EP1171579A1 - Recepteur tr9 humain de facteurs de necrose des tumeurs - Google Patents

Recepteur tr9 humain de facteurs de necrose des tumeurs

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Publication number
EP1171579A1
EP1171579A1 EP00914975A EP00914975A EP1171579A1 EP 1171579 A1 EP1171579 A1 EP 1171579A1 EP 00914975 A EP00914975 A EP 00914975A EP 00914975 A EP00914975 A EP 00914975A EP 1171579 A1 EP1171579 A1 EP 1171579A1
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Prior art keywords
polypeptide
amino acid
seq
sequence
nucleotide sequence
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EP00914975A
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German (de)
English (en)
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EP1171579A4 (fr
Inventor
Jian Ni
Reiner L. Gentz
Guo-Liang Yu
Ping Fan
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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Publication of EP1171579A1 publication Critical patent/EP1171579A1/fr
Publication of EP1171579A4 publication Critical patent/EP1171579A4/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
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/04—Drugs for disorders of the alimentary tract or the digestive system for ulcers, gastritis or reflux esophagitis, e.g. antacids, inhibitors of acid secretion, mucosal protectants
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
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    • A61P19/00—Drugs for skeletal disorders
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/14—Drugs for disorders of the nervous system for treating abnormal movements, e.g. chorea, dyskinesia
    • A61P25/16—Anti-Parkinson drugs
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P27/00—Drugs for disorders of the senses
    • A61P27/02—Ophthalmic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
    • A61P31/12—Antivirals
    • A61P31/20—Antivirals for DNA viruses
    • A61P31/22—Antivirals for DNA viruses for herpes viruses
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/30—Non-immunoglobulin-derived peptide or protein having an immunoglobulin constant or Fc region, or a fragment thereof, attached thereto
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to a novel member of the tumor necrosis factor family of receptors. More specifically, isolated nucleic acid molecules are provided encoding a novel human tumor necrosis factor receptor, TR9 (also known as Death Domain Containing Receptor 6, or simply DR6). TR9 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of the activities of TR9 receptor polypeptides and diagnostic methods for detecting TR9 receptor gene expression. The invention further relates to screening methods for identifying agonists and antagonists of TR9 activity.
  • TR9 also known as Death Domain Containing Receptor 6, or simply DR6
  • TR9 polypeptides are also provided, as are vectors, host cells and recombinant methods for producing the same.
  • the invention also relates to both the inhibition and enhancement of the activities of TR9 receptor polypeptides and diagnostic methods for detecting TR9 receptor gene expression.
  • the invention further relates to screening methods for
  • cytokines Many biological actions, for instance, response to certain stimuli and natural biological processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an intra-cellular response.
  • tumor necrosis factors (TNF) alpha and beta are cytokines, which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease.
  • the TNF molecules belong to the "TNF-ligand” superfamily, and act together with their receptors or counter-ligands, the "TNF-receptor” superfamily. So far, nine members of the TNF ligand superfamily have been identified and ten members of the TNF-receptor superfamily have been characterized.
  • TNF-alpha lymphotoxin-alpha
  • LT-alpha lymphotoxin-alpha
  • LT-beta lymphotoxin-alpha
  • FasL CD40L
  • CD27L CD30L
  • 4-1BBL 4-1BBL
  • OX40L nerve growth factor
  • NGF nerve growth factor
  • the superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein, FAS antigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals 22:291-295 (1994)).
  • TNF-ligand superfamily Many members of the TNF-ligand superfamily are expressed by activated T-cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions. (Meager, A., supra).
  • TNF and LT-alpha are capable of binding to two TNF receptors (the 55- and 75- kd TNF receptors).
  • TNF and LT-alpha are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (Beutler et al., Science 264:667-668 (1994)). Mutations in the p55 receptor cause increased susceptibility to microbial infection.
  • Apoptosis or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms (Steller, Science 267: 1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (Thompson C. B., Science 267: 1456-1462 (1995)).
  • Fas/APO-1 and TNFR-1 are members of the TNF receptor family, which also include TNFR-2, low affinity NGFR, CD40, and CD30, among others (Smith et al., Science 248: 1019-23 (1990); Tewari et al, in Modular Texts in Molecular and Cell Biology; M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995).
  • Fas/APO- 1 and TNFR-1 While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, Fas/APO- 1 and TNFR-1 also share a region of intracellular homology, appropriately designated the "death domain.” which is distantly related to the Drosophila suicide gene, reaper (Golstein et al., Cell 81:185-6 (1995); White et al., Science 264:677-83 (1994)). This shared death domain suggests that both receptors interact with a related set of signal transducing molecules that, until recently, remained unidentified.
  • Fas/APO-1 recruits the death domain-containing adapter molecule FADD/MORT1 (Chinnaiyan et al, Cell 81:505-512 ( 1995); Boldin et al, J. Biol. Chem. 270:7795-8 (1995); Kischkel et al., EMBO 14:5579-5588 ( 1995)). which in turn binds and presumably activates FLICE/MACHl . a member of the ICE/CED-3 family of pro-apoptotic proteases (Muzio et al., Cell 85:817-827 ( 1996); Boldin et al. Cell 85:803-815 ( 1996)).
  • TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kappaB (Tartaglia et al, Immunol Today 13: 151-153 ( 1992)). Accordingly, TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (Hsu et al, Cell 81 :495-504 (1995): Hsu et al., Cell 84:299-308 (1996)).
  • TRADD can signal both apoptosis and NF-kappaB activation (Hsu et al., Cell 84:299-308 (1996); Hsu et al., Immunity 4:387-396 ( 1996)).
  • TNF family ligands and receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefore, for identification and characterization of additional novel TNF receptors and ligands that influence biological activity, both normally and in disease states.
  • the present invention provides isolated nucleic acid molecules, or alternatively consisting of, a polynucleotide encoding the TR9 receptor having the amino acid sequence shown in Figures 1 A-D (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 209037 on May 15, 1997.
  • the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of TR9 receptor polypeptides or peptides by recombinant techniques.
  • the invention further provides an isolated TR9 polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
  • TR9 polypeptides of the invention are administered, to treat, prevent, prognose and/or diagnose an immunodeficiency (e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia.
  • an immunodeficiency e.g., severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia.
  • SCID severe combined immunodeficiency
  • ADA deficiency adenosine deaminase deficiency
  • XLA X
  • X-linked infantile agammaglobulinemia acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hypogammaglobulinemia.
  • transient hypoga maglobulinemia of infancy unspecified hypogammaglobulinemia, agammaglobulinemia, common variable immunodeficiency (CNID) (acquired), Wiskott- Aldrich Syndrome (WAS), X-linked immunodeficiency with hyper IgM, non X-linked immunodeficiency with hyper IgM, selective IgA deficiency, IgG subclass deficiency (with or without IgA deficiency), antibody deficiency with normal or elevated Igs, immunodeficiency with thymoma, Ig heavy chain deletions, kappa chain deficiency, B cell lymphoproliferative disorder (BLPD).
  • BLPD B cell lymphoproliferative disorder
  • IgM immunodeficiency recessive agammaglobulinemia (Swiss type), reticular dysgenesis, neonatal neutropenia, severe congenital leukopenia, thymic alymphoplasia-aplasia or dysplasia with immunodeficiency, ataxia-telangiectasia. short limbed dwarfism, X-linked lymphoproliferative syndrome (XLP), ⁇ ezelof syndrome-combined immunodeficiency with Igs, purine nucleoside phosphorylase deficiency (P ⁇ P), MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.) or conditions associated with an immunodeficiency.
  • XLP X-linked lymphoproliferative syndrome
  • P ⁇ P purine nucleoside phosphorylase deficiency
  • Bare Lymphocyte Syndrome MHC Class II deficiency
  • severe combined immunodeficiency severe combined immunodefic
  • TR9 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose common variable immunodeficiency.
  • TR9 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose X-linked agammaglobulinemia.
  • TR9 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose severe combined immunodeficiency (SCID).
  • SCID severe combined immunodeficiency
  • TR9 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose Wiskott- Aldrich syndrome.
  • TR9 polypeptides or polynucleotides of the invention, or agonists thereof is administered to treat, prevent, prognose and/or diagnose X-linked Ig deficiency with hyper IgM.
  • TR9 antagonists are administered to treat, prevent, prognose and/or diagnose an autoimmune disease (e.g., rheumatoid arthritis, systemic lupus erhythematosus, idiopathic thrombocytopenia purpura, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, rheumatic heart disease, glomerulonephritis (e.g, IgA nephropathy), Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Puipura (e.g., Henloch-Scoenlein purpura).
  • an autoimmune disease e.g., rheumatoid arthritis, systemic lupus erhythematosus
  • Reiter's Disease Stiff-Man Syndrome, Autoimmune Pulmonary Inflammation. Guillain-Barre Syndrome, insulin dependent diabetes mellitis, and autoimmune inflammatory eye, autoimmune thyroiditis, hypothyroidism (i.e., Hashimoto's thyroiditis.
  • Goodpasture's syndrome, Pemphigus, Receptor autoimmunities such as, for example, (a) Graves' Disease , (b) Myasthenia Gravis, and (c) insulin resistance, autoimmune hemolytic anemia, autoimmune thrombocytopenic purpura , schleroderma with anti-collagen antibodies, mixed connective tissue disease, polymyositis/dermatomyositis, pernicious anemia, idiopathic Addison's disease, infertility, glomerulonephritis such as primary glomerulonephritis and IgA nephropathy, bullous pemphigoid, Sjogren's syndrome, diabetes millitus, and adrenergic drug resistance (including adrenergic drug resistance with asthma or cystic fibrosis), chronic active hepatitis, primary biliary cirrhosis, other endocrine gland failure, vitiligo, vasculitis, post-MI, cardiotomy
  • rheumatoid arthritis is treated, prevented, prognosed and/or diagnosed using anti-TR9 antibodies and/or other antagonist of the invention.
  • systemic lupus erythemosus is treated, prevented, prognosed, and/or diagnosed using anti-TR9 antibodies and/or other antagonist of the invention.
  • idiopathic thrombocytopenia purpura is treated, prevented, prognosed, and/or diagnosed using anti- TR9 antibodies and/or other antagonist of the invention.
  • IgA nephropathy is treated, prevented, prognosed and/or diagnosed using anti-TR9 antibodies and/or other antagonist of the invention.
  • the autoimmune diseases and disorders and/or conditions associated with the diseases and disorders recited above are treated, prevented, prognosed and/or diagnosed using anti- TR9 antibodies.
  • compositions comprising a TR9 polynucleotide, a TR9 polypeptide, and/or an anti-TR9 antibody, for administration to cells in vitro, to cells ex vivo, and to cells in vivo, or to a multicellular organism.
  • the compositions of the invention comprise a TR9 polynucleotide for expression of a TR9 polypeptide in a host organism for treatment of disease.
  • the compositions of the invention comprise a TR9 polynucleotide for expression of a TR9 polypeptide in a host organism for treatment of an immunodeficiency and/or conditions associated with an immunodeficiency.
  • a TR9 gene e.g., expression to enhance the normal B-cell function by expanding B cell numbeis oi lncieasmg B cell iespan or expiession to enhance the normal l cell function by expanding T cell numbeis or incieasing T cell iespan
  • expression to enhance the normal B-cell function by expanding B cell numbeis oi lncieasmg B cell iespan or expiession to enhance the normal l cell function by expanding T cell numbeis or incieasing T cell iespan e.g., expression to enhance the normal B-cell function by expanding B cell numbeis oi lncieasmg B cell iespan or expiession to enhance the normal l cell function by expanding T cell numbeis or incieasing T cell iespan
  • the piesent invention also provides a scieenmg method foi identifying compounds capable of enhancing or inhibiting a cellular lesponse induced by the TR9 receptor
  • the method involves contacting cells which expiess the TR9 receptor with the candidate compound, assaying a cellulai response, and comparing the cellular lesponse to a standard cellular lesponse. the standaid being assayed when contact is made in absence of the candidate compound, whereby, an increased cellular lesponse over the standard indicates that the compound is an agonist and a deci eased cellular response over the standard indicates that the compound is an antagonist
  • a screening assay for agonists and antagonists involves determining the effect a candidate compound has on the binding of cellular ligands to TR9 receptois
  • the method involves contacting TR9 receptors with a ligand polypeptide and a candidate compound and determining whether ligand binding to the TR9 receptors is increased or decreased due to the presence of the candidate compound
  • the invention further provides diagnostic assays such as quantitative and diagnostic assays for detecting levels of TR9 receptor protein
  • diagnostic assays such as quantitative and diagnostic assays for detecting levels of TR9 receptor protein
  • a diagnostic assay in accordance with the invention for detecting over-expression of TR9, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors
  • Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, lmmunoregulatory activities, and the transcriptional regulation of several genes
  • Cellular response to TNF-family ligands include not only normal physiological responses, but also diseases associated with increased apoptosis or the inhibition of apoptosis
  • the present invention is directed to a method for inhibiting apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the TR9 polypeptide an effective amount of an antagonist capable of decreasing TR9 mediated signaling.
  • TR9 mediated signaling is decreased to treat, prevent, diagnose, and/or detect a disease wherein increased apoptosis is exhibited.
  • Figures 1A-D show the nucleotide sequence (SEQ ID NO: 1) and deduced amino acid sequence (SEQ ID NO:2) of the TR9 receptor. Analysis using the computer program PSORT reveals that the protein has a predicted leader sequence of about 40 amino acid residues (underlined) and a deduced molecular weight of about 72 kDa.
  • amino acid residues from about 41 to about 350 constitute the extracellular domain (amino acid residues from about 1 to about 310 in SEQ ID NO:2); from about 351 to about 367 the transmembrane domain (amino acid residues from about 311 to about 327 in SEQ ID NO:2); from about 368 to about 655 the intracellular domain (amino acid residues from about 328 to about 615 in SEQ ID NO:2); and from about 429 to about 495 the death domain (amino acid residues from about 389 to about 455 in SEQ ID NO:2).
  • Figure 2 shows the regions of similarity between the amino acid sequences of the TR9 receptor (SEQ ID NO:2) and Fas (SEQ ID NO:3), NGFR p75 (SEQ ID NO:4), and TNFR 1 (SEQ ID NO:5). Residues that match the consensus are shaded.
  • Figure 3 shows an analysis of the TR9 amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, as predicted for the amino acid sequence depicted in Figures 1A-D using the default parameters of the recited computer programs.
  • amino acid residues about 44 to about 121, about 156 to about 31 1, about 323 to about 348, about 376 to about 412, about 433 to about 474, about 485 to about 599, and about 61 1 to about 628 in Figures 1 A-D correspond to the shown highly antigenic regions of the TR9 protein.
  • FIGS 1 A-D correspond to the following fragments, respectively, in SEQ ID NO:2: amino acid residues about 4 to about 81, about 116 to about 271, about 283 to about 308, about 336 to about 372, about 393 to about 434, about 445 to about 559, and about 571 to about 588.
  • Figures 4A-C Highlight the predicted amino acid sequence of TR9.
  • Figure 4A The open reading frame for TR9 defines a type I transmembrane protein of 655 amino acids (SEQ ID NO:2).
  • Application of a computer program other than PSORT has predicted the mature protein to start at amino acid 42 (Gin, indicated by a black triangle).
  • the putative signal peptide and transmembrane domain are single and double underlined, respectively.
  • Six potential N-glycosylation sites are indicated by black dots.
  • the cytoplasmic death domain is boxed.
  • An intracellular region containing a potential leucine-zipper motif overlapping with a proline rich sequence is underlined with a thick line.
  • Figure 4B Sequence alignment of extracellular cysteine-rich domains of TR9 (SEQ ID NO: 19) and osteoprotegerin (SEQ ID NO:20). Alignment was done with Megalign (DNASTAR) software. Shading represents identical residues.
  • Figure 4C Sequence comparison of death domains of TR9 (SEQ ID NO.21), CD95 (SEQ ID NO:22), TNFR1 (SEQ ID NO:23), DR3 (SEQ ID NO:24), DR4 (SEQ ID NO:25), and DR5 (SEQ ID NO:26). Alignment was performed and represented in the same way as in Figure 4B. OPG; osteoprotegerin.
  • TR9 induces apoptosis in mammalian cells. Ectopic expression of
  • TR9 induces apoptosis in Hela cells, but not in MCF7 cells.
  • Hela and MCF7 cells were cotransfected with a empty vector, TR9, TR9 delta, or DR4, together with a beta- galactosidase-expressing reporter construct using a lipofectamine method according to the manufacturer's instructions (BRL).
  • BBL manufacturer's instructions
  • X-Gal 5-bromo-4-chloro-3-indoxyl-beta-D-galactopyranoside
  • TR9 mediates nuclear factor NF-kappaB activation.
  • Cotransfection of 293 cells was performed with the indicated expression constructs and a NF-kappaB luciferase reporter construct. After transfection (at 36 hours), cell extracts were prepared and luciferase activities determined as previously described (Chinnaiyan et al., Science 274:990-992 (1996); and Pan et al., Science 276: 1 11-113 (1997)). Transfection efficiency was monitored by beta-galactosidase activity. A portion of the transfected cells was used to monitor expression of TR9 or TR9 delta. Cell lysates were prepared and immunoprecipitated with FLAG M2 affinity gel and the presence of TR9 or TR9 delta detected by blotting with anti-FLAG.
  • the present invention provides isolated nucleic acid molecules, or alternatively consisting of, a polynucleotide encoding a TR9 receptor polypeptide having the amino acid sequence shown in Figures 1A-C (SEQ ID NO:2), which was determined by sequencing a cloned cDNA As shown in Figuie 2 the TR9 receptoi piotein of the piesent invention shares sequence homology with Fas (SEQ ID NO 3 ) NGFR p75 (SEQ ID NO 4), and TNFR 1 (SEQ ID NO 5) The nucleotide sequence shown in SEQ ID NO 1 was obtained by sequencing a cDNA clone which was deposited on May 15, 1997 at the Amencan Type Cultuie Collection 10801 Univeisity Boulevaid Manassas, Virginia, 201 10-2209, and given accession numbei 209037 The deposited clone is inserted in the pBluescript SK(-)
  • nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc ), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99 9% identical to the actual nucleotide sequence of the sequenced DNA molecule The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art As is also known in the art, a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift m translation of the nucleotide sequence such that the predicted ammo acid sequence encoded by a determined nucleotide sequence will be completely different
  • a nucleic acid molecule of the present invention encoding a TR9 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material Illustra e of the invention, the nucleic acid molecule described in Figures 1A-D (SEQ ID NO 1 ) was discovered in a cDNA library derived from human microvascular endothelial cells The gene was also identified in cDNA libraries from the following tissues human placenta, stromal cells, human amygdala, human umbilical vein endothelial cells, kidney cancel, human gall bladder, reportedly adult brain, normal human liver, hepatocellular tumoi .
  • the determined nucleotide sequence of the TR9 cDNA of Figures 1A-D contains an open reading frame encoding a protein of about 615 amino acid residues, with a predicted leader sequence of about 40 amino acid residues, and a deduced molecular weight of about 72 kDa.
  • the amino acid sequence of the predicted mature TR9 receptor is shown in Figures 1 A-D (SEQ ID NO:2) from amino acid residue about 1 to residue about 615.
  • the TR9 protein shown in Figures 1A-D (SEQ ID NO:2) is about 24% identical and about 43% similar to NGFR ( Figure 2).
  • TR9 induces apoptosis of mammalian cells (see Figure 6). It is expected that TR9-induced apoptosis will be efficiently blocked by inhibitors of death proteases including z-NAD-fmk, an irreversible broad spectrum caspase inhibitor and CrmA, a cowpox virus encoded serpin that preferentially inhibits apical caspases such as FLICE/MACH-1 (caspase-8).
  • the present invention also provides the mature form(s) of the TR9 receptor of the present invention.
  • proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • Most mammalian cells and even insect cells cleave secreted proteins with the same specificity.
  • cleavage of a secreted protein is not entirely uniform, which results in two or more mature species on the protein.
  • the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
  • the present invention provides a nucleotide sequence encoding the mature TR9 receptor polypeptides having the amino acid sequence encoded by the cD ⁇ A clone contained in the host identified as ATCC Deposit No. 209037 and as shown in Figures 1A-D (SEQ ID NO:2).
  • the mature TR9 protein having the amino acid sequence encoded by the cDNA clone contained in the host identified as ATCC Deposit 209037 is meant the mature form(s) of the TR9 receptor produced by expression in a mammalian cell (e.g., COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the clone contained in the vector in the deposited host.
  • the mature TR9 receptor having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 209037 may or may not differ from the predicted "mature" TR9 receptor protein shown in SEQ ID NO:2 (amino acids from about 1 to about 615) depending on the accuracy of the predicted cleavage site based on computer analysis.
  • the predicted amino acid sequence of the complete TR9 polypeptides of the present invention were analyzed by a computer program ( 'PSORT”) (K Nakai and M Kanehisa, Genomics 14 897-91 1 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence
  • 'PSORT a computer program
  • the analysis by the PSORT program predicted the cleavage site between amino acid lesidues 40 and 41 in Figures 1A-D (amino acid residues -1 and 1 in SEQ ID NO 2)
  • the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (- 1 ,-3) rule ot von Heinje von Heinje, supia
  • the leader sequence for the TR9 receptor protein is predicted to consist of amino acid residues from about 1 to 40 in Figures 1 A-D (amino acid residues -40 to about -1 in S
  • the predicted TR9 receptor polypeptide encoded by the deposited cDNA comprises about 655 ammo acids, but may be anywhere in the range of 645-665 amino acids, and the predicted leader sequence of this protein is about 40 amino acids, but may be anywhere in the range of about 30 to about 50 ammo acids
  • the domains described herein have been predicted by computer analysis, and accordingly, that depending on the analytical criteria used for identifying various functional domains, the exact "address" of, for example, the extracelluai domain, intracelluar domain, death domain, cystein- ⁇ ch motifs, and transmembrane domain of TR9 may differ slightly
  • the exact location of the TR9 extracellular domain in Figures 1 A-D may vary slightly (e g , the address may ' shift" by about 1 to about 20 residues, more likely about 1 to about 5 residues
  • the invention further provides polypeptides having va ⁇ ous lesidues deleted horn the N-terminus and/oi C-tei minus ol the complete TR9. including polypeptides lacking one oi moie ammo acids fiom the N-termim of the extiacellulai domain described heiem, which constitute soluble forms ol the extracellulai domain of the TR9 polypeptides As indicated, nucleic acid molecules of the piesent invention may be in the foim of
  • RNA such as mRNA
  • RNA in the foim of DNA. including, foi instance.
  • cDNA and genomic DNA obtained by cloning or pioduced synthetically
  • the DNA may be double- stranded or single-stranded Single-stranded DNA oi RNA may be the coding strand, also known as the sense strand, oi it may be the non-coding strand, also referred to as the anti-sense strand
  • isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA oi RNA, which has been removed from its native environment
  • iecombinant DNA molecules contained in a vector are consideied isolated for the residueposes of the present invention
  • Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution
  • nucleic acid molecules having nucleotide sequences related to extensive portions of the nucleotide sequence in Figures 1A-D of (SEQ ID NO: 1 ), which have been determined from the following related cDNA clones
  • the invention includes a polynucleotide. oi du ely consisting ot. any poition of at least about 30 nucleotides. pieteiably at least about 50 nucleotides.
  • nucleotide sequence disclosed in Figuies 1A-D fiom nucleotides 655 to 907 nucleotides 615 to 867 ot SEQ ID NO 1
  • nucleotide sequence disclosed in Figures 1 A-D fiom nucleotides to 540 to 1020 nucleotides 500 to 980 as depicted in SEQ ID NO. l.
  • the invention provides isolated nucleic acid molecules encoding the TR9 receptor polypeptide having an ammo acid sequence as encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No 209037 on May 15. 1997.
  • nucleic acid molecules are provided encoding the mature TR9 receptor polypeptide or the full-length TR9 receptoi polypeptide lacking the N- terminal methionine.
  • the invention also provides an isolated nucleic acid molecule having the nucleotide sequence shown m Figuies 1 A-D (SEQ ID NO 1) or the nucleotide sequence of the TR9 cDNA contained in the above-descnbed deposited clone, oi a nucleic acid molecule having a sequence complementary to one of the above sequences
  • isolated molecules particularly DNA molecules, have uses which include, but are not limited to, as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TR9 receptor gene in human tissue, for instance, by Northern blot analysis
  • the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
  • fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA (the clone deposited as ATCC Deposit No 209037), or the nucleotide sequence shown in Figures 1 A-D (SEQ ID NO 1), or the complementary strand thereto is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt.
  • fragments have numerous uses which include, but are not limited to, diagnostic probes and primers as discussed herein
  • larger fragments of 50-1500 nt oi 501-1500 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of the deposited cDNA or as shown in Figures 1A-D (SEQ ID NO 1)
  • fragments which include 20 or more contiguous bases from the nucleotide sequence of the deposited cDNA or the nucleotide sequence as shown in Figures 1A-D (SEQ ID NO.1)
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, oi 1) nucleotides, at either terminus or at both termini.
  • TR9 polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1-50, 51-100. 101-150. 151-200. 201-250, 251-300. 301-350, 351-400, 401-450, 445-879, 451-500. 501-550, 551-600. 615-651 , 651-700, 701-750. 751-800, 800-850, 850-867, 851 -900, 901-950.
  • 951 - 1000 1001-1050, 1051-1 100, 1 101-1 150, 1 151-1200, 1201-1250, 1251-1300, 1301 -1350, 1351- 1400, 1401-1450, 1451-1500, 1501-1550. 1551- 1600, 1601-1650, 1651-1700. 1701-1750, 1751-1800, 1801-1850, 1851-1900. 1901-1950, 1951-2000. 2001-2050, 2051-3000, or 3001 to the end of SEQ ID NO: 1 , or the complementary DNA strand thereto, or the cDNA contained in the deposited clone.
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
  • the polynucleotide fragments of the invention encode a polypeptide which demonstrates a TR9 functional activity.
  • a polypeptide demonstrating a TR9 "functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a complete (full-length) or mature TR9 polypeptide.
  • Such functional activities include, but are not limited to, biological activity (e.g., ability to induce apoptosis in cells expressing the polypeptide (see e.g., Example 5), the ability to activate monocytes (e.g., induce TNF-alpha and/or MCP-1 secretion from monocytes), and the ability to increase survival of monocytes (see, e.g., Example 8), antigenicity [ability to bind (or compete with a TR9 polypeptide for binding) to an anti-TR9 antibody], immunogenicity (ability to generate antibody which binds to a TR9 polypeptide), ability to form multimers, and ability to bind to a receptor or ligand for a TR9 polypeptide (e.g., AIM-II and TR9 ligands expressed on the surface of monocytes).
  • biological activity e.g., ability to induce apoptosis in cells expressing the polypeptide (see e.g., Example 5
  • monocytes e.g.
  • TR9 polypeptides and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
  • various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays.
  • immunoradiometric assays gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
  • antibody binding is detected by detecting a label on the primary antibody.
  • the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
  • the secondary antibody is labeled.
  • binding can be assayed, e.g., by means well-known in the art, such as, for example, reducing and non- reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, et al., Microbiol. Rev. 59:94- 123 ( 1995).
  • physiological correlates of TR9 binding to its substrates can be assayed.
  • TR9 polypeptides and fragments, variants derivatives and analogs thereof may routinely be applied to measure the ability of TR9 polypeptides and fragments, variants derivatives and analogs thereof to elicit TR9 related biological activity (e.g., ability to induce apoptosis in cells expressing the polypeptide (see e.g., Example 6), the ability to activate monocytes, and the ability to increase survival of monocytes (see, e.g., Examples 8 and 9) in vitro or in vivo.
  • TR9 related biological activity e.g., ability to induce apoptosis in cells expressing the polypeptide (see e.g., Example 6), the ability to activate monocytes, and the ability to increase survival of monocytes (see, e.g., Examples 8 and 9) in vitro or in vivo.
  • biological activity can routinely be measured using the cell death assays performed essentially as previously described (Chinnaiyan et al., Cell 81 :50
  • plasmids encoding full-length TR9 or a candidate death domain containing receptor are co-transfected with the pLantern reporter construct encoding green fluorescent protein. Nuclei of cells transfected with TR9 will exhibit apoptotic morphology as assessed by DAPI staining.
  • nucleic acid fragments of the present invention include a nucleic acid molecule encoding a member selected from the group: a polypeptide comprising or alternatively, consisting of, the TR9 receptor extracellular domain (predicted to constitute amino acid residues from about 1 to about 310 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, the four TNFR-like cysteine rich motifs of TR9 (amino acid residues 67 to 211 in Figures 1A-D; amino acid residues 27 to 171 in SEQ ID NO:2); a polypeptide comprising or alternatively, consisting of, the TR9 receptor transmembrane domain (predicted to constitute amino acid residues from about 311 to about 327 in SEQ ID NO 2) a polypeptide compnsmg, oi altemativ ely.
  • TR9 functional activity e g . antigenic activity oi biological acitivity
  • a polypeptide comprising or alternatively consisting of the TR9 leceptor mtiacellular domain piedicted to constitute ammo acid residues from about 328 to about 615 in SEQ ID NO 2
  • a polypeptide compnsmg oi alternatively, consisting of, the TR9 receptor extracellular and intracellular domains with all oi part of the transmembrane domain deleted a polypeptide comprising oi alternatively , consisting of, the TR9 leceptor death domain (predicted to constitute ammo acid lesidues fiom about 389 to about 455 in SEQ ID NO 2)
  • the polynucleotides of the invention encode functional att ⁇ butes of TR9
  • Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consist of, alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming legions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophihc regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic legions, flexible regions, suiface-foiming legions and high antigenic index regions of TR9 polypeptides
  • prefe ⁇ ed regions set out in Figuie 3 and in Table I include, but are not limited to, regions ot the afoiementioned types identified by analysis of the amino acid sequence set out in Figures 1A-D As set out in Figure 3 and in Table I, such preferred regions include Gargori-Robson alpha-iegions.
  • polypeptides comprise, or alternatively consist of, regions of TR9 that combine several structural features, such as several (e g , 1, 2, 3, or 4) of the same or diffeient region features set out above
  • the data presented in columns NIII, IX, XIII, and XIN of Table I can routinely be used to determine regions of TR9 which exhibit a high degree of potential for antigenicity Regions of high antigenicity are determined from the data presented in columns NIII, IX, XIII, and/or IN by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response
  • Lys 342 A C 1 08 -0 13 * 0 65 1 01
  • nucleic acid fragments of the present invention also include nucleic acid molecules encoding polypeptides comprising, or alternatively consisting of, one, two, three, four, five, or more epitope-bearing portions of the TR9 receptor protein.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising or alternatively, consisting of, amino acid residues from about 4 to about 81 in SEQ ID NO:2; a polypeptide comprising or alternatively, consisting of, amino acid residues from about 1 16 to about 271 in SEQ ID NO:2; a polypeptide comprising or alternatively, consisting of, amino acid residues from about 283 to about 308 in SEQ ID NO:2; a polypeptide comprising or alternatively, consisting of.
  • polypeptides of the invention are less than
  • polynucleotides of the invention comprise, or alternatively consist of, at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TR9 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide set forth in Figures 1 A-D (SEQ ID NO: 1).
  • polynucleotides of the invention comprise, or alternatively consist of, at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TR9 coding sequence, but do not comprise all or a portion of any TR9 intron.
  • the nucleic acid comprising, or alternatively consisting of, TR9 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TR9 gene in the genome).
  • the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes, preferably under stringent hybridization conditions, to a portion of the polynucleotide sequence of a polynucleotide of the invention such as, for instance, the sequence complementary to the coding and/or noncoding (i.e., transcribed, untranslated) sequence depicted in Figures 1 A-D, the sequence of the cDNA clone contained in ATCC Deposit 209037 and the sequence encoding a TR9 domain or a polynucleotide fragment as described herein.
  • a polynucleotide which hybridizes, preferably under stringent hybridization conditions, to a portion of the polynucleotide sequence of a polynucleotide of the invention such as, for instance, the sequence complementary to the coding and/or noncoding (i.e., transcribed, untranslated) sequence depicted in Figures 1 A
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising: 50% formamide, 5x SSC (750 mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 0.1 x SSC at about 65°C.
  • a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70, or 80-150 nt, or the entire length of the reference polynucleotide.
  • “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These have uses, which include, but are not limited to, as diagnostic probes and primers as discussed above and in more detail below.
  • polynucleotides of the invention hybridize to a complementary strand of a polynucleotide encoding amino acid residues 40-152, 40-48, 40-51, 51-66, 66-73, 73-83, 83-104, 104-1 10, 1 10-128, 128-146, and/or 146-152 as depicted in SEQ ID NO:2.
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly tract of the TR9 receptor cDNA shown in SEQ ID NO: 1
  • a complementary stretch of T (or U) residues would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone generated using oligo dT as a primer).
  • nucleic acid molecules of the present invention which encode a TR9 receptor polypeptide may include, but are not limited to, those encoding the amino acid sequence of the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding the about amino acid leader or secretory sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non- translated sequences that play a role in transcription, mRNA processing, including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; an additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
  • the sequence encoding the polypeptide may be fused to a marker sequence, such as a sequence encoding a peptide which facilitates purification of the fused polypeptide.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available. As described in Gentz et al., Proc. Natl. Acad. Sci. USA 86:821-824 (1989), for instance, hexa-histidine provides for convenient purification of the fusion protein.
  • the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:767-778 (1984).
  • other such fusion proteins include the TR9 receptor fused to Fc at the N- or C-terminus.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs or derivatives of the TR9 receptor. Variants may occur naturally, such as a natural allelic variant. By an "allelic variant” is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism.
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis. site directed mutagenesis (see e.g., Carter et al., Nucl. Acids Res. 13:4331 (1986); and Zoller et al., Nucl. Acids Res.
  • cassette mutagenesis see e.g., Wells et al., Gene 34:315 (1985)
  • restriction selection mutagenesis see e.g., Wells er al., Philos. Trans. R. Soc. London SerA 317:415 (1986)).
  • variants include those produced by nucleotide substitutions, deletions or additions, which may involve one or more nucleotides.
  • the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and functional activities of the TR9 receptor or portions thereof Also especially pietened in this legaid are consetvative substitutions
  • nucleic acid molecules comprising, or alternatively consisting ot a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 80%. 85%, 95% 96%, 97%, 98% oi 99% identical to (a) a nucleotide sequence encoding the polypeptide having the amino acid sequence shown in Figures 1A-D (SEQ ID NO 2), (b) a nucleotide sequence encoding the polypeptide having the ammo acid sequence shown in Figuies 1A-D (SEQ ID NO 2), but lacking the N-terminal methionme, (c) a nucleotide sequence encoding the predicted mature TR9 polypeptide (full-length polypeptide with any attending leader sequence removed) comprising or alternatively consisting of, the amino acid sequence at positions from about 1 to about 615 in SEQ ID NO 2, (d) a nucleotide sequence encoding the TR9 polypeptide having the amino
  • a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a TR9 receptor polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding the TR9 receptor.
  • up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence
  • These mismatches of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere
  • the reference (query) sequence may be the entire TR9 nucleotide sequence shown in Figures 1 A-D (SEQ ID NO: 1) or any fragment (e.g., a polynucleotide encoding the amino acid sequence of a TR9 N and/or C terminal deletion described herein) as described herein.
  • nucleotide sequence shown in Figures 1 A-D (SEQ ID NO: 1 ) or to the nucleotides sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 5371 1). Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of this embodiment.
  • a 90 base subject sequence is compared with a 100 base query sequence. This time the deletions are internal deletions so that there are no bases on the 5' or 3' of the subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected. Once again, only bases 5' and 3' of the subject sequence which are not matched/aligned with the query sequence are manually corrected for. No other manual corrections are made for the purposes of this embodiment.
  • the present application is directed to nucleic acid molecules at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in Figures 1 A-D (SEQ ID NO: 1) or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having TR9 receptor functional activity.
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TR9 receptor functional activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
  • PCR polymerase chain reaction
  • nucleic acid molecules of the present invention that do not encode a polypeptide having TR9 receptor functional activity include, inter alia, (1) isolating the TR9 receptor gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TR9 receptor gene, as described in Nerma et al, Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, ⁇ .Y. (1988); and (3) Northern Blot analysis for detecting TR9 receptor mRNA expression in specific tissues.
  • nucleic acid molecules of the present invention that do not encode a polypeptide having TR9 receptor functional activity include, inter alia, (1) isolating the TR9 receptor gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TR9 receptor gene, as described in
  • polypeptide having TR9 receptor functional activity polypeptides exhibiting activity similar, but not necessarily identical, to a functional activity of the TR9 receptor of the invention (e.g., the full-length (i.e..
  • TR9 polypeptide functional activity can be measured by the ability of a polypeptide sequence described herein to form multimers (e.g.. homodimers and homotrimers) with complete TR9. and to bind a TR9 ligand (e.g., TR9 ligand expressed on the surface of monocytes).
  • TR9 polypeptide functional activity can be also be measured, for example, by determining the ability of a polypeptide of the invention to activate monocytes, increase cell survival of monocytes or to induce apoptosis in cells expressing the polypeptide.
  • TR9 receptor functional activity e.g., biological activity
  • TR9 receptor functional activity can routinely be measured using the cell death assays performed essentially as previously described (Chinnaiyan et al., Cell 81:505-512 (1995); Boldin et al., J. Biol. Chem.
  • plasmids encoding full-length TR9 or a candidate death domain containing receptor are co-transfected with the pLantern reporter construct encoding green fluorescent protein. Nuclei of cells transfected with TR9 will exhibit apoptotic morphology as assessed by DAPI staining.
  • TR9-induced apoptosis will be blocked by the inhibitors of ICE- like proteases, CrmA and z-NAD-fmk.
  • apoptosis induced by TR9 will be blocked by dominant negative versions of FADD (FADD-D ⁇ ) or FLICE (FLICE-D ⁇ /M ACHa 1 C360S) .
  • nucleic acid molecules having a sequence at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identical to, for example, the nucleic acid sequence of the deposited cD ⁇ A or the nucleic acid sequence shown in Figures 1A-D (SEQ ID ⁇ O: l), or fragments thereof, will encode a polypeptide "having TR9 receptor functional activity.”
  • degenerate variants of these nucleotide sequences all encode the same polypeptide. in many instances, this will be clear to the skilled artisan even without performing the above described assay.
  • nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having TR9 receptor functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g., replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
  • This invention is also related to the use of TR9 polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of TR9 associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression, over-expression, or altered expression of TR9 or a soluble form thereof, such as, for example, tumors or autoimmune diseases.
  • Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
  • the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. (Saiki et al, Nature 324: 163-166 ( 1986)).
  • RNA or cDNA may also be used in the same ways.
  • PCR primers complementary to the nucleic acid encoding TR9 can be used to identify and analyze TR9 expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
  • Point mutations can be identified by hybridizing amplified DNA to radiolabeled TR9 RNA or alternatively, radiolabeled TR9 antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing. In addition, cloned DNA segments may be employed as probes to detect specific DNA segments. The sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method.
  • a sequencing pnmei is used with double stianded PCR product oi a single- stianded template molecule geneiated by a modified PCR
  • sequence detei mination is peifoimed by conventional piocedures with ladiolabeled nucleotide or by automatic sequencing procedures with fluoiescent-tags
  • Genetic testing based on DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fiagments in gels, with oi without denatuiing agents Small sequence deletions and insertions can be visualized by high resolution gel electiophoresis DNA fragments of diffeient sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments aie letarded in the gel at different positions according to then specific melting or partial melting tempeiatures (see, e g , Myeis et al , Science 230 1242 ( 1985))
  • Sequence changes at specific locations also may be levealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g , Cotton et al , Pioc Natl Acad Sci USA 85 4397-4401 (1985))
  • the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e g , restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA
  • restriction enzymes e g , restriction fragment length polymorphisms ("RFLP")
  • RFLP restriction fragment length polymorphisms
  • Southern blotting of genomic DNA In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis
  • the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors, or which are otherwise engineered to produce the polypeptides of the invention, and the production of TR9 receptor polypeptides, or fiagments thereof, by recombinant or synthetic techniques
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host Generally, a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged pid If the vector is a virus, it may be packaged in vitro using an appropriate packaging cell line and then transduced into host cells
  • the DNA of the invention is operatively associated with an appropriate heterologous regulatory element (e g , promoter or enhancer), such as, the phage lambda PL promoter, the E coh lac, trp, and tac promoters, the SN40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate heterologous regulatory element e g , promoter or enhancer
  • suitable promoters will be known to the skilled artisan.
  • these constructs will further contain sites for transcription initiation, termination and, in the transcribed region, a ribosome binding site for translation.
  • the coding portion of the mature transcripts expressed by the constructs will preferably include a translation initiating at the beginning and a termination codon (UAA, UGA or UAG) appropriately positioned at the end of the polypeptide to be translated.
  • the expression vectors will preferably include at least one selectable marker.
  • markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture and tetracycline or ampicillin resistance genes for culturing in E. coli and other bacteria.
  • Representative examples of appropriate hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No.
  • insect cells such as Drosophila S2 and Spodoptera Sf9 cells
  • animal cells such as CHO, COS and Bowes melanoma cells
  • plant cells Appropriate culture mediums and conditions for the above-described host cells are known in the art.
  • vectors preferred for use in bacteria include pH ⁇ 4 (ATCC Accession Number 209645, deposited February 25, 1998), pQE70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia.
  • Preferred expression vectors for use in yeast systems include, but are not limited to, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3.5, pHIL-D2, pHIL-S l, pPIC3.5K, pPIC9K, and PAO815 (all available from Invitrogen, Carlsbad, CA).
  • the present invention also relates to host cells containing the vector constructs discussed herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g., promoter and/or enhancer) using techniques known of in the art.
  • the host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g., a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
  • the host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired.
  • Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
  • different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g., phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
  • Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation. transduction. infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al., Basic Methods In Molecular Biology ( 1986).
  • TR9 polypeptides can be recovered and purified from recombinant cell cultures by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • TR9 polypeptides and preferably the secreted form, can also be recovered from: products purified from natural sources, including bodily fluids, tissues and cells, whether directly isolated or cultured; products of chemical synthetic procedures; and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect, and mammalian cells.
  • the yeast Pichia pastoris is used to express TR9 protein in a eukaryotic system.
  • Pichia pastoris is a methylotrophic yeast which can metabolize methanol as its sole carbon source.
  • a main step in the methanol metabolization pathway is the oxidation of methanol to formaldehyde using O 2 . This reaction is catalyzed by the enzyme alcohol oxidase.
  • Pichia pastoris In order to metabolize methanol as its sole carbon source, Pichia pastoris must generate high levels of alcohol oxidase due, in part, to the relatively low affinity of alcohol oxidase for O 2 .
  • alcohol oxidase genes are highly active.
  • alcohol oxidase produced from the AOX1 gene comprises up to approximately 30% of the total soluble protein in Pichia pastoris. See, Ellis, S.B., et al, Mol. Cell. Biol. 5: 1 1 11-21 (1985); Koutz, P.J, et al , Yeast 5 167-77 ( 1989), Tschopp. J F .
  • a heteiologous coding sequence such as, foi example, a TR9 polynucleotide of the piesent invention, undei the ti nscnptional regulation of all or part of the AOXI regulatory sequence is expressed at exceptionally high levels in Pichia yeast grown in the presence of methanol
  • the plasmid vector pPIC9K is used to express DNA encoding a TR9 polypeptide of the invention, as set forth herein, in a Pichea yeast system essentially as described in "Pichia Piotocols Methods in Moleculai Biology," D R Higgins and J Cregg, eds The Humana Press, Totowa. NJ, 1998
  • This expiession vector allows expression and secretion of a TR9 protein of the invention by virtue of the strong AOXI promoter linked to the Pichia pastoi is alkaline phosphatase (PHO) secretory signal peptide (l e , leader) located upstream of a multiple cloning site
  • yeast vectors could be used in place of pPIC9K, such as, pYES2, pYDl, pTEFl/Zeo, pYES2/GS, pPICZ, pGAPZ, pGAPZalpha, pPIC9, pPIC3 5, pHIL- D2, pHIL-S l, pPIC3 5K, and PAO815, as one skilled in the art would readily appreciate, as long as the proposed expression construct piovides appropriately located signals for transcription, translation, secretion (if desired), and the like, including an m- frame AUG as required
  • high-level expression ot a heterologous coding sequence such as, for example, a TR9 polynucleotide of the present invention
  • a heterologous coding sequence such as, for example, a TR9 polynucleotide of the present invention
  • an expression vector such as, for example, pGAPZ or pGAPZalpha
  • the TR9 polypeptides may be glycosylated or may be non-glycosylated
  • TR9 polypeptides may also include an initial modified methionme lesidue, in some cases as a result of host-mediated processes
  • the N-terminal methionme encoded by the translation initiation codon generally is removed with high efficiency from any protein after translation in all eukaryotic cells
  • the N-terminal methionme on most proteins also is efficiently removed in most prokaryotes, for some proteins, this prokaryotic removal process is inefficient, depending on the nature of the amino acid to which the N-terminal methionme is covalently linked
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e g , TR9 coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with TR9 polynucleotides ol the invention and which activates, alteis, and/oi amplifies endogenous TR9 polynucleotides
  • genetic material e.g., heterologous polynucleotide sequences
  • techniques known in the art may be used to operably associate heterologous contiol regions (e g .
  • the polypeptide may be expressed or synthesized in a modified foim, such as a fusion protein (comprising, oi alternatively consisting of, the polypeptide joined via a peptide bond to a heterologous protein sequence (of a different protein)), and may include not only secretion signals, but also additional heterologous functional regions
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubihze proteins
  • EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising vanous portions of constant region of lmmunoglobin molecules together with another human protein or part thereof
  • the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EPA 0 232 262).
  • fusion proteins of the invention comprise, or alternatively, consist of, amino acid residues 1 to 310 of SEQ ID NO:2 fused to an Fc polypepitde sequence.
  • TR9 polypeptides of the present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or may be non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue or alternatively, may be missing the N- terminal methionine, in some cases as a result of host-mediated processes.
  • polypeptides of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and
  • a peptide corresponding to a fragment of the TR9 polypeptides of the invention can be synthesized by use of a peptide synthesizer.
  • nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the TR9 polypeptide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b-methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general. Furthermore, the amino acid
  • Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al, Nucl. Acids Res. 73:4331 (1986); and Zoller et al, Nucl. Acids Res. 70:6487 (1982)), cassette mutagenesis (see, e.g., Wells et al. Gene 34:315 ( 1985)). restriction selection mutagenesis (see, e.g., Wells et al, Philos. Trans. R. Soc. London SerA 377:415 ( 1986)).
  • TR9 proteins which are differentially modified during or after translation, e.g., by glycosylation, acetylation. phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH. acetylation, formylation. oxidation, reduction, metabolic synthesis in the presence of tunicamycin; etc.
  • Additional post-translational modifications encompassed by the invention include, for example, e.g.. N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
  • the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation of the protein.
  • the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
  • the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
  • the polymer may be of any molecular weight, and may be branched or unbranched.
  • the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
  • Other sizes may be used, depending on the desired therapeutic profile (e.g., the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects of the polyethylene glycol to a therapeutic protein or analog).
  • the polyethylene glycol may have an average molecular weight of about 200,
  • the polyethylene glycol may have a branched structure.
  • polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol 56:59-72 (1996); Vorobjev et al, Nucleosides Nucleotides 18:2145-2150 ( 1999); and Caliceti et al, Bioconjug. Chem. 70:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
  • the polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein.
  • polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
  • Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
  • the amino acid residues having a free amino group may include lysine residues and the N-terminal amino acid residues; those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
  • Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
  • polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
  • polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
  • One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
  • polypeptides chemically modified at the N-terminus may specifically desire polypeptides chemically modified at the N-terminus.
  • polyethylene glycol as an illustration of the present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
  • fiom othei monopegylated moieties may be by pu ⁇ fication of the N-teiminally pegylated mate ⁇ al fiom a population of pegylated piotein molecules
  • Selective polypeptides chemically modified at the N-termmus modification may be accomplished by leductive alkylation which exploits differential reactivity of different types of pnmary ammo gioups (lysine versus the N-terminal) available tor de ⁇ vatization in a particular protein Under the appropriate reaction conditions, substantially selective de ⁇ vatization of the protein at the N-teiminus with a carbonyl gioup containing polymei is achieved
  • pegylation of the proteins of the invention may be accomplished by any number of means
  • polyethylene glycol may be attached to the protein either directly or by an intervening hnkei Lmkerless systems foi attaching polyethylene glycol to proteins are described in Delgado et al , C ⁇ it Rev Thera Drug Carriei S ⁇ s 9 249-304 (1992), Francis et al , Intent J ofHematol 68 1- 18 (1998), U S Patent No 4,002,531 , U S Patent No 5,349,052, WO 95/06058, and WO 98/32466, the disclosures of each of which are lncorpoiated herein by reference
  • One system foi attaching polyethylene glycol directly to ammo acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchlo ⁇ de (ClSO 2 CH 2 CF,) Upon
  • Polyethylene glycol can also be attached to pioteins using a number of different intervening linkers
  • U S Patent No 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins
  • Protem-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG-succimmidylsuccmate, MPEG activated with 1 , 1 '-carbonyldnmidazole, MPEG-2,4,5-t ⁇ chloropeny lcarbonate, MPEG-p- mtrophenolcarbonate, and various MPEG-succinate derivatives
  • MPEG-succimmidylsuccmate MPEG activated with 1 , 1 '-carbonyldnmidazole
  • MPEG-2,4,5-t ⁇ chloropeny lcarbonate MPEG-p- mtrophenolcarbonate
  • various MPEG-succinate derivatives A number additional polyethylene glycol derivatives and reaction
  • the number of polyethylene glycol moieties attached to each protein of the invention may also vary
  • the pegylated proteins of the invention may be linked, on average, to 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20. or more polyethylene glycol molecules.
  • the average degree of substitution within ranges such as 1-3. 2-4. 3-5. 4-6, 5-7, 6-8, 7-9. 8- 10. 9-1 1 , 10- 12. 1 1-13. 12-14. 13- 15. 14-16, 15-17, 16-18, 17-19.
  • the TR9 can be recovered and purified by standard methods which include, but are not limited to, ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography. hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
  • HPLC high performance liquid chromatography
  • TR9 receptor polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of TR9.
  • applications in treatment prevention, diagnosis, and/or detection of tumors, resistance to parasites, bacteria and viruses, to induce proliferation of T-cells, endothelial cells and certain hematopoietic cells, to treat, prevent, diagnose, and/or detect restenosis, graft vs. host disease, to regulate anti-viral responses and to prevent certain autoimmune diseases after stimulation of TR9 by an agonist.
  • Additional applications relate to diagnosis and to treatment, prevention. diagnosis, and/or detection of disorders of cells, tissues and organisms. These aspects of the invention are discussed further below.
  • TR9 polypeptides of the invention can also be expressed in transgenic animals.
  • Animals of any species including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
  • techniques described herein or otherwise known in the art are used to express polypeptides of the invention in humans, as part of a gene therapy protocol.
  • transgene i.e., nucleic acids of the invention
  • transgene i.e., nucleic acids of the invention
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson et al, Appl. Microbiol. Biotechnol. 40:691-698 (1994); Carver et al., Biotechnology (NY) 11: 1263-1270 (1993); Wright et al., Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., US Patent Number 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al., Proc. Natl. Acad.
  • transgenic clones containing polynucleotides of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al.. Nature 380:64-66 (1996); Wilmut et al., Nature 385:810-813 (1997)), each of which is herein incorporated by reference in its entirety).
  • the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i.e., mosaic animals or chimeric animals.
  • the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g., head-to-head tandems or head-to-tail tandems.
  • the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci. USA 89:6232-6236 (1992)).
  • the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
  • gene targeting is preferred.
  • vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
  • the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Science 265: 103-106 (1994)).
  • the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
  • the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene-expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
  • founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
  • breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
  • Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of TR9 polypeptides, studying conditions and or disorders associated with aberrant TR9 expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
  • cells that are genetically engineered to express the proteins of the invention, or alternatively, that are genetically engineered not to express the proteins of the invention are administered to a patient in vivo.
  • Such cells may be obtained from the patient (i.e., animal, including human) or an MHC compatible donor and can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc.
  • the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and or endogenous regulatory sequence associated with the polypeptides of the invention, e.g., by transduction (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
  • the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
  • the engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically. e.g., in the circulation, or intraperitoneally. Alternatively, the cells can be incorporated into a matrix and implanted in the body, e.g., genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft. (See, for example, Anderson et al., US Patent Number 5,399.349; and Mulligan & Wilson, US Patent Number 5.460,959, each of which is incorporated by reference herein in its entirety).
  • the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
  • the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
  • the invention further provides for proteins containing polypeptide sequences encoded by polynucleotides of the invention.
  • the TR9 proteins of the invention may be in monomers or multimers (i.e., dimers, trimers. tetramers, and higher multimers). Accordingly, the present invention relates to monomers and multimers of the TR9 proteins of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
  • the polypeptides of the invention are monomers, dimers, trimers or tetramers.
  • the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
  • Multimers encompassed by the invention may be homomers or heteromers.
  • the term homomer refers to a multimer containing only TR9 proteins of the invention (including TR9 fragments, variants, and fusion proteins, as described herein). These homomers may contain TR9 proteins having identical or different polypeptide sequences.
  • a homomer of the invention is a multimer containing only TR9 proteins having an identical polypeptide sequence.
  • a homomer of the invention is a multimer containing TR9 proteins having different polypeptide sequences.
  • the multimer of the invention is a homodimer (e.g., containing TR9 proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing TR9 proteins having identical or diffeient polypeptide sequences)
  • the homomenc multimei of the invention is at least a homodimei, at least a homot ⁇ mer, 01 at least a homotetiamei
  • the term heteiomer reie is to a multimer containing heterologous proteins (1 e , proteins containing only polypeptide sequences that do not conespond to a polypeptide sequences encoded by the TR9 gene) in addition to the TR9 pioteins of the invention
  • the multimer of the invention is a heterodimei.
  • the homomenc multimer of the invention is at least a homodimer at least a homot ⁇ mer, or at least a homotetiamer
  • Multimers of the invention may be the result of hydrophobic hydiophi c, ionic and or covalent associations and/or may be indiiectly linked, by for example, liposome formation
  • multimei s of the invention such as, for example, homodimers or homot ⁇ mers, aie formed when pioteins of the invention contact one another solution
  • heteromultimers of the invention such as, for example, heterot ⁇ meis or heterotetramers are formed when proteins of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution
  • multimers of the invention are formed by covalent associations with and/or between the TR9 proteins
  • the multimei s of the invention may be geneiated using chemical techniques known in the art
  • proteins desned to be contained in the multimei s of the invention may be chemically cioss-hnked using nkei molecules and linkei molecule length optimization techniques known in the art (see, e g , US Patent Number 5,478,925, which is herein mcorpoiated by reference in its entirety)
  • multimei s of the invention may be geneiated using techniques known in the art to form one or more mtei- molecule cioss-links between the cysteine residues located within the polypeptide sequence of the proteins desned to be contained in the multimei (see e g , US Patent Number 5,478.925.
  • proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus oi N-termmus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one oi more of these modified proteins (see, e g , US Patent Number 5 478,925, which is herein incorporated by reference in its entirety) Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer of the invention (see, e g , US Patent Number 5,478,925 which is herein incorporated by leference in its entirety)
  • multimers of the invention may be generated using genetic engineering techniques known in the art
  • proteins contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e g , US Patent Number 5,478,925, which is herein incorporated by reference in its entirety)
  • polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e g , US Patent Number 5,478,925, which is herein incorporated by reference in its entirety)
  • recombinant techniques described herein or otherwise known in the art are applied to generate recombinant poly
  • polypeptide fragments of the present invention include polypeptides compnsmg, or alternatively consisting of, an ammo acid sequence contained in SEQ ID NO 2, encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize (e g , under stringent hyb ⁇ dization conditions) to the nucleotide sequence contained in the deposited clone, oi shown in Figuies 1A-D (SEQ ID NO 1) or the complementary strand thereto Piotem fragments may be ' free-standing or comprised withm a larger polypeptide of which the fiagment forms a part oi region, most preferably as a smgle continuous region
  • Representative examples of polypeptide fragments of the invention include, for example, fragments that
  • polypeptide fragments can be at least about 20, 30, 40, 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, or 150 amino acids in length
  • about includes the particularly recited value, larger or smaller by seveial (5, 4, 3, 2, or 1) ammo acids, at either extreme or at both extremes
  • polypeptide fiagments of the invention comprise, or alternatively, consist of, amino acid residues 40
  • TR9 functional activity e.g., antigenic activity or biological acitivity
  • a polypeptide comprising or alternatively, consisting of, the TR9 receptor intracellular domain predicted to constitute amino acid residues from about 328 to about 615 in SEQ ID NO:2
  • a polypeptide comprising or alternatively, consisting of, the TR9 receptor extracellular and intracellular domains with all or part of the transmembrane domain deleted a polypeptide comprising, or alternatively consisting of, the TR9 receptor death domain (predicted to constitute amino acid residues from about 389 to about 455 in SEQ ID NO:2)
  • a polypeptide comprising, or alternatively, consisting of, one e.g., antigenic activity or biological acitivity
  • a polypeptide comprising or alternatively, consisting of, the TR9 receptor intracellular domain predicted to constitute amino acid residues from about 328 to about 615 in SEQ ID NO:2
  • the polypeptide fragments of the invention comprise, or alternatively, consist of, any combination of 1, 2, 3. 4, 5, 6, 7, or all 8 of the above members.
  • the amino acid residues constituting the TR9 receptor extracellular, transmembrane and intracellular domains have been predicted by computer analysis.
  • the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to about 15 amino acid residues) depending on the criteria used to define each domain.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • TR9 extracellular cysteine-rich motifs of TR9 is important for interactions between TR9 and its ligands.
  • polypeptide fragments of the invention comprise, or alternatively consist of amino acid residues 27 to 65, 66 to 105, 106 to 145, and/or 146 to 171 of SEQ ID NO:2.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination of 1, 2, 3, or all 4 of the extracellular cysteine-rich motifs disclosed in Figures 1A-D and Figure 4B.
  • fragments of the invention are fragments comprising, or alternatively, consisting of structural or functional attributes of TR9.
  • Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta-regions"), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil- regions”), hydrophillic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., regions of polypeptides consisting of amino acid residues having an antigenic index of or equal to greater than 1.5, as identified using the default parameters of the Jameson-Wolf program) of TR9.
  • antigenic index regions i.e., regions of polypeptides consisting of amino acid residues having an antigenic index of or equal to greater than 1.5, as identified using the default parameters of the Jameson-Wolf program
  • Certain preferred regions are those disclosed in Figure 3 and Table I and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in Figuies 1A-D.
  • piefe ⁇ ed legions include, Garmer-Robsonpieicted alpha-iegions. beta-iegions, turn-iegions.
  • the invention provides a peptide or polypeptide compnsmg, oi alternatively, consisting of, one, two. three, four, five or more, epitope-bearing portions of a polypeptide of the invention
  • the epitope of this polypeptide portion is an immunogenic or antigenic epitope of a polypeptide desc ⁇ bed herein
  • An "immunogenic epitope” is defined as a part of a protein that elicits an antibody iesponse when the whole protein is the immunogen
  • a region of a piotem molecule to which an antibody can bind is defined as an ' antigenic epitope
  • the number of immunogenic epitopes of a protein generally is less than the numbei of antigenic epitopes See, for instance, Geysen et al , Proc Natl Acad Sci USA 81 3998- 4002 ( 1983)
  • peptides or polypeptides bearing an antigenic epitope that contain a region of a protein molecule to which an antibody can bind
  • relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein See, for instance.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention See, for instance, Wilson et al , Cell 37 161 -US (1984) at 777.
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TR9 receptor-specific antibodies include, a polypeptide comprising, or alternatively consisting of, amino acid residues from about 4 to about 81 in SEQ ID NO:2, about 1 16 to about 271 in SEQ ID NO:2.
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means.
  • R.A. Houghten "General Method for the Rapid Solid-Phase Synthesis of Large Numbers of Peptides: Specificity of Antigen-Antibody Interaction at the Level of Individual Amino AcidsJ Proc. Natl. Acad. Sci. USA 82:5131-5135 (1985).
  • SMPS Simultaneous Multiple Peptide Synthesis
  • TR9 receptor polypeptides of the present invention and the epitope-bearing fragments thereof described above can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al., Nature 331:84-86 (1988)).
  • Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TR9 protein or protein fragment alone (Fountoulakis et al, J. Biochem. 270:3958-3964 (1995)).
  • TR9 polypeptides protein engineering may be employed.
  • Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability.
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
  • proteins including the extracellular domain of a membrane associated protein or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N- terminus or C-terminus without substantial loss of biological function.
  • TR9 functional activities may still be retained.
  • the ability of the shortened protein to induce and/or bind to antibodies which recognize TR9 will retained irrespective of the size or location of the deletion.
  • polypeptides composed of as few as six TR9 amino acid residues may often evoke an immune response. Whether a particular polypeptide lacking N-terminal and/or C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • N-terminus N-terminus. Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an TR9 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six TR9 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence of the TR9 polypeptide depicted in Figures 1A-D (SEQ ID NO:2) or encoded by the cDNA of the deposited clone.
  • N- terminal deletions of the TR9 polypeptide can be described by the general formula m to 615, where m is a number from -39 to 614 corresponding to the position of amino acid identified in SEQ ID NO:2 and preferably, corresponds to one of the N-terminal amino acid residues identified in the N-terminal deletions specified herein.
  • N-terminal deletions of the TR9 polypeptide of the invention comprise, or alternatively consist of, amino acid residues: Q-2 to L-615; P-3 to L-615; E-4 to L-615; Q-5 to L-615; K-6 to L-615; A-7 to L-615; S-8 to L-615; N-9 to L-615; L-10 to L-615; I- 11 to L-615; G-12 to L-615; T-13 to L-615; Y-14 to L-615; R-15 to L-615; H-16 to L- 615; V-17 to L-615; D-18 to L-615; R-19 to L-615; A-20 to L-615; T-21 to L-615; G-22 to L-615; Q-23 to L-615; V-24 to L-615; L-25 to L-615; T-26 to L-615; C-27 to L-615; D-28 to L-615; K-29 to L-615; C-30 to L-615; P-31 to L-615; A-
  • polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%. 95%, 96%. 97%, 98% or 99% identical to the polynucleotide sequences encoding the TR9 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • N-terminal deletions of the TR9 polypeptide can be described by the general formula m to 310 where m is a number from -40 to 309 corresponding to the amino acid sequence identified in SEQ ID NO:2.
  • N terminal deletions of the TR9 of the invention comprise, or alternatively, consist of, amino acid residues: Q-2 to L-310; P-3 to L-310; E-4 to L-310; Q-5 to L-310; K-6 to L-310: A-7 to L-310; S-8 to L-310; N-9 to L-310; L-10 to L-310; M l to L-310; G-12 to L-310; T-13 to L-310; Y-14 to L-310; R-15 to L-310; H-16 to L-310; V-17 to L- 310; D-18 to L-310; R-19 to L-310: A-20 to L-310; T-21 to L-310; G-22 to L-310; Q-23 to L-310; V-24 to L-310; L-25 to
  • polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TR9 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • TR9 muteins with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities.
  • peptides composed of as few as six TR9 amino acid residues may often evoke an immune response.
  • further embodiments of the invention are directed to C-terminal deletions of the TR9 polypeptide described by the general formula 1 to n, where n is a number from 2 to 614 corresponding to the position of amino acid residue identified in SEQ ID NO:2 and preferably, corresponds to one of the C-terminal amino acid residues identified in the C-terminal deletions specified herein.
  • C terminal deletions of the TR9 polypeptide of the invention comprise, or alternatively, consist of, amino acid residues: A-1 to L-614; A-1 to D-613; A-1 to P-612; A-1 to L-611 ; A-1 to H-610; A-1 to S-609; A-1 to Y-608: A-1 to V-607; A-1 to S-606; A-1 to D-605; A- 1 to L-604; A-1 to L-603; A-1 to T-602; A-1 to Q-601 ; A-1 to S-600; A-1 to A-599; A-1 to E-598; A-1 to Q-597; A-1 to S-596; A-1 to K-595; A-1 to V-594; A-1 to G-593; A-1 to 1-592; A-1 to 1-591; A-1 to E-590; A-1 to F-589; A-1 to L-588; A-1 to R-587; A-1 to D- 586; A-1 to L-585; A-1 to K-584;
  • A--11 ttoE-194 A-l toP-193; A-1 toR-192; A-1 toP-191; A-1 toF-190; A-1 to I- 1 18899; A A--11 tto A-188; A-1 to T-187; A-1 to G-186; A-1 to P-185; A-1 to S-184; A-1 to P-
  • A--1l ttoV-170 A-1 to N-169; A-1 to D-168; A-1 toT-167; A-1 to E-166; A-1 to K-
  • A--1l tto ⁇ G-134 A-1 to R-133; A-1 to A-132; A-1 to C-131; A-1 to Q-130; A-1 to K- 1 12299; A A--11 tto( C-128; A-1 to R-127; A-1 to V-126; A-1 to D-125; A-1 to E-124; A-1 to T-
  • polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively, consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequences encoding the TR9 polypeptides described above, and the polypeptides encoded thereby.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence, and the polypeptides encoded thereby.
  • polypeptide fragments comprising, or alternatively, consisting of, amino acids described by the general formula m to n, where m and n correspond to any one of the amino acid residues specified above for these symbols, respectively.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • a 1 is any integer between 1 to 3437 of SEQ ID NO: 1
  • b 1 is an integer of 15 to 3452, where both a 1 and b 1 correspond to the positions of nucleotide residues shown in SEQ ID NO: l, and where the b' is greater than or equal to a 1 + 14.
  • the polynucleotides of the invention are less than 300 kb, 200 kb, 100 kb, 50 kb, 15 kb, 10 kb, or 7.5 kb in length.
  • polynucleotides of the invention comprise at least 15 contiguous nucleotides of TR9 coding sequence, but do not comprise all or a portion of any TR9 intron.
  • the nucleic acid comprising TR9 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TR9 gene in the genome).
  • the polynucleotides of the invention are less than
  • polynucleotides of the invention comprise at least 15. at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TR9 coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb. 30 kb, 25 kb, 20 kb. 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide sequences set forth in Figures 1 A-D (SEQ ID NO: 1).
  • polynucleotides of the invention comprise at least 15, at least 30, at least 50, at least 100, or at least 250. at least 500, or at least 1000 contiguous nucleotides of TR9 coding sequence, but do not comprise all or a portion of any TR9 intron.
  • the nucleic acid comprising TR9 coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TR9 gene in the genome).
  • the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500. 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
  • the invention further provides isolated TR9 polypeptides having the amino acid sequence encoded by the deposited cDNAs, or the amino acid sequences in Figures 1A-D (SEQ ID NO:2) or a peptide or polypeptide comprising a portion of the above polypeptides.
  • the polypeptides of the invention may be membrane bound or may be in a soluble circulating form. Soluble peptides are defined by amino acid sequence wherein the sequence comprises, or alternatively consists of, the polypeptide sequence lacking the transmembrane domain.
  • the polypeptides of the present invention may exist as a membrane bound receptor having a transmembrane region and an intra- and extracellular region or they may exist in soluble form wherein the transmembrane domain is lacking.
  • a form of the TR9 receptor is the TR9 receptor shown in Figures 1A-D (SEQ ID NO:2) which contains, in addition to a leader sequence, transmembrane, intracellular and extracellular domains.
  • the polypeptide fragments of the invention are not larger than 610, 600, 580, 570, 550, 525, 500, 475, 450, 400, 425, 390, 380, 375, 350, 336, 334, 331, 305, 300, 295, 290, 285, 280, 275, 260, 250, 225, 200, 185, 175, 170, 165, 160. 155, 150, 145, 140, 135, 130. 125, 120, 1 15, 1 10, 105, 100, 90, 80, 75, 60, 50, 40, 30. or 25 amino acid residues in length.
  • the invention fuithei includes va ⁇ ations of the TR9 receptor which show substantial TR9 receptor functional activity (e g , biological activity) oi which include regions of TR9 receptor polypeptide such as the protein portions discussed herein
  • TR9 receptor functional activity e g , biological activity
  • Such mutants include deletions insertions, inversions, repeats, and type substitutions
  • guidance concerning which amino acid changes are likely to be phenotypically silent can be found in Bowie et al , "Deciphering the Message in Protein Sequences Tolerance to Ammo Acid Substitutions," Science 247 1306-1310 (1990)
  • substitutions of charged amino acids with other charged or neutral amino acids which may produce proteins with highly desirable improved characteristics, such as less aggregation Aggreg
  • the fragment, derivative or analog of the polypeptide of Figures 1A-D may be (I) one in which one or more ofthe amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid res ⁇ due(s), and more preferably at least one but less than ten conserved amino acid residues), and such substituted amino acid res ⁇ due(s) may or may not be one encoded by the genetic code, or (n) one in which one or more of the amino acid residues includes a substituent group, or (in) one in which the mature polypeptide is fused with another compound, such as a compound to increase the half-life of the polypeptide (for example, polyethylene glycol), or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is
  • the replacement of amino acids can also change the selectivity of binding to cell surface receptors. Ostade et al., Nature 361 :266-268 ( 1993). describes certain mutations resulting in selective binding of TNF-alpha to only one of the two known types of TNF receptors.
  • the TR9 receptor of the present invention may include one or more amino acid substitutions, deletions, or additions, either from natural mutations or human manipulation.
  • changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table II).
  • the numbei of substitutions, additions oi deletions in the amino acid sequence of Figures 1A-D and/oi any of the polypeptide fragments desc ⁇ bed heie is 75. 70. 60, 50, 40, 35, 30, 25, 20. 15, 10, 9, 8, 7, 6, 5, 4. 3. 2, 1 or 30-20, 20- 15, 20-10, 15-10, 10-1 , 5-10. 1-5, 1-3 or 1-2
  • Amino acids in the TR9 protein of the piesent invention that are essential for function can be identified by methods known in the art, such as site-dnected mutagenesis or alanine-scanmng mutagenesis (Cunningham and Wells, Science 244 1081-1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested foi biological activity such as, receptor/hgand binding or in vitro proliferative, and/or activation activity upon monocytes. Sites that are critical for hgand-receptor binding can also be determined by structural analysis such as crystallization, nucleai magnetic resonance or photoaffinity labeling (Smith et al, J Mol. Biol 224:899-904 (1992) and de Nos et al. Science 255:306-312 (1992))
  • TR9 polypeptides may be employed to improve or alter the characteristics of TR9 polypeptides.
  • Recombinant D ⁇ A technology known to those skilled in the art can be used to create novel mutant proteins or mutems including single or multiple am o acid substitutions, deletions, additions or fusion proteins.
  • modified polypeptides can show, e.g., enhanced activity or increased stability
  • they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions
  • ⁇ on-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al, Nucl. Acids Res 73:4331 (1986); and Zoller et al, Nucl Acids Res. 70-6487 (1982)), cassette mutagenesis (see e.g., Wells et al, Gene 34:315 (1985)), restriction selection mutagenesis (see e.g , Wells et al, Ph ⁇ os. Trans. R. Soc. London SerA 377:415 (1986)).
  • art-known mutagenesis techniques include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et
  • the invention also encompasses TR9 derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate TR9 polypeptides that are better suited for expression, scale up, etc., in the host cells chosen.
  • cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges;
  • ⁇ -hnked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate ⁇ -hnked sites.
  • DNA shuffling may be employed to modulate the activities of TR9 thereby effectively generating agonists and antagonists of TR9. See generally. U.S. Patent Nos. 5.605,793, 5,81 1 ,238, 5,830.721 , 5,834.252, and 5,837,458, and Patten, P. A., et al, Curr. Opinion Biotechnol. 8:724-33 (1997); Harayama, S. Trends Biotechnol. 16(2):76-82 (1998); Hansson, L. O., et al, J. Mol.
  • alteration of TR9 polynucleotides and corresponding polypeptides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments into a desired TR9 molecule by homologous, or site-specific, recombination.
  • TR9 polynucleotides and corresponding polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of TR9 may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • the heterologous molecules are, for example, TNF-alpha, lymphotoxin- alpha (LT-alpha. also known as TNF- beta), LT-beta (found in complex heterotrimer LT- alpha2-beta), OPGL, FasL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF- gamma (International Publication No.
  • WO 96/14328 AIM-I (International Publication No. WO 97/33899), AIM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6): 1185-1 190), endokine-alpha (International Publication No. WO 98/07880), Neutrokine-alpha (International Publication No. WO 98/18921), OPG, OX40, and nerve growth factor (NGF), and soluble forms of Fas, CD30, CD27, CD40 and 4-IBB, DR3 (International Publication No. WO 97/33904), DR4 (International
  • WO 98/32856 discloses a variety of diseases and conditions in which TR5 (International Publication No. WO 98/30693), TR6 (International Publication No. WO 98/30694), TR7 (International Publication No. WO 98/41629), TRANK, TRIO (International Publication No. WO 98/54202), 312C2 (International Publication No. WO 98/06842), TR11, TR1 1SV1, TR11SV2, TR12, and TNF-Rl, TRAMP/DR3/APO-3/WSL/LARD, TRAIL-R 1/DR4/APO-2, TRAIL-R2/DR5, DcRl TRAIL-R3 TRID/LIT, DcR2/TRAIL-R4, CAD. TRAIL, TRAMP, v-FLIP.
  • the heterologous molecules are any member of the TNF family.
  • polypeptides of the present invention are preferably provided in an isolated form.
  • isolated polypeptide is intended a polypeptide removed from its native environment.
  • a polypeptide produced and/or contained within a recombinant host cell is considered isolated for purposes of the present invention.
  • polypeptides that have been purified, partially or substantially, from a recombinant host cell are polypeptides that have been purified, partially or substantially, from a recombinant host cell.
  • a recombinantly produced version of the TR9 receptor can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
  • polypeptides of the present invention include a polypeptide comprising, or alternatively, consisting of the polypeptide encoded by the deposited cDNA including the leader; a polypeptide comprising, or alternatively, consisting of, the mature polypeptide encoded by the deposited cDNA minus the leader (i.e., the mature protein); a polypeptide comprising, or alternatively, consisting of amino acids about -40 to about 615 in SEQ ID NO:2; a polypeptide comprising, or alternatively, consisting of, amino acids about -39 to about 615 in SEQ ID NO:2; a polypeptide comprising, or alternatively, consisting of, amino acids about 1 to about 615 in SEQ ID NO:2; a polypeptide comprising, or alternatively, consisting of, the extracellular domain; a polypeptide comprising, or alternatively, consisting of the four TNFR-like cysteine rich motifs of TR9 (amino acid residues 67 to 211 in Figures 1A-D
  • identical to a reference amino acid sequence of a TR9 receptor polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids of the reference amino acid of the TR9 receptor.
  • up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • These alterations of the reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
  • any particular polypeptide is at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in Figures 1A-D (SEQ ID NO:2), the amino acid sequence encoded by deposited cDNA clone, or fragments thereof, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
  • the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% of the total number of amino acid residues in the reference sequence are allowed.
  • the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:237-245 (1990)).
  • the subject sequence is shorter than the query sequence due to N- or C-terminal deletions, not because of internal deletions, a manual correction is made to the results to take into consideration the fact that the FASTDB program does not account for N- and C-terminal truncations of the subject sequence when calculating global percent identity.
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a conespondmg subject lesidue, as a peicent of the total bases of the query sequence
  • a determination of whethei a lesidue is matched/aligned is determined by results of the FASTDB sequence alignment
  • This peicentage is then subtracted from the percent identity, calculated by the above FASTDB piogram using the specified parameteis, to arrive at a final percent identity scoie
  • This final percent identity score is what is used foi the purposes of this embodiment Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are consideied for the purposes of manually adjusting the percent identity score That is.
  • a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the fust 10 residues at the N-termmus
  • the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program If the remaining 90 residues were perfectly matched the final percent identity would be 90%
  • a 90 residue subject sequence is compared with a 100 residue query sequence This time the deletions are internal deletions so there are no residues at the N- or C-termini of the subject sequence which are not matched/aligned with the query In this case the percent identity calculated by FASTDB is not manually corrected Once again, only residue positions outside the N- and C
  • polypeptides of the present invention have uses which include, but are not limited to, molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns and as a source for generating antibodies that bind the polypeptides of the invention, using methods well known to those of skill in the art
  • the present application is also directed to proteins cotaining polypeptides at least
  • the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the a specific TR9 N- and/or C-terminal deletion recited herein Polynucleotides encoding these polypeptides are also encompassed by the invention
  • TR9 proteins of the invention comprise, or alternatively consist of, fusion proteins, as desc ⁇ bed above, wheiein the TR9 polypeptide component of the fusion protein is one of the polypeptide sequences set forth herein as m n
  • the poh peptide sequence component of the fusion piotem that is homologous to the TR9 polypeptides of the invention is at least 80%, 85%. 90%, 95%, 96%, 97%, 98% or 99% identical to the polypeptide acid sequence of a specific N- and/or C-terminal deletion recited herein Polynucleotides encoding these polypeptides are also encompassed by the invention
  • the present invention encompasses polypeptides comprising, or alternatively, consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO 2, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in ATCC deposit No 209037 or encoded by a polynucleotide that hyb ⁇ dizes to the complement of the sequence of SEQ ID NO 1 or contained in ATCC deposit No 209037 under stringent hybridization conditions or lowei stringency hybridization conditions as defined supi a
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as.
  • epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human
  • the present invention encompasses a polypeptide comprising an epitope. as well as the polynucleotide encoding this polypeptide.
  • An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra (See, for example, Geysen et al .
  • antigenic epitope is defined as a portion of a protein to which an antibody can lmmunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein Immunospecific binding excludes non-specific binding but does not necessarily exclude cross-reactivity with other antigens Antigenic epitopes need not necessarily be immunogenic
  • antigenic epitopes preferably contain a sequence of at least 4, at least 5. at least 6. at least 7, more preferably at least 8. at least 9, at least 10, at least 11, at least 12. at least 13. at least 14, at least 15. at least 20, at least 25, at least 30, at least 40, at least 50, and, most preferably, between about 15 to about 30 amino acids.
  • Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95. or 100 amino acid residues in length. Additional non-exclusive preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as portions thereof. Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Preferred antigenic epitopes include the antigenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these antigenic epitopes. Antigenic epitopes can be used as the target molecules in immunoassays.
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al., supra; Wilson et al., supra; Chow et al., Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al., J. Gen. Virol. 66:2347-2354 (1985).
  • immunogenic epitopes include the immunogenic epitopes disclosed herein, as well as any combination of two, three, four, five or more of these immunogenic epitopes.
  • the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
  • immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
  • Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al., supra; Wilson et al., supra, and Bittle et al., J. Gen. Virol., 66:2347-2354 (1985).
  • animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
  • KLH keyhole limpet hemacyanin
  • peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl- N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such aglutaraldehyde.
  • Animals such as rabbits, rats and mice are immunized with either free or carrier- coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 ⁇ g of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
  • booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody which can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
  • the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • the polypeptides of the present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI , CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
  • Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins. See, e.g., EP 394,827; Traunecker et al., Nature, 331 :84-86 (1988). Enhanced delivery of an antigen across the epithelial barrier to the immune system has been demonstrated for antigens (e.g., insulin) conjugated to an FcRn binding partner such as IgG or Fc fragments (see, e.g., PCT Publications WO 96/22024 and WO 99/04813).
  • antigens e.g., insulin
  • FcRn binding partner such as IgG or Fc fragments
  • IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion desulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al., J. Biochem., 270:3958-3964 (1995). Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g., the hemagglutinin ("HA”) tag or flag tag) to aid in detection and purification of the expressed polypeptide.
  • an epitope tag e.g., the hemagglutinin ("HA") tag or flag tag
  • the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
  • the tag serves as a matrix-binding domain for the fusion protein.
  • Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
  • the TR9 polypeptides of the present invention and the epitope-bearing fragments thereof are fused with a heterologous antigen (e.g.. polypeptide, carbohydrate, phospholipid. or nucleic acid).
  • the heterologous antigen is an immunogen.
  • the heterologous antigen is the gpl20 protein of
  • HIV or a fragment thereof.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the TR9 polypeptides of the present invention and the epitope-bearing fragments thereof are fused with polypeptide sequences of another TNF family member (or biologically active fragments or variants thereof).
  • the TR9 polypeptides of the present invention are fused with a CD40L polypeptide sequence.
  • the CD40L polypeptide sequence is soluble.
  • DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See generally, U.S. Patent Nos. 5,605,793; 5,81 1 ,238; 5,830,721 ; 5,834,252; and 5,837,458, and Patten et al., Curr. Opinion Biotechnol.
  • alteration of polynucleotides corresponding to SEQ ID NO: 1 may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
  • polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
  • one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide encoding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
  • TR9 polypeptides of the invention are fusedwith soluble CD40L polypeptides, or biologically acitve fragments or variants thereof.
  • polypeptides of the invention relate to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, polypeptide fragment, or variant of SEQ ID NO:2, and/or an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigen binding).
  • TCR T-cell antigen receptors
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti- idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, IgG2, IgG3, IgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • Immunoglobulins may have both a heavy and light chain.
  • An array of IgG, IgE, IgM, IgD, IgA, and IgY heavy chains may be paired with a light chain of the kappa or lambda forms.
  • the antibodies are human antigen-binding antibody fragments of the present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a VL or VH domain.
  • Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion of the following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
  • the antibodies of the invention may be from any animal origin including birds and mammals.
  • the antibodies are human, murine (e.g., mouse and rat), donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
  • "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al.
  • the antibodies of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity.
  • Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope. such as a heterologous polypeptide or solid support material.
  • a heterologous polypeptide or solid support material See, e.g., PCT publications WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793: Tutt, et al., J. Immunol. 147:60-69 ( 1991); U.S. Patent Nos. 4.474,893; 4,714,681 ; 4,925,648; 5,573,920; 5,601 ,819; Kostelny et al., J. Immunol. 148: 1547-1553 ( 1992).
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention which they recognize or specifically bind.
  • the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g., by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and Figures.
  • Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog of a polypeptide of the present invention are included. Antibodies that bind polypeptides with at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention. In specific embodiments, antibodies of the present invention cross-react with murine, rat and/or rabbit homologs of human proteins and the corresponding epitopes thereof.
  • Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • the above-described cross-reactivity is with respect to any single specific antigenic or immunogenic polypeptide, or combination(s) of 2, 3, 4, 5, or more of the specific antigenic and/or immunogenic polypeptides disclosed herein.
  • antibodies which bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions are also included in the present invention.
  • Antibodies of the present invention may also be described or specified in terms of their binding affinity to a polypeptide of the invention.
  • Preferred binding affinities include those with a dissociation constant or Kd less than 5 X 10 "2 M, 10 "2 M, 5 X 10 "3 M, lO "3 M, 5 X 10 "4 M.
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
  • the antibody competitively inhibits binding to the epitope by at least 95%, at least 90%, at least 85 %, at least 80%, at least 75%. at least 70%, at least 60%, or at least 50%.
  • Antibodies of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • antibodies of the present invention bind an antigenic epitope disclosed herein, or a portion thereof.
  • the invention features both receptor-specific antibodies and ligand-specific antibodies.
  • the invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.
  • Receptor activation may be determined by techniques described herein or otherwise known in the art. For example, receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
  • phosphorylation e.g., tyrosine or serine/threonine
  • antibodies are provided that inhibit ligand activity or receptor activity by at least 95%, at least 90%, at least 85%, at least 80%, at least 75%, at least 70%, at least 60%, or at least 50% of the activity in absence of the antibody.
  • the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
  • neutralizing antibodies which bind the ligand and prevent binding of the ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
  • antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation, for example, by inducing dimerization of the receptor.
  • the antibodies may be specified as agonists, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See, e.g., PCT publication WO 96/40281; U.S. Patent No. 5,81 1,097; Deng et al., Blood 92(6)1981-1988 (1998); Chen et al., Cancer Res. 58(16):3668-3678 (1998); Harrop et al., J. Immunol. 161(4): 1786-1794 (1998); Zhu et al., Cancer Res. 58(15):3209-3214 (1998); Yoon et al., J. Immunol. 160(7):3170-3179 (1998); Prat et al., J. Cell. Sci. 11 l(Pt2):237-247
  • Antibodies of the present invention may be used, for example, but not limited to, to purify, detect, and target the polypeptides of the present invention, including both in vitro and in vivo diagnostic and therapeutic methods.
  • the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels of the polypeptides of the present invention in biological samples. See, e.g., Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference herein in its entirety).
  • the antibodies of the present invention may be used either alone or in combination with other compositions.
  • the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
  • antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, radionuclides, or toxins. See, e.g., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No.
  • the antibodies of the invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
  • the antibody derivatives include antibodies that have been modified, e.g., by glycosylation, acetylation, pegylation, phosphylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non- classical amino acids.
  • the antibodies of the present invention may be generated by any suitable method known in the art.
  • Polyclonal antibodies to an antigen-of- interest can be produced by various procedures well known in the art.
  • a polypeptide of the invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
  • adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and corynebacterium parvum. Such adjuvants are also well known in the art.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
  • the term “monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “monoclonal antibody” refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced.
  • a “monoclonal antibody” may comprise, or alternatively consist of, two proteins, i.e., a heavy and a light chain.
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes solated from a mouse immunized with an antigen of the invention with myeloma cells -and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide of the invention.
  • Antibody fragments which recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art. In phage display methods, functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • such phage can be utilized to display antigen binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and M13 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
  • the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host, including mammalian cells, insect cells, plant cells, yeast, and bacteria, e.g., as described in detail below.
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region.
  • Methods for producing chimeric antibodies are known in the art. See e.g., Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Gillies et al., (1989) J. Immunol. Methods 125: 191-202; U.S. Patent Nos. 5,807,715; 4,816,567; and 4,816397, which are incorporated herein by reference in their entirety.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • These framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions. (See, e.g., Queen et al., U.S. Patent No.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular assets, such as CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos. 5,225,539; 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 592,106; EP 519,596; Padlan, Molecular
  • Human antibodies are particularly desirable for therapeutic treatment, detection, and/or prevention in human patients.
  • Human antibodies can be made by a variety of methods known in the art including phage display methods described above using antibody libraries derived from human immunoglobulin sequences. See also, U.S. Patent Nos. 4,444,887 and 4,716,111; and PCT publications WO 98/46645, WO 98/50433, WO 98/24893, WO 98/16654, WO 96/34096, WO 96/33735, and WO 91/10741; each of which is incorporated herein by reference in its entirety.
  • Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
  • the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
  • the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
  • the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion of the JH region prevents endogenous antibody production.
  • the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice.
  • the chimeric mice are then bred to produce homozygous offspring which express human antibodies.
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
  • Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
  • the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B cell differentiation, and subsequently undergo class switching and somatic mutation.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g., a mouse antibody
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff J. Immunol. 147(8):2429-2438 (1991)).
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/oi binding domain and, as a consequence, bind to and neutialize polypeptide and/or its ligand
  • Such neutiahzing anti-idiotypes oi Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutialize polypeptide hgand Foi example, such anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its hgands/receptors, and thereby block its biological activity
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof
  • The also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e g , as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO 2
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art For example, if the nucleotide sequence of the antibody is known, a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e g .

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Abstract

Cette invention concerne un nouveau membre de la famille des récepteurs des facteurs de nécrose des tumeurs, en particulier des molécules isolées d'acides nucléiques codant pour le récepteur TR9 humain. L'invention concerne également des poylypeptides en tant que vecteurs et cellules hôtes ainsi que des méthodes recombinantes permettant d'obtenir lesdits vecteurs et cellules hôtes. De plus, elle concerne des méthodes de recherche systématique d'agonistes et d'antagonistes de l'activité du récepteur TR9.
EP00914975A 1999-03-24 2000-03-16 Recepteur tr9 humain de facteurs de necrose des tumeurs Withdrawn EP1171579A4 (fr)

Applications Claiming Priority (5)

Application Number Priority Date Filing Date Title
US12601999P 1999-03-24 1999-03-24
US126019P 1999-03-24
US13422099P 1999-05-14 1999-05-14
US134220P 1999-05-14
PCT/US2000/006831 WO2000056862A1 (fr) 1999-03-24 2000-03-16 Recepteur tr9 humain de facteurs de necrose des tumeurs

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EP1171579A1 true EP1171579A1 (fr) 2002-01-16
EP1171579A4 EP1171579A4 (fr) 2002-07-31

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JP (1) JP2002542771A (fr)
AU (1) AU3628800A (fr)
CA (1) CA2365255A1 (fr)
WO (1) WO2000056862A1 (fr)

Families Citing this family (5)

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Publication number Priority date Publication date Assignee Title
AU7830398A (en) * 1997-06-11 1998-12-30 Human Genome Sciences, Inc. Human tumor necrosis factor receptor tr9
WO2001085209A2 (fr) * 2000-05-10 2001-11-15 Eli Lilly And Company Traitement de maladies induites par les lymphocytes t par modulation de l'activite de dr6
WO2003013585A1 (fr) * 2001-08-08 2003-02-20 Genset S.A. Agonistes et antagonistes de la mifaxine convenant au traitement de troubles du metabolisme au moyen
DK1446733T3 (da) 2001-10-13 2009-10-26 Asterion Ltd Glycosylphosphatidylinositol indeholdende polypeptider
EP1455825A4 (fr) * 2001-12-17 2006-05-31 Lilly Co Eli Traitement de maladies mediees par les lymphocytes b par modulation de l'activite du dr6

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998056892A1 (fr) * 1997-06-11 1998-12-17 Human Genome Sciences, Inc. Recepteur humain tr9 du facteur de necrose tumorale

Family Cites Families (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
IL114417A0 (en) * 1994-07-07 1995-10-31 Res Dev Foundation Tumor necrosis factor receptor-I associated proteins and protein kinase and methods for their use
US5563039A (en) * 1995-03-31 1996-10-08 Tularik, Inc. TNF receptor-associated intracellular signaling proteins and methods of use

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1998056892A1 (fr) * 1997-06-11 1998-12-17 Human Genome Sciences, Inc. Recepteur humain tr9 du facteur de necrose tumorale

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
DATABASE EMBL [Online] 9 September 1998 (1998-09-09) PAN ET AL.: "Homosapiens TNFR-related death receptor-6 (DR6) mRNA, complete cds" Database accession no. AF068868 XP002144008 *
PAN G ET AL: "Identification and functional characterization of DR6, a novel death domain-containing TNF receptor" FEBS LETTERS, ELSEVIER SCIENCE PUBLISHERS, AMSTERDAM, NL, vol. 431, 1998, pages 351-356, XP002113408 ISSN: 0014-5793 *
See also references of WO0056862A1 *

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AU3628800A (en) 2000-10-09
CA2365255A1 (fr) 2000-09-28
WO2000056862A1 (fr) 2000-09-28
JP2002542771A (ja) 2002-12-17
EP1171579A4 (fr) 2002-07-31

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