WO2000028032A2 - Proteines du recepteur de surface des cellules humaines - Google Patents

Proteines du recepteur de surface des cellules humaines Download PDF

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Publication number
WO2000028032A2
WO2000028032A2 PCT/US1999/026742 US9926742W WO0028032A2 WO 2000028032 A2 WO2000028032 A2 WO 2000028032A2 US 9926742 W US9926742 W US 9926742W WO 0028032 A2 WO0028032 A2 WO 0028032A2
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WIPO (PCT)
Prior art keywords
hcsrp
polynucleotide
seq
sequence
sequences
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PCT/US1999/026742
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English (en)
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WO2000028032A3 (fr
Inventor
Y. Tom Tang
Neil C. Corley
Karl J. Guegler
Henry Yue
Mariah R. Baughn
Preeti Lal
Jennifer L. Hillman
Olga Bandman
Yalda Azimzai
Janice Au-Young
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Incyte Pharmaceuticals, Inc.
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Application filed by Incyte Pharmaceuticals, Inc. filed Critical Incyte Pharmaceuticals, Inc.
Priority to JP2000581199A priority Critical patent/JP2005500801A/ja
Priority to EP99958913A priority patent/EP1129189A2/fr
Priority to AU16185/00A priority patent/AU1618500A/en
Priority to CA002349815A priority patent/CA2349815A1/fr
Publication of WO2000028032A2 publication Critical patent/WO2000028032A2/fr
Publication of WO2000028032A3 publication Critical patent/WO2000028032A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • This ent ⁇ on relates to nucleic acid and amino acid sequences of human cell surface receptor proteins and to the use of these sequences in the diagnosis treatment, and of cell proliferate e disorders, immune system disorders infections, and neuronal disorders
  • receptor describes proteins that specifically recognize other molecules
  • the category is broad and includes proteins with a variety of functions
  • the bulk of receptors are cell surface proteins which bind extracellular ligands and produce cellular responses in the areas of growth, differentiation, endocytosis. exocytosis and immune response Central to the function of cell surface receptors is the capacity to adhere or bind to other proteins or ligands through special functional domains
  • Cell surface receptors are typically integral plasma membrane proteins These receptors recognize hormones such as catecholamines, peptide hormones, growth and differentiation factors, small peptide factors such as thyrotropin releasing hormone, galanin. somatostatin. and tachykinins, and circulatory system-borne signaling molecules
  • hormones such as catecholamines, peptide hormones, growth and differentiation factors, small peptide factors such as thyrotropin releasing hormone, galanin. somatostatin. and tachykinins
  • MHC major histocompatibility complex
  • Other cell surface receptors bind ligands to be internalized by the cell This receptor-mediated endocytosis functions in the uptake of low density lipoproteins (LDL), transfernn. glucose- or mannose-terminal glycoproteins. galactose-terminal glycoproteins. immunoglobulins.
  • LDL low density lipoproteins
  • transfernn glucose- or mannose-terminal glycoproteins.
  • growth factor receptors including receptors for epidermal growth factor.
  • These phosphorylated sites are recognition sites for the binding of other cytoplasmic signaling proteins
  • These proteins participate in signaling pathyyays that eventually link the initial receptor activation at the cell surface to the activation of a specific lntraceliular target molecule In the case of tyrosine residue autophosphor lation.
  • these signaling proteins contain a common domain referred to as a src homology
  • SH2 domains are found in a ya ⁇ ety of signaling molecules and oncogenic proteins such as phospholipase C- ⁇ . Ras GTPase activating protein, and pp60 c"src (Lowenstein. E.J. et al. ( 1992)
  • GPCRs G-protein coupled receptors
  • G-protein coupled receptors are integral membrane proteins characterized by the presence of seven hydrophobic transmembrane domains which span the plasma membrane and form a bundle of antiparallel alpha ( ⁇ ) helices. These proteins range in size from under 400 to over 1000 amino acids (Strosberg. A.D. ( 1991 ) Eur. J. Biochem. 196: 1 - 10; Coughlin, S.R. ( 1994) Curr. Opin. Cell Biol. 6: 191 - 197).
  • the amino-terminus of the GPCR is extracellular, of variable length and often glycosylated; the carboxy-terminus is cytoplasmic and generally phosphorylated.
  • Extracellular loops of the GPCR alternate with intracellular loops and link the transmembrane domains.
  • the most conserved domains of GPCRs are the transmembrane domains and the first tyvo cytoplasmic loops.
  • the transmembrane domains account for structural and functional features of the receptor. In most cases, the bundle of alpha helices forms a binding pocket.
  • the extracellular N-terminal segment or one or more of the three extracellular loops may also participate in ligand binding. Ligand binding activates the receptor by inducing a conformational change in intracellular portions of the receptor.
  • the activated receptor interacts yvith an intracellular heterotrimeric guanine nucleotide binding (G) protein complex which mediates further intracellular signaling activities, generally the production of second messengers such as cyclic AMP (cAMP), phospholipase C, inositol triphosphate or ion channel proteins (Baldwin, J.M. ( 1994) Curr. Opin. Cell Biol. 6: 180- 190).
  • GPCRs are the rhodopsin-like GPCRs that transmit extracellular signals of diverse natures including hormones, neurotransmitters and light. Rhodopsin is a photosensitive GPCR in the vertebrate eye.
  • Rhodopsin which defines a conserved subfamily of GPCRs found in animal retinas, is about 350 amino acids in length.
  • rhodopsin molecules are embedded in membranous stacks found in photoreceptor (rod) cells. Each rhodopsin molecule responds to a photon of light by triggering a decrease in cGMP levels which leads to the closure of plasma membrane sodium channels. In this manner, a visual signal is converted to a neural impulse.
  • Other rhodopsin-like GPCRs are directly involved in responding to neurotransmitters. These GPCRs include the receptors for adrenaline (adrenergic receptors), acetylcholine (muscarinic receptors), adenosine.
  • the somatostatin receptor type 4 is another example of a rhodopsin-like GPCR. It is one of several high affinity receptors for somatostatin. a tetrapeptide that inhibits the secretion of growth hormone from the anterior pituitary'. Expression of particular somatostatin receptors has been linked to the efficacy of drug therapy in specific endocrine tumors (Kubota. A. et al. ( 1994) J. Clin. Invest. 93: 1321 - 1325).
  • Another rhodopsin-like GPCR is the prostanoid EP1 receptor that recognizes prostanoids such as prostaglandin to mediate a variety of physiological functions including cardiovascular and immune responses.
  • EP1 receptors have a role in the contraction and relaxation of smooth muscle and can activate the phosphoinositide pathway (Watson, supra, pp. 239-251 ).
  • the prostanoid DP receptor is another rhodopsin-like GPCR that is specific for prostaglandin D2 (PGD2). Expression of the DP receptor has been localized to the mammalian brain and eye tissues and upon activation facilitates elevation of intracellular cAMP and Ca2+ mobilization but does not generate inositol 1,4.5- triphosphate (Boie. Y. et al. ( 1995) J. Biol. Chem. 270: 18910-18916; Gerashchenko. D. et al. ( 1998) J. Neurochem. 71 :937-945).
  • Still another rhodopsin-like GPCR is the endothelin receptor that plays a role in cardiovascular system regulation through endothelins.
  • Endothelins are potent vasoconstrictors that can stimulate cardiac and smooth muscle contraction as well as stimulate secretion in tissues such as kidney, liver and adrenals.
  • Endothelin receptors may have a role in the brain, where they are also found, and there is evidence that endothelins may be associated with pathophysiological conditions such as stress (Watson, supra, pp. 1 1 1-1 16).
  • the secretin receptor is an example of a unique GPCR that responds to secretin, a peptide hormone that stimulates the secretion of enzymes and ions in the pancreas and small intestine
  • EMR1 epidermal growth factor (EGF)-Iike modules at the N-terminus followed by a serine/threonine rich domain. The latter feature is characteristic of mucin-like integral membrane adhesive proteins.
  • GPCRs have been identified which play a roie in the immune response.
  • a new subfamily of GPCRs has been identified from a human monocyte (HM) cDNA library (Nomura. H. et al. (1993) Int. Immunol. 5: 1239- 1249).
  • HM74 human monocyte
  • Most of these GPCRs likely bind to cytokines and other leukocytic signaling molecules.
  • HM74 is particularly unusual in that its N- terminus does not contain N-glycosylation sites.
  • TRs The thrombin receptors
  • ⁇ -thrombin induces production of IL-8 and IL-6 in cultured monocytes and endothelial cells (Johnson. . et al. ( 1998) J. Immunol. 160:5130-5135).
  • ⁇ -thrombin inhibits the action of IL-6. leukemia inhibitory factor, and ciliary neurotrophic factor in Chinese hamster lung fibroblasts (Bhat. G.J. et al. ( 1998) Arch. Biochem. Biophys. 350:307-3 14).
  • ⁇ -thrombin when ⁇ -thrombin binds to the TR it proteolytically cleaves 40 amino acids from the N-terminus ofthe receptor.
  • the cleaved peptide is termed the thrombin receptor agonist peptide and acts as a tethered ligand for the TR to increase the potency of the thrombin- derived signal (Hou, L. Et al. ( 1998) J. Periodontal Res. 33:205-21 1 : Johnson, et al. supra).
  • GPCR mutations may cause loss of function or constitutive activation, have been associated with numerous human diseases (Coughlin. supra). For instance, retinitis pigmentosa may arise from mutations in the rhodopsin gene.
  • Parma, J. et al. 1993, Nature 365:649-651 ) report that somatic activating mutations in the thyrotropin receptor cause hyperfunctioning thyroid adenomas and suggest that certain GPCRs susceptible to constitutive activation may behave as protooncogenes.
  • Cvtokine Receptors Cvtokine Receptors
  • Cytokines comprise a family of signaling molecules that modulate the immune system and the inflammatory response. Cytokines are usually secreted by leukocytes, or white blood cells, in response to injury or infection. However, other tissues are capable of secreting cytokines in response to disease or other physiologic perturbations. Cytokines function as growth and differentiation factors that act primarily on cells of the immune system such as lymphocytes, monocytes, and granulocytes. Like other signaling molecules, cytokines bind to specific plasma membrane receptors and trigger intracellular signal transduction pathways which regulate gene expression, cell proliferation, and cell differentiation.
  • EPO Erythropoietin
  • the EPO receptor is a single-pass transmembrane protein of about 500 amino acids, the intracellular domain of which associates yvith JA 2 kinase. Activated EPO receptor stimulates the phosphorylation activity of JA 2 which triggers gene transcription and mitogenesis.
  • immunoglobulin (Ig) domain for example the Immunoglobulin (Ig) domain
  • the human immune system is responsible for combating infectious agents contracted from the environment.
  • One critical component of the immune system are highly specialized molecules called immunoglobulins (Ig) or antibodies that can recognize and bind to foreign antigens, thereby facilitating their elimination.
  • Characteristic features of immunoglobulins include their structural motifs that include regions for membrane attachment, antigen recognition (variable (V) regions), and polymerization.
  • Polymerized immunoglobulins such as glandularly secreted IgA and IgM must undergo transcellular transport, a process mediated by the poly-immunoglobulin (poly-lg) receptor.
  • the poly-lg receptor is itself a member of the immunoglobulin superfamily having homology to the variable (V) region of immunoglobulins (Hood. L. et al. ( 1985) Cell 40:225-229). Like all immunoglobulin superfamily members, poly-lg receptor is involved in adhesion or binding to other proteins through the conserved immunoglobulin-like domain. This Ig domain is comprised of antiparallel ⁇ sheets joined by a disulfide bond in an arrangement called the Ig fold.
  • Members of the Ig superfamily include T-cell receptors, MHC proteins, CD4, CD8, and CD 28 cell surface proteins, and antibodies.
  • Immunoglobulins are the central components of the humoral immune response.
  • IgG the most common class of immunoglobulin in the circulation, can be described in terms of two main functional domains.
  • Antigen recognition is mediated by the Fab (antigen binding fragment) region ofthe IgG, while effector functions are mediated by the Fc (crystallizable fragment) region.
  • Binding of IgG to an antigen, such as a bacterium triggers the destruction of the antigen by phagocytic yvhite blood cells, such as macrophages and neutrophils.
  • the IgG Fc receptors expressed by phagocytic cells are single- pass transmembrane glycoproteins of about 400 amino acids (Sears. D. W. et al. (1990) J. Immunol. 144:371-378).
  • the extracellular portion o the IgG Fc receptor typically contains two or three Ig domains.
  • T cells play a dual role in the immune system as effectors and regulators, coupling antigen recognition yvith the transmission of signals that induce cell death in infected cells and stimulate other immune cells.
  • T cells collectively recognize a wide range of different antigens, a clonal line of T cells can only recognize a single antigen.
  • the antigen must be presented to the T cell receptor (TCR) as a peptide complexed yvith a major histocompatibility molecule (MHC) on the surface of an antigen-presenting cell.
  • TCR T cell receptor
  • MHC major histocompatibility molecule
  • the TCR on most T cells consists of tyvo polypeptide subunits. ⁇ and ⁇ . yvhich are immunoglobulin-like integral membrane glycoproteins of similar molecular yveight.
  • the TCR ⁇ and TCR ⁇ subunits have an extracellular domain containing both variable and constant regions, a transmembrane domain that traverses the membrane once, and a short intracellular domain (Saito, H. et al. ( 1984) Nature 309:757-762).
  • the genes for the TCR subunits are constructed through somatic rearrangement of different gene segments. Interaction of antigen in the proper MHC context yvith the TCR initiates signaling cascades that induce the proliferation, maturation, and function of cellular components o the immune system (Weiss. A. ( 1991 ) Annu. Rev. Genet. 25: 487- 5 10). Rearrangements in TCR genes and alterations in TCR expression have been noted in lymphomas.
  • the invention features substantially purified polypeptides, human ceil surface receptor proteins, referred to collectively as “HCSRP” and individually as “HCSRP- 1 ,” “HCSRP-2,” “HCSRP-3,” “HCSRP-4,” “HCSRP-5,” “HCSRP-6,” “HCSRP-7,” “HCSRP-8,” “HCSRP-9,” “HCSRP- 10,” “HCSRP- 1 1 ,” “HCSRP- 12,” and “HCSRP- 13.”
  • the invention provides a substantially purified polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 13 and fragments thereof.
  • the invention also includes a polypeptide comprising an amino acid sequence that differs by one or more conservative amino acid substitutions from an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 13.
  • the invention further provides a substantially purified variant having at least 90% amino acid identity to at least one of the amino acid sequences selected from the group consisting of SEQ ID NO: 1- 13 and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: l - 13 and fragments thereof.
  • the invention also includes an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 13 and fragments thereof.
  • the invention provides an isolated and purified polynucleotide which hybridizes under stringent conditions to the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 13 and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide having a sequence which is complementary' to the polynucleotide encoding the polypeptide comprising the amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 13 and fragments thereof.
  • the invention also provides a method for detecting a polynucleotide in a sample containing nucieic acids, the method comprising the steps of: (a) hybridizing the complement of the polynucleotide sequence to at least one of the polynucleotides of the sample, thereby forming a hybridization complex: and (b) detecting the hybridization complex, yvherein the presence of the hybridization complex correlates yvith the presence of a polynucleotide in the sample.
  • the method further comprises amplifying the polynucleotide prior to hybridization.
  • the invention also provides an isolated and purified polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26 and fragments thereof.
  • the invention further provides an isolated and purified polynucleotide variant having at least 90% polynucleotide sequence identity to the polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26 and fragments thereof.
  • the invention also provides an isolated and purified polynucleotide having a sequence yvhich is complementary to the polynucleotide comprising a polynucleotide sequence selected from the group consisting of SEQ ID NO: 14-26 and fragments thereof.
  • the invention further provides an expression vector containing at least a fragment of the polynucleotide encoding the polypeptide comprising an amino acid sequence selected from the group consisting of SEQ ID NO: 1- 13.
  • the expression vector is contained within a host cell.
  • the invention also provides a method for producing a polypeptide. the method comprising the steps of: (a) culturing the host cell containing an expression vector containing a polynucleotide ofthe invention under conditions suitable for the expression of the polypeptide: and (b) recovering the polypeptide from the host cell culture.
  • the invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1- 13 and fragments thereof, in conjunction with a suitable pharmaceutical carrier.
  • the invention further includes a purified antibody which binds to a polypeptide selected from the group consisting of SEQ ID NO: 1 - 13 and fragments thereof.
  • the invention also provides a purified agonist and a purified antagonist to the polypeptide.
  • the invention also provides a method for treating or preventing a disorder associated yvith decreased expression or activity of HCSRP, the method comprising administering to a subject in need of such treatment an effective amount of a pharmaceutical composition comprising a substantially purified polypeptide having the amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 13 and fragments thereof, in conjunction yvith a suitable pharmaceutical carrier.
  • the invention also provides a method for treating or preventing a disorder associated yvith increased expression or activity of HCSRP, the method comprising administering to a subject in need of such treatment an effective amount of an antagonist of a polypeptide having an amino acid sequence selected from the group consisting of SEQ ID NO: 1 - 13 and fragments thereof.
  • Table 1 shows polypeptide and nucleotide sequence identification numbers (SEQ ID NOs), clone identification numbers (clone IDs). cDNA libraries, and cDNA fragments used to assemble full- length sequences encoding HCSRP.
  • Table 2 shows features of each polypeptide sequence, including potential motifs, homologous sequences, and methods, algorithms, and searchable databases used for analysis of HCSRP.
  • Table 3 shows selected fragments of each nucleic acid sequence: the tissue-specific expression patterns of each nucleic acid sequence as determined by northern analysis: diseases, disorders, or conditions associated yvith these tissues; and the vector into which each cDNA was cloned.
  • Table 4 describes the tissues used to construct the cDNA libraries from which cDNA clones encoding HCSRP were isolated.
  • Table 5 shows the tools, programs, and algorithms used to analyze HCSRP, along with applicable descriptions, references, and threshold parameters.
  • a host cell includes a plurality of such host cells
  • an antibody is a reference to one or more antibodies and equivalents thereof knoyvn to those skilled in the art, and so forth.
  • HCSRP refers to the amino acid sequences of substantially purified HCSRP obtained from any species, particularly a mammalian species, including bovine, ovine, porcine, murine. equine, and human, and from any source, whether natural, synthetic, semi-synthetic, or recombinant.
  • agonist refers to a molecule which intensifies or mimics the biological activity of
  • HCSRP HCSRP.
  • Agonists may include proteins, nucleic acids, carbohydrates, small molecules, or any other compound or composition yvhich modulates the activity of HCSRP either by directly interacting with HCSRP or by acting on components of the biological pathway in which HCSRP participates.
  • allelic variant is an alternative form of the gene encoding HCSRP. Allelic variants may result from at least one mutation in the nucleic acid sequence and may result in altered mRNAs or in polypeptides whose structure or function may or may not be altered. A gene may have none, one, or many allelic variants of its naturally occurring form. Common mutational changes which give rise to allelic variants are generally ascribed to natural deletions, additions, or substitutions of nucleotides. Each of these types of changes may occur alone, or in combination yvith the others, one or more times in a given sequence.
  • altered nucleic acid sequences encoding HCSRP include those sequences with deletions, insertions, or substitutions of different nucleotides. resulting in a polypeptide the same as HCSRP or a polypeptide with at least one functional characteristic of HCSRP. Included within this definition are polymorphisms yvhich may or may not be readily detectable using a particular oligonucleotide probe of the polynucleotide encoding HCSRP. and improper or unexpected hybridization to allelic variants, with a locus other than the normal chromosomal locus for the polynucleotide sequence encoding HCSRP.
  • the encoded protein may also be "altered,” and may contain deletions, insertions, or substitutions of amino acid residues which produce a silent change and result in a functionally equivalent HCSRP.
  • Deliberate amino acid substitutions may be made on the basis of similarity in polarity, charge, solubility, hydrophobicity, hydrophilicity, and/or the amphipathic nature of the residues, as long as the biological or immunological activity of HCSRP is retained.
  • negatively charged amino acids may include aspartic acid and glutamic acid
  • positively charged amino acids may include lysine and arginine.
  • Amino acids yvith uncharged polar side chains having similar hydrophilicity values may include: asparagine and glutamine: and serine and threonine.
  • Ammo acids y ⁇ ⁇ th uncharged side chains hay ing similar hydrophilicity values may include leucine, lsoleucine, and valine. gly cine and alanine. and phenylalanine and tyrosine
  • amino acid sequence refers to an oligopeptide. peptide. polypeptide. or protein sequence, or a fragment of any of these, and to naturally occurring or synthetic molecules
  • amino acid sequence and like terms are not meant to limit the amino acid sequence to the complete native ammo acid sequence associated with the recited protein molecule
  • Amplification relates to the production of additional copies of a nucleic acid sequence Amplification is generally carried out using polymerase chain reaction (PCR) technologies well known in the art
  • Antagonist refers to a molecule which inhibits or attenuates the biological activity of HCSRP Antagonists may include proteins such as antibodies, nucleic acids, carbohydrates, small molecules, or any other compound or composition which modulates the activity of HCSRP either by directly interacting with HCSRP or by acting on components ofthe biological pathway in which HCSRP participates
  • antibody refers to intact immunoglobulin molecules as well as to fragments thereof, such as Fab, F(ab') 2 , and Fv fragments, yvhich are capable of binding an epitopic determinant
  • Antibodies that bind HCSRP polypeptides can be prepared using intact polypeptides or using fragments containing small peptides of interest as the immunizing antigen
  • the polypeptide or oligopeptide used to immunize an animal e g , a mouse, a rat, or a rabbit
  • an animal e g , a mouse, a rat, or a rabbit
  • Commonly used carriers that are chemically coupled to peptides include bovine serum albumin, thyroglobulin, and keyhole limpet hemocyanin (KLH) The coupled peptide is then used to immunize the animal
  • antigenic determinant refers to that region of a molecule (i e . an epitope) that makes contact yv ith a particular antibody
  • an antigenic determinant may compete yy ith the intact antigen (l e . the immunogen used to elicit the immune response) for binding to an antibody
  • antisense refers to any composition containing a nucleic acid sequence which is complementary to the "sense " strand of a specific nucleic acid sequence
  • Antisense molecules may be produced by any method including sy nthesis or transcription Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and to block either transcription or translation.
  • the designation “"negative “ or “minus “ can refer to the antisense strand, and the designation "positive” or “plus” can refer to the sense strand.
  • biologically active refers to a protein having structural, regulatory, or biochemical functions of a naturally occurring molecule.
  • immunologically active refers to the capability of the natural, recombinant. or synthetic HCSRP. or of any oligopeptide thereof, to induce a specific immune response in appropriate animals or cells and to bind with specific antibodies.
  • complementarity refers to the natural binding of polynucleotides by base pairing.
  • sequence 5' A-G-T 3'
  • complementary sequence 3' T-C-A 5'.
  • Complementarity between two single-stranded molecules may be "partial.” such that only some of the nucleic acids bind, or it may be "complete,” such that total complementarity exists between the single stranded molecules.
  • the degree of complementarity between nucleic acid strands has significant effects on the efficiency and strength of the hybridization between the nucleic acid strands. This is of particular importance in amplification reactions, which depend upon binding between nucleic acid strands, and in the design and use of peptide nucleic acid (PNA) molecules.
  • PNA peptide nucleic acid
  • composition comprising a given polynucleotide sequence and a “composition comprising a given amino acid sequence” refer broadly to any composition containing the given polynucleotide or amino acid sequence.
  • the composition may comprise a dry formulation or an aqueous solution.
  • Compositions comprising polynucleotide sequences encoding HCSRP or fragments of HCSRP may be employed as hybridization probes.
  • the probes may be stored in freeze-dried form and may be associated with a stabilizing agent such as a carbohydrate.
  • the probe may be deployed in an aqueous solution containing salts (e.g., NaCl). detergents (e.g..).
  • Consensus sequence refers to a nucleic acid sequence yvhich has been resequenced to resolve uncalled bases, extended using the XL-PCR kit (Perkin-Elmer, Norwalk CT) in the 5' and/or the 3' direction, and resequenced. or which has been assembled from the overlapping sequences of one or more Incyte Clones and, in some cases, one or more public domain ESTs. using a computer program for fragment assembly, such as the GELVIEW fragment assembly system (GCG, Madison WI).
  • GELVIEW fragment assembly system GELVIEW fragment assembly system
  • Constant amino acid substitutions are those substitutions that, when made, least interfere yvith the properties of the original protein, i.e.. the structure and especially the function ofthe protein is conserved and not significantly changed by such substitutions.
  • the table below shows amino acids which may be substituted for an original amino acid in a protein and which are regarded as conservative amino acid substitutions.
  • Conservative amino acid substitutions generally maintain (a) the structure of the polypeptide backbone in the area of the substitution, for example, as a beta sheet or alpha helical conformation, (b) the charge or hydrophobicity of the molecule at the site of the substitution, and/or (c) the bulk of the side chain.
  • a “deletion” refers to a change in the amino acid or nucleotide sequence that results in the absence of one or more amino acid residues or nucleotides.
  • derivative refers to the chemical modification of a polypeptide sequence, or a polynucleotide sequence. Chemical modifications of a polynucleotide sequence can include, for example, replacement of hydrogen by an alkyl, acyl, hydroxyl, or amino group.
  • a derivative polynucleotide encodes a polypeptide yvhich retains at least one biological or immunological function ofthe natural molecule.
  • a derivative polypeptide is one modified by glycosylation, pegyiation, or any similar process that retains at least one biological or immunological function of the polypeptide from yvhich it was derived.
  • a “fragment” is a unique portion of HCSRP or the polynucleotide encoding HCSRP which is identical in sequence to but shorter in length than the parent sequence.
  • a fragment may comprise up to the entire length of the defined sequence, minus one nucleotide/amino acid residue.
  • a fragment may comprise from 5 to 1000 contiguous nucleotides or amino acid residues.
  • a fragment used as a probe, primer, antigen, therapeutic molecule, or for other purposes, may be at least 5. 10, 15, 20, 25. 30. 40. 50. 60, 75, 100. 150. 250 or at least 500 contiguous nucleotides or amino acid residues in length. Fragments may be preferentially selected from certain regions of a molecule.
  • a poiypeptide fragment may comprise a certain length of contiguous amino acids selected from the first 250 or 500 amino acids (or first 25% or 50% of a polypeptide) as shown in a certain defined sequence.
  • a fragment of SEQ ID NO: 14-26 comprises a region of unique polynucleotide sequence that specifically identifies SEQ ID NO: 14-26. for example, as distinct from any other sequence in the same genome.
  • a fragment of SEQ ID NO: 14-26 is useful, for example, in hybridization and amplification technologies and in analogous methods that distinguish SEQ ID NO: 14-26 from related polynucleotide sequences.
  • the precise length of a fragment of SEQ ID NO: 14-26 and the region of SEQ ID NO: 14-26 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • a fragment of SEQ ID NO: l - 13 is encoded by a fragment of SEQ ID NO: 14-26.
  • a fragment of SEQ ID NO: 1 - 13 comprises a region of unique amino acid sequence that specifically identifies SEQ ID NO: 1- 13.
  • a fragment of SEQ ID NO: 1- 13 is useful as an immunogenic peptide for the development of antibodies that specifically recognize SEQ ID NO: 1- 13.
  • the precise length of a fragment of SEQ ID NO: 1- 13 and the region of SEQ ID NO: 1- 13 to which the fragment corresponds are routinely determinable by one of ordinary skill in the art based on the intended purpose for the fragment.
  • the term "similarity" refers to a degree of complementarity. There may be partial similarity or complete similarity.
  • the word "identity” may substitute for the word “similarity.”
  • a partially complementary sequence that at least partially inhibits an identical sequence from hybridizing to a target nucleic acid is referred to as “substantially similar.”
  • the inhibition of hybridization of the completely complementary sequence to the target sequence may be examined using a hybridization assay (Southern or northern blot, solution hybridization, and the like) under conditions of reduced stringency.
  • a substantially similar sequence or hybridization probe will compete for and inhibit the binding of a completely similar (identical) sequence to the target sequence under conditions of reduced stringency. This is not to say that conditions of reduced stringency are such that non-specific binding is permitted, as reduced stringency conditions require that the binding of tyvo sequences to one another be a specific (i.e.. a selective) interaction.
  • the absence of non-specific binding may be tested by the use of a second target sequence which lacks even a partial degree of complementarity (e.g., less than about 30% similarity or identity). In the absence of non-specific binding, the substantially similar sequence or probe will not hybridize to the second non-complementary target sequence.
  • the phrases "percent identity " and "% identity.” as applied to polynucleotide sequences refer to the percentage of residue matches betyveen at least tyvo polynucleotide sequences aligned using a standardized algorithm. Such an algorithm may insert, in a standardized and reproducible way, gaps in the sequences being compared in order to optimize alignment betyveen tyvo sequences, and therefore achieve a more meaningful comparison of the two sequences.
  • the "weighted" residue weight table is selected as the default. Percent identity is reported by CLUSTAL V as the "percent similarity" between aligned polynucleotide sequence pairs.
  • NCBI National Center for Biotechnology Information
  • BLAST Basic Local Alignment Search Tool
  • the BLAST software suite includes various sequence analysis programs including "blastn,” that is used to align a known polynucleotide sequence with other polynucleotide sequences from a variety of databases. Also available is a tool called “BLAST 2 Sequences " that is used for direct pairwise comparison of tyvo nucleotide sequences. "BLAST 2 Sequences " can be accessed and used interactively at http:/ ⁇ yvvyv.ncbi.nlm.nih.gov/gorf/bl2.ht ⁇ l. The "BLAST 2 Sequences” tool can be used for both blastn and blastp (discussed below). BLAST programs are commonly used with gap and other parameters set to default settings.
  • BLAST 2 Sequences Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such default parameters may be, for example: Matrix: BLOSUM62 Reward for match: 1 Penalty for mismatch: -2
  • Percent identity may be measured over the length of an entire defined sequence, for example. as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined sequence, for instance, a fragment of at least 20. at least 30. at least 40. at least 50. at least 70. at least 100, or at least 200 contiguous nucleotides.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures, or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • nucleic acid sequences that do not show a high degree of identity may nevertheless encode similar amino acid sequences due to the degeneracy of the genetic code. It is understood that changes in a nucleic acid sequence can be made using this degeneracy to produce multiple nucleic acid sequences that all encode substantially the same protein.
  • NCBI BLAST software suite may be used.
  • BLAST 2 Sequences tool Version 2.0.9 (May-07- 1999) with blastp set at default parameters.
  • Such default parameters may be. for example: Matrix: BLOSUM62 Open Gap: 11 and Extension Gap: 1 penalties
  • Percent identity may be measured over the length of an entire defined polypeptide sequence, for example, as defined by a particular SEQ ID number, or may be measured over a shorter length, for example, over the length of a fragment taken from a larger, defined polypeptide sequence, for instance, a fragment of at least 15. at least 20. at least 30. at least 40. at least 50. at least 70 or at least 150 contiguous residues.
  • Such lengths are exemplary only, and it is understood that any fragment length supported by the sequences shown herein, in the tables, figures or Sequence Listing, may be used to describe a length over which percentage identity may be measured.
  • HACs Human artificial chromosomes
  • HACs are linear microchromosomes which may contain DNA sequences of about 6 kb to 10 Mb in size, and which contain all ofthe elements required for stable mitotic chromosome segregation and maintenance.
  • humanized antibody refers to antibody molecules in which the amino acid sequence in the non-antigen binding regions has been altered so that the antibody more closely resembles a human antibody, and still retains its original binding ability.
  • Hybridization refers to the process by which a polynucleotide strand anneals with a complementary strand through base pairing under defined hybridization conditions. Specific hybridization is an indication that two nucleic acid sequences share a high degree of identity. Specific hybridization complexes form under permissive annealing conditions and remain hybridized after the "washing" step(s). The washing step(s) is particularly important in determining the stringency ofthe hybridization process, with more stringent conditions allowing less non-specific binding, i.e., binding between pairs of nucleic acid strands that are not perfectly matched.
  • Permissive conditions for annealing of nucleic acid sequences are routinely determinable by one of ordinary skill in the art and may be consistent among hybridization experiments, whereas wash conditions may be varied among experiments to achieve the desired stringency, and therefore hybridization specificity. Permissive annealing conditions occur, for example, at 68°C in the presence of about 6 x SSC, about 1 % (w/v) SDS, and about 100 ⁇ g/ml denatured salmon sperm DNA.
  • stringency of hybridization is expressed, in part, with reference to the temperature under which the wash step is carried out.
  • wash temperatures are selected to be about 5°C to 20°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • High stringency conditions for hybridization between polynucleotides ofthe present invention include wash conditions of 68°C in the presence of about 0.2 x SSC and about 0.1 % SDS. for 1 hour. Alternatively, temperatures of about 65°C, 60°C. 55°C. or 42°C may be used. SSC concentration may be varied from about 0. 1 to 2 x SSC. w ith SDS being present at about 0. 1 %.
  • blocking reagents are used to block non-specific hybridization. Such blocking reagents include, for instance. denatured salmon sperm DNA at about 100-200 ⁇ g ml.
  • Organic solvent such as formamide at a concentration of about 35-50% v/v
  • RNA:DNA hybridizations Useful variations on these wash conditions will be readily apparent to those of ordinary skill in the art.
  • Hybridization particularly under high stringency conditions, may be suggestive of evolutionary similarity between the nucleotides. Such similarity is strongly indicative of a similar role for the nucleotides and their encoded polypeptides.
  • hybridization complex refers to a complex formed between two nucleic acid sequences by virtue of the formation of hydrogen bonds between complementary bases.
  • a hybridization complex may be formed in solution (e.g., C 0 t or R o t analysis) or formed between one nucleic acid sequence present in solution and another nucleic acid sequence immobilized on a solid support (e.g., paper, membranes, filters, chips, pins or glass slides, or any other appropriate substrate to which cells or their nucleic acids have been fixed).
  • insertion and “addition” refer to changes in an amino acid or nucleotide sequence resulting in the addition of one or more amino acid residues or nucleotides. respectively.
  • Immuno response can refer to conditions associated with inflammation, trauma, immune disorders, or infectious or genetic disease, etc. These conditions can be characterized by expression of various factors, e.g., cytokines, chemokines, and other signaling molecules, w hich may affect cellular and systemic defense systems.
  • microarray refers to an arrangement of distinct polynucleotides on a substrate.
  • element and “array element” in a microarray context, refer to hybridizable polynucleotides arranged on the surface of a substrate.
  • modulate refers to a change in the activity of HCSRP. For example, modulation may cause an increase or a decrease in protein activity, binding characteristics, or any other biological, functional, or immunological properties of HCSRP.
  • nucleic acid and 'nucleic acid sequence refer to a nucleotide, oligonucleotide. polynucleotide. or any fragment thereof. These phrases also refer to DNA or RNA of genomic or synthetic origin w hich may be single-stranded or double-stranded and may represent the sense or the antisense strand, to peptide nucleic acid (PNA). or to any DNA-like or RNA-like material.
  • PNA peptide nucleic acid
  • operably linked refers to the situation in which a first nucleic acid sequence is placed in a functional relationship with the second nucleic acid sequence.
  • a promoter is operably linked to a coding sequence if the promoter affects the transcription or expression of the coding sequence.
  • operably linked DNA sequences may be in close proximity or contiguous and, where necessary to join two protein coding regions, in the same reading frame.
  • PNA protein nucleic acid
  • PNA refers to an antisense molecule or anti-gene agent which comprises an oligonucleotide of at least about 5 nucleotides in length linked to a peptide backbone of amino acid residues ending in lysine. The terminal lysine confers solubility to the composition. PNAs preferentially bind complementary single stranded DNA or RNA and stop transcript elongation, and may be pegylated to extend their lifespan in the cell.
  • Probe refers to nucleic acid sequences encoding HCSRP, their complements, or fragments thereof, which are used to detect identical, allelic or related nucleic acid sequences.
  • Probes are isolated oligonucleotides or polynucleotides attached to a detectable label or reporter molecule. Typical labels include radioactive isotopes, ligands. chemiluminescent agents, and enzymes.
  • Primary are short nucleic acids, usually DNA oligonucleotides. which may be annealed to a target polynucleotide by complementary base-pairing. The primer may then be extended along the target DNA strand by a DNA polymerase enzyme. Primer pairs can be used for amplification (and identification) of a nucleic acid sequence, e.g., by the polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • Probes and primers as used in the present invention typically comprise at least 15 contiguous nucleotides of a known sequence. In order to enhance specificity, longer probes and primers may also be employed, such as probes and primers that comprise at least 20, 25, 30, 40, 50, 60, 70, 80, 90, 100, or at least 150 consecutive nucleotides ofthe disclosed nucleic acid sequences. Probes and primers may be considerably longer than these examples, and it is understood that any length supported by the specification, including the tables, figures, and Sequence Listing, may be used.
  • PCR primer pairs can be derived from a known sequence, for example, by using computer programs intended for that purpose such as Primer (Version 0.5, 1991 , Whitehead Institute for Biomedical Research. Cambridge MA).
  • Oligonucleotides for use as primers are selected using software known in the art for such pu ⁇ ose. For example, OLIGO 4.06 software is useful for the selection of PCR primer pairs of up to 100 nucleotides each, and for the analysis of oligonucleotides and larger polynucleotides of up to 5,000 nucleotides from an input polynucleotide sequence of up to 32 kilobases. Similar primer selection programs have inco ⁇ orated additional features for expanded capabilities. For example, the PrimOU primer selection program (available to the public from the Genome Center at University' of Texas South West Medical Center. Dallas TX) is capable of choosing specific primers from megabase sequences and is thus useful for designing primers on a genome-wide scope.
  • the Primer3 primer selection program (available to the public from the Whitehead Institute/MIT Center for Genome Research. Cambridge MA) allows the user to input a "mispriming library. " in which sequences to avoid as primer binding sites are user-specified. Primer3 is useful, in particular, for the selection of oligonucleotides for microarrays. (The source code for the latter two primer selection programs may also be obtained from their respective sources and modified to meet the user ' s specific needs.)
  • the PrimeGen program (available to the public from the UK Human Genome Mapping Project Resource Centre, Cambridge UK) designs primers based on multiple sequence alignments, thereby allowing selection of primers that hybridize to either the most conserved or least conserved regions of aligned nucleic acid sequences.
  • this program is useful for identification of both unique and conserved oligonucleotides and polynucleotide fragments.
  • the oligonucleotides and polynucleotide fragments identified by any of the above selection methods are useful in hybridization technologies, for example, as PCR or sequencing primers, microarray elements, or specific probes to identify fully or partially complementary polynucleotides in a sample of nucleic acids. Methods of oligonucleotide selection are not limited to those described above.
  • a "recombinant nucleic acid” is a sequence that is not naturally occurring or has a sequence that is made by an artificial combination of two or more otherwise separated segments of sequence. This artificial combination is often accomplished by chemical synthesis or, more commonly, by the artificial manipulation of isolated segments of nucleic acids, e.g., by genetic engineering techniques such as those described in Sambrook. supra.
  • the term recombinant includes nucleic acids that have been altered solely by addition, substitution, or deletion of a portion of the nucleic acid.
  • a recombinant nucleic acid may include a nucleic acid sequence operably linked to a promoter sequence. Such a recombinant nucleic acid may be part of a vector that is used, for example, to transform a cell.
  • such recombinant nucleic acids may be part of a viral vector, e.g.. based on a vaccinia virus, that could be use to vaccinate a mammal wherein the recombinant nucleic acid is expressed, inducing a protective immunological response in the mammal.
  • a sample suspected of containing nucleic acids encoding HCSRP, or fragments thereof, or HCSRP itself may comprise a bodily fluid: an extract from a cell, chromosome, organelle. or membrane isolated from a cell; a cell; genomic DNA. RNA, or cDNA, in solution or bound to a substrate; a tissue; a tissue print: etc.
  • 'specific binding and “specifically binding” refer to that interaction between a protein or peptide and an agonist, an antibody, an antagonist, a small molecule, or any natural or synthetic binding composition. The interaction is dependent upon the presence of a particular structure ofthe protein, e.g.. the antigenic determinant or epitope. recognized by the binding molecule. For example, if an antibody is specific for epitope "A.” the presence of a polypeptide containing the epitope A. or the presence of free unlabeled A, in a reaction containing free labeled A and the antibody will reduce the amount of labeled A that binds to the antibody.
  • substantially purified refers to nucleic acid or amino acid sequences that are removed from their natural environment and are isolated or separated, and are at least about 60% free, preferably about 75% free, and most preferably about 90% free from other components with which they are naturally associated.
  • substitution refers to the replacement of one or more amino acids or nucleotides by different amino acids or nucleotides. respectively.
  • Substrate refers to any suitable rigid or semi-rigid support including membranes, filters, chips, slides, wafers, fibers, magnetic or nonmagnetic beads, gels, tubing, plates, polymers, microparticles and capillaries.
  • the substrate can have a variety of surface forms, such as wells, trenches, pins, channels and pores, to which polynucleotides or polypeptides are bound.
  • Transformation describes a process by which exogenous DNA enters and changes a recipient cell. Transformation may occur under natural or artificial conditions according to various methods well known in the art, and may rely on any known method for the insertion of foreign nucleic acid sequences into a prokaryotic or eukaryotic host cell.
  • the method for transformation is selected based on the type of host cell being transformed and may include, but is not limited to. viral infection, electroporation. heat shock, lipofection, and particle bombardment.
  • the term “transformed " cells includes stably transformed cells in which the inserted DNA is capable of replication either as an autonomously replicating plasmid or as part of the host chromosome, as well as transiently transformed cells which express the inserted DNA or RNA for limited periods of time.
  • a “variant" of a particular nucleic acid sequence is defined as a nucleic acid sequence having at least 40% sequence identity to the particular nucleic acid sequence over a certain length of one of the nucleic acid sequences using blastn with the "BLAST 2 Sequences " tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of nucleic acids may show, for example, at least 50%, at least 60%, at least 70%, at least 80%. at least 85%, at least 90%, at least 95% or at least 98% or greater sequence identity over a certain defined length.
  • a variant may be described as. for example, an "allelic " (as defined above), “splice, " "species. “ or “polymo ⁇ hic " variant.
  • a splice variant may have significant identity to a reference molecule, but will generally have a greater or lesser number of polynucleotides due to alternate splicing of exons during mRNA processing.
  • the corresponding polypeptide may possess additional functional domains or lack domains that are present in the reference molecule.
  • Species variants are polynucleotide sequences that vary from one species to another. The resulting polypeptides generally will have significant amino acid identity relative to each other.
  • a polymorphic variant is a variation in the polynucleotide sequence of a particular gene between individuals of a given species.
  • Polymorphic variants also may encompass ""single nucleotide polymorphisms " (SNPs) in which the polynucleotide sequence varies by one nucleotide base.
  • SNPs single nucleotide polymorphisms
  • the presence of SNPs may be indicative of, for example, a certain population, a disease state, or a propensity for a disease state.
  • a "variant" of a particular polypeptide sequence is defined as a polypeptide sequence having at least 40% sequence identity to the particular polypeptide sequence over a certain length of one of the polypeptide sequences using blastp with the "BLAST 2 Sequences" tool Version 2.0.9 (May-07- 1999) set at default parameters.
  • Such a pair of polypeptides may show, for example, at least 50%. at least 60%, at least 70%, at least 80%. at least 90%, at least 95%. or at least 98% or greater sequence identity over a certain defined length of one of the polypeptides.
  • the invention is based on the discovery of new human cell surface receptor proteins (HCSRP), the polynucleotides encoding HCSRP. and the use of these compositions for the diagnosis, treatment, or prevention of cell proliferative disorders, immune system disorders, infections, and neuronal disorders.
  • HCSRP human cell surface receptor proteins
  • Table 1 lists the Incyte clones used to assemble full length nucleotide sequences encoding HCSRP. Columns 1 and 2 show the sequence identification numbers (SEQ ID NOs) of the polypeptide and nucleotide sequences, respectively. Column 3 shows the clone IDs of the Incyte clones in which nucleic acids encoding each HCSRP were identified, and column 4 shows the cDNA libraries from which these clones were isolated. Column 5 shows Incyte clones and their corresponding cDNA libraries. Clones for which cDNA libraries are not indicated were derived from pooled cDNA libraries. The Incyte clones in column 5 were used to assemble the consensus nucleotide sequence of each HCSRP and are useful as fragments in hybridization technologies.
  • SEQ ID NOs sequence identification numbers
  • column 1 references the SEQ ID NO: column 2 shows the number of amino acid residues in each polypeptide; column 3 shows potential phosphorylation sites: column 4 shows potential glycosylation sites; column 5 shows the amino acid residues comprising signature sequences and motifs: column 6 shows homologous sequences as identified by BLAST analysis: and column 7 shows analytical methods used to characterize each polypeptide through sequence homoiogy and protein motifs.
  • the amino acid sequence of SEQ ID NO: l from about amino acid residue 30 to about 81 is distinct from the tethered ligand thrombin receptor agonist peptide of the N-terminus of the human thrombin receptor and the amino acid sequence of SEQ ID NO:2 from about amino acid residue 1 15 to about 140 is distinct from the C-terminus joining and constant regions of the human TCR ⁇ subunit.
  • the columns of Table 3 show the tissue-specificity and diseases, disorders, or conditions associated with nucleotide sequences encoding HCSRP. The first column of Table 3 lists the nucleotide SEQ ID NOs. Column 2 lists fragments of the nucleotide sequences of column 1.
  • fragments are useful, for example, in hybridization or amplification technologies to identify SEQ ID NO: 14-26 and to distinguish between SEQ ID NO: 14-26 and related polynucleotide sequences.
  • the polypeptides encoded by these fragments are useful, for example, as immunogenic peptides.
  • Column 3 lists tissue categories which express HCSRP as a fraction of total tissues expressing HCSRP.
  • Column 4 lists diseases, disorders, or conditions associated with those tissues expressing HCSRP as a fraction of total tissues expressing HCSRP.
  • Column 5 lists the vectors used to subclone each cDNA library.
  • HCSRP HCSRP in cancer, autoimmune and inflammatory response, and in lung, thymus, bladder, seminal vesicle, and penile tissues, and in rheumatoid arthritis.
  • SEQ ID NO: 14 is expressed primarily in tumor-associated epithelial tissues and SEQ ID NO: 15 is expressed primarily in growth- and tumor-associated epithelial tissues and in immune response tissues.
  • Table 4 show descriptions ofthe tissues used to construct the cDNA libraries from which cDNA clones encoding HCSRP were isolated.
  • Column 1 references the nucleotide SEQ ID NOs
  • column 2 shows the cDNA libraries from which these clones were isolated
  • column 3 shows the tissue origins and other descriptive information relevant to the cDNA libraries in column 2.
  • the invention also encompasses HCSRP variants.
  • a preferred HCSRP variant is one which has at least about 80%, or alternatively at least about 90%. or even at least about 95% amino acid sequence identity to the HCSRP amino acid sequence, and which contains at least one functional or structural characteristic of HCSRP.
  • the invention also encompasses polynucleotides which encode HCSRP.
  • the invention encompasses a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 14-26. which encodes HCSRP.
  • the invention also encompasses a variant of a polynucleotide sequence encoding HCSRP.
  • such a variant polynucleotide sequence will have at least about 80%. or alternatively at least about 90%. or even at least about 95% polynucleotide sequence identity to the polynucleotide sequence encoding HCSRP.
  • a particular aspect ofthe invention encompasses a variant of a polynucleotide sequence comprising a sequence selected from the group consisting of SEQ ID NO: 14-26 which has at least about 80%. or alternatively at least about 90%, or even at least about 95% polynucleotide sequence identity to a nucleic acid sequence selected from the group consisting of SEQ ID NO: 14-26.
  • Any one of the polynucleotide variants described above can encode an amino acid sequence which contains at least one functional or structural characteristic of HCSRP. It will be appreciated by those skilled in the art that as a result ofthe degeneracy ofthe genetic code, a multitude of polynucleotide sequences encoding HCSRP.
  • the invention contemplates each and every possible variation of polynucleotide sequence that could be made by selecting combinations based on possible codon choices. These combinations are made in accordance with the standard triplet genetic code as applied to the polynucleotide sequence of naturally occurring HCSRP, and all such variations are to be considered as being specifically disclosed.
  • nucleotide sequences which encode HCSRP and its variants are generally capable of hybridizing to the nucleotide sequence of the naturally occurring HCSRP under appropriately selected conditions of stringency, it may be advantageous to produce nucleotide sequences encoding HCSRP or its derivatives possessing a substantially different codon usage, e.g., inclusion of non- naturally occurring codons. Codons may be selected to increase the rate at which expression of the peptide occurs in a particular prokaryotic or eukaryotic host in accordance with the frequency with which particular codons are utilized by the host.
  • RNA transcripts having more desirable properties such as a greater half-life, than transcripts produced from the naturally occurring sequence.
  • the invention also encompasses production of DNA sequences which encode HCSRP and HCSRP derivatives, or fragments thereof, entirely by synthetic chemistry.
  • the synthetic sequence may be inserted into any of the many available expression vectors and cell systems using reagents well known in the art.
  • synthetic chemistry' may be used to introduce mutations into a sequence encoding HCSRP or any fragment thereof.
  • polynucleotide sequences that are capable of hybridizing to the claimed polynucleotide sequences, and. in particular, to those shown in SEQ ID NO: 14-26 and fragments thereof under various conditions of stringency.
  • Hybridization conditions including annealing and wash conditions, are described in "Definitions. "
  • sequence preparation is automated with machines such as the MICROLAB 2200 liquid transfer system (Hamilton, Reno NV), PTC200 thermal cycler (MJ Research.
  • the nucleic acid sequences encoding HCSRP may be extended utilizing a partial nucleotide sequence and employing various PCR-based methods known in the art to detect upstream sequences, such as promoters and regulatory elements
  • restriction-site PCR uses universal and nested primers to amplify unknown sequence from genomic DNA within a cloning vector (See, e g , Sarkar, G (1993) PCR Methods Applic 2 318-322 )
  • Another method, inverse PCR uses primers that extend in divergent directions to amplify unknown sequence from a circularized template
  • the template is derived from restriction fragments comprising a known genomic locus and surrounding sequences (See, e g , T ⁇ glia, T et al ( 1988) Nucleic Acids Res 16 8186 )
  • primers may be designed using commercially available software, such as OLIGO 4 06 Primer Analysis software (National Biosciences.
  • Plv mouth MN or another appropriate program to be about 22 to 30 nucleotides in length, to have a GC content of about 50% or more, and to anneal to the template at temperatures of about 68°C to 72°C
  • libraries that hav e been size-selected to include larger cDNAs
  • random-primed libraries which often include sequences containing the 5' regions of genes, are preferable for situations in w hich an oligo d(T) library does not yield a full-length cDNA Genomic libraries may be useful for extension of sequence into 5' non-transcribed regulatory regions
  • Capillary electrophoresis systems which are commercially available mav be used to analyze the size or confirm the nucleotide sequence of sequencing or PCR products
  • capillary sequencing may employ flowable polymers for electrophoretic separation, four different nucleotide- specific.
  • laser-stimulated fluorescent dy es, and a charge coupled device camera for detection of the emitted wavelengths
  • Output/light intensity may be converted to electrical signal using appropriate software (e g . GENOTYPER and SEQUENCE NAVIGATOR, Perkin-Elmer).
  • nucleotide sequences ofthe present invention can be engineered using methods generally known in the art in order to alter HCSRP-encoding sequences for a variety of pu ⁇ oses including, but not limited to.
  • oligonucleotide-mediated site-directed mutagenesis may be used to introduce mutations that create new restriction sites, alter glycosy lation patterns, change codon preference, produce splice variants, and so forth
  • sequences encoding HCSRP may be synthesized, in whole or in part, using chemical methods well known in the art (See. e g . Caruthers, M H et al ( 1980) Nucleic Acids Symp Ser 7 215-223. and Horn. T et al ( 1980) Nucleic Acids S>mp Ser 7 225-232 )
  • HCSRP itself or a fragment thereof may be synthesized using chemical methods
  • peptide synthesis can be performed using various solid-phase techniques (See.
  • .__ proteins, or any part thereof, to produce a variant polypeptide.
  • the peptide may be substantially purified by preparative high performance liquid chromatography. (See. e.g.. Chiez, R.M. and F.Z. Regnier ( 1990) Methods Enzymol. 182:392-421.)
  • the composition of the synthetic peptides may be confirmed by amino acid analysis or by sequencing. (See, e.g., Creighton. T. ( 1984) Proteins. Structures and Molecular Properties. WH Freeman, New York NY.)
  • the nucleotide sequences encoding HCSRP or derivatives thereof may be inserted into an appropriate expression vector, i.e., a vector which contains the necessary elements for transcriptional and translational control of the inserted coding sequence in a suitable host.
  • these elements include regulatory sequences, such as enhancers, constitutive and inducible promoters, and 5' and 3' untranslated regions in the vector and in polynucleotide sequences encoding HCSRP. Such elements may vary in their strength and specificity.
  • Specific initiation signals may also be used to achieve more efficient translation of sequences encoding HCSRP. Such signals include the ATG initiation codon and adjacent sequences, e.g. the Kozak sequence.
  • microorganisms such as bacteria transformed with recombinant bacteriophage.
  • plasmid. or cosmid DNA expression vectors yeast transformed with yeast expression vectors: insect cell systems infected with viral expression vectors (e.g.. baculovirus); plant cell systems transformed with viral expression vectors (e.g., cauliflower mosaic virus. CaMV, or tobacco mosaic virus. TMV) or with bacterial expression vectors (e.g.. Ti or pBR322 plasmids): or animal cell systems.
  • the invention is not limited by the host cell employed.
  • cloning and expression vectors may be selected depending upon the use intended for polynucleotide sequences encoding HCSRP.
  • routine cloning, subcloning, and propagation of polynucleotide sequences encoding HCSRP can be achieved using a multifunctional E. coli vector such as PBLUESCRIPT (Stratagene. La Jolla CA) or PSPORT1 plasmid (Life Technologies). Ligation of sequences encoding HCSRP into the vector ' s multiple cloning site disrupts the lac ⁇ gene, allowing a colorimetric screening procedure for identification of transformed bacteria containing recombinant molecules.
  • these vectors may be useful for in vitro transcription, dideoxy sequencing, single strand rescue with helper phage, and creation of nested deletions in the cloned sequence.
  • vectors which direct high level expression of HCSRP may be used.
  • vectors containing the strong, inducible T5 or T7 bacteriophage promoter may be used.
  • Yeast expression systems may be used for production of HCSRP.
  • a number of vectors containing constitutive or inducible promoters may be used in the yeast Saccharomvces cerevisiae or Pichia pastoris.
  • constitutive or inducible promoters such as alpha factor, alcohol oxidase, and PGH promoters
  • such vectors direct either the secretion or intracellular retention of expressed proteins and enable integration of foreign sequences into the host genome for stable propagation.
  • Plant systems may also be used for expression of HCSRP. Transcription of sequences encoding HCSRP may be driven viral promoters, e.g., the 35S and 19S promoters of CaMV used alone or in combination with the omega leader sequence from TMV (Takamatsu, N. ( 1987) EMBO J. 3:17-31 1 ). Alternatively, plant promoters such as the small subunit of RUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. et al. ( 1984) EMBO J. 3: 1671- 1680; Broglie. R. et al. ( 1984) Science 224:838-843; and Winter, J. et al.
  • a number of viral-based expression systems may be utilized.
  • sequences encoding HCSRP may be ligated into an adenovirus transcription/translation complex consisting ofthe late promoter and tripartite leader sequence. Insertion in a non-essential E l or E3 region of the viral genome may be used to obtain infective virus which expresses HCSRP in host cells.
  • transcription enhancers such as the Rous sarcoma virus (RSV) enhancer, may be used to increase expression in mammalian host cells.
  • SV40 or EBV- based vectors may also be used for high-level protein expression.
  • Human artificial chromosomes (HACs) may also be employed to deliver larger fragments of
  • HACs DNA than can be contained in and expressed from a plasmid.
  • HACs of about 6 kb to 10 Mb are constructed and delivered via conventional delivery methods (liposomes. polycationic amino polymers, or vesicles) for therapeutic purposes.
  • liposomes. polycationic amino polymers, or vesicles for therapeutic purposes.
  • HCSRP in cell lines is preferred.
  • sequences encoding HCSRP can be transformed into cell lines using expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • expression vectors which may contain viral origins of replication and/or endogenous expression elements and a selectable marker gene on the same or on a separate vector.
  • cells may be allowed to grow for about I to 2 days in enriched media before being switched to selective media.
  • the pu ⁇ ose ofthe selectable marker is to confer resistance to a selective agent, and its presence allows growth and recovery of cells which successfully express the introduced sequences.
  • Resistant clones of stably transformed cells may be propagated using tissue culture techniques appropriate to the cell type.
  • any number of selection systems may be used to recover transformed cell lines. These include, but are not limited to, the herpes simplex virus thymidine kinase and adenine phosphoribosyltransferase genes, for use in tkr and apr ⁇ cells, respectively. (See. e.g., Wigler, M. et al. ( 1977) Cell 1 1 :223-232; Lowy, I. et al. ( 1980) Cell 22:81 7-823.) Also, antimetabolite. antibiotic, or herbicide resistance can be used as the basis for selection.
  • dhfr confers resistance to methotrexate
  • neo confers resistance to the aminoglycosides neomycin and G-41 8:
  • als and pat confer resistance to chlorsulfuron and phosphinotricin acetyltransferase, respectively.
  • ⁇ glucuronidase and its substrate ⁇ -glucuronide or luciferase and its substrate luciferin may be used. These markers can be used not only to identify transformants. but also to quantify the amount of transient or stable protein expression attributable to a specific vector system. (See, e.g.. Rhodes. CA. ( 1995) Methods Mol. Biol. 55: 121 - 13 1 .)
  • marker gene expression suggests that the gene of interest is also present, the presence and e.xpression of the gene may need to be confirmed.
  • sequence encoding HCSRP is inserted within a marker gene sequence
  • transformed cells containing sequences encoding HCSRP can be identified by the absence of marker gene function.
  • a marker gene can be placed in tandem with a sequence encoding HCSRP under the control of a single promoter. Expression ofthe marker gene in response to induction or selection usually indicates expression of the tandem gene as well.
  • host cells that contain the nucleic acid sequence encoding HCSRP and that express HCSRP may be identified by a variety of procedures known to those of skill in the art. These procedures include, but are not limited to. DNA-DNA or DNA-RNA hybridizations. PCR amplification, and protein bioassay or immunoassay techniques which include membrane, solution, or chip based technologies for the detection and/or quantification of nucleic acid or protein sequences. Immunological methods for detecting and measuring the expression of HCSRP using either specific polyclonal or monoclonal antibodies are known in the art. Examples of such techniques include enzyme-linked immunosorbent assays (ELISAs), radioimmunoassays (RIAs), and fluorescence activated cell sorting (FACS).
  • ELISAs enzyme-linked immunosorbent assays
  • RIAs radioimmunoassays
  • FACS fluorescence activated cell sorting
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering epitopes on HCSRP is preferred, but a competitive binding assay may be employed.
  • assays are well known in the art. (See, e.g., Hampton, R. et al. ( 1990) Serological Methods, a Laboratory Manual, APS Press, St. Paul MN, Sect. IV: Coligan, J.E. et al. ( 1997) Current Protocols in Immunology, Greene Pub. Associates and Wiley-Interscience, New York NY: and Pound, J.D. (1998) Immunochemical Protocols, Humana Press, Totowa NJ.)
  • Means for producing labeled hybridization or PCR probes for detecting sequences related to polynucleotides encoding HCSRP include oligolabeling, nick translation, end-labeling, or PCR amplification using a labeled nucleotide.
  • the sequences encoding HCSRP. or any fragments thereof may be cloned into a vector for the production of an mRNA probe.
  • RNA polymerase such as T7, T3, or SP6 and labeled nucleotides.
  • T7, T3, or SP6 RNA polymerase
  • reporter molecules or labels which may be used for ease of detection include radionuclides. enzymes, fluorescent, chemiluminescent, or chromogenic agents, as well as substrates, cofactors. inhibitors, magnetic particles, and the like.
  • Host cells transformed with nucleotide sequences encoding HCSRP may be cultured under conditions suitable for the expression and recovery' of the protein from cell culture.
  • the protein produced by a transformed cell may be secreted or retained intracellularly depending on the sequence and/or the vector used.
  • e.xpression vectors containing polynucleotides which encode HCSRP may be designed to contain signal sequences which direct secretion of HCSRP through a prokaryotic or eukaryotic cell membrane.
  • a host cell strain may be chosen for its ability to modulate expression of the inserted sequences or to process the expressed protein in the desired fashion.
  • modifications of the polypeptide include, but are not limited to. acetylation. carboxylation, glycosylation, phosphorylation, lipidation, and acylation.
  • Post-translational processing which cleaves a " prepro " or "pro” form of the protein may also be used to specify protein targeting, folding, and/or activity.
  • Different host cells which have specific cellular machinery and characteristic mechanisms for post-translational activities (e.g., CHO, HeLa. MDCK, HEK293.
  • HCSRP American Type Culture Collection
  • ATCC Manassas VA
  • WI38 World Health Organization
  • ATCC Manassas VA
  • natural, modified, or recombinant nucleic acid sequences encoding HCSRP may be ligated to a heterologous sequence resulting in translation of a fusion protein in any of the aforementioned host systems.
  • a chimeric HCSRP protein containing a heterologous moiety that can be recognized by a commercially available antibody may facilitate the screening of peptide libraries for inhibitors of HCSRP activity.
  • Heterologous protein and peptide moieties may also facilitate purification of fusion proteins using commercially available affinity matrices.
  • Such moieties include, but are not limited to. glutathione S-transferase (GST), maltose binding protein (MBP). thioredoxin (Trx). calmodulin binding peptide (CBP), 6-His. FLAG, c- yc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and 6-His enable purification of their cognate fusion proteins on immobilized glutathione. maltose, phenylarsine oxide, calmodulin. and metal- chelate resins, respectively. FLAG, c- yc.
  • fusion protein may also be engineered to contain a proteolytic cleavage site located between the HCSRP encoding sequence and the heterologous protein sequence, so that HCSRP may be cleaved away from the heterologous moiety following purification. Methods for fusion protein expression and purification are discussed in Ausubel ( 1995. supra, ch. 10). A variety of commercially available kits may also be used to facilitate expression and purification of fusion proteins.
  • synthesis of radiolabeled HCSRP may be achieved in vitro using the TNT rabbit reticulocyte lysate or wheat germ extract system (Promega). These systems couple transcription and translation of protein-coding sequences operablv associated w ith the T7, T3. or SP6 promoters Translation takes place in the presence of a radiolabeled ammo acid precursor, for example 5 S-meth ⁇ on ⁇ ne
  • HCSRP fragments of HCSRP may be produced not only by recombinant means, but also bv direct peptide synthesis using solid-phase techniques (See, e g . Creighton. supra, pp 55-60 ) Protein synthesis may be performed by manual techniques or by automation Automated synthesis may be achieved, for example, using the ABI 43 1 A peptide synthesizer (Perkin-Elmer) Various fragments of HCSRP may be synthesized separately and then combined to produce the full length molecule THERAPEUTICS Chemical and structural similarity, e g . in the context of sequences and motifs, exists between regions of HCSRP and human cell surface receptor proteins In addition, the expression of HCSRP is closely associated with lung, thymus.
  • HCSRP appears to play a role in cell proliferative disorders, immune system disorders, infections, and neuronal disorders In the treatment of disorders associated with increased HCSRP expression or activity, it is desirable to decrease the expression or activity of HCSRP In the treatment of disorders associated with decreased HCSRP expression or activity, it is desirable to increase the expression or activity of HCSRP
  • HCSRP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCSRP
  • disorders include, but are not limited to, a cell proliferative disorder such as actinic keratosis. arteriosclerosis, atherosclerosis, bursitis. cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myeiofibrosis, paroxysmal nocturnal hemoglobinu ⁇ a. polycythemia vera, psoriasis, primary thrombocythemia, and cancers including adenocarcinoma, leukemia, lymphoma.
  • a cell proliferative disorder such as actinic keratosis. arteriosclerosis, atherosclerosis, bursitis. cirrhosis, hepatitis, mixed connective tissue disease (MCTD), myeiofibrosis, paroxysmal nocturnal hemoglobinu ⁇ a.
  • melanoma myeloma, sarcoma, teratocarcinoma. and, in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia.
  • AIDS acquired immunodeficiency syndrome
  • Addison s disease adult respiratory distress syndrome, allergies, ankylosing spondyiitis, amyloidosis, anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thvroiditis, bronchitis, bursitis. cholecystitis, cirrhosis, contact dermatitis. Crohn's disease, atopic dermatitis, dermatomyositis. diabetes melhtus. emphysema, erythroblastosis fetalis. erythema nodosum. atrophic gastritis, glomeruloneph ⁇ tis. Goodpasture s syndrome, gout. Graves disease.
  • Hashimoto s thvroiditis paroxysmal nocturnal hemoglobinu ⁇ a. hepatitis, hypereosinophi a, irritable bowel syndrome, episodic Iv mphopenia with lv mphocv toto ⁇ ns. mixed connectiv e tissue disease (MCTD). multiple sclerosis, myasthema gravis. mvocardial or pe ⁇ cardial inflammation, myelofibrosis. osteoarthritis, osteoporosis, pancreatitis, poiycvthemia vera, polymyositis. psoriasis. Reiter s syndrome, rheumatoid arthritis, scleroderma.
  • filovirus hepadnavirus, he ⁇ esvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus. paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus. rhabdovirus. or togavirus, an infection caused by a bacterial agent classified as pneumococcus. staphylococcus. streptococcus, bacillus, corynebacterium, clost ⁇ dium. meningococcus. gonococcus. liste ⁇ a, moraxella. kingella, haemophilus, legionella, bordetella, gram-negative enterobacte ⁇ um including shigella.
  • trypanosoma toxoplasma, pneumocystis ca ⁇ nii, intestinal protozoa such as giardia, t ⁇ chomonas, tissue nematodes such as trichinella.
  • intestinal nematodes such as ascaris, lymphatic fila ⁇ al nematodes, trematodes such as schistosoma. or cestrodes such as tapeworm, and a neuronal disorder such as akathesia.
  • Alzheimer ' s disease amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia, cerebral neoplasms, dementia, depression, diabetic neuropathy, Down s syndrome, tardive dyskmesia, dystonias, epilepsy, Huntmgton ' s disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis.
  • Parkinson ' s disease paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder
  • a vector capable of expressing HCSRP or a fragment or derivative thereof may be administered to a subject to treat or prevent a disorder associated with decreased expression or activ ity of HCSRP including, but not limited to. those described above
  • a pharmaceutical composition comprising a substantially purified
  • HCSRP in conjunction with a suitable pharmaceutical carrier may be administered to a subject to treat or prevent a disorder associated with decreased expression or activity of HCSRP including, but not limited to. those provided above
  • an agonist which modulates the activity of HCSRP may be administered to a subject to treat or prevent a disorder associated with decreased e.xpression or activity of HCSRP including, but not limited to. those listed above.
  • an antagonist of HCSRP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HCSRP.
  • disorders include, but are not limited to. those cell proliferative disorders, immune system disorders, infections, and neuronal disorders described above.
  • an antibody which specifically binds HCSRP may be used directly as an antagonist or indirectly as a targeting or delivery mechanism for bringing a pharmaceutical agent to ceils or tissues which express HCSRP.
  • a vector expressing the complement ofthe polynucleotide encoding HCSRP may be administered to a subject to treat or prevent a disorder associated with increased expression or activity of HCSRP including, but not limited to, those described above.
  • any of the proteins, antagonists, antibodies, agonists, complementary sequences, or vectors of the invention may be administered in combination with other appropriate therapeutic agents. Selection of the appropriate agents for use in combination therapy may be made by one of ordinary skill in the art. according to conventional pharmaceutical principles.
  • the combination of therapeutic agents may act synergistically to effect the treatment or prevention o the various disorders described above. Using this approach, one may be able to achieve therapeutic efficacy with lower dosages of each agent, thus reducing the potential for adverse side effects.
  • HCSRP An antagonist of HCSRP may be produced using methods which are generally known in the art.
  • purified HCSRP may be used to produce antibodies or to screen libraries of pharmaceutical agents to identify those which specifically bind HCSRP.
  • Antibodies to HCSRP may also be generated using methods that are well known in the art. Such antibodies may include, but are not limited to. polyclonal. monoclonal, chimeric. and single chain antibodies, Fab fragments, and fragments produced by a Fab expression library. Neutralizing antibodies (i.e., those which inhibit dimer formation) are generally preferred for therapeutic use.
  • various hosts including goats, rabbits, rats, mice, humans, and others may be immunized by injection with HCSRP or with any fragment or oligopeptide thereof yvhich has immunogenic properties.
  • various adjuvants may be used to increase immunological response.
  • adjuvants include, but are not limited to. Freund's. mineral gels such as aluminum hydroxide, and surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides. oil emulsions. KLH, and dinitrophenol.
  • BCG Bacilli Calmette-Guerin
  • Corvnebacterium parvum are especially preferable.
  • the oligopeptides. peptides. or fragments used to induce antibodies to HCSRP have an amino acid sequence consisting of at least about 5 amino acids, and generally will consist of at least about 10 amino acids. It is also preferable that these oligopeptides. peptides. or fragments are identical to a portion of the amino acid sequence of the natural protein and contain the entire amino acid sequence of a small, naturally occurring molecule. Short stretches of HCSRP amino acids may be fused with those of another protein, such as KLH. and antibodies to the chimeric molecule may be produced.
  • Monoclonal antibodies to HCSRP may be prepared using any technique which provides for the production of antibody molecules by continuous cell lines in culture. These include, but are not limited to, the hybridoma technique, the human B-cell hybridoma technique, and the EBV-hybridoma technique.
  • the hybridoma technique the human B-cell hybridoma technique
  • EBV-hybridoma technique See, e.g.. Kohler, G. et al. ( 1975) Nature 256:495-497: Kozbor, D. et al. ( 1985) J. Immunol. Methods 81 :31-42; Cote, R.J. et al. (1983) Proc. Natl. Acad. Sci. USA 80:2026-2030; and Cole, S.P. et al. ( 1984) Mol. Cell Biol. 62: 109-120.)
  • Antibodies with related specificity, but of distinct idiotypic composition may be generated by chain shuffling from random combinatorial immunoglobulin libraries. (See, e.g., Burton, D.R. (1991 ) Proc. Natl. Acad. Sci. USA 88: 10134-10137.)
  • Antibodies may also be produced by inducing in vivo production in the lymphocyte population or by screening immunoglobulin libraries or panels of highly specific binding reagents as disclosed in the literature. (See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. USA 86:3833-3837; Winter, G. et al. ( 1991 ) Nature 349:293-299.) Antibody fragments which contain specific binding sites for HCSRP may also be generated.
  • fragments include, but are not limited to, F(ab') 2 fragments produced by pepsin digestion of the antibody molecule and Fab fragments generated by reducing the disulfide bridges of the F(ab')2 fragments.
  • Fab expression libraries may be constructed to allow rapid and easy identification of monoclonal Fab fragments with the desired specificity. (See. e.g., Huse. W.D. et al. ( 1989) Science 246: 1275-1281.)
  • immunoassays may be used for screening to identify antibodies having the desired specificity.
  • Numerous protocols for competitive binding or immunoradiometric assays using either polyclonal or monoclonal antibodies with established specificities are well known in the art.
  • Such immunoassays typically involve the measurement of complex formation between HCSRP and its specific antibody
  • a two-site, monoclonal-based immunoassay utilizing monoclonal antibodies reactive to two non-interfering HCSRP epitopes is generally used, but a competitive binding assay may also be employed (Pound, supra)
  • K ⁇ which is defined as the molar concentration of HCSRP-antibody complex divided by the molar concentrations of free antigen and free antibody under equilibrium conditions
  • the K a determined for a preparation of monoclonal antibodies, which are monospeciflc for a particular HCSRP epitope represents a true measure of affinity High-affinity antibody preparations with K a ranging from about 10 9 to 10 12 L/mole are preferred for use in immunoassays in which the HCSRP-antibody complex must withstand rigorous manipulations Low-affinity antibody preparations with K a ranging from about !
  • polyclonal antibody preparations may be further evaluated to determine the quality and suitability of such preparations for certain downstream applications
  • a polyclonal antibody preparation containing at least 1 -2 mg specific antibody/mi, preferably 5- 10 mg specific antibody/ml is generally employed in procedures requiring precipitation of HCSRP- antibody complexes
  • Procedures for evaluating antibody specificity, titer. and avidity, and guidelines for antibody quality and usage in various applications, are generally available (See, e g , Catty, supra, and Coligan et al supra )
  • the polynucleotides encoding HCSRP may be used for therapeutic pu ⁇ oses
  • the complement ofthe polynucleotide encoding HCSRP may be used in situations in which it would be desirable to block the transcription of the mRNA
  • cells may be transformed with sequences complementary to polynucleotides encoding HCSRP
  • complementary molecules or fragments may be used to modulate HCSRP activ ity. or to achieve regulation of gene function
  • sense or antisense oligonucleotides or larger fragments can be designed from various locations along the coding or control regions of sequences encoding HCSRP Expression vectors derived from retroviruses.
  • adenoviruses. or he ⁇ es or vaccinia viruses, or from various bacterial plasmids. may be used for delivery of nucleotide sequences to the targeted organ, tissue, or cell population. Methods which are well known to those skilled in the art can be used to construct vectors to express nucleic acid sequences complementary to the polynucleotides encoding HCSRP. (See, e.g.. Sambrook. supra: Ausubel. 1995. supra.)
  • Genes encoding HCSRP can be turned off by transforming a cell or tissue with expression vectors which express high levels of a polynucleotide. or fragment thereof, encoding HCSRP. Such constructs may be used to introduce untranslatable sense or antisense sequences into a cell. Even in the absence of integration into the DNA. such vectors may continue to transcribe RNA molecules until they are disabled by endogenous nucleases. Transient expression may last for a month or more with a non-replicating vector, and may last even longer if appropriate replication elements are part of the vector system.
  • modifications of gene expression can be obtained by designing complementary sequences or antisense molecules (DNA, RNA, or PNA) to the control, 5', or regulatory regions of the gene encoding HCSRP.
  • Oligonucleotides derived from the transcription initiation site e.g., between about positions - 10 and +10 from the start site, may be employed.
  • inhibition can be achieved using triple helix base-pairing methodology.
  • Triple helix pairing is useful because it causes inhibition ofthe ability of the double helix to open sufficiently for the binding of polymerases, transcription factors, or regulatory molecules. Recent therapeutic advances using triplex DNA have been described in the literature. (See, e.g., Gee, J.E. et al.
  • a complementary sequence or antisense molecule may also be designed to block translation of mRNA by preventing the transcript from binding to ribosomes.
  • Ribozymes enzymatic RNA molecules, may also be used to catalyze the specific cleavage of RNA.
  • the mechanism of ribozyme action involves sequence-specific hybridization ofthe ribozyme molecule to complementary target RNA, followed by endonucleolytic cleavage.
  • engineered hammerhead motif ribozyme molecules may specifically and efficiently catalyze endonucleolytic cleavage of sequences encoding HCSRP.
  • RNA target Specific ribozyme cleavage sites within any potential RNA target are initially identified by scanning the target molecule for ribozyme cleavage sites, including the following sequences: GUA, GUU, and GUC. Once identified, short RNA sequences of between 15 and 20 ribonucleotides. corresponding to the region of the target gene containing the cleavage site, may be evaluated for secondary structural features which may render the oligonucleotide inoperable. The suitability of candidate targets may also be evaluated by testing accessibility to hybridization with complementary oligonucleotides using ⁇ bonuclease protection assay s
  • RNA molecules may be generated bv in vitro and in vivo transcription of DNA sequences encoding HCSRP Such DNA sequences may be incorporated into a wide variety of vectors with suitable RNA polymerase promoters such as T7 or SP6 Alternatively, these cDNA constructs that synthesize complementary RNA.
  • RNA molecules may be modified to increase intracellular stability and half-life Possible modifications include, but are not limited to. the addition of flanking sequences at the 5' and/or 3' ends of the molecule, or the use of phosphorothioate or 2' O-methyl rather than phosphodiesterase linkages within the backbone ofthe molecule This concept is inherent in the production of PNAs and can be extended in all of these molecules by the inclusion of nontraditional bases such as inosine, queosine.
  • vectors may be introduced into stem cells taken from the patient and clonal ly propagated for autologous transplant back into that same patient Delivery by transfection, by liposome injections, or by polycationic amino polymers may be achieved using methods which are well known in the art (See, e g , Goldman, C K et al ( 1997) Nat Biotechnol 15 462-466 )
  • any of the therapeutic methods described above may be applied to any subject in need of such therapy, including, for example, mammals such as humans, dogs, cats, cows, horses, rabbits, and monkeys
  • compositions may consist of HCSRP, antibodies to HCSRP, and mimetics, agonists, antagonists, or inhibitors of HCSRP
  • compositions may be administered alone or in combination with at least one other agent, such as a stabilizing compound, which may be administered in any sterile, biocompatible pharmaceutical carrier including, but not limited to. saline, buffered saline, dextrose, and water
  • a stabilizing compound such as a stabilizing compound
  • the compositions may be administered to a patient alone, or in combination with other agents, drugs, or hormones
  • compositions utilized in this invention mav be administered by any number of routes including, but not limited to. oral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal. intraventricular. transdermal, subcutaneous, intraperitoneal. intranasal, enteral. topical, sublingual. or rectal means.
  • these pharmaceutical compositions may contain suitable pharmaceutically-acceptable carriers comprising e.xcipients and auxiliaries which facilitate processing of the active compounds into preparations which can be used pharmaceutically. Further details on techniques for formulation and administration may be found in the latest edition of Remington's Pharmaceutical Sciences (Maack Publishing. Easton PA).
  • compositions for oral administration can be formulated using pharmaceutically acceptable carriers well known in the art in dosages suitable for oral administration.
  • Such carriers enable the pharmaceutical compositions to be formulated as tablets, pills, dragees, capsules. liquids, gels, syrups, slurries, suspensions, and the like, for ingestion by the patient.
  • compositions for oral use can be obtained through combining active compounds with solid excipient and processing the resultant mixture of granules (optionally, after grinding) to obtain tablets or dragee cores.
  • auxiliaries can be added, if desired.
  • Suitable excipients include carbohydrate or protein fillers, such as sugars, including lactose, sucrose, mannitol, and sorbitol; starch from corn, wheat, rice, potato, or other plants; cellulose, such as methyl cellulose, hydroxypropyimethyl-cellulose, or sodium carboxymethylcellulose; gums, including arabic and tragacanth: and proteins, such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar. and alginic acid or a salt thereof, such as sodium alginate.
  • Dragee cores may be used in conjunction with suitable coatings, such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone. carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • suitable coatings such as concentrated sugar solutions, which may also contain gum arabic, talc, polyvinylpyrrolidone. carbopol gel, polyethylene glycol, and/or titanium dioxide, lacquer solutions, and suitable organic solvents or solvent mixtures.
  • Dyestuffs or pigments may be added to the tablets or dragee coatings for product identification or to characterize the quantity of active compound, i.e., dosage.
  • Push-fit capsules made of gelatin, as well as soft, sealed capsules made of gelatin and a coating, such as glycerol or sorbitol.
  • Push-fit capsules can contain active ingredients mixed with fillers or binders, such as lactose or starches, lubricants, such as talc or magnesium stearate. and. optionally, stabilizers.
  • the active compounds may be dissolved or suspended in suitable liquids, such as fatty oils, liquid, or liquid polyethylene glycol with or without stabilizers.
  • compositions suitable for parenteral administration may be formulated in aqueous solutions, preferably in physiologically compatible buffers such as Hanks' solution. Ringer's solution, or physiologically buffered saline.
  • Aqueous injection suspensions may contain substances which increase the viscosity of the suspension, such as sodium carboxymethyl cellulose, sorbitol, or dextran.
  • suspensions of the active compounds may be prepared as appropriate oily injection suspensions.
  • Suitable lipophilic solvents or vehicles include fatty oils, such as sesame oil, or synthetic fatty acid esters, such as ethyl oleate. triglycerides, or liposomes.
  • Non-lipid polycationic amino polymers may also be used for delivery.
  • the suspension may also contain suitable stabilizers or agents to increase the solubility of the compounds and allow for the preparation of highly concentrated solutions.
  • penetrants appropriate to the particular barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art.
  • compositions ofthe present invention may be manufactured in a manner that is known in the art, e.g., by means of conventional mixing, dissolving, granulating, dragee-making, levigating, emulsifying, encapsulating, entrapping, or lyophilizing processes.
  • the pharmaceutical composition may be provided as a salt and can be formed with many acids, including but not limited to, hydrochloric, sulfuric. acetic, lactic, tartaric. malic, and succinic acids. Salts tend to be more soluble in aqueous or other protonic solvents than are the corresponding free base forms.
  • the preparation may be a Iyophilized powder which may contain any or all of the following: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7% mannitol, at a pH range of 4.5 to 5.5, that is combined with buffer prior to use.
  • compositions suitable for use in the invention include compositions wherein the active ingredients are contained in an effective amount to achieve the intended pu ⁇ ose.
  • the determination of an effective dose is well within the capability of those skilled in the art.
  • the therapeutically effective dose can be estimated initially either in cell culture assays, e.g., of neoplastic cells, or in animal models such as mice, rats, rabbits, dogs, or pigs. An animal model may also be used to determine the appropriate concentration range and route of administration. Such information can then be used to determine useful doses and routes for administration in humans.
  • a therapeutically effective dose refers to that amount of active ingredient, for example HCSRP or fragments thereof, antibodies of HCSRP. and agonists, antagonists or inhibitors of HCSRP, which ameliorates the symptoms or condition.
  • Therapeutic efficacy and toxicity may be determined by standard pharmaceutical procedures in cell cultures or with experimental animals, such as by calculating the ED 50 (the dose therapeutically effective in 50% of the population) or LD 50 (the dose lethal to 50% o the population) statistics.
  • the dose ratio of toxic to therapeutic effects is the therapeutic index, which can be expressed as the LD ⁇ ( ED ⁇ 0 ratio.
  • Pharmaceutical compositions which exhibit large therapeutic indices are preferred.
  • the data obtained from cell culture assays and animal studies are used to formulate a range of dosage for human use.
  • the dosage contained in such compositions is preferably within a range of circulating concentrations that includes the ED 50 with little or no toxicity. The dosage varies within this range depending upon the dosage form employed, the sensitivity of the patient, and the route of administration.
  • Dosage and administration are adjusted to provide sufficient levels of the active moiety or to maintain the desired effect. Factors which may be taken into account include the severity of the disease state, the general health of the subject, the age, weight, and gender of the subject, time and frequency of administration, drug combination(s), reaction sensitivities, and response to therapy. Long-acting pharmaceutical compositions may be administered every 3 to 4 days, every week, or biweekly depending on the half-life and clearance rate of the particular formulation.
  • Normal dosage amounts may vary from about 0.1 ⁇ g to 100,000 ⁇ g, up to a total dose of about 1 gram, depending upon the route of administration.
  • Guidance as to particular dosages and methods of delivery is provided in the literature and generally available to practitioners in the art. Those skilled in the art will employ different formulations for nucleotides than for proteins or their inhibitors. Similarly, delivery of polynucleotides or polypeptides will be specific to particular cells, conditions, locations, etc. DIAGNOSTICS
  • antibodies which specifically bind HCSRP may be used for the diagnosis of disorders characterized by expression of HCSRP, or in assays to monitor patients being treated with HCSRP or agonists, antagonists, or inhibitors of HCSRP.
  • Antibodies useful for diagnostic pu ⁇ oses may be prepared in the same manner as described above for therapeutics. Diagnostic assays for HCSRP include methods which utilize the antibody and a label to detect HCSRP in human body fluids or in extracts of cells or tissues.
  • the antibodies may be used with or without modification, and may be labeled by covalent or non-covalent attachment of a reporter molecule.
  • a wide variety of reporter molecules, several of which are described above, are known in the art and may be used.
  • HCSRP human serum
  • the polynucleotides encoding HCSRP may be used for diagnostic purposes.
  • the polynucleotides which may be used include oligonucleotide sequences, complementary RNA and DNA molecules, and PNAs.
  • the polynucleotides may be used to detect and quantify gene expression in biopsied tissues in which expression of HCSRP may be correlated with disease.
  • the diagnostic assay may be used to determine absence, presence, and excess expression of HCSRP, and to monitor regulation of HCSRP levels during therapeutic intervention.
  • hybridization with PCR probes which are capable of detecting polynucleotide sequences, including genomic sequences, encoding HCSRP or closely related molecules may be used to identify nucleic acid sequences which encode HCSRP.
  • the specificity of the probe whether it is made from a highly specific region, e.g., the 5' regulatory region, or from a less specific region, e.g., a conserved motif, and the stringency of the hybridization or amplification will determine whether the probe identifies only naturally occurring sequences encoding HCSRP, allelic variants, or related sequences. Probes may also be used for the detection of related sequences, and may have at least 50% sequence identity to any of the HCSRP encoding sequences.
  • the hybridization probes ofthe subject invention may be DNA or RNA and may be derived from the sequence of SEQ ID NO: 14-26 or from genomic sequences including promoters, enhancers, and introns of the HCSRP gene.
  • Means for producing specific hybridization probes for DNAs encoding HCSRP include the cloning of polynucleotide sequences encoding HCSRP or HCSRP derivatives into vectors for the production of mRNA probes.
  • Such vectors are known in the art. are commercially available, and may be used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.
  • Hybridization probes may be labeled by a variety of reporter groups, for example, by radionuclides such as 32 P or 35 S, or by enzymatic labels, such as alkaline phosphatase coupled to the probe via avidin/biotin coupling systems, and the like.
  • Polynucleotide sequences encoding HCSRP may be used for the diagnosis of disorders associated with expression of HCSRP.
  • disorders include, but are not limited to. a cell proliferative disorder such as actinic keratosis, arteriosclerosis, atherosclerosis, bursitis, cirrhosis, hepatitis, mixed connective tissue disease (MCTD).
  • myelofibrosis paroxysmal nocturnal hemogiobinuria. poiycythemia vera. psoriasis, primary thrombocythemia. and cancers including adenocarcinoma.
  • leukemia lymphoma, melanoma, myeloma, sarcoma, teratocarcinoma. and. in particular, cancers of the adrenal gland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder, ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle, ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin, spleen, testis. thymus, thyroid, and uterus: an immune system disorder such as inflammation, actinic keratosis.
  • AIDS acquired immunodeficiency syndrome
  • Addison ' s disease adult respiratory distress syndrome
  • allergies ankyiosing spondylitis
  • amyloidosis anemia, arteriosclerosis, asthma, atherosclerosis, autoimmune hemolytic anemia, autoimmune thyroiditis.
  • bronchitis bursitis.
  • cholecystitis cirrhosis
  • contact dermatitis Crohn's disease
  • atopic dermatitis dermatomyositis
  • diabetes mellitus emphysema
  • erythroblastosis fetalis erythema nodosum.
  • atrophic gastritis glomerulonephritis, Goodpasture's syndrome, gout, Graves ' disease, Hashimoto ' s thyroiditis, paroxysmal nocturnal hemogiobinuria, hepatitis, hypereosinophilia, irritable bowel syndrome, episodic lymphopenia with lymphocytotoxins.
  • MCTD mixed connective tissue disease
  • multiple sclerosis myasthenia gravis, myocardial or pericardial inflammation, myelofibrosis, osteoarthritis, osteoporosis, pancreatitis, poiycythemia vera, polymyositis, psoriasis, Reiter ' s syndrome, rheumatoid arthritis, scleroderma, Sjogren ' s syndrome, systemic anaphyiaxis, systemic lupus erythematosus, systemic sclerosis, primary thrombocythe ia, thrombocytopenic pu ⁇ ura, ulcerative colitis, uveitis, Werner syndrome, complications of cancer, hemodialysis, and extraco ⁇ oreal circulation, trauma, and hematopoietic cancer including lymphoma, leukemia, and myeloma; an infection caused by a viral agent classified as adenovirus, arenavirus, bunyavirus,
  • coronavirus coronavirus, filovirus, hepadnavirus, he ⁇ esvirus, flavivirus, orthomyxovirus, parvovirus, papovavirus, paramyxovirus, picornavirus, poxvirus, reovirus, retrovirus, rhabdovirus, or togavirus; an infection caused by a bacterial agent classified as pneumococcus, staphylococcus. streptococcus, bacillus, corynebacterium. clostridium, meningococcus, gonococcus, listeria, moraxella, kingeila, haemophilus, legionella, bordetella, gram-negative enterobacterium including shigella.
  • malasezzia histoplasma, or other fungal agents causing various mycoses: an infection caused by a parasite classified as plasmodium or malaria-causing, parasitic entamoeba, leishmania, trypanosoma, toxoplasma, pneumocystis carinii, intestinal protozoa such as giardia, trichomonas, tissue nematodes such as trichinella. intestinal nematodes such as ascaris. lymphatic filarial nematodes. trematodes such as schistosoma. or cestrodes such as tapeworm: and a neuronal disorder such as akathesia.
  • Alzheimer ' s disease amnesia, amyotrophic lateral sclerosis, bipolar disorder, catatonia. cerebral neoplasms, dementia, depression, diabetic neuropathy. Dow n ' s syndrome, tardive dyskinesia. dystonias. epilepsy. Huntington ' s disease, peripheral neuropathy, multiple sclerosis, neurofibromatosis. Parkinson ' s disease, paranoid psychoses, postherpetic neuralgia, schizophrenia, and Tourette's disorder.
  • the polynucleotide sequences encoding HCSRP may be used in Southern or northern analysis, dot blot, or other membrane-based technologies: in PCR technologies: in dipstick, pin, and multiformat ELISA-like assays: and in microarrays utilizing fluids or tissues from patients to detect altered HCSRP expression. Such qualitative or quantitative methods are well known in the art.
  • the nucleotide sequences encoding HCSRP may be useful in assays that detect the presence of associated disorders, particularly those mentioned above.
  • the nucleotide sequences encoding HCSRP may be labeled by standard methods and added to a fluid or tissue sample from a patient under conditions suitable for the formation of hybridization complexes.
  • the sample is washed and the signal is quantified and compared with a standard value. If the amount of signal in the patient sample is significantly altered in comparison to a control sample then the presence of altered levels of nucleotide sequences encoding HCSRP in the sample indicates the presence of the associated disorder.
  • assays may also be used to evaluate the efficacy of a particular therapeutic treatment regimen in animal studies, in clinical trials, or to monitor the treatment of an individual patient.
  • a normal or standard profile for expression is established. This may be accomplished by combining body fluids or cell extracts taken from normal subjects, either animal or human, with a sequence, or a fragment thereof, encoding HCSRP. under conditions suitable for hybridization or amplification. Standard hybridization may be quantified by comparing the values obtained from normal subjects with values from an experiment in which a known amount of a substantially purified polynucleotide is used. Standard values obtained in this manner may be compared with values obtained from samples from patients who are symptomatic for a disorder. Deviation from standard values is used to establish the presence of a disorder.
  • hybridization assays may be repeated on a regular basis to determine if the level of expression in the patient begins to approximate that which is observed in the normal subject.
  • the results obtained from successive assays may be used to show the efficacy of treatment over a period ranging from several days to months.
  • the presence of an abnormal amount of transcript (either under- or overexpressed) in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior to the appearance of actual clinical symptoms.
  • a more definitive diagnosis of this type may allow health professionals to employ preventative measures or aggressive treatment earlier thereby preventing the development or further progression ofthe cancer.
  • oligonucleotides designed from the sequences encoding HCSRP may involve the use of PCR. These oligomers may be chemically synthesized, generated enzymatically, or produced in vitro. Oligomers will preferably contain a fragment of a polynucleotide encoding HCSRP, or a fragment of a polynucleotide complementary to the polynucleotide encoding HCSRP, and will be employed under optimized conditions for identification of a specific gene or condition. Oligomers may also be employed under less stringent conditions for detection or quantification of closely related DNA or RNA sequences.
  • Methods which may also be used to quantify the expression of HCSRP include radiolabeling or biotinylating nucleotides. coamplification of a control nucleic acid, and inte ⁇ olating results from standard curves. (See, e.g., Melby. P.C et al. ( 1993) J. Immunol. Methods 159:235-244; Duplaa. C. et al. (1993) Anal. Biochem. 212:229-236.) The speed of quantitation of multiple samples may be accelerated by running the assay in a high-throughput format where the oligomer of interest is presented in various dilutions and a spectrophotometric or colorimetric response gives rapid quantitation.
  • oligonucleotides or longer fragments derived from any of the polynucleotide sequences described herein may be used as targets in a microarray.
  • the microarray can be used to monitor the expression level of large numbers of genes simultaneously and to identify genetic variants, mutations, and polymorphisms. This information may be used to determine gene function, to understand the genetic basis of a disorder, to diagnose a disorder, and to develop and monitor the activities of therapeutic agents.
  • Microarrays may be prepared, used, and analyzed using methods known in the art. (See, e.g., Brennan, T.M. et al. ( 1995) U.S. Patent No. 5,474,796; Schena, M. et al. ( 1996) Proc. Natl. Acad. Sci.
  • nucleic acid sequences encoding HCSRP may be used to generate hybridization probes useful in mapping the naturally occurring genomic sequence.
  • the sequences may be mapped to a particular chromosome, to a specific region of a chromosome, or to artificial chromosome constructions, e.g., human artificial chromosomes (HACs), yeast artificial chromosomes (YACs), bacterial artificial chromosomes (BACs), bacterial PI constructions, or single chromosome cDNA libraries.
  • HACs human artificial chromosomes
  • YACs yeast artificial chromosomes
  • BACs bacterial artificial chromosomes
  • PI constructions or single chromosome cDNA libraries.
  • Fluorescent in situ hybridization may be correlated with other physical chromosome mapping techniques and genetic map data.
  • FISH Fluorescent in situ hybridization
  • Examples of genetic map data can be found in various scientific journals or at the Online Mendelian Inheritance in Man (OMIM) World Wide Web site. Correlation between the location of the gene encoding HCSRP on a physical chromosomal map and a specific disorder, or a predisposition to a specific disorder, may help define the region of DNA associated with that disorder.
  • the nucleotide sequences of the invention may be used to detect differences in gene sequences among normal, carrier, and affected individuals.
  • In situ hybridization of chromosomal preparations and physical mapping techniques may be used for extending genetic maps. Often the placement of a gene on the chromosome of another mammalian species, such as mouse, may reveal associated markers even if the number or arm of a particular human chromosome is not known. New sequences can be assigned to chromosomal arms by physical mapping. This provides valuable information to investigators searching for disease genes using positional cloning or other gene discovery techniques.
  • any sequences mapping to that area may represent associated or regulatory genes for further investigation.
  • the nucleotide sequence of the subject invention may also be used to detect differences in the chromosomal location due to translocation, inversion, etc., among normal, carrier, or affected individuals.
  • HCSRP its catalytic or immunogenic fragments, or oligopeptides thereof
  • the fragment employed in such screening may be free in solution, affixed to a solid support, borne on a cell surface, or located intracellularly. The formation of binding complexes between HCSRP and the agent being tested may be measured.
  • Another technique for drug screening provides for high throughput screening of compounds having suitable binding affinity to the protein of interest.
  • This method large numbers of different small test compounds are synthesized on a solid substrate. The test compounds are reacted with HCSRP, or fragments thereof, and washed. Bound HCSRP is then detected by methods well known in the art. Purified HCSRP can also be coated directly onto plates for use in the aforementioned drug screening techniques. Alternatively, non-neutralizing antibodies can be used to capture the peptide and immobilize it on a solid support.
  • the nucleotide sequences which encode HCSRP may be used in any molecular biology techniques that have yet to be developed, provided the new techniques rely on properties of nucleotide sequences that are currently known, including, but not limited to, such properties as the triplet genetic code and specific base pair interactions.
  • RNA was purchased from Clontech or isolated from tissues described in Table 4. Some tissues were homogenized and lysed in guanidinium isothiocyanate, while others were homogenized and lysed in phenol or in a suitable mixture of denaturants, such as TRIZOL (Life Technologies), a monophasic solution of phenol and guanidine isothiocyanate. The resulting lysates were centrifuged over CsCI cushions or extracted with chloroform. RNA was precipitated from the lysates with either isopropanol or sodium acetate and ethanol. or by other routine methods. Phenol extraction and precipitation of RNA were repeated as necessary to increase RNA purity. In some cases, RNA was treated with DNase.
  • RNA was isolated using oligo d(T)-coupled paramagnetic particles (Promega), OLIGOTEX latex particles (QIAGEN, Chatsworth CA), or an OLIGOTEX mRNA purification kit (QIAGEN).
  • RNA was isolated directly from tissue lysates using other RNA isolation kits, e.g.. the POLY(A)PURE mRNA purification kit (Ambion Austin TX)
  • SEPHAROSE CL2B, or SEPHAROSE CL4B column chromatography (Amersham Pharmacia Biotech) or preparative agarose gel electrophoresis cDNAs were ligated into compatible restriction enzyme sites of the polylinker of a suitable plasmid, e g , PBLUESCRIPT plasmid (Stratagene) PSPORT1 plasmid (Life Technologies) or pINCY (Incyte Pharmaceuticals. Palo Alto CA) Recombinant plasmids were transformed into competent E coli cells including XL l -Blue, XL l -BlueMRF, or SOLR from Stratagene or DH5 ⁇ . DH 10B. or ElectroM AX DH 10B from Life Technologies II. Isolation of cDNA Clones
  • Plasmids were recovered from host ceils by in vivo excision using the UNIZAP vector system (Stratagene) or by cell lysis Plasmids were purified using at least one of the following a Magic or WIZARD Min ⁇ reps DNA purification system (Promega), an AGTC Miniprep purification kit (Edge Biosystems, Gaithersburg MD), and QIAWELL 8 Plasmid, QIAWELL 8 Plus Plasmid.
  • plasmid DNA was amplified from host cell lysates using direct link PCR in a high-throughput format (Rao. V B ( 1994) Anal Biochem 216 1 - 14) Host cell lysis and thermal cycling steps were carried out in a single reaction mixture Samples were processed and stored in 384-weli plates, and the concentration of amplified plasmid DNA was quantified fluoromet ⁇ cally using PICOGREEN dye (Molecular Probes, Eugene OR) and a FLUOROSKAN II fluorescence scanner (Labsystems Oy, Helsinki. Finland) III.
  • PICOGREEN dye Molecular Probes, Eugene OR
  • FLUOROSKAN II fluorescence scanner Labsystems Oy, Helsinki. Finland
  • Sequencing and Analysis cDNA sequencing reactions were processed using standard methods or high-throughput instrumentation such as the ABI CATALYST 800 (Perkin-Elmer) thermal cy cler or the PTC-200 thermal cvcler (MJ Research) in conjunction with the HYDRA microdispenser (Robbins Scientific) or the MICROLAB 2200 (Hamilton) liquid transfer sy stem cDNA sequencing reactions were prepared using reagents provided by Amersham Pharmacia Biotech or supplied in ABI sequencing kits such as the ABI PRISM BIGDYE Terminator cycle sequencing ready reaction kit (Perkin-Elmer).
  • Electrophoretic separation of cDNA sequencing reactions and detection of labeled polynucleotides were carried out using the MEGABACE 1000 DNA sequencing system (Molecular Dynamics); the ABI PRISM 373 or 377 sequencing system (Perkin-Elmer) in conjunction with standard ABI protocols and base calling software; or other sequence analysis systems known in the art. Reading frames within the cDNA sequences were identified using standard methods (reviewed in Ausubel, 1997, supra, unit 1.1). Some of the cDNA sequences were selected for extension using the techniques disclosed in Example V. The polynucleotide sequences derived from cDNA sequencing were assembled and analyzed using a combination of software programs which utilize algorithms well known to those skilled in the art.
  • Table 5 summarizes the tools, programs, and algorithms used and provides applicable descriptions, references, and threshold parameters.
  • the first column of Table 5 shows the tools, programs, and algorithms used, the second column provides brief descriptions thereof, the third column presents appropriate references, all of which are inco ⁇ orated by reference herein in their entirety, and the fourth column presents, where applicable, the scores, probability values, and other parameters used to evaluate the strength of a match between two sequences (the higher the score, the greater the homology between two sequences).
  • Sequences were analyzed using MACDNASIS PRO software (Hitachi Software Engineering, South San Francisco CA) and LASERGENE software (DNASTAR).
  • Polynucleotide and polypeptide sequence alignments were generated using the default parameters specified by the clustal algorithm as inco ⁇ orated into the MEGALIGN multisequence alignment program (DNASTAR). which also calculates the percent identity between aligned sequences.
  • the polynucleotide sequences were validated by removing vector, linker, and polyA sequences and by masking ambiguous bases, using algorithms and programs based on BLAST, dynamic programing, and dinucleotide nearest neighbor analysis. The sequences were then queried against a selection of public databases such as the GenBank primate, rodent, mammalian, vertebrate, and eukaryote databases, and BLOCKS. PRINTS, DOMO. PRODOM, and PFAM to acquire annotation using programs based on BLAST, FASTA. and BLIMPS. The sequences were assembled into full length polynucleotide sequences using programs based on Phred. Phrap, and Consed.
  • HMM Hidden Markov Model
  • Northern analysis is a laboratory technique used to detect the presence of a transcript of a gene and involves the hybridization of a labeled nucleotide sequence to a membrane on which RNAs from a particular cell type or tissue have been bound. (See, e.g., Sambrook. supra, ch. 7; Ausubel, 1995, supra, ch. 4 and 16.)
  • % sequence identity x % maximum BLAST score 100 The product score takes into account both the degree of similarity between two sequences and the length of the sequence match. For example, with a product score of 40. the match will be exact within a 1 % to 2% error, and. with a product score of 70. the match will be exact. Similar molecules are usually identified by selecting those which show product scores between 15 and 40, although lower scores may identify related molecules. The results of northern analyses are reported as a percentage distribution of libraries in which the transcript encoding HCSRP occurred. Analysis involved the categorization of cDNA libraries by organ/tissue and disease. The organ/tissue categories included cardiovascular, dermatologic, developmental, endocrine, gastrointestinal, hematopoietic/immune.
  • the disease/condition categories included cancer, inflammation, trauma, cell proliferation, neurological, and pooled. For each category, the number of libraries expressing the sequence of interest was counted and divided by the total number of libraries across all categories. Percentage values of tissue-specific and disease- or condition-specific expression are reported in Table 3.
  • the full length nucleic acid sequences of SEQ ID NO 14-26 were produced bv extension of an appropriate fragment o the full length molecule using oligonucleotide primers designed from this fragment
  • One primer was synthesized to initiate 5' extension of the known fragment and the other primer, to initiate 3' extension of the known fragment
  • the initial primers w ere designed using OLIGO 4 06 software (National Biosciences).
  • Step 4 68°C, 2 min, Step 5 Steps 2, 3, and 4 repeated 20 times.
  • Step 6 68°C, 5 mm.
  • the parameters for primer pair T7 and SK+ were as follows Step 1 94°C, 3 mm, Step 2 94°C 15 sec. Step 3 57°C, 1 min. Step 4 68°C, 2 mm. Step 5 Steps 2. 3, and 4 repeated 20 times, Step 6 68°C, 5 mm, Step 7 storage at 4°C
  • the concentration of DNA in each well was determined by dispensing 100 ⁇ l PICOGREEN quantitation reagent (0 25% (v/v) PICOGREEN, Molecular Probes, Eugene OR) dissolved in I X TE and 0 5 ⁇ l of undiluted PCR product into each well of an opaque fluo ⁇ meter plate (Corning Costar, Acton MA), allowing the DNA to bind to the reagent The plate was scanned in a Fluoroskan II (Labsystems Oy. Helsinki.
  • nucleotide sequences of SEQ ID NO: 14-26 are used to obtain 5' regulatory sequences using the procedure above, oligonucleotides designed for such extension, and an appropriate genomic library.
  • Hybridization probes derived from SEQ ID NO: 14-26 are employed to screen cDNAs. genomic DNAs, or mRNAs. Although the labeling of oligonucleotides, consisting of about 20 base pairs, is specifically described, essentially the same procedure is used with larger nucleotide fragments. Oligonucleotides are designed using state-of-the-art software such as OLIGO 4.06 software (National Biosciences) and labeled by combining 50 pmol of each oligomer. 250 uCi of [ ⁇ - 3: P] adenosine triphosphate (Amersham Pharmacia Biotech), and T4 polynucleotide kinase (DuPont NEN. Boston MA). The labeled oligonucleotides are substantially purified using a
  • SEPHADEX G-25 superfine size exclusion dextran bead column (Amersham Pharmacia Biotech). An aliquot containing 10 7 counts per minute of the labeled probe is used in a typical membrane- based hybridization analysis of human genomic DNA digested with one of the following endonucleases: Ase I. Bgl II. Eco RI, Pst I, Xba I. or Pvu II (DuPont NEN). The DNA from each digest is fractionated on a 0.7% agarose gel and transferred to nylon membranes (Nytran Plus. Schleicher & Schuell. Durham NH). Hybridization is carried out for 16 hours at 40°C To remove nonspecific signals, blots are sequentially washed at room temperature under conditions of up to. for example. 0.1 x saline sodium citrate and 0.5% sodium dodecyl sulfate. Hybridization patterns are visualized using autoradiography or an alternative imaging means and compared.
  • a chemical coupling procedure and an ink jet device can be used to synthesize array elements on the surface of a substrate.
  • An array analogous to a dot or slot blot may also be used to arrange and link elements to the surface of a substrate using thermal. UV. chemical, or mechanical bonding procedures.
  • a typical array may be produced by hand or using available methods and machines and contain any appropriate number of elements.
  • nonhybridized probes are removed and a scanner used to determine the levels and patterns of fluorescence. The degree of complementarity and the relative abundance of each probe which hybridizes to an element on the microarray may be assessed through analysis of the scanned images.
  • Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereof may comprise the elements of the microarray. Fragments suitable for hybridization can be selected using software well known in the art such as LASERGENE software (DNASTAR). Full-length cDNAs, ESTs, or fragments thereof corresponding to one of the nucleotide sequences of the present invention, or selected at random from a cDNA library relevant to the present invention, are arranged on an appropriate substrate, e.g., a glass slide. The cDNA is fixed to the slide using, e.g., UV cross-linking followed by thermal and chemical treatments and subsequent drying. (See, e.g., Schena, M. et al.
  • Fluorescent probes are prepared and used for hybridization to the elements on the substrate.
  • the substrate is analyzed by procedures described above.
  • Sequences complementary to the HCSRP-encoding sequences, or any parts thereof, are used to detect, decrease, or inhibit expression of naturally occurring HCSRP. Although use of oligonucleotides comprising from about 15 to 30 base pairs is described, essentially the same procedure is used with smaller or with larger sequence fragments. Appropriate oligonucleotides are designed using OLIGO 4.06 software (National Biosciences) and the coding sequence of HCSRP. To inhibit transcription, a complementary oligonucleotide is designed from the most unique 5' sequence and used to prevent promoter binding to the coding sequence. To inhibit translation, a complementary oligonucleotide is designed to prevent ribosomal binding to the HCSRP-encoding transcript.
  • HCSRP HCSRP
  • cDNA is subcloned into an appropriate vector containing an antibiotic resistance gene and an inducible promoter that directs high levels of cDNA transcription
  • promoters include, but are not limited to. the trp-lac (tat ) hybrid promoter and the T5 or T7 bacteriophage promoter in conjunction w ith the lac operator regulatory element Recombinant vectors are transformed into suitable bacterial hosts, e g .
  • HCSRP isopropyl beta-D- thiogalactopyranoside
  • IPTG isopropyl beta-D- thiogalactopyranoside
  • GST S-transferase
  • GST S-transferase
  • a peptide epitope tag such as FLAG or 6-H ⁇ s
  • HCSRP activ ity measures the expression of HCSRP on the cell surface cDNA encoding HCSRP is transfected into an appropriate mammalian cell line Cell surface proteins are labeled with biotm as described (de la Fuent M A et al ( 1997) Blood 90 2398-2405)
  • Immunoprecipitations are performed using HCSRP-speciflc antibodies, and immunoprecipitated samples are analyzed using SDS-PAGE and immunoblotting techniques The ratio of labeled immunoprecipitant to unlabeled immunoprecipitant is proportional to the amount of HCSRP expressed on the cell surface
  • An undo e assay for HCSRP activ itv is based on a prototy pical assay for ligand/receptor- mediated modulation of cell proliferation This assay measures the amount of newly sy nthesized DNA in Swiss mouse 3T3 cells expressing HCSRP
  • An appropriate mammalian expression vector containing cDNA encoding HCSRP is added to quiescent 3T3 cultured cells using transfection methods well known in the art The transfected cells are incubated in the presence of [ ⁇ ]thym ⁇ d ⁇ ne and vary ing amounts of HCSRP ligand Inco ⁇ oration of [ Hjthy midine into acid-precipitabie DNA is measured over an appropriate time interval using a tritium radioisotope counter, and the amount inco ⁇ orated is directly proportional to the amount of newly synthesized DNA
  • a linear dose- response curv e over at least a hundred-fold HCSRP ligand concentration range is indicative of receptor activity
  • HCSRP function is assessed by expressing the sequences encoding HCSRP at physiologically elevated levels in mammalian cell culture systems cDNA is subcloned into a mammalian expression vector containing a strong promoter that drives high levels of cDNA expression
  • Vectors of choice include pCMV SPORT (Life Technologies) and pCR3 1 (Invitrogen, Carlsbad CA), both of which contain the cytomegalovirus promoter 5- 10 ⁇ g of recombinant vector are transiently transfected into a human cell line, for example, an endothelial or hematopoietic cell line, using either liposome formulations or electroporation 1 -2 ⁇ g of an additional plasmid containing sequences encoding a marker protein are co-transfected Expression of a marker protein provides a means to distinguish
  • HCSRP human immunoglobulin G
  • Transfected cells are efficiently separated from nontransfected cells using magnetic beads coated with either human IgG or antibody against CD64 (DYNAL. Lake Success NY)
  • mRNA can be purified from the cells using methods well know n by those of skill in the art Expression of mRNA encoding HCSRP and other genes of interest can be analyzed by northern analysis or microarray techniques XII. Production of HCSRP Specific Antibodies
  • HCSRP substantially purified using polyacrylamide gel electrophoresis (PAGE, see, e g , Harrington, M G ( 1990) Methods Enzymol 1 82 488-495), or other purification techniques, is used to immunize rabbits and to produce antibodies using standard protocols
  • HCSRP amino acid sequence is analyzed using LASERGENE software (DNASTAR) to determine regions of high immunogenicity, and a corresponding oligopeptide is synthesized and used to raise antibodies by means known to those of skill in the art Methods for selection of appropriate epitopes, such as those near the C-terminus or in hydrophilic regions are well described in the art (See, e g , Ausubel, 1995. supra, ch 1 1 )
  • oligopeptides of about 15 residues in length are synthesized using an ABI 431 A peptide synthesizer (Perkin-Elmer) using fmoc-chemistry and coupled to KLH (Sigma-Aldrich, St Louis MO) by reaction with N-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increase immunogenicity (See, e g . Ausubel, 1995.
  • ABI 431 A peptide synthesizer Perkin-Elmer
  • KLH Sigma-Aldrich, St Louis MO
  • MBS N-maleimidobenzoyl-N-hydroxysuccinimide ester
  • Rabbits are immunized w ith the oligopeptide- KLH complex in complete Freund's adjuvant Resulting antisera are tested for antipeptide and anti- HCSRP activity by, for example, binding the peptide or HCSRP to a substrate, blocking with 1 % BSA. reacting with rabbit antisera. washing, and reacting w ith radio-iodmated goat anti-rabbit IgG XIII. Purification of Naturally Occurring HCSRP Using Specific Antibodies
  • Naturally occurring or recombinant HCSRP is substantially purified by lmmunoaffinrty chromatography using antibodies specific for HCSRP
  • An immunoaffinity column is constructed by covalently coupling anti-HCSRP antibody to an activated chromatographic resin, such as CNBr-activated SEPHAROSE (Amersham Pharmacia Biotech) After the coupling, the resin is blocked and washed according to the manufacturer's instructions
  • HCSRP Media containing HCSRP are passed over the immunoaffinity column and the column is washed under conditions that allow the preferential absorbance of HCSRP (e g . high ionic strength buffers in the presence of detergent)
  • the column is eluted under conditions that disrupt
  • HCSRP HCSRP. or biologically active fragments thereof, are labeled with l2 'I Bolton-Hunter reagent (See, e g . Bolton A E and W M Hunter ( 1973) Biochem J 133 529-539 )
  • Candidate molecules previously arrayed in the wells of a multi-well plate are incubated with the labeled HCSRP, washed, and any wells w ith labeled HCSRP complex are assayed
  • Data obtained using different concentrations of HCSRP are used to calculate values for the number, affinity, and association of HCSRP with the candidate molecules
  • ABI/PARACEL FDF A Fast Data Finder useful in comparing mid annotating Perkin-Elmer Applied Biosystems, Mismatch ⁇ 50% amino acid or nucleic acid sequences Foster City, CA, Paracel Inc , Pasadena, CA
  • Phred A base-calling algorithm that examines automated EwLng, B et al (1998) Genome sequencer traces with high sensitivity and probability Res 8 175-185, Ewing, B and P Green (1998) Genome Res 8 186- 194
  • Motifs A program that searches amino acid sequences for patterns Bairoch et al supra. Wisconsin that matched those defined in Prosite Package Program Manual, version 9, page M51-59, Genetics Computer Group, Madison, WI

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Abstract

Cette invention a trait à des protéines du récepteur de surface des cellules humaines (HCSRP) ainsi qu'à des polynucléotides les identifiant et les codant. Elle concerne également des vecteurs, des cellules hôtes, des anticorps, des agonistes et des antagonistes. Elle porte, de surcroît, sur des méthodes de diagnostic, de traitement ou de prévention d'états pathologiques associés à l'expression des HCSRP.
PCT/US1999/026742 1998-11-12 1999-11-12 Proteines du recepteur de surface des cellules humaines WO2000028032A2 (fr)

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AU16185/00A AU1618500A (en) 1998-11-12 1999-11-12 Human cell surface receptor proteins
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EP1514933A1 (fr) * 1999-07-08 2005-03-16 Research Association for Biotechnology Protéine sécrétoire ou protéine de membrane
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WO2001094385A2 (fr) * 2000-06-05 2001-12-13 Bayer Aktiengesellschaft Regulation du recepteur couple a la proteine humaine g de type hm74
EP1339871A1 (fr) * 2000-08-15 2003-09-03 The Johns Hopkins University School Of Medicine Diagnostic et traitement des troubles associes aux suppresseurs de tumeur
EP1339871A4 (fr) * 2000-08-15 2005-08-17 Johns Hopkins Universityschool Diagnostic et traitement des troubles associes aux suppresseurs de tumeur
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