present invention also contemplates the use of protein derivatives other than modifications in amino acid sequence or glycosylation. Such derivatives may include covalent or cohesive bonds to the chemical moiety. Covalent or aggregative derivatives are useful as immunogens, as reagents in immunoassays, or in purification methods such as for affinity purification of binding partners (eg, other antigens). The desired protein can be purified by covalent attachment to a solid support (eg, cyanogen bromide-activated Sepharose), by methods well known in the art, or by adsorption to polyolefin surfaces with or without glutaraldehyde crosslinking. Can be immobilized for assay or purification or use in other binding compositions. The protein can also be labeled with a detectable group, for example, for use in a diagnostic assay. Purification can be effected by immobilized antibodies or complementary binding partners. Conversely, immunoadsorption or immunodepletion (im
A technology may be developed.

The solubilized proteins or fragments of the invention can be used as immunogens for the production of antisera or antibodies specific for binding to an antigen or a fragment thereof. Purified antigen is an antigen-binding fragment of a natural antibody (eg, F
ab, Fab ', F (ab) 2, etc.). Purified proteins are also produced in response to the presence of elevated levels of antigens or cellular fragments containing antigens, both of which can diagnose abnormal or specific physiological or disease states. It can be used as a reagent for detecting antibodies. The present invention contemplates antibodies raised against the amino acid sequence encoded by the described nucleotide sequences, or fragments of proteins containing the same. In particular, the invention contemplates antibodies that have binding affinity for, or are raised against, a particular fragment, for example, where the fragment comprises a lipid bilayer (both extracellular or intracellular). ) Is expected to exist outside.

The present invention contemplates the isolation of additional closely related species variants. Southern blot analysis and Northern blot analysis should establish that similar genetic entities are present in other mammals. These proteins appear to be widespread in species variants (eg, rodents, lagomorphs, carnivores, artiodactyla, tepidotes, and primates).

The present invention also provides a means to isolate a panel of related antigens that display both distinctions and similarities in structure, expression, and function. The elucidation of many of the physiological effects of molecules is greatly accelerated by the isolation and characterization of their further distinct species variants. In particular, the present invention provides useful probes for identifying additional homologous genetic entities in different species.

An isolated gene allows for the transformation of cells that lack expression of the corresponding protein (eg, either species or cells that lack the corresponding antigen and exhibit negative background activity). . This should allow for an analysis of the function of the gene in comparison to untransformed control cells.

The analysis of key structural elements responsible for various activation or differentiation functions mediated through these antigens can be performed using standard techniques of modern molecular biology.
This is especially true when comparing members of related classes. For example, Cunni
(1989) Homolog scanning mutagenesis technology described in Science 243: 1339-1336; and O'Dowd et al. (19)
88) J. C .; Biol. Chem. 263: 15985-15992; and Lechleiter et al. (1990) EMBO J.
9: 4381-4390.

The present invention also relates to related organic anion transporters (eg, other vertebrate prostaglandin transporters) that exhibit both distinction and similarity in structure, expression, and function in the context of DC-PGT. Provide a means for isolating a group of Elucidation of many of the physiological effects of this antigen is greatly accelerated by the isolation and characterization of distinct species variants. In particular, the present invention provides probes useful for identifying additional homologous genetic entities in different species. The above results indicate that sufficiently homologous genes are present in other species where cross-hybridization appears to allow successful cloning.

An isolated gene allows for the transformation of cells lacking the expression of the described genes (eg, prostaglandin transporters). Various species or cells lacking the corresponding protein can be isolated and should show negative background activity. Expression of the transformed gene allows the isolation of antigenically pure cell lines using defined or single species variants. This approach allows for more sensitive detection and differentiation of the physiological effects of the gene (eg, prostaglandin transporter). Intracellular fragments, such as cytoplasts or membrane fragments, can be isolated and used.

The DC-PGT gene may also be useful for increasing the rate of transport of a desired prostaglandin into transformed cells. Thus, the transporter can be transformed into cells for targeting uptake of the desired substrate or analog. For example, it may be useful to incorporate certain modified prostaglandins into these cells. These cells may be more sensitive to other treatments or may be directly affected. Thus, a particular subset of dendritic cells can be transformed to be more sensitive to prostaglandins or particular substrates. Conversely, such cells may be useful screening targets to identify entities that can block substrate transport and thereby interfere with uptake.

[0151] Structural studies of transporters have led to the design of new variants, particularly analogs that exhibit altered binding affinities, or perhaps, altered rates of transporter activity. This can be combined with previously described screening methods to isolate variants that exhibit the desired spectrum of activity. Alternatively, many different prostaglandins and their analogs can be screened for either transporter binding affinity or transporter import. The transporter may require a direct energy source (eg, ATP or other nucleotide triphosphate), or may depend on an ionic gradient, as described above.

In the context of Dub and cyclin E2, intracellular functions probably include segments of the antigen that can normally reach the cytosol, as well as segments of the receptor. However, protein internalization can occur under certain circumstances, and interactions between intracellular and "extracellular" components can occur. Particular segments that interact with proteins with other intracellular components can be identified by mutagenesis or direct biochemical means, such as cross-linking or affinity methods.

[0153] Structural analysis by crystallographic or other physical methods is also applicable. Further studies of the mechanism of signal transduction are based on affinity methods or genetic means (
(Eg, complementation analysis of variants) involves the study of related components that may be isolable.

Further studies of protein expression and regulation are sought. The control elements associated with the antigen should exhibit differential physiological, developmental, tissue-specific, or other expression patterns. Upstream or downstream genetic regions (eg, control elements) are of interest. In particular, physiological or developmental variants (
For example, a plurality of selectively processed antigen forms) can be found. Therefore,
Differential splicing of messengers can result in a combination of membrane-bound, soluble, and modified versions of the antigen.

Structural studies of antigens have led to the design of novel antigens, particularly analogs that exhibit agonist or antagonist properties of the molecule. This can be combined with the screening methods described above to isolate antigens that exhibit the desired spectrum of activity.

V. Antibodies Antibodies occur naturally against a variety of described polypeptides, including, for example, species, polymorphic, or allelic variants, and fragments thereof. It can be raised both in the form and in their recombinant form. In addition, antibodies can be raised against any of the proteins, including the native or denatured versions, which are either active or inactive. Anti-idiotype antibodies are also contemplated.

Antibodies, including binding fragments and single-chain versions, to predetermined fragments of the antigen, can be raised by immunizing animals with conjugates of the fragment with an immunogenic protein. Monoclonal antibodies are prepared from cells secreting the desired antibody. These antibodies can be screened for binding to a normal or defective polypeptide, or screened for agonistic or antagonistic activity. Antibodies can be agonistic or antagonistic, for example, by steric blocking partner or substrate binding. These monoclonal antibodies will usually have at least about 1 mM, more usually at least about 300 μM, typically at least about 10 μM.
0 μM, more typically at least about 30 μM, preferably at least about 10 μM
M, and more preferably, a K _D of at least about 3 μM or less. More preferred embodiments are even higher affinity (eg, at least 300 nM, 30
nM, 3 nM, or even picomolar affinities).

The term “binding composition” refers to, for example, in an antibody-antigen interaction, for example, DC
-Refers to a molecule that binds to PGT with affinity and selectivity. However, other compounds, such as accessory proteins, can also specifically and / or selectively bind antigens to exclude other molecules. Typically, the association is at a natural, physiologically relevant protein-protein interaction (either covalent or non-covalent) and a member of a multiprotein complex (
(Including carrier compounds or dimerization partners). This molecule is
It can be a polymer or a chemical reagent. If the antigen is a convex shaped molecule (eg, ligand-
Suggestions as to whether or not a receptor interaction needs to be a ligand or receptor can be found in the table except that the interaction exhibits similar specificity (eg, specific or selective affinity). No need to be done. A functional analog can be a polypeptide having a structural modification (eg, mutein) or can be an entirely unrelated molecule (eg, it has a molecular shape that interacts with the appropriate ligand binding determinant). Have). The ligand can function as an agonist or antagonist of the receptor. For example, Goodman et al., Goodman
&Gilman's: The Pharmacological Bases
of Therapeutics (current version) Pergamon Press, T
early, N.M. Y. checking ...

The term “binding agent: antigen complex” as used herein refers to a complex of a binding agent and an antigen (eg, a DC formed by the specific binding of a binding agent to an antigen).
-PGT protein). Specific or selective binding of a binding agent means that the binding agent has a specific binding site (eg, an antigen binding site that recognizes a site on an antigen). For example, antibodies raised against a DC-PGT protein and recognizing an epitope on that protein may allow the formation of a binder: DC-PGT protein complex by specific selective binding. Typically, formation of a binder: DC-PGT protein complex allows for qualitative or quantitative measurement of DC-PGT protein in a mixture of other proteins and biologics. The term "antibody: DC-PGT protein complex" refers to embodiments in which the binding agent is, for example, an antigen-binding portion derived from an antibody. The antibody can be monoclonal, polyclonal, or a binding fragment of an antibody (eg, Fab or F (ab
) 2 fragments). This antibody is preferably a polyclonal antibody for the purpose of cross-reactivity testing.

The phrase “specifically binds to an antibody” or “specifically immunoreactive with”
When referred to a protein or peptide, it refers to a binding reaction that determines the presence of that protein in the presence of a heterogeneous population of other proteins and other biological components. Thus, under the indicated immunoassay conditions, certain antibodies bind to certain proteins and do not significantly bind to 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. Often, serum can be immunoselected or immunodepleted to minimize cross-reactivity with a particular target protein.

A polyclonal antibody raised against an antibody (eg, a defined immunogen (eg, an immunogen consisting of the amino acid sequence of SEQ ID NO: 2 or a fragment thereof, or a polypeptide generated from the nucleic acid of SEQ ID NO: 1) DC-PGT polypeptides that specifically bind to, or are specifically immunoreactive with, such antibodies) are typically determined in an immunoassay. Included in the boundaries of the invention are the primitive DC-Ps as structurally and functionally defined herein.
These nucleic acid sequences (including functional variants) described herein that encode a polypeptide that selectively binds to a polyclonal antibody raised against the GT polypeptide. Immunoassays typically use a polyclonal antiserum raised against, for example, the protein of SEQ ID NO: 2. The antiserum is selected to have low cross-reactivity to other suitable PGT family members, preferably from the same species, and any such cross-reactivity is determined prior to use in the immunoassay. , Is removed by immunoadsorption. Suitable selective serum preparations can be isolated and characterized.

[0162] Purified proteins or defined peptides are useful for generating antibodies by standard methods, as described above. Synthetic peptides or purified proteins can be presented to the immune system to generate monoclonal or polyclonal antibodies. For example, Coligan (1991) Current
Protocols in Immunology Wiley / Greene
And Harlow and Lane (1989) Antibodies: A
Laboratory Manual, Cold Spring Harbo
See rPress. Alternatively, HDTEA84 can be used as a specific binding reagent, and the specificity advantages of its binding can be exploited, much like antibodies are used.

For example, the specific binding composition is HDTEA84, HSLJD37R, or R
It can be used for screening of expression libraries made from cell lines expressing ANKL. The screening may be by standard staining of the surface-expressed antigen construct or by panning. Screening for intracellular expression can also be performed by various staining or immunofluorescence procedures. The binding composition can be used to affinity purify or sort cells that express the protein.

To generate antisera for use in immunoassays, the protein (
For example, the protein of SEQ ID NO: 2) is isolated as described herein. For example, a recombinant protein can be produced in a mammalian cell line. Suitable hosts (eg, inbred strains of mice such as Balb / c) can be prepared using standard adjuvants (eg, Freund's adjuvant) and standard mouse immunization protocols (eg, Freund's adjuvant).
(See Harlow and Lane)). Alternatively, substantially full length synthetic peptides derived from the sequences disclosed herein can be used as immunogens. Polyclonal sera is collected and titrated against immunogenic proteins in an immunoassay (eg, a solid-phase immunoassay using an immunogen immobilized on a solid support). 10 ⁴
Polyclonal antisera with above titers were selected and used to identify other PGT family members (eg, humans) using competitive binding immunoassays such as those described in Harlow and Lane, supra, pp. 570-573. P
(GT or rat PGT). Preferably, at least two PGT family members are used in this determination along with the target. These PGT family members are identified using standard molecular biology and protein chemistry techniques as described herein.
It can be produced as a recombinant protein and isolated. Thus, antibody preparations with the desired selectivity or specificity for a subset of PGT family members can be identified or produced.

[0165] Immunoassays in a competitive binding format can be used for cross-reactivity determinations. For example, the protein of SEQ ID NO: 2 can be immobilized on a solid support. The proteins added to this assay compete with the binding of the antiserum to the immobilized antigen. The ability of the above proteins to compete with the binding of antisera to the immobilized protein is compared to the protein of SEQ ID NO: 2. The percent cross-reactivity for the above proteins is calculated using standard calculations. These antisera with less than 10% cross-reactivity with each of the above listed proteins,
Selected and pooled. Cross-reactive antibodies are then removed from the pooled antisera by immunoadsorption or immunodepletion with the proteins listed above.

[0166] The immunosorbed and pooled antisera are then used in a competitive binding immunoassay as described above to compare the immunogenic protein with a second protein. To make this comparison, 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 antiserum to the immobilized protein is determined. You. If the amount of the second protein required is, for example, two times less than the amount of the protein of SEQ ID NO: 2, then the second protein is specific for antibodies raised against the immunogen. It is said to be combined.

The antibodies of the present invention may also be useful in diagnostic applications. As capture or non-neutralizing antibodies, they can be screened for the ability to bind to the antigen without inhibiting binding by the partner. As neutralizing antibodies, they can be useful in competitive binding assays. They are also useful in detecting or quantifying the described proteins or their binding partners. For example, Chan
(1987) Immunology: A Practical Guide,
Academic Press, Orlando, FL; Price and Ne
wman (1991) Principles and Practice o
f Immunoassay, Stockton Press, N.M. Y. And Ngo ed. (1988) Nonisotopic Immunoassay,
Plenum Press, N.M. Y. checking ... Cross-adsorption or cross-depletion and other tests identify antibodies that exhibit a spectrum of different specificities (eg, unique or shared species specificity).

In addition, the antibodies of the present invention, including antigen-binding fragments, are potent antibodies that bind antigen and inhibit functional binding or the ability of a binding partner to elicit a biological response. It may be an antagonist. They can also be useful as non-neutralizing antibodies, and can be conjugated to toxins or radionuclides, such that when the antibody binds to an antigen, for example, cells that express the antigen on its surface are killed. In addition, these antibodies can be conjugated, directly or indirectly by a linker, to a drug or other therapeutic agent, and can result in drug targeting. They can be labeled for histological evaluation.

Antigen fragments can be conjugated to other substances, particularly polypeptides (such as fused or covalently conjugated polypeptides for use as immunogens). Antigens and fragments thereof can be obtained from various immunogens (eg, keyhole limpet hemocyanin, bovine serum albumin, tetanus toxoid, etc.)
Or can be covalently linked. For a description of how to prepare polyclonal antisera, see Microbiology, Hoeber Medical.
Division, Harper and Row, 1969; Landste
inner (1962) Specificity of Serological
Reactions, Dover Publications, New Yo
rk; Williams et al. (1967) Methods in Immunol.
ogy and Immunochemistry, Volume 1, Academic
Press, New York; and Harlow and Lane (19)
88) Antibodies: A Laboratory Manual, CS
See H Press, NY.

In some instances, it is desirable to prepare monoclonal antibodies from various mammalian hosts (eg, mice, rodents, primates, humans, etc.). Descriptions of techniques for preparing such monoclonal antibodies are described, for example, in Stite
(eds) Basic and Clinical Immunology (4th edition), Language Medical Publications, Los A
Itos, CA, and references cited therein; Harlow and Lan.
e (1988) Antibodies: A Laboratory Manua
1, CSH Press; Goding (1986) Monoclonal A
ntbodies: Principles and Practice (second
Edition), Academic Press, New York; and, in particular, K
Ohler and Milstein (1975) Nature 256: 495.
-497, which discusses one method of producing a monoclonal antibody
Can be found in

Other suitable techniques include exposing lymphocytes to antigenic polypeptides in vitro, or alternatively, exposing lymphocytes to selection of a library of antibodies in phage or similar vectors. Is included. Huse et al. (1989) "Genera.
Tion of a Large Combinatorial Library
y of the Immunoglobulin Repertoire i
n Page Lambda ", Science 246: 1275-128.
1; and Ward et al. (1989) Nature 341: 544-546. The polypeptides and antibodies of the present invention can be used with or without modification, including chimeric or humanized antibodies. Frequently, polypeptides and antibodies will be labeled by attaching, either covalently or non-covalently, a substance that provides a detectable signal. A wide variety of labeling and conjugation techniques are known and have been widely reported 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. 3,817,837;
No. 850,752; No. 3,939,350; No. 3,996,345; No. 4,277,437; No. 4,275,149; and No. 4,366.
No. 241. Also, recombinant immunoglobulins can be produced. Cabi
lly, U.S. Patent No. 4,816,567; Moore et al., 4,642,3.
No. 34; and Queen et al. (1989) Proc. Nat'l Acad. S
ci. USA 86: 10029-10033.

The antibodies of the present invention can also be used for affinity chromatography in isolating proteins. A column can be prepared where the antibody is bound to a solid support (eg, particles such as agarose, Sephadex, etc.), where the cell lysate can pass through the column, and the column is washed, Increase the concentration of mild detergent, thereby releasing the purified protein. For example, Wilc
Hek et al. (1984) Meth. Enzymol. 104: 3-55.

Antibodies raised against each protein are also useful in raising anti-idiotype antibodies. These are useful in detecting or diagnosing various immunological conditions related to the expression of each antigen.

VI. Nucleic Acids The described peptide sequences and related reagents can be used, for example, from natural sources,
For example, DC-PGT polypeptide, HDTEA84 polypeptide, HSLJD
37R polypeptide, RANKL polypeptide, HCC5 polypeptide, Dub
11 polypeptide, Dub12 polypeptide, MD-1 polypeptide, MD-2
It is useful in detecting, isolating, or identifying a polypeptide, or a DNA clone encoding a cyclin E2 polypeptide. Typically, nucleic acids, especially naturally occurring genes, are useful in isolating genes from mammals, and similar modes are used for other species, such as warm-blooded animals (eg, birds and mammals). Applied to isolate genes from They are domestic pets (eg dogs and cats)
And livestock (eg, horses, pigs, cows, sheep, chickens, turkeys,
(Eg, fish). Cross-hybridization makes it possible to isolate the respective counterpart gene from other species. A number of different approaches should be available to successfully isolate a suitable nucleic acid clone.

The peptide sequence allows for the preparation of peptides to generate antibodies for recognizing such segments, and a variety of different methods can be used to prepare such peptides. As used herein, for example, the term prostaglandin transporter, when used in the context of a protein, includes a protein having the amino acid sequence shown in Table 1 or a significant amount of such a protein. Including a fragment. It also displays vertebrates (eg, humans, including humans) that exhibit similar biological functions, eg, are antigenic or interact with prostaglandin transporter-specific binding components (eg, specific antibodies). Animal). These binding components (eg, antibodies)
Is typically high affinity (eg, at least about 100 nM, usually about 30 nM)
Less, preferably less than about 10 nM, and more preferably about 3 nM
Less) with the prostaglandin transporter. Still higher affinities (eg, 100 pM, 30 pM, 100 fM, etc.) are possible.

The present invention relates to structurally related (eg, antigenically related) or biologically active proteins (eg, substrate binding or transport, prostaglandin transporters, TNF receptor-like proteins) , Chemokines, Dubs, surface receptors, or cell cycle regulatory proteins, or polypeptide fragments thereof) are contemplated for use of the isolated DNA or fragments of the present invention. In addition, the present invention relates to a single protein that encodes a structurally related or biologically active protein or polypeptide and that can hybridize under appropriate conditions to a DNA sequence described herein. Includes isolated or recombinant DNA. The biologically active protein or polypeptide is
It can be an intact antigen or fragment and has the amino acid sequence disclosed in Tables 1-13. Further, the present invention relates to isolated DNA or recombinant DN.
A, or the use of fragments thereof. These encode proteins homologous to the respective gene or isolated using the cDNA encoding the protein as a probe. Preferably, such homologous genes or proteins are in natural form isolated from other vertebrates (eg, warm-blooded animals, including mammals such as primates). The isolated DNA contains the respective regulatory sequences (eg, promoter, enhancer,
Poly-A addition signal).

An “isolated” nucleic acid is a nucleic acid that is substantially separated from other components (eg, ribosomes, polymerase, and flanking genomic sequences from the original species) that accompany the native sequence natively. For example, RNA, DNA, or a mixed polymer). The term includes nucleic acid sequences that have been removed from their naturally occurring intracellular milieu, and are recombinant or cloned DNA isolates, and biologically synthesized by chemically synthesized analogs or heterologous systems. Includes chemically synthesized analogs. A substantially pure molecule includes the form of the molecule once isolated or the form of the molecule that is currently isolated. Alternatively, the purified species can be separated from host components from a recombinant expression system. Generally, nucleic acids are less than about 50 kb, usually about 30 kb.
less than about 10 kb, typically less than about 10 kb, and preferably less than about 6 kb,
Present in vector or fragment.

An isolated nucleic acid is generally a homogenous composition of the molecule, but, in some embodiments, contains minor heterogeneity. This heterogeneity is typically found at the ends or portions of the polymer that are not important for the desired biological function or activity.

[0179] Peptide segments can also be used to predict appropriate oligonucleotides to screen libraries. The genetic code (eg, back translation) can be used to select appropriate oligonucleotides useful as probes for screening. For example, SEQ ID NOs: 1, 5, 7, 9, 11, 1
6, 18, 20, 22, 24, 31, 33, 35, 37, 41, 43, 47, or 53. In combination with polymerase chain reaction (PCR) technology,
Synthetic oligonucleotides are useful in selecting the correct clone from a library. Complementary sequences are also used as probes, primers, or antisense strands. A variety of fragments (eg, an anchored vector or poly-A complementary PCR technique, or a fragment ligated with the complementary DNA of another peptide) should be particularly useful.

The present invention relates to isolated D-proteins which encode the corresponding biologically active polypeptides.
The use of NA or fragments is contemplated. In addition, the present invention relates to isolated DNA or recombinant DNs encoding biologically active proteins or polypeptides capable of hybridizing under appropriate conditions to the DNA sequences described herein.
A. The biologically active protein or polypeptide can be an intact antigen or fragment and can be, for example, SEQ ID NOs: 2, 6, 8,
10, 12, 17, 19, 21, 23, 25, 32, 34, 36, 38, 42,
44, 46, 48, 49, or 54. Further, the invention includes the use of isolated or recombinant DNA, or fragments thereof, which encode proteins that are homologous to the desired protein, or encode such proteins. Encodes a protein isolated using the cDNA as a probe. The isolated DNA is 5
The 'and 3' flanking regions may have their respective regulatory sequences (eg, promoter, enhancer, polyA addition signal, etc.).

A “recombinant” nucleic acid is defined either by its method of production or by its structure. With respect to the method of production (eg, the product made by one process), the process involves the use of recombinant nucleic acid technology (eg, including human intervention in the nucleotide sequence, typically selection or production). is there. Or,
It can be a nucleic acid made by generating a sequence that contains a fusion of two fragments that are not naturally adjacent to each other, but is a natural product (eg,
(Naturally occurring variants). Thus, for example, a product made by transforming a cell with any non-naturally occurring vector, such as a nucleic acid comprising a sequence derived using any synthetic oligonucleotide process, is included. Such are often made to replace codons with degenerate codons encoding the same or conserved amino acids, but typically introduce or remove a sequence recognition site.

Alternatively, linking nucleic acid segments of desired function is performed to produce a single genetic entity containing a combination of desired functions not found in commonly available natural forms. Restriction enzyme recognition sites are often targets for such artificial manipulation, but other site-specific targets (eg, promoters, DNA replication sites, regulatory sequences, control sequences, or other useful features) may be designed. Can be incorporated. A similar concept is contemplated for recombinant (eg, fusion) polypeptides. Due to the degeneracy of the genetic code, synthetic nucleic acids encoding polypeptides similar to fragments of these antigens, and fusions of sequences from a variety of different species variants are specifically included.

[0183] In the context of nucleic acids, a significant "fragment" is at least about 17 nucleotides, generally at least about 22 nucleotides, usually at least about 29 nucleotides, more often at least about 35 nucleotides, typically at least about 35 nucleotides. 41 nucleotides, usually at least about 47 nucleotides, preferably at least about 55 nucleotides, and in a particularly preferred embodiment at least about 60 nucleotides
It is a continuous segment of the above nucleotides.

DC-PGT protein, HDTEA84 protein, HSLJD37R protein, RANKL protein, HCC5 protein, Dub protein, MD
DNA encoding the -1 protein, MD-2 protein, or cyclin E2 protein may be, inter alia, a DNA species encoding a related protein or homologous protein, as well as a DNA encoding a homologous protein from a different species, mRN.
Useful for identifying species A and cDNA species. Homologs appear to exist in other species, including primates, rodents, birds, and fish. Such various proteins should be homologous and are included herein. However, even genes encoding proteins with a more distant evolutionary relationship to this antigen, if they are sufficiently homologous, can be readily isolated under appropriate conditions using these sequences. Can be done. Primate proteins are proteins of particular interest.

Recombinant clones derived from genomic sequences, including, for example, introns, are useful for transgenic studies, including, for example, transgenic cells and organisms, and for gene therapy. For example, Go
odnow (1992) "Transgenic Animals" Roitt
(Ed.) Encyclopedia of Immunology, Academ
ic Press, San Diego, pp. 1502-1504; Travis
(1992) Science 256: 1392-1394; Kuhn et al. (19
91) Science 254: 707-710; Capecchi (1989)
) Science 244: 1288; Robertson (1987, ed.) T.
eratocarcinomas and Embryonic Stem C
cells: A Practical Approach, IRL Press,
Oxford; and Rosenberg (1992) J. Mol. Clinical
See Oncology 10: 180-199.

[0186] Substantial homology in the context of nucleic acid sequence comparison, when compared, when either the segment or its complement, with the appropriate nucleotide insertions or deletions, is optimally aligned, At least about 50%, typically at least about 58%, usually at least about 65%, often at least about 71% of the nucleotides
Typically at least about 77%, usually at least about 85%, preferably at least about 95-98% or more, and in certain embodiments, about 9% of the nucleotides.
It is high up to 9% or more, which means that they are the same. Alternatively, the substantial homology is
The segment is present when it hybridizes under selective hybridization conditions to the strand or its complement, typically using the sequence of DC-PGT (eg, in SEQ ID NO: 1). Typically, selective hybridization will be at least about 55% homology over a stretch of at least about 30 nucleotides, preferably at least about 75% homology over a stretch of about 25 nucleotides, and most preferably about 20 nucleotides. Occurs when there is at least about 90% homology over Kanehisa (19
84) Nuc. Acids. Res. 12: 203-213. The length of the homology comparison as described can span a longer stretch, and in certain embodiments, is at least about 17 nucleotides, usually at least about 28 nucleotides.
It spans a stretch of nucleotides, typically at least about 40 nucleotides, and preferably at least about 75 to 100 or more nucleotides.

[0187] Stringent conditions when referring to homology in the context of hybridization include the use of salts, temperatures, organic solvents, and other parameters, typically other parameters that are controlled in the hybridization reaction. , Stringent combination conditions. Stringent temperature conditions are generally above about 30 ° C, usually above about 37 ° C, typically about 55 ° C.
, Preferably greater than about 70 ° C. Stringent salt conditions are usually less than about 1000 mM, usually less than about 400 mM, typically less than about 2 mM.
It is less than 50 mM, preferably less than about 150 mM. However, the combination of parameters is much more important than the measurement of any single parameter. See, for example, Wetmur and Davidson (1968) J. Mol. Mol. Biol.
31: 349-370. Hybridization under stringent conditions is above background and at least 2 background.
Times, preferably at least 3-5 times or more.

[0188] DC-PGT, HDTEA84, HSLJD37R, R from other mammalian species
ANKL, HCC5, Dub11, Dub12, MD-1, MD-2, or cyclin E2 can be cloned and isolated by cross-hybridization between closely related species. Between closely related species, the homology can be relatively low, and therefore the hybridization of relatively closely related species is
Be wise. Alternatively, preparation of an antibody preparation that exhibits lower species specificity may be useful in an expression cloning approach.

VII. Production of Proteins; Mimics Nucleic acids encoding the described proteins or fragments thereof can be prepared by chemical synthesis, screening of cDNA libraries, or genomes prepared from a wide variety of cell lines or tissue samples. It can be obtained by screening libraries. See, for example, Okayama and Berg (1982) Mol. Cell
. Biol. 2: 161-170; Gubler and Hoffman (198
3) Gene 25: 263-269; and Glover (1984, eds.)
DNA Cloning: A Practical Approach, IRL
See Press, Oxford. Alternatively, the sequences provided herein provide useful PCR primers or synthetic or other preparations (including naturally occurring embodiments) of the appropriate genes encoding the receptor.
Enable.

[0190] DNA can be expressed in a wide variety of host cells for synthesis of full-length proteins or fragments. This protein or fragment can then be used, for example, to produce polyclonal or monoclonal antibodies; for binding studies; for construction and expression of the modified molecule; for structure / function studies; and detection assays. Can be used for controls in Each antigen or fragment thereof can be expressed in a host cell transformed or transfected with a suitable expression vector. These molecules can be substantially purified to be free of proteins or cellular contaminants other than those derived from the recombinant host, and thus, when combined with a pharmaceutically acceptable carrier and / or diluent. Particularly useful in pharmaceutical compositions. The antigen or a portion thereof can be expressed as a fusion with another protein.

As used herein, a vector includes plasmids, viruses, bacteriophages, integrable DNA fragments, and other vehicles,
They allow integration of the DNA fragment into the genome of the host. For example, Pouwels et al. (1985 and addendum) Cloning Vectors:
A Laboratory Manual, Elsevier, N.W. Y. And Rodriguez et al. (1988, eds.) Vectors: A Survey
of Molecular Cloning Vectors and The
See ir Uses, Buttersworth, Boston, MA. Expression vectors are typically self-replicating DNA or RNA constructs containing the desired antigen gene or fragment thereof, and are usually operably linked to the appropriate gene control elements recognized in the appropriate host cell. Is done.
These control elements can effect expression in a suitable host. The particular type of control element required to effect expression will depend on the ultimate host used.
In general, a gene control element may include a prokaryotic or eukaryotic promoter expression control system, and typically includes a transcriptional promoter, any promoter that controls initiation of transcription, the level of mRNA expression. Includes transcriptional enhancers that elevate, sequences encoding appropriate ribosome binding sites, 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.

For the purposes of the present invention, DNA sequences are operably linked when they are functionally related to each other. For example, DNA for a presequence or secretory leader is operably linked to a polypeptide when it is expressed as a preprotein or directs the polypeptide to the cell membrane or participates in secretion of the polypeptide. Is done. A promoter is operably linked to a coding sequence when it controls transcription of a polypeptide; a ribosome binding site is operably linked to a coding sequence when it is positioned to permit translation. Be linked. Operably linked usually means flanking and in reading frame, but certain genetic elements (eg, repressor genes)
Not linked adjacently, but still binds to an operator sequence that controls expression. See, for example, Rodriguez et al., Chapter 10, 205-236; Bal.
Bas and Bolivar (1990) Methods in Enzymo.
l. 185: 14-37; and Ausubel et al. (1993) Current.
Protocols in Molecular Biology, Green
See ne and Wiley, NY.

Representative examples of suitable expression vectors include pCDNA1; pCD (Okayama
a, et al. (1985) Mol. Cell Biol. 5: 1136-1142); pMC1neo Poly-A (Thomas et al. (1987) Cel.
l 51: 503-512); and pAC 373 or pAC.
Baculovirus vectors such as 610. For example, Miller
(1988) Ann. Rev .. Microbiol. 42: 177-199. Usually, expression vectors are designed for their stable replication in host cells, or for amplification to greatly increase the total number of copies of the desired gene per cell. It is not always necessary to require that the expression vector replicate in the host cell. For example, vectors that do not contain an origin of replication recognized by the host cell can be used to provide for transient expression of the antigen or fragment thereof in various hosts. It is also possible to use vectors that cause the integration of the gene or fragment thereof into the host DNA by recombination, or to integrate a promoter that controls the expression of the endogenous gene.

Adenovirus technology is available for expression of genes in various cells and organs. See, eg, Hitt et al. (1997) Adv. Pharmacol
. 40: 137-195; and Quantum Biotechnology
See es, Montreal, Canada. Animals can be useful for determining the effect of a gene on various developmentally or physiologically functional animal systems.

[0195] Suitable host cells include prokaryotes, lower eukaryotes, and higher eukaryotes. Prokaryotes include both gram negative and gram positive organisms (eg, E. coli).
And B. subtilis). Lower eukaryotes are yeast (eg, Sc
erevisiae and Pichia, and Dictyostelium
Genus). Higher eukaryotes are of both non-mammalian origin (eg, insect cells and birds) and mammalian origin (eg, human, primate, and rodent).
Includes established tissue culture cell lines derived from animal cells.

[0196] Prokaryotic host vector systems include a wide variety of vectors for many different species. As used herein, E.I. E. coli and its vectors are used genetically to include equivalent vectors used in other prokaryotes. D
An exemplary vector for amplifying NA is pBR322 or many of its derivatives. Vectors that can be used to express the prostaglandin transporter or fragments thereof include the lac promoter (pUC series)
A trp promoter (pBR322-trp); an Ipp promoter (pIN
Series); a vector containing a lambda-pP or pR promoter (pOTS); or a hybrid promoter such as ptac (pDR540). Brosius et al. (1988)
Expression Vectors Employing Lambda
, Trp-, lac-, and Ipp-derived Promoters "
Rodriguez and Denhardt (eds.) Vectors: A Sur
bay of Molecular Cloning Vectors and
See Their Uses, Butterworth, Boston, Chapter 10, pages 205-236, which is incorporated herein by reference.

[0197] Lower eukaryotes, such as yeast and Dictyostelium, can be transformed with vectors encoding vertebrate prostaglandin transporters. For the purposes of the present invention, most common lower eukaryotic hosts are baker's yeast,
Saccharomyces cerevisiae. It is commonly used on behalf of lower eukaryotes, but numerous other strains and species are also available. Yeast vectors typically contain an origin of replication (if not integrated), a selection gene, a promoter, DNA encoding the desired protein or fragment thereof, and sequences for translation termination, polyadenylation, and transcription termination. Become. Suitable expression vectors for yeast are constitutive promoters, such as the gene promoters for 3-phosphoglycerate kinase and various other glycolytic enzymes, or inducible promoters, such as the alcohol dehydrogenase 2 promoter or the metallothionein promoter. including. Suitable vectors include the following types of derivatives: self-replicating low copy number (eg, YRp series), self-replicating high copy number (eg, YEp series);
Or mini-chromosomes (eg, YCp series).

Higher eukaryotic tissue culture cells are preferred host cells for the expression of functionally active prostaglandin transporters. In principle, most higher eukaryotic tissue culture cell lines (eg, insect baculovirus expression systems) can be used whether from invertebrate or vertebrate sources. . However, mammalian cells are preferred because processing (both concurrent and post-translation) has a strong tendency to mimic the natural form. Transformation or transfection and propagation of such cells has become a routine procedure. Examples of useful cell lines include HeLa cells, Chinese hamster ovary (CHO) cell line, baby rat kidney (BRK) cell line, insect cell line, avian cell line, and monkey (COS)
) Cell lines. Expression vectors for such cell lines usually include an origin of replication, a promoter, a translation initiation site, an RNA splicing site (genomic DNA).
Is used), a polyadenylation site, and a transcription termination site. These vectors also usually contain a selection gene or an amplification gene. A suitable expression vector can be, for example, a plasmid, virus, or retrovirus having a promoter from a source such as from adenovirus, SV40, parvovirus, vaccinia virus, or cytomegalovirus. Representative examples of suitable expression vectors include pCDNA1; pCD (Okayama
a, et al. (1985) Mol. Cell. Biol. 5: 1136-1142); pMC1neo Poly-A (Thomas et al. (1987) Cel.
l 51: 503-512); and pAC373 or pAC6.
Baculovirus vectors such as 10 are included.

In a system that provides a specific or defined glycosylation pattern, DC
Expressing a PGT polypeptide, HDTEA84 polypeptide, HSLJD37R polypeptide, RANKL polypeptide, HCC5 polypeptide, Dub11 polypeptide, Dub12 polypeptide, MD-1 polypeptide, MD-2 polypeptide, or cyclin E2 polypeptide. Is often desired. For example, Luckow and Summers (1988) Bio / Technol.
6: 47-55; and Kaufman (1990) Meth. Enz
ymol. 185: 487-511. Preferred prokaryotic forms lack eukaryotic glycosylation patterns. However, the pattern can be altered by exposure to the polypeptide (eg, from an unglycosylated form to a suitable glycosylated protein introduced into a heterologous expression system). For example,
The desired gene can be co-transformed with one or more genes encoding a mammalian or other glycosylation enzyme. Using this approach, specific mammalian glycosylation patterns can be achieved or approached in prokaryotic or other cells.

DC-PGT, HDTEA84, HSLJD37R, RANKL, HCC5,
Dub11, Dub12, MD-1, MD-2, or cyclin E2, or a fragment thereof, is bound to cell membrane-bound phosphatidylinositol (PI)
However, it can be removed from the membrane by treatment with a phosphatidylinositol cleaving enzyme (eg, phosphatidylinositol phospholipase C). This releases the antigen, which is biologically active, and allows purification by standard procedures of protein chemistry. For example, Low (1989) Bi
ochim. Biophys. Acta 988: 427-454; Tse et al. (
1985) Science 230: 1003-1008; and Brunne.
(1991) J. R. et al. Cell. Biol. 114: 1275-1283.

The transformed cells are preferably mammalian cells, typically transformed or transfected with a vector comprising a prostaglandin transporter gene, constructed using recombinant DNA technology. Including cells. A transformed host cell usually expresses the antigen or a fragment thereof, but need not express the protein for the purposes of cloning, amplifying, and manipulating the DNA. The invention further contemplates culturing the transformed cells in a nutrient medium, thus allowing the protein or soluble fragment thereof to accumulate in the culture. Soluble proteins can be recovered from either culture or culture medium, and membrane-bound proteins can be prepared from the appropriate subcellular fraction.

[0202] Now that the genes have been characterized, their fragments or derivatives can be prepared by conventional processes for peptide synthesis. These include
Stewart and Young (1984) Solid Phase Pep
tide Synthesis, Pierce Chemical Co. , R
Ockford, IL; Bodanszky and Bodanszky (198
4) The Practice of Peptide Synthesis,
Springer-Verlag, New York; and Bodansz
ky (1984) The Principles of Peptide Sy
nthesis, Springer-Verlag, New York. For example, azide process, acid chloride process, acid anhydride process, mixed anhydride process, active ester process (eg, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethyl ester), carbodiimidazole process, Redox process or dicyclohexylcarbodiimide (DCCD) /
Additional processes may be used. Both solid phase and liquid phase synthesis are applicable for the process described above.

A protein, fragment, or derivative typically involves the condensation of amino acids to terminal amino acids, one at a time, in sequence, according to the processes described above as utilized in peptide synthesis. Prepared either by a so-called step-wise process or by coupling peptide fragments to terminal amino acids. Amino groups not used in the coupling reaction are typically protected to prevent mis-coupling.

When solid phase synthesis is employed, the C-terminal amino acid is attached to the insoluble carrier or support through its carboxyl group. The insoluble carrier is not particularly limited as long as it has an ability to bind to a reactive carboxyl group. Examples of such insoluble carriers include halomethyl resins (eg, chloromethyl or bromomethyl resins), hydroxymethyl resins, phenolic resins, tert-alkyloxycarbonyl-hydrazide resins, and the like.

An amino acid protected with an amino group can be obtained by condensation of the activated carboxyl group,
And through a reactive amino group of a previously formed peptide or chain, linked in sequence to synthesize the peptide stepwise. After synthesizing the complete sequence, the peptide is separated from the insoluble carrier to produce the peptide. This solid-phase approach is generally described in Merrifield et al. (1963) J. Mol. Am. Chem
. Soc. 85: 2149-2156, which is incorporated herein by reference.

The prepared proteins and fragments thereof can be isolated and purified from the reaction mixture by peptide separation, for example, by extraction, precipitation, electrophoresis, and various forms of chromatography. The proteins of the present invention can be obtained in varying degrees of purity depending on the desired use. Purification can be by use of the protein purification techniques disclosed herein or by use of the antibodies described herein in immunosorbent affinity chromatography.
Can be achieved. This immunosorbent affinity chromatography involves first binding the antibody to a solid support, and then binding the bound antibody to a solubilized lysate of cells from a suitable source, lysates of other cells expressing the protein, Alternatively, it is performed by contacting (see below) with cell lysates or supernatants that produce the desired protein as a result of DNA technology. Detergents may need to be included in the method to maintain protein solubility.

VIII. Uses The invention is described elsewhere herein (eg, in the general description of cell-mediated conditions or in the following description of kits for diagnosis). Such reagents find use in diagnostic applications. This gene is useful in forensic analysis (eg, identifying species or diagnosing different cell subsets or cell types).

[0208] DC-PGT, when used to remove prostaglandins (PG) and other metabolically active organic anions from the body (liver, fetal liver, lung, and placenta), is responsible for this mechanism. It is easy to assume that changes in performance can enhance an allergic response. Prostaglandin PGF ₂ alpha and PGD _2, and PGG ₂ and thromboxane A ₂ is particularly can cause airway obstruction in peripheral lung while PGE ₂ and PGI ₂ is a bronchodilator. Use of the transporters of the present invention may assist in the transport or removal of these prostaglandins to modify airway obstruction.

In addition, prostaglandins play an important role in the secondary immunosuppression seen after surgical stress. Alexander (1990) J. Am. Trau
ma 30: S70; Faist et al. Trauma 27:83
7; Nineman et al. Trauma 24: 201; Woo
d, et al. (1987) Arch. Surg. 122: 179; Polk et al., Erem.
in and Sewell (eds. 1992) The Immunological
Basis of Surgical Science and Practi
ce, Oxford U.S. Press. In particular, PGE2 inhibits lymphocyte proliferation, reduces IL-2 release, reduces response to IL-2, inhibits natural killer cells, and activates suppressor cells. The major injury is IL
It has been shown to result in decreased production of PGE2 from inhibitory macrophages, as well as reduced production of -1 and IL-2. This effect can last for 7-10 days after a major injury. Other studies did not show an increase in circulating PGE2 following burns, but found increased local production with increased sensitivity of lymphocytes to the effects of PGE2.

Prostaglandin E2 may play a role in rheumatoid arthritis by promoting the entry of inflammatory cells into the joint through locally produced vasodilatory effects. Once in synovial fluid, polymorphonuclear leukocytes can take up immune complexes, which in turn cause neutrophils to produce reactive oxygen metabolites and other inflammatory mediators, leading to more inflammatory Strengthen the cascade. Henson et al. (1
987) J. Clin. Invest. 79: 699.

Thus, it is possible to use the present invention to modulate prostaglandins in subjects suffering from trauma, injury, disease, or in post-surgical treatment.

[0212] Immune system cells can synthesize PG, and thus it may be useful to use DC-PGT in the role of efflux to remove PG from the intracellular space. Similarly,
DC-PGT can transport certain organic anions. Abnormal growth, reproduction, degeneration,
And atrophy can be controlled by appropriate therapeutic treatment, using the compositions provided herein. For example, a disease or disorder associated with aberrant prostaglandin transporter function appears to be a target for a substrate or blocking substrate. Alternatively, transporters may be useful tools for providing important metabolites or metabolite blockers to each cell.

For example, transformation with a transporter can increase the availability of a substrate for a cell. In certain situations, the prostaglandin analog may be advantageously provided to the cell. The prostaglandin analog may confer high sensitivity (eg, radiosensitivity or other sensitivity) to further treatment, or may directly affect normal metabolism (eg, nucleic acid-related enzymes). Alternatively, the transporter may be useful for screening for antagonists or inhibitors that may be effective in blocking the normal availability of natural substrates to cells. Methods for screening for such prostaglandin analogs are provided.

Screening using the prostaglandin transporter for binding metabolites or compounds with binding affinity for the transporter, including isolation of the relevant components, can be performed. Subsequent biological assays then determine whether the compound has an intrinsic biological activity and, therefore, whether it is an agonist or antagonist to block the activity of the transporter. Can be used for In particular, prostaglandin analogs may be useful in blocking the binding of a natural target, or otherwise in blocking transporter activity. Alternatively, various other analogs may be useful in blocking ion transporters or organic anion sources. The invention further contemplates the therapeutic use of antibodies to the prostaglandin transporter as antagonists. This approach is particularly useful with other prostaglandin transporter species variants and other members of the family.

Antagonists of transporter activity (eg, antibodies that block transport)
Can be useful in a variety of medical conditions. These include immune, inflammatory, or allergic abnormalities, all of which are important when transport of organic anions occurs. Certain congenital disorders of prostaglandin physiology are susceptible to such therapeutic approaches.

HDTEA84, HSLJD37R, RANKL, HCC5, MD-1, or MD-2 (naturally occurring or recombinant), fragments thereof, and antibodies to them are identified as having binding affinity for them Aberrant growth (e.g., a cancerous condition, or a degenerative condition), along with the administered compound.
Useful in treating conditions associated with abnormal physiology or development. In particular, modulation of lymphocyte development is achieved by appropriate therapeutic treatment using the compositions provided herein. For example, a disease or disorder associated with abnormal expression or abnormal signaling by a ligand or receptor appears to be a target for an agonist or antagonist of the antigen. The antigen may be a hematopoietic cell (eg,
Plays a role in the regulation or development of lymphocytes that affect immunological responses (eg, autoimmune disorders).

In particular, the antigen may provide a costimulatory signal for activation of the cell, or may be involved in regulating the growth or differentiation of the cell. Therefore, HDTEA84, HSLJD
37R, RANKL, HCC5, MD-1, or MD-2 appear to regulate cells that have receptors for them (eg, T cell-mediated interactions with other cell types). These interactions, in certain circumstances, lead to the regulation of cell proliferation, cytokine synthesis by these or other cells, or the development of certain effector cells.

Furthermore, HDTEA84, HSLJD37R, RANKL, HCC5, MD-
1, or MD-2, or an antagonist may redirect a T cell response (
For example, during Th1 and Th2 polarization or with Th0 cells), or may affect B cells or other lymphocyte subsets. Among these agonists are, for example, various antibodies that recognize appropriate epitopes that mimic the binding of a ligand or receptor to its partner. Alternatively, they can bind sterically to epitopes that can block receptor binding. Bone morphogenesis can be regulated by these receptor segments.

Ligands or receptors are associated with abnormal conditions (eg, rheumatoid arthritis, systemic lupus erythematosus (SLE), autoimmune disorders including Hashimoto's autoimmune thyroiditis,
And selective and potent methods for modulating the immune response in acute and chronic inflammatory responses, where T cell activation, expansion, and / or immunological T cell memory play an important role I can do it. Samter et al. (Eds.) Immunol
optical Diseases Volumes 1 and 2, Little, Bro
wn and Co. See also HDTEA84, HSLJD37R,
Modulation of bone morphogenesis, T cell activation, expansion, and / or cytokine release by a naturally occurring secreted form of RANKL, HCC5, MD-1, or MD-2 or an antagonist thereof may be effected.

[0220] In addition, certain combination compositions with other modulators of signaling, especially TNF receptor-like genes, are useful. Such other signaling molecules include, for example, TCR reagents, CD40, CD40L, CTLA-8, CD28,
SLAM, FAS, osteoprotegerin
And their respective antagonists (including antibodies).

The cyclin E2 nucleotides (eg, human cyclin E2 DNA or R
NA) can be used as a component in forensic assays. For example, the provided nucleotide sequence can be labeled using, for example, ³² P or biotin,
It can then be used to probe standard restriction fragment polymorphism blots that provide measurable characteristics and aid in discrimination between individuals. Such probes can be used in well-known forensic techniques, such as genetic fingerprinting. In addition, nucleotide probes generated from cyclin E2 sequences can be used in in situ assays to detect chromosomal abnormalities. For example, rearrangements in the human chromosome encoding the cyclin E2 gene can be detected via well-known in situ techniques using a cyclin E2 probe along with other known chromosomal markers. The cyclin E2 gene is
In a variety of cancers, such as breast cancer, it may have prognostic utility.

Antibodies and other binding agents directed against cyclin E2 proteins or nucleic acids can be used to purify the corresponding cyclin E2 molecules. As described in the examples below, antibody purification of the cyclin E2 protein component is both possible and practical. Antibodies and other binding agents may also be diagnostic to determine whether a cyclin E2 protein component is present in a tissue sample or cell population using well-known techniques described herein. Can be used in any manner. The ability of the binding agent to bind to the cyclin E2 protein
It provides a means for diagnosing disorders associated with dysregulation of cyclin E2 protein. Antibodies and other cyclin E2 protein binding agents may also be useful as histological markers. As described in the Examples below, cyclin E2 protein expression is restricted to certain tissue types. By directing the probe (eg, an antibody or nucleic acid against the cyclin E2 protein), it is possible to use the probe to distinguish tissue and cell types in situ or in vitro.

[0223] The invention also provides reagents of significant therapeutic value. Cyclin E
2 proteins (naturally occurring or recombinant), fragments thereof, and antibodies thereto, along with compounds identified as having binding affinity for the cyclin E2 protein, may be associated with abnormal growth (eg, cancerous conditions). Or degenerative conditions), including conditions associated with abnormal physiology or development.
Abnormal growth, regeneration, degeneration, and atrophy can be modulated by appropriate therapeutic treatment using the compositions provided herein. For example, diseases or disorders associated with aberrant expression or aberrant signaling by the cyclin E2 protein are targets for agonists or antagonists of this protein. This protein appears to play a role in the regulation or development of nerve cells or hematopoietic cells (eg, lymphocytes, which affect immunological responses).

Various abnormal conditions can be detected by Northern blot analysis, for example, HDTEA8
4. Known in each of the cell types shown to have mRNA. Ber
kow (ed.) The Merck Manual of Diagnostics
and Therapy, Merck & Co. , Rahway, NJ; Th
Orn et al., Harrison's Principles of Intern
al Medicine, McGraw-Hill, NY; and Weather.
rall et al. (eds.) Oxford Textbook of Medicine,
See Oxford University Press, Oxford. Many other medical conditions and diseases involve or are T-cell mediated, and many of these are responsive to treatment with the agonists or antagonists provided herein. obtain. For example, States and Te
rr (1991) Basic and Clinical Immunolo
gy Appleton and Lange, Norwalk, CT; and Samter et al. (eds.) Immunological Diseases Lite.
tle, Brown and Co. checking ... These problems should be susceptible to prophylaxis or treatment using the compositions provided herein.

[0225] Certain or selective antibodies are purified, and then purified to a patient (veterinary animal or human).
Can be administered. These reagents may be used for therapeutic use with additional active or inactive ingredients such as conventional pharmaceutically acceptable carriers or diluents such as physiologically non-toxic stabilizers, excipients, Or immunogenic adjuvants with preservatives))). These combinations can be sterile filtered and placed into dosage forms such as by lyophilization in dosage vials or by storage in stabilized aqueous preparations. The invention also contemplates the use of antibodies or binding fragments thereof, including forms that are not complement-fixed.

Drug screening using proteins or fragments thereof, including isolation of relevant components, can be performed to identify compounds that have binding affinity or other relevant biological effects on antigen function. . Subsequent biological assays are then utilized to determine whether the compound has an intrinsic stimulatory activity, or because the compound blocks the activity of an antigen, the compound is a blocker or antagonist (eg, a mutein antagonist). Or not. Similarly, compounds having an intrinsic stimulatory activity may activate the signaling pathway, and thus the compounds are agonists, as they overcome any blocking activity of these soluble forms of the receptor. The invention further relates to agonists or antagonists and to stimulatory molecules (eg, muteins as agonists)
Therapeutic use of blocking antibodies to ligands or receptors is contemplated. This approach using other soluble receptor species variants is particularly useful.

Another therapeutic approach encompassed by the present invention includes immune disorders, including, but not limited to, any of the conventional administration techniques, including but not limited to local injection, inhalation, or systemic administration. Direct administration of the reagents or compositions to a subject having an allergic disorder or traumatic disorder is included. Reagents, formulations or compositions included within the boundaries of the invention can also be targeted to specific cells or transporters by the methods described herein. The actual dose of a reagent, formulation, or composition that modulates an immune, allergic, or traumatic disorder will depend on many factors, including the size and health of the organism, but one of skill in the art The following teachings describing methods and techniques for determining a clinical dose may be used. For example, Spilker (1984) Guide to Clinical Stu
dies and Developing Protocols, Raven
Press Books, Ltd. , New York, pp. 7-13, 54-6.
Page 0; Spicker (1991) Guide to Clinical Tri
als, Raven Press Books, Ltd. , New York,
93-101; Craig and Stitzel, eds. (1986) Modern.
Pharmacology, 2nd edition, Little Brown and C
o. , Boston, pp. 127-33; edited by Speed (1987) Aver.
y's Drug Treatment: Principles and Pr
actice of Clinical Pharmacology and
Therapeutics, Third Edition, Williams and Wilkin
s, Baltimore, pp. 50-56; Tallalida et al. (1988) P.
rinciples in General Pharmacology, Sp
See riger-Verlag, New York, pp. 18-20. In general, the dose will be in a pharmaceutically acceptable carrier such that the final concentration administered per day to the adult is between about 0.5 fg / ml and 500 μg / ml, inclusive. is there.

The amount of reagents required for effective treatment depends on a number of different factors (means of administration, target site,
(Including the patient's physiological condition and other medications administered). Therefore,
Treatment dose determinations should be titrated to optimize safety and efficacy.
Typically, dosage determinations used in vitro can provide useful guidance in useful amounts of these reagents for in situ administration. Animal testing of effective doses for treatment of a particular disorder will provide additional putative indicators of human dosing. Various studies are described, for example, in Gilman et al. (1990), Goodman.
and Gilman: The Pharmacological Base
s of Therapeutics, 8th Edition, Pergamon Press
; And Remington's Pharmaceutical Scie
nces, 17th edition (1990), Mack Publishing Co. ,
Easton, Penn. . For example, administration methods such as oral, intravenous, intraperitoneal, or intramuscular administration, and transdermal diffusion are discussed. Pharmaceutically acceptable carriers include water, saline, buffers, and, for example, Merck Index, M
erck & Co. , Rahway, New Jersey. The dosage form ranges from a concentration of less than 1 mM, typically about 10 μm.
It is generally expected that concentrations below about M, usually about 100 nM Myori, preferably below about 10 pM (picomolar), and most preferably about 1 fM (femtomolar), with the appropriate carrier. Sustained-release formulations or devices are often utilized for continuous or long-term administration. For example, Langer (1
990) Science 249: 1527-1533.

The ligands, receptors, enzymes, fragments thereof, and antibodies thereto or fragments thereof, antagonists, and agonists can be administered directly to the host to be treated, or, depending on the size of the compound, Previously, it may be desirable to be conjugated to a carrier protein such as ovalbumin or serum albumin. Therapeutic formulations can 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. The formulations typically comprise at least one active ingredient (as defined above) together with one or more of its acceptable carriers. Each carrier should be acceptable in both a pharmaceutical and physiological sense, in the sense that it is compatible with the other ingredients and is not harmful to the patient. Formulations include those suitable for oral, rectal, nasal, topical, or parenteral (including subcutaneous, intramuscular, intravenous and intradermal) administration. Such formulations may conveniently be presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. For example, Gil
(1990) Goodman and Gilman: The Phar.
macological Bases of Therapeutics, No. 8
Edition, Pergamon Press; and Remington's Pha
rmaceutical Sciences, 17th edition (1990), Mack
Publishing Co. Easton, Penn. Avis et al. (1
993) Pharmaceutical Dosage Forms: Par
enteral Medicines, Dekker, New York;
Lieberman et al. (1990) Pharmaceutical Dosa
ge Forms: Tablets, Dekker, New York; and Lieberman et al. (1990) Pharmaceutical Dos.
age Forms: Disperse Systems, Dekker, Ne
See w York. Therapies of the present invention may include other agents, such as other modulators of cell activation (eg, CD40, CD40 ligand, CD28, CTLA-4,
B7, B70, SLAM, T cell receptor signaling entity, or their respective antagonists), or may be used in association therewith.

[0230] Both naturally occurring and recombinant forms of the proteins of the present invention are particularly useful in kits and assay methods that can screen compounds for binding activity to the protein. Several methods of automated assays have been developed in recent years, enabling the screening of tens of thousands of compounds in a short time. See, e.g., Fodor et al. (1991) Science 251: 767-773. It describes a means of testing binding affinity with a plurality of predetermined polymers synthesized on a solid substrate. The development of a suitable assay can be greatly facilitated by the availability of large amounts of purified, soluble proteins or nucleic acids, as provided by the present invention.

[0231] Other methods can be used to determine key residues in a substrate, ligand, or receptor binding interaction. Mutation analysis (eg, Somoza et al. (199)
3) J.I. Expo. Med. 178: 549-558) can be performed to determine particular residues important in interaction and / or signal transduction. This allows the study of both extracellular domains (involved in soluble ligand interactions) or transmembrane forms of intracellular domains (providing important interactions in intracellular signaling).

For example, once the antigen is structurally defined, eg, by three-dimensional structural data, an antagonist can usually be found. Testing for potential interacting analogs is now possible based on the development of highly automated assay methods using purified proteins. In particular, novel agonists and antagonists are discovered by using the screening techniques described herein. Of particular interest are compounds that have been found to have complex binding affinity for a range of protein molecules, for example, compounds that can act as antagonists on species variants of an antigen.

One method of drug screening involves recombinant DN expressing the desired protein.
Eukaryotic or prokaryotic host cells stably transformed with the A molecule are utilized. Upon isolation from other molecules, cells expressing the selected protein can be isolated. Such cells, whether in viable or fixed form, can be used in standard binding partner binding assays. Par
ce et al. (1989) Science 246: 243-247; and Owick.
ki et al. (1990) Proc. Nat'l. Acad. Sci. USA 87:
See also 4007-4011. These describe sensitive methods for detecting cellular responses.

Another technique for drug screening includes approaches that provide high-throughput screening for compounds with the appropriate binding affinity for the desired target protein, as described in Geysen, European Patent Application No. 84 / 0
No. 3564 (published September 13, 1984). First, a large number of different small peptide test compounds are synthesized on a solid substrate, such as a plastic pin or some other suitable surface (see Fodor et al. (1991)). The pins are then reacted with either the solubilized unpurified target protein or the solubilized purified target protein and washed.
The next step involves detecting the bound protein.

[0235] Rational drug design can also be based on the molecular shape of the protein and structural studies of other effectors or analogs. Effectors can be other proteins that mediate other functions in response to binding, or other proteins that normally interact. One means of determining which sites interact with certain other proteins is physical structure determination (eg, X-ray crystallography or two-dimensional NMR techniques). These provide guidance as to which amino acid residues form a molecular contact region. For a detailed description of protein structure determination, see, eg, Blundel
1 and Johnson (1976) Protein Crystallogr
aphy, Academic Press, New York.

IX. Kits The present invention also relates to the use of the proteins, fragments thereof, peptides and fusion products thereof of the present invention in various diagnostic kits and methods for detecting, for example, the presence of proteins or binding partners. Intended. Typically, the kit has a compartment containing either the described polypeptide or gene segment or a reagent that recognizes one or the other, for example, a fragment or an antibody. Alternatively, kits can be based on nucleic acids.

For example, kits for determining the binding affinity of a test compound for HDTEA84 typically include: a test compound; a labeled compound (eg, HDTEA84).
Binding partners or antibodies with known binding affinity for A84); HD
Source of TEA84 (naturally occurring or recombinant); and means to separate bound compound from free labeled compound (eg, solid phase to immobilize this molecule). Once the compounds have been screened, those having the appropriate binding affinity for this antigen can be evaluated in appropriate biological assays as is well known in the art, and these may be agonists or antagonists for the HDTEA84 signaling pathway. Or not. Recombinant HDT
The availability of an EA84 polypeptide also provides well-defined standards for calibrating such assays.

For example, a preferred kit for determining the concentration of HDTEA84 in a sample typically comprises: a labeled compound (eg, a binding partner or antibody) with a known binding affinity for the antigen; Source of antigen (naturally occurring or recombinant), as well as means for separating bound compounds from free bound compounds (eg, solid phase to immobilize HDTEA84). A compartment containing the reagents, as well as instructions, will usually be provided.

For example, antibodies specific to HDTEA84 or fragments (including antigen-binding fragments) are useful in diagnostic applications to detect the presence of elevated levels of HDTEA84 and / or fragments thereof. Such diagnostic assays may use lysates, live cells, fixed cells, immunofluorescence, cell cultures, body fluids, and may further include the detection of antigens related to antigens in serum. Diagnostic assays can be homogenous (without the step of separating the complex between free reagent and antigen binding partner) or heterogeneous (with the step of separation). There are a variety of commercially available assays, for example: radioimmunoassay (RIA), enzyme linked immunosorbent assay (ELISA), enzyme immunoassay (EIA), enzyme amplified immunoassay (EMIT), substrate Labeled fluorescent immunoassay (SLFIA) and the like. For example, V
and Vunakis et al. (1980) Meth Enzymol. 70: 1 to 1
525; Harlow and Lane (1980) Antibodies: A
Laboratory Manual, CSH Press, NY; and C
Oligan et al. (1993) Current Protocols in Im
See Munology, Greene and Wiley, NY.

Anti-idiotypic antibodies have similar uses for diagnosing the presence of antibodies to the described proteins. This is because antibodies against the described proteins can be characteristic of various abnormal conditions. Overproduction of the prostaglandin transporter can reflect a variety of medical conditions,
It may be characteristic of an abnormal physiological condition, especially a proliferative cell condition (eg, cancer or abnormal differentiation). For example, leukemias and lymphomas can exhibit altered transporter expression, which can reflect their altered physiology and provide a means for selective targeting. Alternatively, HDTEA84, HSLJD37R,
Overproduction of RANKL, HCC5, MD-1 or MD-2 can result in abnormal physiological conditions, the production of various immunological responses, which are particularly proliferative cell conditions such as cancer. Or abnormal activation or differentiation).
The expression level of DC-PGT, Dub, or cyclin E2 may also be characteristic (advantageous or disadvantageous) for a particular therapeutic condition.

[0241] Frequently, reagents for diagnostic assays are provided in kits to optimize the sensitivity of the assay. For the present invention, depending on the nature of the assay, protocols and labels (either labeled or unlabeled, antibodies or binding partners, or labeled HDTEA84) are provided. This usually involves other additives (eg, buffers, stabilizers, substances necessary for signal generation (eg, substrates for enzymes), etc.). Preferably, the kit also includes instructions for proper use and disposal of the contents after use. Typically, the kit has a compartment for each useful reagent. Desirably, the reagent is provided as a freeze-dried powder, wherein the reagent can be reconstituted in an aqueous medium to provide the appropriate concentration of reagent to perform the assay.

Many of the above-described components of drug screening and diagnostic assays can be used without modification or can be modified in various ways. For example, labeling can be accomplished by covalently or non-covalently attaching a moiety that directly or indirectly provides a detectable signal. In these assays,
A binding partner, a test compound, HDTEA84, or an antibody thereto,
It can be labeled either directly or indirectly. Possibilities for direct labeling include the following labeling groups: radioactive labels (eg, ¹²⁵ I), enzymes (US Pat. No. 3,645,090) (eg, peroxidase and alkaline phosphatase), and fluorescence intensity; Fluorescent labels that can monitor wavelength shifts or changes in fluorescence polarization (US Pat. No. 3,940,475). Possibilities for indirect labeling include biotinylation of one component followed by binding to avidin attached to one of the above labeling groups.

There are a number of ways to separate bound from free polypeptide or a number of ways to separate bound from free test compound. Polypeptides can be immobilized on various matrices and subsequently washed. Suitable matrices include plastics (eg, ELISA plates), filters, and beads. For example, Coligan et al. (1993) Current P
rotocols in Immunology, Volume 1, Chapter 2, Green
See e and Wiley, NY. Other suitable separation techniques include, but are not limited to: Rattle et al. (1984) Clin.
Chem. 30: 1457-1461, Fluorescein antibody magnetizable particle method, and bi-antibody magnetic particle separation as described in U.S. Patent No. 4,659,678.

[0244] Methods of attaching proteins or fragments thereof to various labels have been widely reported in the literature and do not require detailed description herein. Many of these techniques are either through the use of carbodiimides or active esters to form peptide bonds, through the reaction of mercapto groups with activated halogens (eg, chloroacetyl) to form thioesters, Through a reaction for coupling with an olefin (eg, maleimide)
Includes the use of activated carboxyl groups. Fusion proteins also find use in these applications.

Another diagnostic aspect of the invention involves the use of oligonucleotide or polynucleotide sequences obtained from the sequences of the described proteins. These sequences can be used as probes to detect levels of message in samples from patients suspected of having an abnormal condition (eg, cancer or developmental problems). Since antigens are markers of activation, it can be useful to determine the number of activated T cells, for example, to determine when further suppression is needed. The preparation of nucleotide sequences, both RNA and DNA, labeling of these sequences, and the preferred size of these sequences, is well-documented and discussed in the literature. For example, Langer-Safer et al. (1982) Pro.
c. Nat'l. Acad. Sci. 79: 4381-4385; Caske
y (1987) Science 236: 962-967; and Wilch
ek et al. (1988) Anal. Biochem. 171: 1-32.

Alternatively, an antibody that can recognize a specific duplex (including a DNA duplex, an RNA duplex, a DNA-RNA hybrid duplex, or a DNA-protein duplex) can be used. The antibody can then be labeled and an assay is performed in which the duplex is bound to the surface, such that upon formation of the duplex on the surface, the presence of the antibody bound to the duplex Can be detected. The use of probes for novel antisense RNAs can be performed using any of the conventional techniques (eg, nucleic acid hybridization, plus and minus screening, recombination screening).
al probing), hybrid released translation (HRT)
) And hybrid inhibitory translation (HRT)). This also includes amplification techniques such as the polymerase chain reaction (PCR).

Diagnostic kits that also test for the qualitative or quantitative presence of other markers are also contemplated. Diagnosis or prognosis may depend on a combination of multiple indicators used as markers. Thus, the kit may test for a combination of markers.
For example, Vialet et al. (1989) Progress in Growth.
Factor Res. 1: 89-97. Other kits can be used to evaluate a subset of T cells.

X. Method of Isolating Substrate / Specific Partner DC-PGT should interact with its substrate target. This substrate is similar to an organic molecule that is subjected to transport. Dub and cyclin E2 are also screened for substrate identification.

The HDTEA84, HSLJD37R, and RANKL proteins interact with TNF ligands based on, for example, similarities in their structure and function against other cell surface antigens displaying similar structures, and cell type specificity of expression Should do it. The MD-1 and MD-2 antigens are associated with known proteins, which interact with B cell antigens. Methods for isolating ligands are made available by the ability to generate purified proteins for screening programs. Similar techniques are applicable to HCC5 chemokines, and MD-1 and MD-2 surface receptors.

The sequences provided herein allow for the screening or isolation of particular ligands. Many methods exist for expression cloning, panning, affinity isolation, or other means for identifying ligands. A two-hybrid selection system can also be applied, and appropriate constructs with available sequences can be made as appropriate. For example, Fields and Song (1989) Na
340: 245-246.

The broad scope of the present invention is best understood by reference to the following examples. This example is not intended to limit the invention to any particular embodiment.

EXAMPLES General Methods Some of the standard methods are described or referenced below:
For example, Maniatis et al. (1982) Molecular Cloning.
, A Laboratory Manual, Cold Spring Har
Bor Laboratory, Cold Spring Harbor Pr
ess; Sambrook et al. (1989) Molecular Cloning.
: A Laboratory Manual (2nd edition) Volumes 1-3, CSH P
Ausubel et al., Biology, Green Publ.
ising Associates, Brooklyn, NY; or Au
subel et al. (1987 and addendum) Current Protocols i.
n Molecular Biology, Greene and Wiley
New York; Innis et al. (1990) PCR Protocols.
: A Guide to Methods and Applications
Academic Press, N .; Y. . Methods for protein purification include methods such as ammonium sulfate precipitation, column chromatography, electrophoresis, centrifugation, and crystallization. See, for example, Ausubel et al. (1
987 and periodic supplements); Deutscher (1990) “Guidet
o Protein Purification ”Methods in En
zymol. Vol. 182, and other volumes in this series; and manufacturer's literature on the use of protein purification products (eg, Pharmacia, Pisc)
ataway, N.M. J. , Or Bio-Rad, Richmond, CA).
Combination with recombinant techniques allows fusion with an appropriate segment, such as the FLAG sequence or an equivalent that can be fused via a protease-removable sequence. See, for example, Hochuli (1990) "Purific.
ation of Recombinant Proteins with M
et al Chelate Absorbent "Setlow, Genet
ic Engineering, Principle and Methods
12: 87-98, Plenum Press, N.M. Y. ; And Crow
e et al. (1992) QIAexpress: The High Level Ex.
Pression & Protein Purification System
, QIAGEN, Inc. Chatsworth, CA. Cell culture technology is D
Oyle et al. (1994) Cell and Tissue Culture:
Laboratory Procedures, John Wiley and
Sons, NY.

[0253] Standard immunological techniques are described, for example, in: Hertzenbe
rg et al. (1996) Weir's Handbook of Experim
ental Immunology, Volumes 1-4, Blackwell Sci
ence; Colligan (1991) Current Protocols
in Immunology, Wiley / Greene, NY; and Me
volumes in Enzymology, Volume 70, Volume 73, Volume 74, Volume 84, Volume 92, Volume 93, Volume 108, 116, Volume 121, Volume 132, Volume 150, and Volume 16
2 volumes, and 163 volumes.

The FACS analysis is described below: Melamed et al. (1990) Flo
w Cytometry and Sorting, Wiley-Liss, I
nc. , New York, NY; Shapiro (1988) Practic.
al Flow Cytometry, Liss, New York, NY; and Robinson et al. (1993) Handbook of Flow C.
ymetry Methods, Wiley-Liss, New York
, NY. Fluorescent labeling of the appropriate reagents was performed by standard methods.

FASTA (Pearson and Lipman, 1988) and BLAS
T (Altschul et al. (1990) J. Mol. Biol. 215: 403).
-410) The program uses non-overlapping protein and nucleotide databases (Benson et al. (1994) Nucl. Acids Res).
. 22: 3441-3444; Bairoch and Boeckmann (1
994) Nucl. Acids Res. 22: 3578-3580) was used to search using the resulting cDNA sequence and the encoded protein sequence. Altschul et al. (1994) Nature Genet. 6:
119-129; and Koonin et al. (1994) EMBO J. 13: 4
A sensitive search strategy of 93-503 served as an example of how to locate distant structural homologs of protein chains. Multiple alignments of the collected homologs are described by ClustalW (Thompson et al. (1994) Comp. A.
pplic. Biosci. 10: 19-29) and MACAW (Schul
(1991) Proteins 9: 180-190).

The protein (eg, DC-PGT) membrane topology, as well as the cohort of putative homologs, is composed of domains (eg, hydrophobic transmembrane helices, and likely cytoplasmic or extracellular exposures of hydrophilic binding loops). Were analyzed by various methods that attempted to determine the consensus number. For single sequence analysis, ALO
M and MTOP (Klein et al. (1985) Biochem. Biophys).
. Acta 815: 468-476; and Hartmann et al. (1989).
Proc. Natl. Acad. Sci. USA 86: 5786-5790).
PSORT World-Wide Web site (Nakai
And Kanehisa (1991) Proteins 11: 95-110;
And Nakai and Kanehisa (1992) Genomics 1
4: 897-911) and then access to the TopPredII program (Claros and von Heijne (1994) Comp. Appl.
ic. Biosci. 10: 686-686; MacIntosh PPC version) is used to dissect the chains into possible hydrophobic transmembrane and loop regions of DC-PGT, and furthermore, to position these latter regions with charged residues. (Von Heijne (1992) J. Mol.
Biol. 225: 487-494; and Sippos and von He.
ijne (1993) Eur. J. Biochem. 213: 1333-134
0). MEMSAT (Jones et al. (1994) Biochem. 33: 303).
8-3049; MS-DOS PC version is used as well, with individual sequences fitting to a statistically based topological model, which makes decisions on transmembrane and loop chain segments of the membrane Was done. Two Web-accessible programs that allow the use of evolutionary data by analyzing multiple aligned sequences are described in PHD (Rost et al. (1994) Comp. Applic.
Biosci. 10: 53-60; and Rost et al. (1995) Protei.
n Sci. 4: 521-533) and TMAP (Persson and A).
rgos (1994) J. Mol. Mol. Biol. 237: 182-192); the former utilizes a neutral network system to accurately predict the shared location of the transmembrane segment of the helix in the protein family. Similar analysis of other proteins can be performed.

(I. Production of Dendritic Cells) Human CD34 + cells were obtained as follows. For example, Caux et al. (1995)
), Bauchereau and Schmitt, Dendrick Cells
in Fundamental and Clinical Immunol
, Plenum Press, NY, pages 1-5. Peripheral or umbilical cord blood cells (sometimes selected CD34 +) are transferred to stem cell factor (Stem
10% (v / v) heat-inactivated fetal bovine serum (FBS; Flow Labor) in the presence of Cell Factor (SCF), GM-CSF, and TNF-α.
atories, Irvine, CA), 10 mM HEPES, 2 mM L-
Glutamine, 5 × 10 ⁻⁵ M 2-mercaptoethanol, penicillin (100 μm)
g / ml) supplemented with endotoxin-free RPMI 1640 medium (GI
(BCO, Grand Island, NY). This is called a complete medium.

For expansion, CD34 + cells were transferred to 25-75 cm ² flasks (Corning
, NY) at 2 × 10 ⁴ cells / ml. Optical conditions are maintained by splitting these cultures on days 5 and 10 with medium containing fresh GM-CSF and TNF-α (1-3 × 10 ⁵ cells / ml). I do. In some cases, at about day 6, the cells are FACS sorted for CD1a expression.

In some cases, cells are routinely harvested after 12 days in culture, and finally, adherent cells are harvested using a 5 mM EDTA solution. In other cases, CD1
a cells were resuspended at 5 × 10 ⁶ cells / ml in complete medium and 1 μg / ml
Phorbol 12-myristic acid 13-acetic acid (PMA, Sigma) and 100
ng / ml ionomycin (Calbiochem, La Jolla, CA)
Activation for an appropriate period of time (eg, 1 hour or 6 hours). These cells are grown for an additional 6 days and RNA is isolated for cDNA library preparation.

[0260] Other specific cell types can be isolated as well.

II. RNA Isolation and Library Construction Total RNA was prepared as described, for example, in Chirgwin et al. (1978) Biochem. 18
: Guanidine thiocyanate / as described by 5294-5299 /
Isolate using the CsCl gradient procedure.

Alternatively, poly (A) + RNA can be purified using the OLIGOTEX mRNA isolation kit (
QIAGEN). Double-stranded cDNA is generated for cDNA synthesis and plasmid cloning, for example, using the SUPERSCRIPT plasmid system (Gibco BRL, Gaithersburg, MD). The resulting double-stranded cDNA is unidirectionally cloned into, for example, pSport1 and electroporated to ELECTROMAX DH1 by electroporation.
Transfect OBTM cells (Gibco BRL, Gaithersburg, MD).

III. Sequencing DNA isolated from randomly picked clones, or DNA after subtractive hybridization using inactivated cells, was ligated to nucleotides using standard techniques. Subject to sequence analysis. Alternatively, selected and isolated clones can be selected. Taq DiDeoxy Terminator Cycle Sequencing Kit (Applied Biosystems, Foster)
City, CA) may be used. Labeled DNA fragments are separated using an appropriately automated sequencer DNA sequencing gel. Alternatively, the isolated clone is sequenced as described below: eg, Mani
atis et al. (current version) Molecular Cloning, A Labora
tory Manual, Cold Spring Harbor Labor
atmory, Cold Spring Harbor Press; Sambr
OK et al. (current version) Molecular Cloning: A Laborato
ry Manual (2nd edition) Volumes 1-3, CSH Press, NY; Aus
Ubel et al., Biology, Green Publishing Assoc.
iates, Brooklyn, NY; or Ausubel et al. (current edition and addendum) Current Protocols in Molecular Bi.
ology, Green / Wiley, New York. For example, Maxim
And chemical sequencing methods using Gilbert's sequencing technology are also available.

(IV. Construction of Recombinant Gene) Poly (A) + RNA was prepared using, for example, a FastTrack mRNA kit (In
(vitrogen, San Diego, CA) using the appropriate cell population. Samples are electrophoresed, for example, in a 1% agarose gel containing formaldehyde and GeneScreen membrane (NEN Re
search products, Boston, Mass.). Hybridization is performed, for example, with 0.5 M NaHPO ₄ pH 7.2, 7% SD
S, at 1 mM EDTA, and 65 ° C. in 1% BSA (Fraction V), ³² P-
Performed using dCTP-labeled DC gene cDNA at 10 ⁷ cpm / ml. After hybridization, the filters were washed at 50 ° C. with 0.2 × SSC, 0.1
Wash three times in% SDS, for example for 30 minutes, and expose to film for 24 hours. Positive signals are typically above 2x background, preferably 5-25x.

The recombinant gene construct is used to generate a probe for detecting the message. Inserts can be cut out and used in the detection methods described above. Various standard methods for interspecies hybridization and washing are well known in the art. See, for example, Sambrook et al. And Ausu.
See bel.

V. Gene Cloning HDTEA84 was collected 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 were designed and synthesized, and PCR products were obtained from either of these libraries. This product was used as a hybridization clone to screen these libraries for full-length clones that could contain the transmembrane segment.

Similarly, HSLJD37R is a cDNA library from smooth muscle, pancreatic tumor, adipocytes, HUVEC cells, adult lung, endothelial cells, prostate cell line PC3, microblood endothelial cells, fetal heart, and dendritic cells. It was identified from the sequence of origin. GenBank report by Pan et al. Was presented. GenBank registration number 3549
See H.263. Other sequences include multiple sclerosis injury, breast,
Detected in libraries from kidney and germinal center B cells. RT-PC
R represents B cells, PBL, granulocytes, T cells, monocytes, dendritic cells, U937 cells treated with PMA / ionomycin, JY cells, MRC5 cells, CHA, Jurkat
, And YC1 cells. This suggests that the transcript is widely expressed.

RANKL has also been identified in cDNA libraries from specific tissues, as described. Similarly, the HCC5 chemokine sequence was identified. Dub
The 11 gene and the Dub12 gene were partially identified from their similarity to the known Dub1 and Dub2 genes. MD-1 and MD-2
Were partially identified from their similarity to the ligand for the RP105 gene. Cyclin E2 was identified based on its similarity to cyclin E.

(VI. Expression Profile) To test DC-PGT mRNA expression, RT-
Standard Northern blot analysis using PC fragments was performed on human tissues. For example, a Northern blot containing about 10-20 μg of total RNA is run by standard methods in a formaldehyde gel and transferred to a Nytran membrane (Schleicher & Schuell, Keene, NH), and the blot is DC- Hybridized with a labeled PCR fragment of PGT and washed at 65 ° C. The cDNA was converted to an RNAzol solution (T
el-test, Inc. , Friendswood, TX) can be used to isolate cells, embryonic tissues, and adult organs using the manufacturer's instructions. Large amounts of plasmid DNA containing the differential display PCR product were purified according to the manufacturer's instructions using the QIAGEN Plasmid Maxi Kit (Q
(UIAGEN). Plasmid DNA was transferred to EcoRI (Boe).
hringer Mannheim) or BstXI (NE Biolabs)
, Mass), gel extracted using a QIAEX gel extraction kit (QIAGEN), and a Prime-It II kit (Stratagene, La.
(Jolla, Calif.) Using [ ³² P] dCTP (Amersham) at random (all according to manufacturer's instructions). Various primers can be used to quantify the expression of the message. The means for blocking DNA hybridization signals or RNA isolation may be any suitable RNA
It can be applied to approximate quantification of the expression level of.

The results show that one band of mRNA is about 9.0 kB and another band is about 3.0 kB.
And 4.4 kB, which is consistent with the predicted size of the nucleic acid of SEQ ID NO: 1. The smaller mRNA product band could be an alternative priced form of SEQ ID NO: 1. DC-PGT is activated and non-activated dendritic cells (D
C), both are highly expressed in activated monocytes, activated granulocytes, and adult lung. No expression was found in T cells or PBL cells (either activated or non-activated cells). Minor expression was detected in B cells (both activated and non-activated cells) and restricted expression was detected in the brain. Northern analysis results, in particular,
The absence of expression in L suggests expression in macrophages (Kupfer cells in liver, microglia in brain, alveolar macrophages in lung) rather than monocytes. Southern expression analysis performed using conventional techniques confirmed the expression pattern shown in the Northern analysis.

For example, DC-PGT tissue distribution is likely to be highest in kidney, placenta, liver, bone marrow, thymus, spleen, lung, and have some mRNA levels in testis. This tissue distribution is consistent with organs having particularly important ion exchange properties (eg, Na, K, or Ca), or hematopoietic organs. In general, expression was higher in fibroblasts and hematopoietic cells were comparable to neuronal cells.

CDNA encoding HDTEA84, HSLJD37R, or RANKL
Is used to determine the tissue distribution of the antigen-encoding message. Using standard hybridization probes, Northern analysis of RNA from the following suitable sources can be performed: cells (eg, stimulated cells or cells of various physiological conditions), various tissues ( For example, spleen, liver, thymus,
Lung) or cells of various species. Southern analysis of a cDNA library can also provide valuable distribution information. Standard tissue blots or species blots are commercially available. Similar techniques are useful for assessing diagnostic or medical conditions that may be associated with expression in various cell types.

[0273] PCR analysis using appropriate primers can also be used. Antibody analysis, including immunohistochemistry or FACS, can be used to determine cell or tissue distribution.

Southern blot analysis of the primate cDNA library is performed, for example, on: U937 promonocyte strain, resting (M100); 4, 16 h LPS, IFN
γ, pooled eluted monocytes activated with anti-IL-10 (M106); pooled eluted monocytes activated with LPS, IFNγ, IL-10 for 4, 16 hours (M107)
Eluted monocytes activated with LPS for 1 hour (M108); eluted monocytes activated with LPS for 6 hours (M109); CD34 + GM-CSF, DC 30% CD1a + from day 12 of TNFα (rest); CD34 + GM-CSF, TNFα1
Dendritic cells (DC) 70% CD1a + (rest) from day 2; CD34 + GM-C
SF, DC 70% CD1a + (rest) from day 12 of TNFα (D101); 1
Time PMA and CD34 + GM-CSF activated with ionomycin, T
NFα 12 days DC 70% CD1a + (D102); 6 hours CDA + GM-CSF activated with PMA and ionomycin, TNFα 12 days DC 70% CD1a + (D103); 1 hour or 6 hours activation with PMA and ionomycin Pooled CD34 + GM-CSF, TNFα
DC 95% CD1a + from day 12; monocyte GM-CSF (IL-45 (
DC from rest))) (D107); monocyte GM-CSF (IL-45 days (rest)
)), DC (D108); 4,16 h activated TNFα, monocyte activated and pooled monocyte GM-CSF, IL-4 DC from 5 days (D110); EBV
Transformed B cell line (resting); splenocytes, resting; splenocytes activated with PMA and ionomycin; pooled 20NK clones (resting) 20NK clones activated with PMA and ionomycin; IL-2 treated NKL clone;
NK cytotoxic clone (rest); fetal adipose tissue 28-week male (O108); fetal brain 28-week male (O104); fetal gall bladder 28-week male (O106); fetal heart 28-week male (O
103); fetal small intestine 28 week male (O107); fetal kidney 28 week male (O100); fetal liver 28 week male (O102); fetal lung 28 week male (O101); fetal ovary 25 week female (O109); adult placenta 28 weeks (O113); fetal spleen tissue 28 weeks male (O112)
Fetal testis tissue 28 weeks male (O111); fetal uterus 25 weeks female (O110); TH
0 clone Mot72, resting (T102); 3,6,12 hours anti-CD28 and anti-CD3 activated pooled T cells, TH0 clone Mot72 (T10
3); T cells cloned by anergy treatment with a specific peptide for 2, 7, 12 hours, TH0 clone Moto72 (T104); T cells, TH1 clone HY06
Resting (T107); 3,6,12 hours anti-CD28 and anti-CD3 activated and pooled T cells, TH1 clone HY06 (T108); 2,6,12 hours anergy treated with specific peptide and pooled T cells, TH1 clone HY
06 (T109); Th1 with reduced rest from activation; T cells, TH2 clone H
Y935, resting (T110); pooled T cells activated with anti-CD28 and anti-CD3 for 2, 7, 12 hours, TH2 clone HY935 (T111); and Th2 with reduced resting from activation.

Samples of mouse mRNA distribution may include: for example, resting mouse fibroblast L cell line (C200); Braf: ER (fusion of Braf to estrogen receptor) transfected cells, control (C201) ; T
H1-polarized T cells (Me114 Bright, spleen-derived CD4 + cells polarized with INF-γ and anti-IL-4 for 7 days; T200); TH2-polarized T cells (Mel14 Bright, spleen-derived CD4 + cells, polarized with IL-4 and anti-IFN-γ for 7 days; T201); highly TH1-polarized T cells (Openshaw et al. (1995) J. Exp. Med. 182: 1357-).
1367; activated with anti-CD3 for 2, 6, 16 hours and pooled; T202); highly TH2-polarized T cells (Openshaw et al. (1992).
5) J.I. Exp. Med. 182: 1357-1367; anti-CD3
Activated and pooled for 2, 6, 16 hours; T203); CD44-CD25 + pre-T cells sorted from thymus (T204); TH1 T cells resting for 3 weeks after last stimulation with antigen Clone D1.1 (T205); 10 μg
T1 cell clone D1.1 (T2
06); TH2 T cell clone CDC35 (T207) resting for 3 weeks after last stimulation with antigen; T stimulated with 10 μg / ml ConA for 15 hours.
H2 T cell clone CDC35 (T208); Mel 14+ native T cells (T209) from spleen, resting; pooled for 6, 12, 24 hours with IFN-γ / IL-12 / anti-IL-4 against Th1 Mel 14+ T cells (
T210); Mel 14+ T cells polarized and pooled with IL-4 / anti-IFN-γ for Th2 for 6, 13, 24 hours (T211); Unstimulated mature B-cell leukemia cell line A20 (B200); Stimulated B cell line CH12 (B201); unstimulated giant B cells from spleen (B202); B from whole spleen activated with LPS
Cells (B203); Metrizamide-enriched dendritic cells from spleen, resting (D200);
Bone marrow derived dendritic cells, resting (D201); Monocyte cell line RAW 264.7 (M200) activated with LPS for 4 hours; BM and M-CSF induced bone marrow macrophages (M201); macrophage cell line J774, pause (M202)
Macrophage cell line J774 pooled at 0.5, 1, 3, 6, 12 hours;
+ LPS + anti-IL-10 (M203); pooled macrophage cell line J774 + LPS + IL-10 (M204) at 0.5, 1, 3, 5, 12 hours; aerosol challenged mouse lung tissue, Th2 primer, 7, 14, Pooled for 23 hr aerosol OVA challenge (Garlisi et al. (1995)
Clinical Immunology and Immunopathol
org 75: 75-83; X206); Nipponstrong.
ulus infected lung tissue (Coffman et al. (1989) Science 2
45: 308-310; X200); adult normal whole lung (O20).
0); whole lung, rag-1 (Schwarz et al. (1993) Immunodef.
ciency 4: 249-252; O205); IL-10.
K. O. Spleen (see Kuhn et al. (1991) Cell 75: 263-274; X201); adult normal whole spleen (O201); whole spleen, rag-
1 (O207); IL-10 K.I. O. Peyer's patch (O202); whole normal Peyer's patch (O210); O. Mesenteric lymph nodes (X203); whole normal mesenteric lymph nodes (0211); O. Colon (X203)
NOD mouse pancreas (Makino et al. (1980);
) Jikken Dobutsu 29: 1-13; X205);
Whole thymus, rag-1 (O208); whole kidney, rag-1 (O209); whole heart, rag-1 (O202); whole brain, rag-1 (O203); whole testis,
rag-1 (O204); whole liver, rag-1 (O206); rat normal joint tissue (O300); and rat arthritis joint tissue (X300).

A. Direct Protein Detection with Antibodies Various cells, tissues, and developmental stages are stained with labeled antibodies. This detection may be by immunohistochemistry by FACS in dispersed cells and by other methods appropriate for other sample types. Using antibodies specific for various forms,
One can distinguish between membrane-associated and soluble fragments. Various amplification means can be combined to increase sensitivity.

B. Functional Detection Certain neutralizing antibodies should provide a means to specifically block the biological activity of the prostaglandin transporter. The activity associated with prostaglandin binding, or the activity associated with prostaglandin transport, can be measured by means of sensitivity based on knowledge of various forms of natural biological function.

Further testing of cell populations (eg, hematopoietic precursors), or other cells or tissue types, is useful to further determine distribution and likely function. Other tissue types at defined developmental stages, and pathological samples, can be screened to determine whether the pathological state or stage can advantageously match the biological activity of the transporter. .

VII. Protein Expression PCR is used to generate constructs containing open reading frames, preferably operably linked to appropriate promoter, selection, and regulatory sequences. The resulting expression plasmid is transformed into a suitable cell type (eg, Top
p5, E.C. E. coli strain (Stratagene, La Jolla, CA). Ampicillin resistant (50 μg / ml) transformants were transformed at 550 nm
Grow at 37 ° C. in Luria Broth (Gibco) until the optical density at 0.7 is reached. The recombinant protein is induced with 0.4 mM isopropyl-βD-thiogalacto-pyranoside (Sigma, St. Louis, MO) and the cell incubation is continued at 20 ° C. for an additional 18 hours. Cells from the 1 liter culture are harvested by centrifugation and, for example, 200 ml of ice-cold 30% sucrose, 50 mM Tris HCl pH 8.0, 1 mM
Resuspend in ethylenediaminetetratetraacetic acid. After 10 minutes on ice, add ice-cold water to a total volume of 2 liters. After 20 minutes on ice, the cells are harvested by centrifugation and the supernatant is collected using 5 μM Millipak 60 (Millipak).
ore Corp. , Bedford, MA).

[0280] Recombinant proteins are purified by standard purification methods (eg, various ion exchange chromatography methods). Immunoaffinity methods using the antibodies described below can also be used. Affinity methods in which the epitope tag is engineered into the expression construct can be used herein.

A similar method is used for the preparation of expression constructs and cells in eukaryotic cells. Eukaryotic promoters and expression vectors can be purified as described above.

Further studies of prostaglandin transporter expression and control will continue. The control elements bound to the antigen may exhibit different developmental, tissue-specific, or other expression patterns. Upstream or downstream of the gene region (eg, regulatory sequences) is of interest.

Multiple transfected cell lines are screened for cells that express antigenic, membrane-bound, or soluble forms at higher levels compared to other cells.
Various cell lines are screened and selected for their advantageous properties, which are easy to handle. Natural proteins can be isolated from natural sources or by expression from transformed cells using a suitable expression vector. Purification of the expressed protein can be accomplished by standard procedures, or can be combined with engineered means for efficient purification with high efficiency from cell lysates or supernatants. FLAG or His6 segments may be used for such purification features.

VIII. Protein Purification The prostaglandin transporter is a prostaglandin transporter specific so that specific binding reagents can be combined with protein purification techniques to allow separation from other proteins and contaminants. Isolated by a combination of affinity chromatography using a specific binding composition (eg, an antibody). Various surfactant combinations can be tested to determine which combination solubilizes contaminants while retaining biological activity. Purification can track biological activity (eg, binding or transport of prostaglandins to membranes) or can be tracked by ELISA or other structural binding reagents.

A similar method applies to the purification of other polypeptides.

IX. Isolation of Homolog Genes The described genes (eg, cDNA) can be used as hybridization probes to screen libraries from desired sources (eg, primate cell cDNA libraries). Can be used. Many different species can be screened using probes for both the stringency and presence required for easy hybridization. Appropriate hybridization conditions are used to select those clones that exhibit the specificity of cross-hybridization.

[0287] A cDNA library from the desired species is recovered from the appropriate cell type. Screening by hybridization or PCR, using degenerate probes based on the peptide sequence, also allows for the isolation of appropriate clones. Alternatively, the use of appropriate primers for PCR screening improves the quality of appropriate nucleic acid clones.

Similar methods are applicable to the isolation of any species, polymorphic, or allelic variant. Species variants are cross-species based on the isolation of full-length isolates or fragments from one species as probes.
Isolated using hybridization techniques.

Alternatively, using antibodies raised against the protein, a suitable, eg, c
From the DNA library, cells expressing the cross-reactive protein are screened. Purified proteins or defined peptides are useful for the production of antibodies using standard methods, as described above. Synthetic peptides or purified proteins are presented to the immune system to produce monoclonal or polyclonal antibodies. For example, Coligan (1991) Current Pro
tocols in Immunology Wiley / Green; and Harlow and Lane (1989) Antibodies: A Labo.
rattro Manual, Cold Spring Harbor Pre
See ss. The resulting antibodies are used, for example, for screening, panning, or sorting.

X. Antibody Preparations Synthetic peptides or purified proteins (natural or recombinant) are presented to the immune system to produce monoclonal or polyclonal antibodies. For example, Coligan (1991) Current Protocols
in Immunology Wiley / Green; and Harlow and Lane (1989) Antibodies: A Laboratory.
See Manual, Cold Spring Harbor Press. Polyclonal sera, or hybridomas may be prepared. In appropriate contexts, the binding reagent is either labeled (eg, fluorescent or other, as described above) or immobilized on a substrate for a panning method.

XI. Chromosome Mapping DNA isolation, restriction enzyme digestion, agarose gel electrophoresis, Southern blot transcription and hybridization are performed according to standard techniques. Jenkins et al. Virol. 43: 26-36. The blot is
It can be prepared using a ybond-N-nylon membrane (Amersham). The probe is labeled with ³² P-dCTP; washing is performed, for example, with 0.1 × SS
C, against 0.1% SDS, 65 ° C. final stringency.

Alternatively, BIOS Laboratories (New Haven, CT)
) Mouse somatic cell hybrid panel can be combined with the PCR method. See Fan et al. (1996) Immunogenetics 44: 97-103.

Prepare a chromosome spread. In situ hybridization was performed for 72 hours.
Performed on chromosome preparations obtained from plant hemagglutinin-stimulated human lymphocytes cultured for hours. 5-bromodeoxyuridine is added for the last 7 hours of culture (60 μg / ml of medium) to ensure good chromosome banding after hybridization.

The PCR fragment amplified with the primers is cloned into an appropriate vector. This vector is labeled by nick translation with ³ H. This radiolabeled probe is described in Mattei et al. (1985) Hum. Ge
net. Hybridize to a metaphase spread at a final concentration of 200 ng / ml in the hybridization solution as described in 69: 327-331.

After coating with a nuclear tracking emulsion (KODAK NTB ₂ ), the slides are exposed. During the banding procedure, the chromosomal spread was first stained with a buffered Giemsa solution to avoid any slippage of the silver grains,
And take photos of metaphase. R-banding is then performed by the fluorochrome-photolysis-Giemsa (FPG) method, and metaphase pictures are retaken before analysis.

Using these techniques, the DC-PGT gene was transformed into the resulting rodscore 10
00.0 was used to map to the marker SHGC-3911 on chromosome 11q13. Other markers within the SHGC-3911 region of chromosome 11q13 include the FcεRI receptor alleged to be associated with an allergic condition. In comparison with the location of DC-PGT, the ubiquitously expressed human PGT homolog of Lu et al. (Described above) is located on chromosome 7.

XII. Biochemical Characterization For example, constructs for the expression of DC-PGT include a tag (FLAG) sequence introduced into the protein (Hopp et al. (1988) Biotechnology (NY).
6): 1205-1210). DC-PGT of SEQ ID NO: 1
The open reading frame of the cDNA is converted to a suitable PC using standard methods.
Amplified with R primer, FLAG peptide sequence (IBI, New Have
n, CT) at the C-terminus of the protein. For example, 12 cycle PCR: 9
4 ° C., 30 seconds; 55 ° C., 1 minute; 72 ° C., 4 minutes along with PFU enzyme (Srata
gene). The PCR construct was constructed using the XhoI and XbaI sites incorporated into the 5 ′ and 3 ′ primers, respectively, using the PME18X vector (DN
AX).

The COS-7 cells were cultured in DMEM, 10% FCS, 4 mM L-glutamine (J
RH Biosciences, Lenexa, KS), 100 U / ml penicillin, and 100 μg / ml streptomycin. Electroporation of plasmid DNA (BIORAD, Hercules, CA)
(20 μg / 1 × 10 ⁷ cells) and plated in tissue culture dishes. The medium is changed after 24 hours and the cell lysate and medium are collected 3 days after transfection. Lysis buffer (25 mM H
epes 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 plate. Keep plate on ice for 45 minutes. The lysate is centrifuged for 15 minutes to remove cell debris. The centrifuged cell lysate supernatant and sterile filtered media from cultured cells are incubated overnight at 4 ° C. with anti-FLAG M2 Affinity Gel (IBI) and washed four times with PBS. The immunoprecipitate is eluted in Econocolumn (BIORAD) at 2.5 M glycine pH 2.5. Neutralize the eluate with Hepes pH 7.4 (JRH Biosciences),
And 24% TCA and 2% deoxycholic acid sodium salt (Sigma
Concentrate by precipitation using Pellets are mixed with 2 × Sample Buffer (
NOVEX, San Diego, CA), electrophoresed on a 4-20% Tris-glycine gel (Novex), and a PVDF membrane (Imm
obilon-P, Millipore Corporation, Bedfo
rd, MA). The membrane is exposed to 3% skim milk for 1 hour at 37 ° C. Anti-FLAG M2 antibody is used as recommended (IBI). Anti-mouse Ig horseradish peroxidase conjugate (Amersham) was used at a dilution of 1: 2,000 and the peroxidase detection was performed using an ELC detection reagent (Amersham).
).

[0299] Other fusion proteins can be produced. For example, a recombinant prostaglandin transporter construct is prepared, for example, as a fusion product with a useful affinity reagent (eg, a FLAG peptide). The peptide segment may be useful for purifying the expression product of the construct. For example, Crowe et al. (1992) QIAexpr
ess: The High Level Expression & Prot
ein Purification System QUIAGEN, Inc.
Chatsworth, CA; and Hopp et al. (1988) Bio / Tech.
No. 6: 1204-1210. A membrane containing a transporter,
Assay to determine prostaglandin's natural substrate. Prostaglandins are most likely to be uracil-related prostaglandins, but may include pyrimidine or purine analogs with varying levels of binding or transport efficiency. For example, Goodman and Gilman (current version), The
Pharmacological Basis of Therapeutic
s; Lukovics and Zablocka, Nucleoside Syn
thesis: Organosilicon Methods, Ellis H
orwood, N.M. Y. Townsend, Chemistry of Nu;
cleosides and Nucleotides, Vol. 1-3, Plen
um Press, N.M. Y. Munch-Partson (1983) Met;
abolism of Nucleotides, Nucleosides, a
nd Prostaglandins in Microorganisms
Academic Press, NY; Gehrke (1990) Chroma
toggle & Modification of Nucleosid
es, Volumes A, B, and C, Elsevier; Bloch (1975) Ch.
emistry, Biology, & Clinical Uses of N
nucleoside Analogs, Annals NY Acad. Sci
Ulbricht (1964) Purines, Pyrimidines,
& Nucleotides, Franklin Co. checking ...

XIII. Expression Cloning; Partner Screening A. Antibody and Flow Cytometry Classification Expression cloning of cells transformed with an appropriate cDNA library is classified by FACS using the antibody reagents described above. The sorted cells are isolated and grown and subjected to multiple sorting cycles, leading to a high percentage of cells expressing the desired DNA.

B. Antibodies and Staining Antibodies (eg, against DC-PGT) are used for screening libraries made from cell lines expressing polypeptides. Standard staining techniques are used to detect or classify intracellularly or surfacely expressed ligand, or to screen for surface-expressing transformed cells by panning. Screening for intracellular expression is performed by various staining or immunofluorescence procedures. M
cMahan et al., (1991) EMBO J. See also 10: 2821-2832.

For example, on day 0, 2-chamber permanox slides were loaded with 1 ml of fibronectin per chamber (10 ng / ml in PBS).
) For 30 minutes at room temperature. Rinse once with PBS. Then, COS
Cells are plated in 1.5 ml of culture medium at 2-3 × 10 ⁵ cells per chamber. Incubate overnight at 37 ° C.

On day 1, for each sample, 66 μg / ml in DME without serum
DEAE-dextran, 66 μM chloroquine, and 0.1 μg of 4 μg DNA.
Prepare 5 ml of solution. For each set, for example, one dilution and 1/200
Prepare positive control and negative mock of diluted huIL-10-FLAG cDNA. Rinse cells with serum free DME. DN
Solution A is added and incubated at 37 ° C. for 5 hours. The medium is removed and 0.5 ml of 10% DMSO in DME is added for 2.5 minutes. Remove and wash once with DME. Add 1.5 ml of growth medium and incubate overnight.

On the second day, change the medium. On day 3 or 4, cells are fixed and stained. The cells are rinsed twice with Hank's buffered saline (HBSS) and fixed in 4% paraformaldehyde (PFA) / glucose for 5 minutes in HB.
Wash 3 times (3x) with SS. The slides were removed after removing all liquid by -8.
It can be stored at 0 ° C. For each chamber, a 0.5 ml incubation is performed as follows. HBSS / saponin (0.1%) containing 32 μl / ml 1 M NaN ₃ is added for 20 minutes. The cells were then washed once with HBSS / saponin (
1 ×) Wash. Add soluble antibody to cells and incubate for 30 minutes. Wash cells twice with HBSS / saponin. A second antibody (eg, Vecto)
r anti-mouse antibody (1/200 dilution)) and incubate for 30 minutes. Prepare an ELISA solution (eg, Vector Elite ABC horseradish peroxidase solution) and pre-incubate for 30 minutes.
For example, use one drop of solution A (avidin) and one drop of solution B (biotin) per 2.5 ml of HBSS / saponin. Wash cells twice with HBSS / saponin. Add ABC HRP solution and incubate for 30 minutes. The cells are washed twice with HBSS (second wash for 2 minutes), thereby closing the cells.
Then Vector Diaminobenzoic acid (DAB) is added for 5-10 minutes. 5m
Use 2 drops of buffer + 4 drops of DAB + 2 drops of H ₂ O ₂ per liter of glass distilled water. Carefully remove the chamber and rinse the slides in water. Air dry for a few minutes, then add a drop of Crystal Mount and add a coverslip. Bake at 85-90 ° C for 5 minutes.

Alternatively, antibodies to the selected protein are used to affinity purify the antigen or sort cells that express the antigen. For example, Sambrook
See k et al. or Ausubel et al., which are incorporated herein by reference. Antigens are typically expressed on the cell surface.

[0306] Hybridization approaches can also be utilized to find closely related variants of an antigen based on nucleic acid hybridization.

XIV. Screening for DC-PGT Substrate Specificity The type of organic anion transported by the DC-PGT of the present invention can be tested directly using standard methods. For example, DC-PGT cDNA is DC-PGT cDNA.
Various tracer-labeled substrates of GT (eg, PGE1, PGE2, PG
E2a, prostaglandins such as PGD2, thromboxane such as TxB2, or glutathione, p-aminohippuric acid, taurocholic acid, uric acid, unconjugated and conjugated bilirubin, and estradiol glucuronide. Such non-prostaglandins (an anionic substrate) can be expressed in HeLa cell monolayers or Xenopus oocytes. For example, for oocyte expression, water or complementary RNA (cRNA) (transcribed and capped in vitro from DC-PGT cDNA) can be added to Xenopus oocytes at about 50 ng cRNA per oocyte. Inject. Uptake studies were performed two to three days after injection, the oocytes were washed three times in Waymouth solution and at various times.
Incubate at 7 ° C. with radioactive substrate (about 0.25 μCi / ml; total concentration, about 1 nM), wash three times with ice-cold Waymouth solution, and
Perform by dissolving in 1% 10% SDS. The radioactivity bound to the oocytes is determined by liquid scintillation spectroscopy. For HeLa cell expression, cells are grown on a 35 mm dish to about 80% confluence, and then expanded to Fuesst et al. (1986) Proc. Nat'l Acad. Sci. U
Infect 10 plaque forming units per cell with the recombinant vaccine virus vTF7-3 according to the method of SA 83: 8122-8126. 30 minutes after infection,
Cells were isolated from Blakely (1991) Anal. Biochem. 194: 30
Transfect with DC-PGT cDNA (10 μg / ml) + Lipofectin (20 μg / ml) according to the method of 2-310. After a 3 hour incubation, the vaccinia virus and DNA-lipofectin complex are removed and the cells are maintained overnight in Dulbecco's modified Eagle's medium supplemented with 5% fetal calf serum. Uptake studies are performed 19 hours after transfection. The monolayer was washed three times with serum-free culture medium and incubated at 27 ° C. for various times.
Radioactive substrate at 0.5 μCi / ml per dish; total concentration, about 0.2 n
Incubate with M). For uptake, cells were washed once with ice-cold Waymouth solution (containing 5% fetal bovine serum albumin) and then
Stop by washing 4 times with the out solution alone. Cells are discarded and bound radioactivity is measured by liquid scintillation spectroscopy.

(XV. Measurement of DC-PGT Substrate Uptake Kinetics) Various prostaglandins or thromboxanes (eg, PGE1, PGE
2, Competitive tracer uptake kinetics using DC-PGT comparing PGE2a, PGD2 or TxB2) is determined using a standard competitive transport assay. For example, to determine the time-dependent uptake of tracer-labeled prostaglandin into HeLa cells expressing the DC-PGT clone, the following final concentrations of ³ [H] -PG are used (New England Nucl):
ear, Boston, MA): PEG ₂ : 0.7 nM (176 cpm / fmo)
l); PEG ₁ : 0.6 nM (62 cpm / fmol); PGD ₂ : 0.9 nM (
126 cpm / fmol); PGF ₂ α: 0.6 nM (185 cpm / fmol)
); TXB2: 1.0 nM (114 cpm / fmol); PGI ₂ analog ³ [H
] -Iloprost (Amersham Corp., Arlington He)
lights, IL): 7.9 nM (14 cpm / fmol).

(XVI. Measurement of DC-PGT Uptake Inhibition) A composition that inhibits DC-PGT uptake can also be measured. For example, to measure inhibition of tracer PGE _2, at 10-minute intervals uptake (0.2 nM ³ [
H] -PGE2) was produced at various concentrations of the unlabeled prostanoid PGE ₂ , P
_{GE 1, PGD 2, PGF 2} α, TXB 2, PGI 2, (100~500nM; Ca
yman Chemical, Ann Arbor MI) or furosemide, probenecid, and indomethacin (10-100 μM, SigmaCh).
electronic Co. , St. (Louis, MO) with or without an inhibitor, one or two separate transfections are measured in duplicate for a given transfection. Since the concentration of the substrate is at least 500 times lower than the unlabeled prostanoid concentration, the apparent affinity constant K _1/2
See Name and Richards (1972) Elementary K.
inetics of Membrane Carrier Transport
t, John Wiley & Sons, as described by New York, is determined from the following _{equation: K 1/2 = [v i /} (v-v i)] [i] with no where v = inhibitor Uptake, vi = uptake with inhibitor,
And i = inhibitor concentration.

XVII. Screening for Agonists or Antagonists Using the HeLa or Xenopus systems (described above), or comparison systems, one of skill in the art can screen for inhibitors or agonists of DC-PGT mediated tracer transport. , The DC-PGT of the present invention may be used. The potency of potential antagonists can be compared to known PG transport inhibitors such as furosemide, probenecid, or indomethacin. A potential agonist or antagonist composition is prepared using the system described above and a test composition and D
Incubate for a time sufficient to allow binding to the C-PGT transporter. An increase or decrease in the degree of tracer uptake may be correlated with the particular composition being tested. Thus, a compound or composition that modulates organic anion transport via the DC-PGT transporter of the present invention can be used to prepare prostaglandins or thromboxanes in the presence or absence of the compound or composition being tested. Can be identified by assessing the uptake of various anions such as Similar methods can be used to screen for substrates for this enzyme, such as Dub and cyclin E2.

XVIII. Isolation of Receptor Ligands The construct for expression of the product can be used as a specific binding reagent, which takes advantage of its binding specificity in the same way that an antibody is used This identifies its binding partner (eg, ligand). The receptor reagent is either labeled (eg, fluorescent or other) as described above, or is immobilized on a substrate for a panning method. Anderson et al. (1997) Nature.
See also 390: 175-179, which is incorporated herein by reference.

[0312] The binding compositions are used to screen expression libraries made from cell lines that express a binding partner (eg, a TNF family ligand). Standard staining techniques are used to detect or classify intracellularly or surfacely expressed receptors, or to screen for surface expressing transformed cells by panning. Screening for intracellular expression is performed by various staining or immunofluorescence procedures. McMahan et al. (1991) EMBO J. 10: 2
See also 821-2832.

Alternatively, receptor reagents are used to affinity purify or sort cells that express the receptor. For example, Sambrook et al. Or Aus
See ubel et al.

Another strategy is to screen for membrane bound ligand by panning. A cDNA containing the ligand cDNA is constructed as described above. The ligand can be immobilized and used to immobilize the expressing cells. Immobilization can be achieved, for example, by using an appropriate antibody that recognizes the FLAG sequence or receptor fusion construct, or by using an antibody raised against the first antibody. The recursive cycle of selection and amplification leads to the enrichment of appropriate clones and the final isolation of ligand-expressing clones.

A phage expression library can be screened with the receptor. Appropriate labeling techniques (eg, anti-FLAG antibodies) allow specific labeling of appropriate clones.

IX. Chemotaxis Assay Chemokine proteins are produced in COS cells transfected with a plasmid containing the chemokine cDNA, for example, by electroporation.
Hara et al. (1992) EMBO J .; 10: 1875-1884. Physical analysis methods (eg, CD analysis) have been applied to compare the tertiary structure to other chemokines and to assess whether the protein appears to be folded into an active conformation. You. Culture supernatants are collected after transfection and subjected to bioassay. Mock control (eg, luciferase c
Plasmid with DNA). de Wet (1987) Mol. C
ell. Biol. 7: 725-757. Typically, a positive control (for example, R & D System (Minneapolis, MN)
)) Is used. Similarly, antibodies can be used to block biological activity (eg, as a control).

The lymphocyte migration assay has been described previously (eg, Bacon et al., (198)
8) Br. J. Pharmacol. 95: 966-974). Other trafficking assays are also available. See, for example, Quidling-Jarlink et al. (1995) Eur. J. Immun
ol. 25: 322-327; Koch et al. (1994) J. Mol. Clinical
Investigation 93: 921-928; and Antony et al. (
1993). Immunol. 151: 7216-7223. R
. Coffman and A.J. O'Gara (DNAX, Palo Alto,
Mouse Th2 T cell clone, CDC-25 (
(1985) J. Tony et al. Exp. Med. 161: 223-241) and HDK-1 (Cherwinski et al. (1987) J. Exp. M).
ed. 166: 1229-1244) are used as controls.

The Ca 2+ flux upon chemokine stimulation was determined by Bacon et al.
Immunol. 154: 3654-3666.

The maximum number of migrating cells responding to MIP-1α typically occurs at a concentration of 10 ⁻⁸ M, which is consistent with the first report on the CD4 + population of human T cells. Sc
hall (1993) J. Mol. Exp. Med. 177: 1821-1826. A dose response curve is determined, which preferably provides a characteristic bell-shaped dose response curve.

Following stimulation with CC chemokines, lymphocytes generally have measurable intracellular Ca
Shows 2+ flux. MIP-1α may induce an immediate transient of calcium mobilization. Typically, the chemokine levels used in these assays are comparable to those used in the chemotaxis assay (1/1000 dilution of conditioned supernatant).

XX. Biological Activity As described above, assays are selected that are robust and sensitive to, for example, immune cells, neuronal cells, or stem cells. Chemokines are added to this assay in increasing doses to see if a dose response is detected. For example, in a proliferation assay, cells are plated out on a plate. Provide appropriate culture media and add chemokines to the cells in various amounts. Proliferation is monitored over a period of time, which detects either a direct effect on the cells or an indirect effect of the chemokine.

Alternatively, activation or trigger assays are used. Select the appropriate cell type: for example, hematopoietic cells, myeloid cells (macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphocytes (T cells, B cells, or NK cells), neural cells (
Neurons, glia, oligodendrocytes, astrocytes, etc.), or stem cells (eg, precursor cells that differentiate into other cell types (eg, gut crypt cells (gut c
rypt cell) and undifferentiated cell type)).

A retroviral infection assay has also been described, which includes, for example, CCR
One receptor, the CCR3 receptor, and the CCR5 receptor are used. R
These receptors that bind to ANTES and MIP-1 related chemokines are:
It also appears to be a receptor for HCC5. These chemokine receptors in the retroviral infection process, and related RANTES and MIP-1
Recent descriptions of effects by chemokines suggest that HCC5 has a similar effect. For example, Balter (1996) Science 272: 1.
740 (describes evidence for chemokine receptors as coreceptors for HIV); and Deng et al. (1996) Nature 381: 661-666.

Chemokines are also described, for example, in H. et al. It can be assayed for activity in hematopoietic assays as described by Broxmeyer. Bellido et al.
(1995) J. Mol. Clinical Investigation 95:28
86-2895; and Jilka et al. (1995) Expt'l Hemato.
See logy 23: 500-506. These are also, for example, Str
eiter et al. (1992) Am. J. Pathol. 141: 1279-128
4, can be assayed for angiogenic activity. For a role in inflammation, see Wakefield et al. Surgi
cal Res. 64: 26-31.

Other assays include those that have been demonstrated with other chemokines. For example, Schall and Bacon (1994) Current Op.
inion in Immunology 6: 865-873; and Ba
con and Schall (1996) Int. Arch. Allergy &
Immunol. 109: 97-109.

The DUB gene can be screened for deubiquitinating activity as described. See, for example, Hochstrasser (1995) Curr. Op
in. Cell Biol. 7: 215-223; Wilkinson et al. (19
95) Biochemistry 34: 14535-14546; Baker
(1992) J. et al. Biol. Chem. 267: 23364-23375; B
aek et al. (1998) J. Am. Biol. Chem. 272: 25560-2556
5; and Papa and Hochstrasser (1993) Nature.
e 366: 313-319. For example, for an in vitro assay for UBP activity, ¹²⁵ I-labeled Ub-PESTc was purchased from Woo et al. (1995).
J.). Biol. Chem. 270: 18766-18773,
Used as a substrate. The reaction mixture (0.1 ml) was 100 mM Tris
HCl (pH 7.8), 1 mM dithiothreitol, 1 mM EDTA and 5% glycerol with appropriate amount of enzyme preparation and 10-30 μg of ¹²⁵ I-labelled Ub-PESTc. After incubating the mixture for the appropriate time, 50 μl of 40% (w / v) trichloroacetic acid and 50 μl of 1.2% (w / v)
v) Stop the reaction by adding bovine serum albumin. The sample is centrifuged, and the resulting supernatant is counted for its radioactivity using a counter. The enzymatic activity was measured as the percentage of hydrolyzed acid lysate of ¹²⁵ I-labeled Ub-PESTc (%
). Ub-NH-carboxy extended protein and His-di-Ub
Incubation is performed as described above, except that in the presence of 5 μg of substrate. After incubation for an appropriate period of time, samples were prepared according to Baek et al. Biol. Chem. 272: 25
Subject to discontinuous gel electrophoresis as described in 560-25565. Then
The proteins in the gel were visualized by staining with Coomassie Blue R-250 or by exposing to X-ray film (Fuji) at 70 ° C. To prepare a ¹²⁵ I-labeled poly-Ub-NH-lysozyme conjugate,
2 μg of ¹²⁵ I-labeled lysozyme (5 × 10 ⁵ cpm) was added to 10 μg of Ub, 120
μg of Fraction II as well as 10 mM Tris-HCl (pH 7.8)
), 15 units / ml creatine phosphokinase, 6.5 mM phosphocreatine, 1.5 mM ATP, 1 mM dithiothreitol, 0.5 mM Mg
An ATP regeneration system consisting of Cl ₂ and 1 mM KCl was placed in a final volume of 0.05 ml.
Incubate with Incubations are performed for 2 hours at 37 ° C. in the presence of 1 mM hemin, preventing proteolysis of the ubiquitinated protein conjugate by 26S proteosomes. After the incubation, the sample is washed with endogenous U.
Heat at 55 ° C. for 10 minutes for BP inactivation. Alternatively, Dub11 or Dub12 can be prepared as described by Zhu et al. (1997) J. Am. Biol. Chem. 272: 51
-57 can be expressed as a GST fusion protein. This method involves cloning into an appropriate expression vector and subsequently, Ub-Met-β
It is due to co-transformation plasmids encoding gal, wherein the ubiquitin is fused with the NH ₂ -terminus of β- galactosidase, and tested for cleavage.

However, deubiquitinase has also been reported to have additional functions in addition to deubiquitination. For example, Hochstrasser (1996)
Cell 84: 815-815; Hicke and Riezman (1996)
) Cell 84: 277-287; and Chen et al. (1996) Cell.
84: 853-862.

[0330] MD gene products are screened for cell signaling activity. See, for example, Miyake et al. Immunol. 161: 1348-1
353; Kobe and Deisenhofer (1994) Trends B
iochem. Sci. 19: 412.

XXI. Antagonism of Cyclin E2 Protein Inhibition of cell cycle progression is particularly important for control of hyperproliferative diseases such as cancer. Several methods are available to achieve this control. The ability to bind cyclins is inhibited, for example, by the use of antibodies raised against cyclin binding proteins. Other elements include, for example, peptidomimetics, which are peptides designed to mimic the binding sites of cyclin binding proteins and disrupt the interaction of these proteins with cyclins. The most effective ways to block cell cycle progression are, for example, to block the interaction of the binding protein with cyclins or to use Hung et al. (1996) C
The downstream activity of the binding protein as described in hemistry and Biology 3: 623-639.
the use of small molecules to block activity. Exposure of cells to these permeable small molecules should result in conditional loss of target protein function.

Also included in this category is the use of gene therapy to block expression of cyclin binding proteins or gene transcription factors. How to use gene therapy
For example, Goodnow (1992) "Transgenic Animals"
"Roitt ed., Encyclopedia of Immunology, A
Cademic Press, San Diego, pp. 1502-1504; T
ravis (1992) Science 256: 1392-1394; Kuh
(1991) Science 254: 707-710; Capecchi.
(1989) Science 244: 1288; Robertson (198
7) Hen, Teratocarcinomas and Embronic S
tem Cells: A Practical Approach, IRL P
ress, Oxford; and Rosemberg (1992) J. Am. Cli
natural Oncology 10: 180-199. Also included is the use of antisense RNA in gene therapy to block target gene expression or proper splicing of gene transcripts.

All citations herein are incorporated by reference to the same extent as if each individual publication or patent application was specifically and individually indicated to be incorporated by reference. Incorporated.

Many modifications and variations of this invention can be made without departing from the spirit and scope of this invention, which will be apparent to those skilled in the art. The specific embodiments described herein are provided for purposes of illustration only, and the present invention is not limited to the terms of the following claims, and is intended to cover such claims. It is limited by the full scope of equivalents.

[Sequence list]

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) C07K 16/18 C07K 16/40 16/24 19/00 16/28 C12N 1/15 16/40 1/19 19/00 1/21 C12N 1/15 C12P 21/02 C 1/19 C12Q 1/68 A 1/21 G01N 33/566 5/10 33/68 C12P 21/02 C12N 15/00 ZNAA C12Q 1/68 A61K 37/02 G01N 33/566 37/54 33/68 C12N 5/00 A (31) Priority claim number 60 / 093,897 (32) Priority date July 23, 1998 (July 23, 1998) ( 33) Priority claim country United States (US) (31) Priority claim number 09 / 132,968 (32) Priority date August 12, 1998 (August 12, 1998) (33) Priority claim country United States ( (US) (31) Priority claim number 09 / 136,214 (32) Priority date August 18, 1998 (1998) 8.18) (33) Priority claim country United States (US) (31) Priority claim number 60 / 099,999 (32) Priority date September 11, 1998 (September 11, 1998) (33) Priority Claimed State United States (US) (81) Designated State EP (AT, BE, CH, CY, DE, DK, ES, FI, FR, GB, GR, IE, IT, LU, MC, NL, PT, SE ), OA (BF, BJ, CF, CG, CI, CM, GA, GN, GW, ML, MR, NE, SN, TD, TG), AP (GH, GM, KE, LS, MW, SD, SL, SZ, UG, ZW), EA (AM, AZ, BY, KG, KZ, MD, RU, TJ, TM), AE, AL, AM, AT, AU, AZ, BA, BB, BG, BR , BY, CA, CH, CN, CZ, DE, DK, EE, ES, FI, GB, GD, GE, HR, HU, ID, IL, IN, IS, JP, K , KR, KZ, LC, LK, LR, LT, LU, LV, MD, MG, MK, MN, MX, NO, NZ, PL, PT, RO, RU, SE, SG, SI, SK, SL, TJ, TM, TR, TT, UA, UZ, VN, YU, ZA (71) Applicant 2000 Galloping Hill Road, Kenilworth, New Jersey 07033-0530, U.S.A. S. A (72) Inventors Lebec, Sergi Jay. E. France F-69380 Cibriau Dazerg, Shemin de Marand, 514 (72) Inventor Murphy, Erin E. United States California 94301, Palo Alto, Emerson Street 180 (72) Inventors Mattson, Jinin D. United States California 94114, San Francisco, Alvarado Street 559 (72) Inventors Gorman, Daniel M. United States California 94560, Newark, Central Avenue 6371 (72) Inventor Hedrick, Joseph A. United States New Jersey 08536, Plainsborough, Quail Ridge Drive 52-08 (72) Inventor One, Lucan United States New Jersey 08816, East Brunswick, Hollis Road 21 (72) Inventor Zlotonic, Albert United States California 94306, Palo Alto, Algar Drive 507 ( 72) Inventors Margolo, Nicholas J. United States New Jersey 07946, Millington, Rolling Hill Drive 99 (72) Inventor Green, Jonathan Earl. United States New Jersey 07079, South Orange, Tyrow Road 457 (72) Inventor Johnston, James A. United States California 95032, Los Gatos, Mary Alice Drive 205 (72) Inventor Bazan, Jose Fernando United States California 94025, Menlo Park, University Drive 775 (72) Inventor Mahoney, Daniel United States of America New York 10016, New York, East 39T. Street number 21-A 330 (72) Inventors Leeds, Emma M. United States of America California 94115, San Francisco, Washington Street 3107 (54) [Title of the Invention] Mammalian gene; dendritic cell prostaglandin-like transporter (DC-PGT), HDTE A, HSLJD37R and RANKL, HCC5 chemokine, deubiquitin Chemical formulas 11 and 12 (DUB11, DUB12), MD-1, MD-2 and cyclin E2, related reagents and methods

Claims (84)

[Claims] 1. An isolated or purified, antigenic, polypeptide comprising:
The following: a) a plurality of different segments, each of said segments having identity to at least 12 contiguous amino acids from mature SEQ ID NO: 2; or b) the mature SEQ ID NO: 2 At least 17 contiguous amino acids from the polypeptide. 2. The polypeptide of claim 1, wherein said plurality of segments comprises: a) one of at least 19 contiguous amino acids; or b) two of at least 15 contiguous amino acids. A polypeptide comprising: 3. The polypeptide of claim 1, wherein the polypeptide comprises: a) comprises the mature SEQ ID NO: 2; b) is specific for a polyclonal antibody that specifically binds to SEQ ID NO: 2. Or c) the polypeptide is: i) is a natural allelic variant of SEQ ID NO: 2; ii) is at least 30 amino acids in length; iii) at least 2 specific for SEQ ID NO: 2 Iv) is a synthetic polypeptide; v) is attached to a solid support; or vi) is a conservative substitution no more than 5-fold from SEQ ID NO: 2. 4. A fusion protein comprising a first part and a second part, wherein said first part comprises the polypeptide of claim 1 and said second part is detectable. Fusion proteins containing various markers. 5. A pharmaceutical composition comprising the sterile polypeptide of claim 1 in a pharmaceutically acceptable carrier. 6. An isolated or recombinant polynucleotide encoding a polypeptide according to claim 1. 7. The polynucleotide of claim 6, wherein the polynucleotide comprises: a) a portion encoding the mature polypeptide of SEQ ID NO: 1; or b) a mature SEQ ID NO: 2 Encoding a polynucleotide. 8. The polynucleotide of claim 6, wherein the polynucleotide is: a) a PCR product; b) a hybridization probe; c) a mutagenic primer. Or d) a polynucleotide produced by chemical synthesis. 9. The polynucleotide of claim 6, wherein said polynucleotide is: a) detectably labeled; b) deoxyribonucleic acid; or c) double stranded. nucleotide. 10. An expression vector comprising the polynucleotide according to claim 6. 11. The expression vector according to claim 10, wherein said polypeptide specifically binds to a polyclonal antibody raised against the immunogen of mature SEQ ID NO: 2. 12. The expression vector according to claim 10, wherein: a) a target polynucleotide sequence having at least 60 contiguous nucleotides derived from SEQ ID NO: 1 under stringent hybridization conditions. B) encodes a polypeptide having at least 50 contiguous amino acid residues from mature SEQ ID NO: 2, or c) transfers to a prokaryotic or eukaryotic host cell. An expression vector, which is suitable for transfection. 13. The cell of claim 13, wherein the host cell is: a) a mammalian cell; b) a bacterial cell; c) an insect cell; d) a prokaryote; e) a eukaryote; or f) a COS cell. 13. The expression vector according to 12. 14. A method for producing a polypeptide, comprising a step of expressing the vector according to claim 13 in the host cell. 15. An isolated or recombinant hybridizing under stringent hybridization conditions to the coding portion of SEQ ID NO: 1 and washing conditions of at least 50 ° C., a salt concentration of less than 400 mM and 50% formamide. Polynucleotide. 16. An expression vector comprising the polynucleotide according to claim 15. 17. The expression vector according to claim 16, wherein the expression vector has stringent hybridization conditions and at least 60
An expression vector that hybridizes to the coding portion of SEQ ID NO: 1 under wash conditions at <RTIgt; 0 C, </ RTI> a salt concentration of less than 200 mM and 50% formamide. 18. The expression vector of claim 25, which encodes a polypeptide that specifically binds to an antibody raised against mature SEQ ID NO: 2. 19. The polynucleotide of claim 15, which hybridizes to SEQ ID NO: 1, wherein the polynucleotide is: a) a PCR product; b) a hybridization probe; c) A polynucleotide which is a mutagenic primer; or d) produced by chemical synthesis. 20. A method of regulating the physiology or development of a cell, comprising contacting the cell with an agonist or antagonist of the polypeptide of claim 1. 21. A method for detecting the presence of a complementary polynucleotide in a sample, comprising the steps of: selectively hybridizing the polynucleotide of claim 6 to said complementary polynucleotide in said sample. Contacting with a specific polynucleotide to form a detectable duplex, thereby indicating the presence of said polynucleotide in said sample. 22. A method for identifying a compound that binds to a polypeptide according to claim 1, comprising the steps of: a) interacting said compound and a component comprising said polypeptide with said compound. Incubating under conditions sufficient to allow for the determination of the binding of the compound to the polypeptide. 23. An isolated or recombinant polynucleotide encoding an antigenic polypeptide comprising: a) at least 17 contiguous amino acids from the mature polypeptide from SEQ ID NO: 6; b) SEQ ID NO: At least 17 contiguous amino acids from the mature polypeptide from SEQ ID NO: 10; c) at least 17 contiguous amino acids from the mature polypeptide from SEQ ID NO: 10; d) at least 17 contiguous amino acids from the mature polypeptide from SEQ ID NO: 12 E) at least 17 contiguous amino acids from the mature polypeptide from SEQ ID NO: 17; f) at least 17 contiguous amino acids from the mature polypeptide from SEQ ID NO: 19; g) the mature poly from SEQ ID NO: 21 At least 17 contiguous amino acids from the peptide; or h) SEQ ID NO: 3, at least 17 contiguous amino acids from the mature polypeptide from. 24. The polynucleotide of claim 23, which encodes all of the following polypeptides: a) the signal processed SEQ ID NO: 6; b) the signal processed SEQ ID NO: 8; c) the signal processed D) SEQ ID NO: 12 in which the signal was processed; e) SEQ ID NO: 17 in which the signal was processed; f) SEQ ID NO: 19; g) SEQ ID NO: 21; or h) SEQ ID NO: 23. 25. Less than 500 mM salt at 55 ° C., and 50
24. The polynucleotide of claim 23, which hybridizes with% formamide: a) the mature protein coding portion of SEQ ID NO: 5; b) the coding portion of the signal processed SEQ ID NO: 7; c) the signal of SEQ ID NO: 9. D) the coding part of the signal of SEQ ID NO: 11 processed; e) the mature protein coding part of SEQ ID NO: 16; f) the polypeptide coding part of SEQ ID NO: 18; g) the polypeptide of SEQ ID NO: 20 Coding portion; or h) the polypeptide coding portion of SEQ ID NO: 22. 26. The polynucleotide of claim 25 comprising at least 35 contiguous nucleotides: a) the mature protein coding portion of SEQ ID NO: 5; b) the signal processed coding portion of SEQ ID NO: 7; c) the coding portion of the signal of SEQ ID NO: 9 processed; d) the coding portion of the signal of SEQ ID NO: 11 processed; e) the mature protein coding portion of SEQ ID NO: 16; f) the polypeptide coding portion of SEQ ID NO: 18; g) the polypeptide coding portion of SEQ ID NO: 20; or h) the polypeptide coding portion of SEQ ID NO: 22. 27. An expression vector comprising the polynucleotide according to claim 23. 28. A host cell comprising the expression vector of claim 27, wherein the host cell includes a eukaryotic cell. 29. A method for producing an antigenic polypeptide, comprising:
A method comprising expressing the recombinant polynucleotide according to 1. 30. A method for detecting a polynucleotide according to claim 23, comprising: a) a mature protein coding portion of SEQ ID NO: 5; b) a coding portion where the signal of SEQ ID NO: 7 is processed; c) the coding portion of the signal of SEQ ID NO: 9 processed; d) the coding portion of the signal of SEQ ID NO: 11 processed; e) the mature protein coding portion of SEQ ID NO: 16; f) the polypeptide coding portion of SEQ ID NO: 18; g) a polypeptide coding portion of SEQ ID NO: 20; or h) a probe that hybridizes under stringent conditions to at least 25 contiguous nucleotides of the polypeptide coding portion of SEQ ID NO: 22; Contacting to form a duplex, wherein the duplex is detected. Wherein the reading is indicative of the presence of the polynucleotide. 31. A kit for the determination of the polynucleotide of claim 23, wherein the kit comprises under stringent hybridization conditions at least 17 consecutive nucleotides of the polynucleotide of claim 1. A kit comprising a compartment containing a probe that hybridizes to form a duplex. 32. The kit of claim 31, wherein said probe is detectably labeled. 33. A binding compound comprising an antibody binding site that specifically binds to a polypeptide comprising at least 17 consecutive amino acids from: a) a signal processed SEQ ID NO: 6; b) a signal processed C) SEQ ID NO: 10 in which the signal was processed; d) SEQ ID NO: 12 in which the signal was processed; e) SEQ ID NO: 17 in which the signal was processed; f) SEQ ID NO: 19; g) SEQ ID NO: 21; h) SEQ ID NO: 23. 34. The binding compound according to claim 33, wherein: a) the antibody binding site is 1) or less: a) SEQ ID NO: 6 where the signal was processed; b) the signal was processed C) SEQ ID NO: 10 in which the signal was processed; d) SEQ ID NO: 12 in which the signal was processed; e) SEQ ID NO: 17 in which the signal was processed; f) SEQ ID NO: 19; g) SEQ ID NO: 21; h) selectively immunoreactive with SEQ ID NO: 23; 2) purified or recombinantly produced, raised against human HDTEA84 protein; 3) purified or recombinant Produced against human HSLJD37R protein; or 4) monoclonal antibody, Fab or F (a Or b) the binding compound is: 1) an antibody molecule; 2) a polyclonal antiserum; 3) detectably labeled; 4) sterile; or 5) A binding compound present in the buffered composition. 35. A method of using the binding compound of claim 33, wherein the binding compound is contacted with a biological sample containing the antigen, thereby forming a binding compound: antigen complex. A method comprising the steps of: 36. The method of claim 35, wherein said biological sample is from a human and said binding compound is an antibody. 37. The binding compound of claim 34, and comprising: a) instructions for using the binding compound for the detection; or b) a compartment providing separation of the binding compound. Detection kit. 38. A binding composition according to claim 33, and further comprising:
The following: a) SEQ ID NO: 6 in which the signal was processed; b) SEQ ID NO: 8 in which the signal was processed; c) SEQ ID NO: 10 in which the signal was processed; d) SEQ ID NO: 12 in which the signal was processed; e) F) SEQ ID NO: 19; g) SEQ ID NO: 21; or h) SEQ ID NO: 23, a substantially pure or isolated, antigenic polymorph comprising at least 17 consecutive amino acids peptide. 39. The polypeptide of claim 38, wherein the polypeptide comprises: a) at least one fragment of at least 25 contiguous amino acid residues from the primate HDTEA84 protein; b) comprises at least one fragment of at least 25 contiguous amino acid residues from a primate HSLJD37R protein; c) comprises a fragment of at least 25 contiguous amino acid residues from a rodent or primate RANKL protein. Contains at least one; d) is a soluble polypeptide; e) is detectably labeled; f) is present in a sterile composition; g) is present in a buffered composition; I) recombinantly produced; or j) a naturally occurring polypeptide sequence To, polypeptide. 40. The polypeptide of claim 39, wherein the polypeptide comprises: a) SEQ ID NO: 6 wherein the signal is processed; b) SEQ ID NO: 8 wherein the signal is processed; c) the signal is D) the signal processed SEQ ID NO: 12; e) the signal processed SEQ ID NO: 17; f) SEQ ID NO: 19; g) SEQ ID NO: 21; or h) SEQ ID NO: 23 A polypeptide comprising at least 17 contiguous amino acids. 41. A method of regulating the physiology or function of a precursor cell, comprising: a) a binding compound that binds to the cell according to claim 38; b) an HDTEA84 polypeptide; c) Contacting an HSLJD37R polypeptide; or d) a RANKL polypeptide. 42. The method of claim 41, wherein said contacting is in combination with a TNF family ligand or an antagonist of said TNF family ligand. 43. The following: a) a substantially pure or recombinant HCC5 polypeptide exhibiting identity to SEQ ID NO: 25 over a length of at least 12 amino acids; b) the mature SEQ ID NO: 25 An isolated native sequence HCC5; c) a fusion protein comprising the HCC5 sequence; d) substantially pure, exhibiting identity to SEQ ID NO: 32 or SEQ ID NO: 34 over a length of at least about 12 amino acids. Or a recombinant Dub11 polypeptide; e) an isolated native sequence Dub11 of the mature SEQ ID NO: 32 or SEQ ID NO: 34; f) a fusion protein comprising the Dub11 sequence; g) at least a SEQ ID NO: 36 or SEQ ID NO: 38 Substantially pure or recombinant, Dub12, showing identity over a length of about 12 amino acids
H) an isolated native sequence Dub12 of mature SEQ ID NO: 36 or SEQ ID NO: 38; i) a fusion protein comprising the Dub12 sequence; j) identical to SEQ ID NO: 42 over a length of at least about 12 amino acids. A) a substantially pure or recombinant MD-1 polypeptide; k) an isolated native sequence MD-1 of the mature SEQ ID NO: 42; l) a fusion protein comprising a primate MD-1 sequence M) a substantially pure or recombinant MD-2 polypeptide exhibiting identity over a length of at least about 12 amino acids to SEQ ID NO: 44 or SEQ ID NO: 46; n) the mature SEQ ID NO: 44) SEQ ID NO: 44 or SEQ ID NO: 46; o) a fusion protein comprising the vMD-2 sequence; A substantially pure or recombinant MD-2 polypeptide exhibiting identity over a length of at least about 12 amino acids; q) the isolated native sequence MD-2 of mature SEQ ID NO: 48; r) a fusion protein comprising a mouse MD-2 sequence. 44. A Dub11 poly, substantially pure or isolated, comprising: a) an HCC5 polypeptide, wherein said length is at least 17 amino acids; b) a Dub11 poly, wherein said length is at least 17 amino acids. A peptide; c) the Dub12 polypeptide, wherein said length is at least 17 amino acids; d) a primate MD-1 polypeptide, wherein said length is at least 17 amino acids; e) said length is at least 17 amino acids; 44. The composition of claim 43, wherein the primate MD-2 polypeptide; or f) the rodent MD-2 polypeptide, wherein said length is at least 17 amino acids. 45. An HCC5 polypeptide, substantially pure or isolated, comprising: a) said HCC5 polypeptide, wherein said length is at least 21 amino acids; b) Dub11 poly, wherein said length is at least 21 amino acids. A peptide; c) the Dub12 polypeptide, wherein said length is at least 21 amino acids; d) said primate MD-1 polypeptide, wherein said length is at least 21 amino acids; e) said length is at least 21 amino acids; 45. The composition of claim 44, wherein the primate MD-2 polypeptide; or f) the rodent MD-2 polypeptide, wherein said length is at least 21 amino acids. 46. The composition of claim 43, wherein: a) the HCC5 polypeptide is: i) from a primate, including a human; ii) at least one of SEQ ID NO: 25 Iii) presenting multiple parts exhibiting said identity; iv) being a natural allelic variant of HCC5; v) having a length of at least about 30 amino acids; vi. ) Exhibits at least two non-overlapping epitopes that are specific for primate HCC5; vii) exhibits sequence identity to HCC5 over a length of at least 35 amino acids; viii) is glycosylated; ix) is synthetic X) attached to a solid support; xi) conjugated to another chemical moiety; xii) natural Xiii) a deletion or insertion variant from the native sequence; b) the Dub11 polypeptide is from a primate, including a human; ii) a sequence. Comprises at least one polypeptide segment set forth in SEQ ID NO: 32 or SEQ ID NO: 34; iii) presents a plurality of parts exhibiting said identity; iv) is a natural allelic variant of Dub11; Vi) exhibit at least two non-overlapping epitopes that are specific for primate Dub11; vii) exhibit sequence identity to Dub11 over a length of at least 35 amino acids Viii) is glycosylated; ix) is a synthetic polypeptide; x) is attached to a solid support Xi) conjugated to another chemical moiety; xii) no more than 5 fold substitution from the native sequence; or xiii) a deletion or insertion variant from the native sequence; c) I) the Dub12 polypeptide is derived from a primate, including a human; ii) comprises at least one polypeptide segment set forth in SEQ ID NO: 36 or SEQ ID NO: 38; Iv) is a natural allelic variant of Dub12; v) has at least about 30 amino acids in length; vi) exhibits at least two non-overlapping epitopes specific for primate Dub12 Or vii) show sequence identity to Dub12 over a length of at least 35 amino acids; viii) glycosylation Ix) is a synthetic polypeptide; x) is attached to a solid support; xi) is conjugated to another chemical moiety; xii) is a 5-fold or less substitution from the native sequence Or xiii) a deletion or insertion variant from a native sequence; d) said primate MD-1 polypeptide is; i) human-derived; ii) at least one polypeptide according to SEQ ID NO: 42 Iii) showing a plurality of parts exhibiting said identity; iv) is a natural allelic variant of primate MD-1; v) has a length of at least about 30 amino acids Vi) exhibiting at least two non-overlapping epitopes that are specific for primate MD-1; vii) over a length of at least 35 amino acids for primate MD-1 Viii) is glycosylated; ix) is a synthetic polypeptide; x) is attached to a solid support; xi) is conjugated to another chemical moiety; xiii. X)) a deletion or insertion variant from the native sequence; e) the primate MD-2 polypeptide is derived from a human; ii) comprises at least one polypeptide segment set forth in SEQ ID NO: 44 or SEQ ID NO: 46; iii) exhibits multiple parts exhibiting said identity; iv) natural allelic modification of primate MD-2 V) have at least about 30 amino acids in length; vi) exhibit at least two non-overlapping epitopes that are specific for primate MD-2; vi. i) exhibits sequence identity to primate MD-2 over a length of at least 35 amino acids; viii) is glycosylated; ix) is a synthetic polypeptide; x) is attached to a solid support Xi) is conjugated to another chemical moiety; xii) is a 5-fold or less substitution from the native sequence; or xiii) is a deletion or insertion variant from the native sequence; or f) I) the rodent MD-2 polypeptide is derived from a mouse; ii) comprises at least one polypeptide segment set forth in SEQ ID NO: 48 or SEQ ID NO: 49; iii) a plurality that exhibits the identity Iv) is a natural allelic variant of rodent MD-2; v) has a length of at least about 30 amino acids; vi) rodent MD Exhibits at least two non-overlapping epitopes that are specific for -2; vii) exhibits sequence identity to rodent MD-2 over a length of at least 35 amino acids; viii) is glycosylated; ix) is a synthetic polypeptide; x) is attached to a solid support; xi) is conjugated to another chemical moiety; xii) is a 5-fold or less substitution from the native sequence; or xiii) The composition of claim 43, which is a deletion or insertion variant from the native sequence. 47. The polypeptide is: a) an HCC5 polypeptide; b) a Dub11 polypeptide; c) a Dub12 polypeptide; d) an MD-1 polypeptide; or e) an MD-2 polypeptide. A composition comprising the sterile polypeptide of claim 43. 48. The following: a) said HCC5 polypeptide and the following: 1) a carrier comprising: a) an aqueous compound comprising water, saline and / or a buffer;
Or b) a carrier formulated for oral, rectal, nasal, topical or parenteral administration; 2) another chemokine, including a chemokine selected from the group consisting of HCC1, HCC2, HCC3 and HCC4; Or 3) an antibody antagonist to a chemokine, including a chemokine selected from the group consisting of HCC1, HCC2, HCC3 and HCC4; b) said Dub11 polypeptide and a carrier, wherein said carrier is: a) Aqueous compounds including water, saline and / or buffers; and /
Or b) a carrier, formulated for oral, rectal, nasal, topical or parenteral administration; c) said Dub12 polypeptide and carrier, wherein the carrier comprises: a) water , An aqueous compound comprising saline and / or a buffer; and /
Or b) a carrier formulated for oral, rectal, nasal, topical or parenteral administration; d) said MD-1 polypeptide and a carrier, wherein said carrier comprises: a A) aqueous compounds including water, saline and / or buffers;
Or b) a carrier formulated for oral, rectal, nasal, topical or parenteral administration; e) said MD-2 polypeptide and carrier, wherein said carrier comprises: a A) aqueous compounds including water, saline and / or buffers;
Or b) a carrier formulated for oral, rectal, nasal, topical or parenteral administration. 49. The following: a) the mature protein sequence of the sequence of Table 7; b) the mature protein sequence of the sequence of Table 9; c) the mature protein sequence of the sequence of Table 11; d) the FLAG, His6, or Ig sequence. Including detection or purification tags;
Or e) the sequence of another chemokine protein having said protein in a). 50. A kit, comprising the polypeptide of claim 43, and: a) a compartment containing the polypeptide; and / or b) instructions for using or disposing of reagents in the kit. A kit comprising: 51. A binding compound comprising an antigen-binding portion derived from an antibody, the binding compound comprising: a) the HCC5 polypeptide of claim 43, wherein the antibody is: Has been raised against the peptide sequence of the described mature polypeptide sequence; ii) has been raised against mature HCC5; iii) has been raised against purified HCC5; iv) has been immunoselected. V) is a polyclonal antibody; vi) binds to denatured HCC5; or vii) exhibits a Kd for the antigen of at least 30 μM; b) the Dub11 polypeptide of claim 43, wherein The antibodies are: i) raised against a peptide sequence of the mature polypeptide sequence listed in Table 9; ii) raised against mature Dub11. Iii) raised against purified Dub11; iv) immunoselected; v) a polyclonal antibody; vi) binding to denatured DUB11; or vii) at least 30 μM, 49) indicates the Kd for the antigen; c) the Dub12 polypeptide of claim 43, wherein said antibody is raised against: i) the peptide sequence of the mature polypeptide sequence set forth in Table 9; ii) Iii) raised against purified Dub12; iv) immunoselected; v) polyclonal antibody; vi) binding to denatured Dub12 Or vii) exhibiting a Kd for the antigen of at least 30 μM; d) a primate M according to claim 43; A D-1 polypeptide, wherein said antibody is raised against a peptide sequence of a mature polypeptide sequence listed in Table 11; ii) raised against mature MD-1; iii. Iv) raised against purified MD-1; iv) immunoselected; v) polyclonal antibody; vi) binding to denatured MD-1; or vii) at least 30 μM E) a Kd against an antigen; e) the primate MD-2 polypeptide of claim 43, wherein said antibody is: i) raised against a peptide sequence of a mature polypeptide sequence set forth in Table 11. Ii) has been raised against mature MD-2; iii) has been raised against purified MD-2; iv) has been immunoselected; 44) a rodent MD-2 polypeptide according to claim 43, which is a null antibody; vi) binds to denatured MD-2; or vii) exhibits a Kd for the antigen of at least 30 μM, or f). Wherein said antibody is raised against a peptide sequence of a mature polypeptide sequence set forth in Table 11; ii) raised against a mature rodent MD-2; iii) purification Iv) is immunoselected, v) is a polyclonal antibody, vi) binds to a modified rodent MD-2, or vii A) exhibiting a Kd for the antigen of at least 30 μM; 52. The binding compound of claim 51, wherein: a) said polypeptide is from a primate or rodent; b) said binding compound is an Fv, Fab or Fab2 fragment. C) whether the binding compound is conjugated to another chemical moiety; d) is attached to a solid support containing beads or plastic membrane; e) is present in a sterile composition; f) A binding compound that is detectably labeled, including a radioactive or fluorescent label. 53. A binding compound according to claim 51, and: a) a compartment containing the binding compound; b) a compartment containing the purified antigen; and / or c) the use or disposal of reagents in the kit. Kit containing instructions for use. 54. A method for generating an antigen: antibody complex, comprising the antibody of claim 51, and: a) a primate HCC5 polypeptide; b) a primate Dub11 polypeptide; c) a primate Dub12. Contacting a polypeptide; d) a primate MD-1 polypeptide; e) a primate MD-2 polypeptide; or f) a rodent MD-2 polypeptide, thereby forming the complex. A method comprising: 55. The binding compound of claim 51 and: 1) a carrier, wherein the carrier is: a) an aqueous compound comprising water, saline and / or a buffer;
Or b) a carrier formulated for oral, rectal, nasal, topical or parenteral administration; or 3) another chemokine, including a chemokine selected from the group consisting of HCC1, HCC2, HCC3 and HCC4. An antibody antagonist to the composition. 56. An isolated or recombinant nucleic acid encoding a polypeptide or fusion protein according to claim 43, wherein: A) said HCC5 is a) Or b) the nucleic acid encodes the following: i) the antigenic HCC5 peptide sequence of Table 7; ii) the plurality of antigenic peptide sequences of Table 7; iii) the HCC5 At least 2 in the natural cDNA encoding the segment
Iv) is a hybridization probe for the gene encoding the HCC5 polypeptide; or vii) comprises a chemokine selected from the group consisting of HCC1, HCC2, HCC3 and HCC4. B) said Dub11 is: a) the polypeptide is from a primate, including a human; or b) the nucleic acid is: i) the antigenic Dub11 peptide sequence of Table 9 Ii) encodes multiple of the antigenic peptide sequences of Table 9; iii) exhibits identity to the native cDNA encoding the Dub11 segment for at least 25 nucleotides; or iv) the Dub11 poly. Hybridization to peptide-encoding gene C) the Dub12 is: a) the polypeptide is from a primate, including a human; or b) the nucleic acid encodes the following: i) the antigenic Dub12 peptide sequence of Table 9; ii. Iii) encodes more than one of the antigenic peptide sequences of Table 9; iii) exhibits at least 25 nucleotide identity to the native cDNA encoding the Dub12 segment; or iv) a gene encoding the Dub12 polypeptide D) the primate MD-1 is: a) the polypeptide is from a primate, including a human; or b) the nucleic acid is: i) an antigenic primate of Table 11 Encodes the MD-1 peptide sequence; ii) encodes more than one of the antigenic peptide sequences of Table 11; iii) the primate MD Exhibits identities over at least 25 nucleotides to the native cDNA encoding one segment; or iv) is a hybridization probe for the gene encoding the primate MD-1 polypeptide; E) said primate MD -2; a) the polypeptide is of human origin; or b) the nucleic acid encodes the following: i) the antigenic primate MD-2 peptide sequence of Table 11; ii) the antigenicity of Table 11 Iii) exhibits at least 25 nucleotide identity to the native cDNA encoding the primate MD-2 segment; or iv) encodes the primate MD-2 polypeptide F) said rodent MD-2 comprises: a) a polypeptide; Is the mouse derived; or b) the nucleic acid encodes the following: i) encodes the antigenic rodent MD-2 peptide sequence of Table 11; ii) encodes multiple of the antigenic peptide sequence of Table 11 Iii) exhibits identity to the native cDNA encoding the rodent MD-2 segment for at least 25 nucleotides; or iv) a high sequence for the gene encoding the rodent MD-2 polypeptide. A nucleic acid which is a hybridization probe. 57. The nucleic acid of claim 56, wherein: a) is an expression vector; b) further comprises an origin of replication; c) is derived from a natural source; d) is detectable E) contains a synthetic nucleotide sequence; f) is less than 6 kb, preferably less than 3 kb; g) is derived from a primate, including a human; h) contains a natural full-length coding sequence Or i) a nucleic acid that is a PCR primer, a PCR product or a mutagenesis primer. 58. A cell or tissue comprising the recombinant nucleic acid of claim 56, wherein the cell or tissue is: a) a prokaryotic cell; b) a eukaryotic cell; c) a bacterial cell; d). E) insect cells; f) mammalian cells; g) mouse cells; h) primate cells; or i) human cells. 59. A kit, the nucleic acid of claim 56; and a) a compartment containing the nucleic acid; b) a compartment containing a nucleic acid encoding another chemokine, including HCC1, HCC2, HCC3 and HCC4; or c) A kit comprising instructions for using or processing the reagents in the kit. 60. A nucleic acid, which: a) hybridizes to the polypeptide coding portion of SEQ ID NO: 24 under salt washing conditions of less than 45 ° C. and less than 2M; b) less than 45 ° C. and less than 2M Hybridizes to the polypeptide-encoding portion of SEQ ID NO: 31 or SEQ ID NO: 33 under salt washing conditions of c); D) hybridizes to the polypeptide coding portion of SEQ ID NO: 41 at 45 ° C. and under salt washing conditions of less than 2M; e) at 45 ° C. and 2M Hybridizes to the polypeptide-encoding portion of SEQ ID NO: 43 or SEQ ID NO: 45 under less than salt washing conditions; or f) at 45 ° C. A nucleic acid that hybridizes to the polypeptide-encoding portion of SEQ ID NO: 47 under washing conditions of less than and 2M salt. 61. The nucleic acid of claim 57, wherein: a) said washing conditions are 55 ° C and / or 500 mM salt; or b) said washing conditions are 65 ° C and / or A nucleic acid that is a 150 mM salt. 62. A method of regulating the physiology or development of a cell or tissue culture cell, said method comprising the step of inhibiting the agonist or antagonist of HCC5, primate MD-1, primate MD-2 or rodent MD-2. Exposing the cells,
Method. 63. A method for detecting specific binding to a compound, comprising the following steps: a) the following: i) an antigen binding site that specifically binds to HCC5 chemokine; ii) a specific binding to Dub11 Iii) an antigen binding site that specifically binds to Dub12; iv) an antigen binding site that specifically binds to primate MD-1; v) an antigen binding site that specifically binds to primate MD-2 Vi) an antigen-binding site that specifically binds to rodent MD-2; vii) an expression vector encoding HCC5 chemokine or a fragment thereof; viii) an expression vector encoding Dub11 or a fragment thereof; ix) an expression vector of Dub12 or a fragment thereof. An expression vector encoding primate MD-1 or a fragment thereof. Xi) an expression vector encoding a primate MD-2 or a fragment thereof; xii) an expression vector encoding a rodent MD-2 or a fragment thereof; xiii) specifically recognized by the antigen-binding site of (i). Xiv) a substantially pure protein specifically recognized by the antigen-binding site of (ii); xv) a protein specifically recognized by the antigen-binding site of (iii) A substantially pure protein; xvi) a substantially pure protein specifically recognized by the antigen binding site of (iv); xvii) a substantially specifically recognized by the antigen binding site of (v) Xviiii) a substantially pure protein specifically recognized by the antigen binding site of (vi); xix) 45. A qualitatively pure HCC5 chemokine or peptide thereof, or a fusion protein comprising a 30 amino acid sequence portion of the HCC5 chemokine sequence; xx) Dub11 or a peptide thereof of claim 43, which is substantially pure. Or a fusion protein comprising a 30 amino acid sequence portion of the Dub11 sequence; xxii) a substantially pure Dub12 or peptide thereof according to claim 43, or a fusion protein comprising a 30 amino acid sequence portion of the Dub12 sequence; xxii) substantially 44. A primate MD-1 or peptide thereof according to claim 43, or a fusion protein comprising a 30 amino acid sequence portion of the primate MD-1 sequence; xxiii) a substantially pure protein according to claim 43. Primate MD-2 or a peptide thereof, or primate M Or a fusion protein comprising a 30 amino acid sequence portion of the -2 sequence; or xxiv) a substantially pure, 30 amino acid sequence of the rodent MD-2 or a peptide thereof, or the rodent MD-2 sequence of claim 43. Contacting the compound with a composition selected from the group consisting of: a fusion protein comprising a moiety; and b) detecting binding of the compound to the compound. 64. An isolated or recombinant polynucleotide, comprising: a) encoding at least 17 contiguous amino acid residues of SEQ ID NO: 54; b) at least one of SEQ ID NO: 54 A polynucleotide encoding at least two different segments of 10 contiguous amino acid residues; or c) comprising one or more segments of at least 21 contiguous nucleotides of SEQ ID NO: 53. 65. The following: a) a polypeptide, comprising the step of expressing the expression vector of claim 64 to produce said polypeptide; b) a double-stranded nucleic acid, wherein: Contacting the polynucleotide of claim 64 with a complementary nucleic acid to effect the production of the double-stranded nucleic acid; c) the synthetic polynucleotide of claim 64, wherein the nucleotide is 65. A method of making the polynucleotide of claim 64, comprising the step of chemically polymerizing to produce the polynucleotide; d) comprising using the PCR method. 66. at least the following: a) one segment comprising at least 17 contiguous amino acids from SEQ ID NO: 54; or b) at least 2 of at least 11 contiguous amino acids from SEQ ID NO: 54
An isolated or recombinant antigenic polypeptide comprising two different segments. 67. The antigenic polypeptide of claim 66, comprising at least one segment comprising at least 17 contiguous amino acids from SEQ ID NO: 54. 68. The antigenic polypeptide of claim 66, wherein said polypeptide exhibits at least two non-overlapping one epitopes that are selective for the primate protein of SEQ ID NO: 54. 69. The antigenic polypeptide of claim 66, wherein the polypeptide is: a) no more than 5 fold substitution from the native sequence, or b) a deletion modification from the native sequence. An antigenic polypeptide which is a body or insertional variant. 70. A kit comprising: the polypeptide of claim 66; and instructions for using or disposing of the polypeptide or other reagents of the kit. 71. The antigenic polypeptide of claim 66, comprising at least two different segments of at least 11 contiguous amino acids from SEQ ID NO: 54. 72. The antigenic polypeptide of claim 71, comprising: a) the following from SEQ ID NO: 54: KERYVHD (residues 120-127), DKHFEVLH (residues 127-1)
34), HSDLEPQM (residues 134-141), QKDINKNM (residues 1
77-184), YAPKLQEF (residues 203-210), SEEDILRM
(Residues 219-226), LRMELIIL (residues 224-231), ELCP
VTII (residues 237-244), and LFLQVDAL (residues 249-25)
6) comprising at least one sequence of SEQ ID NO: 54; and / or b) wherein said segment of at least 11 consecutive amino acids comprises one such segment having at least 14 consecutive amino acids from SEQ ID NO: 54; Antigenic polypeptide. 73. The antigenic polypeptide of claim 71, wherein said antigenic polypeptide exhibits at least two non-overlapping epitopes that are selective for the primate protein of SEQ ID NO: 54. Polypeptide. 74. The antigenic polypeptide of claim 71, wherein the antigenic polypeptide comprises: a) the mature sequence of SEQ ID NO: 2; b) the immunogen of SEQ ID NO: 54 C) comprises a polypeptide segment of 17 contiguous amino acids of SEQ ID NO: 54; or d) a natural allelic modification set forth in SEQ ID NO: 54. An antigenic polypeptide that is the body. 75. The antigenic polypeptide of claim 71, wherein said polypeptide is: a) present in a sterile composition; b) is at least 30 amino acids in length; c. D) denatured; e) synthetic polypeptide; f) attached to a solid substrate; or g) a detection tag comprising the sequence of FLAG, His6 or Ig. Or an antigenic polypeptide that is a fusion protein with a purification tag. 76. The antigenic polypeptide of claim 71, wherein said antigenic polypeptide is: a) less than or equal to 5-fold substitution from the native sequence, or b) deleted from the native sequence. An antigenic polypeptide that is a variant or insertion variant. 77. A kit, comprising: the antigenic polypeptide of claim 71; and instructions for using or disposing of the antigenic polypeptide or other reagents in the kit. 78. The use of an antigenic polypeptide according to claim 71, comprising: a) labeling said antigenic polypeptide, wherein said antigenic polypeptide is labeled with a radiolabel. B) separating the antigenic polypeptide from another polypeptide in a mixture, wherein the mixture is flowed over a chromatography matrix, thereby separating the antigenic polypeptide C) for identifying a compound that selectively binds to the antigenic polypeptide, wherein the compound is incubated under appropriate conditions with the antigenic polypeptide, thereby Causing the compound to bind to the antigenic polypeptide; or d) conjugating the antigenic polypeptide to a matrix. Wherein the step of derivatizing the antigenic polypeptide with a reactive reagent and the step of conjugating the matrix and the polypeptide are included; or e) for the antigenic polypeptide A method for inducing an antibody response, comprising introducing the antigenic polypeptide as an antigen into the immune system, thereby inducing the response. 79. A binding compound comprising an antigen-binding portion derived from an antibody that selectively binds to the antigenic polypeptide of claim 66. 80. A method for evaluating the selectivity of binding of a compound to cyclin E2, comprising contacting the compound with a recombinant cyclin E2 polypeptide and at least one other cyclin; Comparing the binding to the plurality of cyclins. 81. The antigenic polypeptide of claim 67, comprising: a) the following from SEQ ID NO: 54: KERYVHD (residues 120-127), DKHFEVLH (residues 127-1)
34), HSDLEPQM (residues 134-141), QKDINKNM (residues 1
77-184), YAPKLQEF (residues 203-210), SEEDILRM
(Residues 219-226), LRMELIIL (residues 224-231), ELCP
VTII (residues 237-244), and LFLQVDAL (residues 249-25)
And / or b) wherein said segment comprising at least 17 contiguous amino acids represents at least 23 contiguous amino acids from SEQ ID NO: 54. 82. The antigenic polypeptide of claim 67, wherein the antigenic polypeptide comprises: a) the mature sequence of SEQ ID NO: 54; b) the immunogen of SEQ ID NO: 54 C) comprises a plurality of polypeptide segments comprising at least 17 contiguous amino acids of SEQ ID NO: 54; or d) a native polypeptide of SEQ ID NO: 54. An antigenic polypeptide that is an allelic variant. 83. The antigenic polypeptide of claim 67, wherein said antigenic polypeptide is: a) present in a sterile composition; b) is at least 30 amino acids in length. C) unglycosylated; d) denatured; e) synthetic polypeptide; f) attached to a solid substrate; or g) comprising the sequence of FLAG, His6 or Ig; An antigenic polypeptide that is a fusion protein with a detection tag or a purification tag. 84. The use of an antigenic polypeptide according to claim 67, comprising: a) labeling said antigenic polypeptide, wherein said antigenic polypeptide is labeled with a radiolabel. B) separating the antigenic polypeptide from another polypeptide in a mixture, wherein the mixture is flowed over a chromatography matrix, thereby separating the antigenic polypeptide C) for identifying a compound that selectively binds to the antigenic polypeptide, wherein the compound is incubated under appropriate conditions with the antigenic polypeptide, thereby Causing the binding of the compound to the antigenic polypeptide; d) a method for conjugating the antigenic polypeptide to a matrix. Derivatizing the antigenic polypeptide with a reactive reagent, and conjugating the matrix to the polypeptide; or e) determining the antibody response to the antigenic polypeptide. A method for inducing, wherein the antigenic polypeptide is introduced into the immune system as an antigen, thereby inducing the response.