EP1165185A2 - Menschlischer tumor necrosis faktor rezeptor änlicher rezeptor 2 - Google Patents

Menschlischer tumor necrosis faktor rezeptor änlicher rezeptor 2

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Publication number
EP1165185A2
EP1165185A2 EP00916583A EP00916583A EP1165185A2 EP 1165185 A2 EP1165185 A2 EP 1165185A2 EP 00916583 A EP00916583 A EP 00916583A EP 00916583 A EP00916583 A EP 00916583A EP 1165185 A2 EP1165185 A2 EP 1165185A2
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Prior art keywords
seq
amino acid
antibody
sequence
polypeptides
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French (fr)
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Jian Ni
Craig A. Rosen
Reiner L. Gentz
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Human Genome Sciences Inc
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Human Genome Sciences Inc
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K39/395—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum
    • A61K39/39533—Antibodies; Immunoglobulins; Immune serum, e.g. antilymphocytic serum against materials from animals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P19/00—Drugs for skeletal disorders
    • A61P19/02—Drugs for skeletal disorders for joint disorders, e.g. arthritis, arthrosis
    • 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
    • A61P35/00—Antineoplastic agents
    • 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
    • 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
    • A61P37/02—Immunomodulators
    • A61P37/04—Immunostimulants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K39/00—Medicinal preparations containing antigens or antibodies
    • A61K2039/505—Medicinal preparations containing antigens or antibodies comprising antibodies
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/60—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments
    • C07K2317/62—Immunoglobulins specific features characterized by non-natural combinations of immunoglobulin fragments comprising only variable region components
    • C07K2317/622—Single chain antibody (scFv)
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • 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
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to novel members of the Tumor Necrosis Factor (TNF) receptor family. More specifically, isolated nucleic acid molecules are provided encoding a human TNF receptor-related protein, referred to herein as the TR2 receptor of FIG. 1A-1B, having considerable homology to murine CD40. Two different TR2 splice variants, referred to as TR2-SV1 and TR2-SV2, are also provided. TR2 polypeptides are also provided with homology to human type 2 TNF receptor (TNF-RII) Further provided are vectors, host cells and recombinant methods for producing the same. The invention also relates to both the inhibition and enhancement of functional activities of TR2 receptor polypeptides and diagnostic methods for detecting TR2 receptor gene expression.
  • TNF Tumor Necrosis Factor
  • TNF- ⁇ and ⁇ are related members of a broad class of polypeptide mediators, which includes the interferons, interleukins and growth factors, collectively called cytokines (Beutler, B. and Cerami, A., Anmi. Rev. Immunol 7:625-655 (1989)).
  • Tumor necrosis factor (TNF- ⁇ and TNF- ⁇ ) was originally discovered as a result of its anti-tumor activity, however, now it is recognized as a pleiotropic cytokine playing important roles in a host of biological processes and pathologies.
  • TNF- ⁇ Tumor necrosis factor
  • TNF- ⁇ lymphotoxin- ⁇
  • LT- ⁇ LT- ⁇
  • TRAIL ligands for the Fas receptor
  • CD30, CD27, CD40, OX40 and 4- IBB receptors These proteins have conserved C- terminal sequences and variable N-terminal sequences which are often used as membrane anchors, with the exception of TNF- ⁇ .
  • TNF- ⁇ and TNF- ⁇ function as homotrimers when they bind to TNF receptors.
  • TNF is produced by a number of cell types, including monocytes, fibroblasts, T-cells, natural killer (NK) cells and predominately by activated macrophages.
  • TNF- ⁇ has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata et al, J. Immunol. 136(7):24S3 (1987)), growth regulation, vascular endothelium effects and metabolic effects.
  • TNF- ⁇ also triggers endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF and
  • TNF- ⁇ up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and NCAM-1.
  • T ⁇ F- ⁇ and the Fas ligand have also been shown to induce programmed cell death.
  • T ⁇ F- ⁇ has many activities, including induction of an antiviral state and tumor necrosis, activation of polymorphonuclear leukocytes, induction of class I major histocompatibility complex antigens on endothelial cells, induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle, ⁇ . and Homer, R., Prog. Allergy JO. 162-182 (1988)).
  • T ⁇ F- ⁇ and T ⁇ F- ⁇ are involved in growth regulation and interact with hemopoietic cells at several stages of differentiation, inhibiting proliferation of various types of precursor cells, and inducing proliferation of immature myelomonocytic cells.
  • mice deficient in T ⁇ F- ⁇ production show abnormal development of the peripheral lymphoid organs and morphological changes in spleen architecture (reviewed in Aggarwal et al,
  • T ⁇ F- ⁇ -/- mice Eur Cytokine Netw, 7(2).93-124 (1996)).
  • peripheral blood from T ⁇ F- ⁇ -/- mice contained a three fold reduction in white blood cells as compared to normal mice.
  • Peripheral blood from TNF- ⁇ -/- mice contained four fold more B cells as compared to their normal counterparts.
  • TNF- ⁇ in contrast to TNF- ⁇ has been shown to induce proliferation of EBN-infected B cells.
  • T ⁇ F-RI 55-KDa
  • T ⁇ F-RII 75-KDa
  • human and mouse cD ⁇ As corresponding to both receptor types have been isolated and characterized (Loetscher et al, Cell, 61 51 (1990)).
  • T ⁇ F-Rs share the typical structure of cell surface receptors including extracellular, transmembrane and intracellular regions.
  • T ⁇ F-RI and T ⁇ F-RII share 28% identity and are characterized by four repeated cysteine-rich motifs with significant intersubunit sequence homology.
  • the majority of cell types and tissues appear to express both T ⁇ F receptors and both receptors are active in signal transduction, however, they are able to mediate distinct cellular responses.
  • T ⁇ F-RII was shown to exclusively mediate human T-cell proliferation by T ⁇ F as shown in PCT WO 94/09137.
  • T ⁇ F-RI dependent responses include accumulation of C-FOS, IL-6, and manganese superoxide dismutase rnR ⁇ A, prostaglandin E2 synthesis, IL-2 receptor and MHC class I and II cell surface antigen expression, growth inhibition, and cytotoxicity.
  • T ⁇ F-RI also triggers second messenger systems such as phospholipase A 2 , protein kinase C, phosphatidylcholine-specific phospholipase C and sphingomyelinase (Pfefferk et al, Cell, 73:457-467 (1993)).
  • phospholipase A 2 protein kinase C
  • phosphatidylcholine-specific phospholipase C sphingomyelinase
  • Retinoic acid for example, has been shown to induce the production of TNF receptors in some cells type while down regulating production in other cells.
  • TNF- ⁇ has been shown to affect the localization of both types of receptor.
  • TNF- ⁇ induces intemalization of TNF-RI and secretion of TNF-RII (reviewed in Aggarwal et al, supra).
  • TNF-Rs are regulated by a variety of agents.
  • yeast two hybrid system and co-precipitation and purification have been used to identify ligands which associate with both types of the TNF-Rs (reviewed in Aggarwal et al., supra and Vandenabeele et al, Trends in Cell Biol.
  • the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, polynucleotides encoding TR2 receptors and splice variants thereof having the amino acid sequences shown in SEQ ID NO:26, HG. 1A-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ ED NO:5) and FIG.7A-7C (SEQ ID NO:8) or the amino acid sequence encoded by the cDNA encoding the TR2 receptors deposited as ATCC Deposit Numbers 97059, 97058 and 97057 on February 13, 1995.
  • 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 TR2 polypeptides or peptides by recombinant techniques.
  • TR2 polypeptides having amino acid sequences encoded by the polynucleotides described herein.
  • the present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting a cellular response induced by TR2 receptors, which involves contacting cells which express TR2 receptors with the candidate compound, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased cellular response over the standard indicates that the compound is an agonist and a decreased 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 TR2 receptors.
  • the method involves contacting TR2 receptors with a ligand polypeptide and a candidate compound and determining whether ligand binding to the TR2 receptors is increased or decreased due to the presence of the candidate compound.
  • the invention further provides a diagnostic method useful during diagnosis or prognosis of a disease states resulting from aberrant cell proliferation due to alterations in TR2 receptor expression.
  • An additional aspect of the invention is related to a method for treating an individual in need of an increased level of a TR2 receptor activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of isolated TR2 polypeptides of the invention or an agonist thereof.
  • a still further aspect of the invention is related to a method for treating an individual in need of a decreased level of a TR2 receptor activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of a TR2 receptor antagonist.
  • the invention additionally provides soluble forms of the polypeptides of the present invention Soluble peptides are defined by amino acid sequences wherein the sequence comprises, or alternatively consists of, the polypeptide sequences lacking a transmembrane domain Such soluble forms of the TR2 receptors are useful as antagonists of the membrane bound forms of the receptors
  • FIG 1A-1B shows the nucleotide (SEQ ID NO 1) and deduced amino acid (SEQ ID NO 2) sequences of a TR2 receptor
  • the protein has a predicted leader sequence of about 36 amino acid residues (underlined) (amino acid residues -36 to -l in SEQ ID NO 2) and a deduced molecular weight ofabout 30,417 kDa
  • amino acid residues from about 37 to about 200 constitute the extracellular domain, from about 201 to about 225 (amino acid residues 165 to 189 in SEQ ID NO 2) the transmembrane domain (underlined), and from about 226 to about 283 (amino acid residues 190 to 247 in SEQ ID NO 2) the intracellular domain
  • amino acid residues from about 37 to about 200 constitute the extracellular domain, from about 201 to about 225 (amino acid residues 165 to 189 in SEQ ID NO 2) the transmembrane domain (underlined), and from about 226 to about 283 (amino acid residues 190 to 247 in SEQ ID NO 2) the intracellular domain
  • Two potential asparagine-linked glycosylation sites are located at amino acid positions 1 10 and 173 (amino acid residues 74 to 137 in SEQ ID NO 2)
  • FIG 2 shows the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG 1 A-1B and a murine CD40 protein (SEQ ID NO 3)
  • FIG 3 shows an analysis of the TR2 receptor amino acid sequence of FIG 1A-1B Alpha, beta, turn and coil regions, hydrophilicity and hydrophobicity, amphipathic regions, flexible regions, antigenic index and surface probability are shown In the "Antigenic Index - Jameson-Wolf graph, amino acid residues 39 to 70, 106 to 120, 142 to 189 and 276 to 283 in FIG 1 A- IB (amino acid residues
  • FIG. 4A-4C shows the nucleotide (SEQ ID NO:4) and deduced amino acid (SEQ ID NO:5) sequences of the TR2-SV1 receptor.
  • the protein has a predicted leader sequence ofabout 36 amino acid residues (underlined) (amino acidresidues -36 to - 1 in SEQ ID NO :5) and a deduced molecular weight ofabout 19.5 kDa.
  • FIG.5 shows the regions of similarity between the amino acid sequences of the full-length TR2-SV1 receptor protein and a human type 2 TNF receptor (SEQ ID NO.6).
  • FIG. 6 shows an analysis of the TR2-SV1 receptor amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • amino acid residues 39 to 70, 99 to 136 and 171 to 185 in FIG. 4A-4C amino acid residues 3 to 34, 63 to 100 and 135 to 149 in SEQ ID NO:5 correspond to the shownhighly antigenic regions of the TR2-SV1 receptor protein.
  • FIG. 7A-7C shows the nucleotide (SEQ ID NO:7) and deduced amino acid (SEQ ID NO:8) sequences of the TR2-SV2 receptor.
  • This protein lacks a putative leader sequence and has a deduced molecular weight ofabout 14 kDa.
  • FIG.8 shows the regions of similarity between the amino acid sequences of the TR2-SV2 receptor protein and a human type 2 TNF receptor (SEQ ID NO: 1
  • FIG.9 shows an analysis of the TR2-SV2 receptor amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • amino acid residues 56 to 68 and 93 to 136 in FIG. 7A-7C correspond to the shown highly antigenic regions of the TR2-SV2 receptor protein.
  • FIG. 10 sho s the regions of similarity between the amino acid sequences of the TR2 receptor protein of FIG. 1 A-IB and the TR2-SV1 receptor protein of FIG. 4A-4C.
  • FIG. 11 shows the regions of similarity between the amino acid sequences ofthe TR2 receptor protein of FIG. 1 A- IB and the TR2-SV2 receptor protein of
  • FIG. 7A-7C are identical to FIG. 7A-7C.
  • FIG. 12 shows the regions of similarity between the amino acid sequences of the TR2-SV1 and the TR2-SV2 receptor proteins.
  • FIG. 13A-13D shows the regions of similarity between the nucleotide sequences encoding the TR2 receptor protein of FIG. 1A-1B and the TR2-SV1 receptor protein of FIG. 4A-4C.
  • FIG. 14A-14D shows the regions of similarity between the nucleotide sequences encoding the TR2 receptor protein of FIG. 1A-1B and the TR2-SV2 receptor protein of FIG. 7A-7C.
  • FIG. 15A-15F shows the regions of similarity between the nucleotide sequences encoding the TR2-SV1 and the TR2-SV2 receptor proteins.
  • FIG. 16 shows an alignment of the amino acid sequence of the TR2 receptor of FIG. 1 A-IB (SEQ ID NO:2) with other TNFR family members.
  • the amino acid sequence of TR2 was aligned with those of TNFR-I (SEQ ID NO: 10), TNFR-II (SEQ ED NO: 11), CD40 (SEQ ID NO: 12) and 4-1BB (SEQ DD NO: 13) on the basis of sequence homology and conserved cysteine residues.
  • Cysteine repeat regions are defined by amino acid residues 5 to 40, 41 to 84, 85 to 127, and 128 to 166 in SEQ ID NO:2, respectively referred to as cysteine repeat regions A-D.
  • FIG. 17 shows the effect of TR2 on B cell in vitro proliferation.
  • B lymphocytes were purified from human tonsils by immunomagnetic selection. Cells were cultured for 72 hours followed by a 24 hour 3 H thymidine pulse in RPMI1640 medium added with 10% FBS, 4 m 1- glutamine, 5 X 10 J M2ME, 100 U/ml Penicillin, 100 ⁇ g/ml Streptomycin, and the indicated factors-
  • the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, polynucleotides encoding a TR2 polypeptide (FIG. 1 A-lB (SEQ ID NO:2)) and splice variants thereof, TR2-SV1 (FIG. 4A-4C (SEQ ID NO:5)) and TR2-SV2 (FIG. 7A-7C (SEQ ID NO.8)), the amino acid sequences of which were determined by sequencing cDNAs.
  • the TR2 protein shown in FIG. 1A-1B shares sequence homology with the murine CD40 receptor (FIG. 2 (SEQ ID NO:3)).
  • the TR2 receptors of the present invention include several allelic variants containing alterations in at least four nucleotides and two amino acids.
  • Nucleotide sequence variants which have been identified include either guanine or adenine at nucleotide 314 and either thymine or cytosine at nucleotides 386, 624 and 627 shown in FIG. 1A-1B (SEQ ID NO:l). While the identified alteration at nucleotides 624 and 627 are silent, the alteration at nucleotide 386 results in the codon at nucleotides 385 to 387 encoding either serine or phenylalanine and the alteration at nucleotide 314 results in the codon at nucleotides 313 to 315 encoding either lysine or arginine.
  • FIG.4A-4C SEQ ID NO:4
  • FIG. 7A-7C SEQ ID NO:7
  • TR2-SV1 accession numbers 97058 and 97057 (TR2-SV2), respectively.
  • TR2-SV2 accession numbers 97058 and 97057 (TR2-SV2), respectively.
  • the deposited cDNAs are contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).
  • splice variant refers to cDNA molecules produced from a RNA molecules initially transcribed from the same genomic DNA sequence which have undergone alternative RNA splicing.
  • Alternative RNA splicing occurs when a primary RNA transcript undergoes splicing, generally for the removal of introns, which results in the production of more than one mRNA molecule each of which may encode different amino acid sequences.
  • the term “splice variant” also refers to the proteins encoded by the above cDNA molecules.
  • TR2 proteins refer to all proteins resulting from the alternate splicing of the genomic DNA sequences encoding proteins having regions of amino acid sequence identity and receptor activity which correspond to the proteins shown in SEQ ID NO:26, HG. 1 A-IB (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5) or FIG. 7A-7C (SEQ ID NO:8), as well as TR2 allellic variants.
  • the TR2 proteins shown in SEQ IDNO:26 andFIG. 1A-1B, the TR2-SV1 protein shown FIG.4A- 4C, and the TR2-S V2 protein shown in FIG.7A-7C are examples of such receptor proteins.
  • 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
  • RECTIFIED SHEET (RULE 91) ISA/EP manual DNA sequencing methods well known in the art.
  • a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
  • nucleic acid molecules of the present invention encoding TR2 polypeptides may be obtained using standard cloning and screening procedures, such as those used for cloning cDNAs using mRNA as starting material.
  • nucleic acid molecule described in FIG. 1A-IB SEQ ID NO:l
  • FIG. 4A-4C SEQ ID NO:4
  • FIG. 7A-7C SEQ ID NO:
  • TR2 mRNA was detected in numerous tissues including lung, spleen and thymus and may be ubiquitously expressed in human cells. TR2 RNA was also found to be expressed in B lymphocytes (CD 19 * ), both
  • CD4* Tr ⁇ and T ro clones CD8 + T lymphocytes, monocytes and endothelial cells.
  • TR2 mRNA As also noted in Example 6, the production of TR2 mRNA was inducible in MG 63 cells by TNF ⁇ . Further, the accumulation of TR2 mRNA was observed in HL60, U937 and THP1 cells upon PMA or DMSO treatment. PMA and
  • DMSO are agents known to induce differentiation of these three cell types.
  • the determined nucleotide sequence of the TR2 cDNA of FIG. 1A-1B contains an open reading frame encoding a protein ofabout 283 amino acid residues, with a predicted leader sequence of about 36 amino acid
  • RECTIFIED SHEET (RULE 91) ISA/EP residues, and a deduced molecular weight of bout 30,417 kDa.
  • the amino acid sequence of the predicted mature TR2 receptor is shown in FIG. 1A-IB from amino acid residue about 37 to residue about 283 (amino acid residues 1 to 247 in SEQ ID NO:2).
  • “about” includes the particularly recited value and values larger or smaller by several (5, , 3, 2, or 1 ) amino acids.
  • Example 6 the location of the leader sequence cleavage site was confirmed for a TR2-Fc fusion protein and found to be between amino acids 36 and 37 shown in FIG. 1A-1B (amino acid residues -1 to 1 in SEQ ID NO:2).
  • the TR2 protein shown in FIG. 1A-1B (SEQ ED NO:2) is about 29% identical and about 47% similar to the murine CD40 protein shown in SEQ ID NO:3 (see FIG.2).
  • the determined cDNA nucleotide sequences of the TR2-SV1 splice variant of TR2 contains an open reading frame encoding a protein of about 185 amino acid residues, with a predicted leader sequence of about 36 amino acidresidues, and a deduced molecular weight ofabout 19.5 kDa.
  • the amino acid sequence of the predicted mature TR2-SV1 receptor is shown in FIG.4A-4C (SEQ ID NO:5) from amino acid residue about 37 to residue about 185 (amino acid residues 1 to 149 in (SEQ ID NO:5).
  • TR2-SV1 protein shown in FIG.4A- 4C (SEQ ID NO:5) is about 25% identical and about 48% similar to the human type 2 TNF receptor protein shown in SEQ ID NO:6 (see FIG.5).
  • the determined cDNA nucleotide sequences of the TR2-SV2 splice variant of TR2 contains an open reading frame encoding a protein ofabout 136 amino acid residues, without a predicted leader sequence, and a deduced molecular weight of about 14 kDa.
  • the amino acid sequence of the predicted TR2-SV2 receptor is shown in FIG. 7A-7C (SEQ ID NO:8) from amino acid residue about 1 to residue about 136.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.
  • RECTIFIED SHEET (RULE 91) ISA/EP (SEQ ID NO:8) is about 27% identical and about 45% similar to the human type 2 TNF receptor protein shown in SEQ ID NO:9 (see FIG. 8).
  • FIG. 1 A-IB A comparison of both the nucleotide and amino acid sequences of the TR2, TR2-SV1 and TR2-SV2 receptor proteins shown in FIG. 1 A-IB, FIG.4A- 4C and FIG.7A-7C shows several regions of near identity. While the amino acid sequence of the TR2 receptor protein, shown in FIG. 1A-1B (SEQ ID NO:2), is about 60% identical and about 73% similar to the amino acid sequence of the TR2-SV1 receptor protein, shown in FIG. 4A-4C (SEQ ID NO:5), in approximately the first one hundred amino acids of their respective sequences the two proteins differ in one location (FIG. 10).
  • 1A-1B (SEQIDNO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino acid sequence of the TR2-SV2 receptor protein, shown in FIG. 7A-7C (SEQ ID NO:2) is about 60% identical and about 71% similar to the amino acid sequence of
  • TR2-SV1 and TR2-SV2 proteins are only about 20% identical and about 38% similar at the amino acid level to each other. Unlike the comparisons of either of these proteins to the TR2 protein shown in FIG. 1 A-IB
  • TR2 cDNAs With respect to their nucleotide sequences of the cDNAs encoding the disclosed TR2 proteins, a comparison of these sequences indicates that the TR2 cDNAs share large regions of near identity at the nucleic acid level (FIG. 13A-13D,FIG. 14A-14DandFIG.15A-15F).
  • the cDNA sequences encoding the TR2 and TR2-SV 1 proteins for example, share large regions of near identity in their nucleotide sequences which encode both the N termini of the respective proteins and their 5' and 3' noncoding regions (FIG. 13A-13D).
  • splice variants proteins which are produced from alternately spliced RNA, referred to as splice variants.
  • the transcript of the src gene undergoes alternate RNA splicing to produce cell type specific products.
  • the Src protein consists of 533 amino acids while in nerve cells an additional short exon is included in the mRNA resulting in a protein of 539 amino acids. See Alberts, B. et al, MOLECULAR BIOLOGY OF THE CELL (3rd Edition, Garland Publishing, Inc., 1994), 455.
  • sex specific mRNA transcripts have been identified in Drosophila where alternate mRNA splicing results in a protein named Dsx which is approximately 550 amino acids in length in males and 430 amino acids in length in females. These two splice variant proteins share a common core sequence ofabout 400 amino acids. See id. at 457.
  • the TR2 receptor protein shown in FIG. 1 A-IB (SEQ ID NO:2) is believed to be the full-length polypeptide encoded by the RNA from which the TR2 receptor proteins are translated.
  • the RNA encoding the TR2-SV1 splice variant shown in FIG. 4A-4C (SEQ ID NO:5) is believed to contain an insertion in the region encoding amino acid residue 102 of the amino acid sequence shown in FIG. I A-IB and a deletion in the region encoding amino
  • RNA encoding the TR2-SV2 splice variant shown in FIG. 7A-7C is believed to begin with die nucleotide sequence encoding amino acid residue 102 of the amino acid sequence shown in FIG. 1 A- IB and contain insertions in the regions encoding amino acid residues 184 and 243 of the ami- ⁇ o acid sequence shown in FIG.
  • the present invention also provides the mature forms of the TR2 receptors 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, mat is, it is inherent in the amino acid sequence of the polypeptide.
  • the present invention provides nucleotide sequences encoding mature TR2 polypeptides having the amino acid sequences encoded by the cDNAs contained in the deposits identified as ATCC Deposit Numbers 97059 and 97058 and as shown in SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-.26, FIG. 1 A-IB (SEQ ID NO-
  • FIG. 4A-4C SEQ ID NO :5
  • the mature TR2 polypeptides having the amino acid sequences encoded by the cDNAs contained in the deposits identified as ATCC Deposit Numbers 97059 and 97058 is meant the mature form(s) of the TR2 receptors 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 cDNA contained in the deposited plasmids.
  • the invention also provides nucleic acid sequences encoding the TR2-S V2 receptor protein of FIG. 7A-7C (SEQ ID NO:8), having the amino acid sequence
  • Acids Res. 74:4683-4690 (1986)) can be used.
  • the accuracy of predicting the cleavage points of nown mammalian secretory proteins for each of these methods is in the range of 75-80%. von Heinje, supra.
  • the two methods do not always produce the same predicted cleavage point(s) for a given protein.
  • TR2 polypeptides shown in FIG. 1 A-IB (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5) and FIG. 7A-7C (SEQ ID NO:8) were analyzed by a computer program ("PSORT) (K. Nakai and M. Kanehisa, Genomics 74:897- 11 (1992)), which is an expert system for predicting the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated. The analysis by the PSORT program predicted the cleavage sites between amino acids -1 and 1 in SEQ ID NO:2 and SEQ ID NO:5.
  • the leader sequences for the TR2 protein shown in SEQ ID NO:2 and the TR2-SV1 protein are predicted to consist of amino acid residues -36 to -1 in both SEQ ID NO:2 and SEQ ID NO:5, while the predicted mature TR2 proteins consist of amino acid residues 1 to 247 for the TR2 protein shown in SEQ EO NO:2 and residues 1 to 149 for the TR2-SV1 protein shown in SEQ ID NO:5.
  • Example 6 the cleavage site of the leader sequence of a TR2- Fc fusion protein was corifirmed using amino acid analysis of the expressed fusion protein. This fusion protein was found to begin at amino acid 37, which corresponds to amino acid 1 in SEQ ED NO:2 and SEQ ID NO:5, indicating that
  • cleavage site of the leader sequence is between amino acids 36 and 37 in this protein (corresponding to amino acid residues -1 to 1 in SEQ ID NO.2 and SEQ ID NO:5)
  • ATCC Deposit Number 97059 comprises about 283 amino acids, but may be anywhere in the range of 250 to 316 amino acids, and the leader sequence of this protein is about 36 amino acids, but may be anywhere in the range ofabout 30 to about 42 amino acids
  • the TR2-S VI receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97058 comprises about 185 amino acids, but may be anywhere in the range of 163-207 amino acids, and the leader sequence of this protein is about 36 amino acids, but may be anywhere in the range of about 30 to about 42 amino acids
  • the TR2-SV2 receptor polypeptide encoded by the cDNA of ATCC Deposit Number 97057 comprises about 136 amino acids, but may be anywhere in the range of 120- 152 amino acids
  • the leader sequences for the TR2 protein shown in SEQ ID NO 26 is predicted to consist of amino acid residues -38 to -1 in SEQ ID NO.26, while the predicted mature TR2 protein consists of amino acid residues 1 to 245 in SEQ ID NO.26
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically
  • RNA such as mRNA
  • DNA including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically
  • DNA may be double-stranded or single-stranded Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or 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 or RNA, which has been removed from its native environment
  • recombinant DNA molecules contained in a vector are considered isolated for the purposes 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
  • Isolated RNA molecules include m vivo or in vitro RNA transcripts of the DNA molecules of the present invention
  • Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically
  • isolated nucleic acid molecules may be produced naturally, recombinantly, or synthetically
  • Isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in SEQ ID NO.26 or FIG 1A-1B (SEQ ID NO 1), DNA molecules comprising, or alternatively consisting of, the coding sequence for the mature TR2 receptor shown in SEQ ID NO 26 (last 245 amino acids) or FIG 1 A- IB (SEQ ID NO 2) (last 247 amino acids), and DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above but which, due to the degeneracy of the genetic code, still encode the TR2 receptor protein shown in SEQ ID NO 26 or FIG 1A-1B (SEQ ID NO 2)
  • ORF open reading frame
  • isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in FIG.
  • isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame
  • the invention provides isolated nucleic acid molecules encoding the TR2, TR2-SV1 and TR2-SV2 polypeptides having the amino acid sequences encoded by the cDNAs contained in the plasmid deposited as ATCC Deposit No. 97059, 97058 and 97057, respectively, on February 13, 1995.
  • these nucleic acid molecules will encode a mature polypeptide ortiae full-length polypeptide lacl ⁇ n ⁇
  • the invention further provides isolated nucleic acid molecules having the nucleotide sequences shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:l), FIG. 4A-4C (SEQE>NO:4), and FIG.7A-7C (SEQ ID NO:7); the nucleotide sequences of the cDNAs contained in the above-described deposited cDNAs; or nucleic acid molecules having a sequence complementary to one of the above sequences.
  • Such isolated molecules, particularly DNA molecules are useful, for example, as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TR2 receptor genes of the present invention in human tissue, for instance, by Northern blot analysis.
  • nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to (a) a nucleotide sequence encoding the TR2 polypeptide having the complete amino acid sequence shown in SEQ ID O:26,FIG. 1A-1B (amino acidresidues -36 to 247 in SEQ ID NO.2), FIG. 4A-4C (amino acid residues -36 to 149 in SEQ ID NO:5), or FIG.
  • FIG. 7A-7C amino acid residues 2 to 136 in SEQ ID NO:8 but lacking the N-te ⁇ ninal methionine; (c) a nucleotide sequence encoding the mature TR2 receptors (full-length polypeptide with any attending leader sequence removed) having the amino acid sequence at positions from about 1 to about 245 in SEQ ID NO:26, from about 37 to about 283 in FIG.
  • amino acid residues 1 to 247 in SEQ ID NO:2 amino acid residues 1 to 247 in SEQ ID NO:2
  • amino acid sequence at positions from about 37 to about 185 in FIG.4A-4C amino acid residues 1 to 149 in SEQ ID NO:5
  • RECTIFIED SHEET (RULE 91) ISA/EP 0) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), or (i).
  • “about” includes the particularly recited value and values larger or smaller by several (5, , 3, 2, or 1) amino acids.
  • 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 a TR2 receptor.
  • a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence 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 1 terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
  • the reference (query) sequence may be the entire TR2 receptor encoding nucleotide sequence shown in SEQ ID NO:25, FIG. 1 A-IB
  • any TR2 receptor polynucleotide fragment e.g., a polynucleotide encoding the amino acid sequence of any of the TR2 receptor N- and/or C- terminal deletions described herein, variant, derivative or analog, as described herein.
  • any particular nucleic acid molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the encoding nucleotide sequence shown in SEQ ID NO:25, FIG. 1 A- IB (SEQ ID NO: 1), FIG.4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ID NCc7), or to the nucleotide sequence of the deposited cDNAs, can be determined
  • RECTIFIED SHEET (RULE 91) ISA/EP 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 53711) 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
  • Bestfit program Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711
  • 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
  • Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to a reference sequence according to the present invention, 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
  • 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.
  • 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. Only bases outside the 5' and 3' bases of the subject sequence, as displayed by the FASTDB alignment, which are not matched/aligned with the query sequence, are calculated for the purposes of manually adjusting the percent identity score. For example, a 90 base subject sequence is aligned to a 100 base query sequence to determine percent identity. The deletions occur at the 5' end of the subject sequence and therefore, the FASTDB alignment does not show a matched/alignment of the first 10 bases at
  • the 10 unpaired bases represent 10% of the sequence (number of bases at the 5' and 3' ends not matched/total number of bases in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 bases were perfectly matched the final percent identity would be 90%.
  • 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%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein, (e.g., encoding a polypeptide having the amino acid sequence of an N and/or C terminal deletion disclosed herein, such as, for example, a nucleic acid molecule encoding amino acids 50 to 283 of SEQ ID NO:2), irrespective of whether they encode a polypeptide having a TR2 receptor functional activity.
  • nucleic acid molecules of the present invention that do not encode a polypeptide having TR2 receptor activity include, inter alia, (1) isolating a TR2 receptor gene or allelic or splice variants thereof in a cDNA library; (2) in situ hybridization (e.g.
  • FISH fluorescence in situ hybridization
  • nucleic acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:l), FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ED NO:7) or to the nucleic acid sequence of the deposited cDNAs which do, in fact, encode a polypeptide having TR2 receptor activity.
  • the polynucleotide fragments of the invention encode a polypeptide which demonstrates a TR2 functional activity.
  • a polypeptide having TR2 receptor activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TR2 receptors of the present invention (e.g., complete (full-length) TR2 receptor polypeptides, mature TR2 receptor polypeptides, secreted TR2 receptor polypeptides, and soluble TR2 receptor polypeptides (e.g., having sequences contained in the extracellular domain of a TR2 receptor ) as measured, for example, in a particular imrounoassay or biological assay.
  • a TR2 receptor activity can routinely be measured by determining the ability of a TR2 receptor polypeptide to bind a TR2 receptor ligand (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin- , and the Herpes virus protein HSVl gD).
  • TR2 receptor activity can routinely be measured by determining the ability of a TR2 receptor polypeptide to bind a TR2 receptor ligand (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin- , and the Herpes virus protein HSVl gD).
  • RECTIFIED SHEET (RULE 91) ISA/EP can be measured by dete ⁇ ni- ⁇ ing the ability of a polypeptide-Fc fusion protein to inhibit lymphocyte proliferation as described below in Example 6.
  • TR2 receptor activity may also be measured by determining the ability of a polypeptide, such as cognate ligand which is free or expressed on a cell surface, to confer proliferatory activity in intact cells expressing the receptor.
  • nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence of the deposited cDNAs or the nucleic acid sequences shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:l), FIG. 4A-4C (SEQ ID NO:4), or HG.
  • the present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of the isolated nucleic acid molecules
  • RECTIFIED SHEET (RULE 91) ISA/EP described herein.
  • a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNAs or the nucleotide sequence shown in SEQ ID NO:25, HG. 1 A-IB (SEQ ID NO:l), FIG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ED NO:7) is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) nucleotides.
  • 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 50, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825 or 848 nt in length are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequences of the deposited cDNAs or as shown in SEQ ID NO:25, FIG. 1 A-IB (SEQ ID NO: 1), FIG. 4A-4C (SEQ ID NO:4), or HG. 7A-7C (SEQ ED
  • fragments at least 20 nt in length are intended fragments which include 20 or more contiguous bases from the nucleotide sequences of the deposited cDNAs or the nucleotide sequences as shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:l), FIG. 4A-4C (SEQ ID NO.4), or FIG. 7A-7C (SEQ ED NO:7).
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding polypeptides comprising, or alternatively consisting of, the mature TR2-SV1 receptor (predicted to constitute amino acid residues from about 37 to about 185 in FIG. 4A-4C (amino acid residues 1 to 149 in SEQ ID NO:5)) and the complete TR2-SV2 receptor (predicted to constitute amino acid residues from about I to about 136 in FIG. 7A-7C (SEQ ED NO:8)).
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.
  • amino acid residues constituting the extracellular, transmembrane and intraceUular domains have been predicted by computer analysis
  • 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
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids
  • Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding a polypeptide comprising, or alternatively consisting of, the TR2 receptor protein of FIG 1A-1B (SEQ ID NO 2) extracellular domain (predicted to constitute amino acid residues from about 37 to about 200 in FIG 1A-1B (amino acid residues 1 to 164 in SEQ ID NO 2)), a polypeptide comprising, or alternatively consisting of, the TR2 receptor transmembrane domain (amino acid residues 201 to 225 in FIG 1A-1B (amino acid residues 165 to 189 in SEQ ID NO 2)), a polypeptide comprising, or alternatively consisting of, the TR2 receptor intraceUular domain (predicted to constitute amino acid residues from about 226 to about 283 in FIG 1A-1B (amino acid residues 190 to 247 in SEQ ID NO 2)), and a polypeptide comprising, or alternatively consisting of, the TR2 receptor protein of FIG 1A-1B (
  • Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding amino acid residues the extracellular domain of the TR2 protein having the amino acid sequence set out in SEQ ID NO 26, both with and without the associated leader sequence (amino acid residues -38 to 162 of SEQ ID NO 26 and amino acid residues 1 to 162 of SEQ ID NO 26, respectively)
  • Preferred nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of the TR2 receptor proteins.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising, or alternatively consisting of, one, two, three, four, five or more amino acid sequences selected from amino acid residues from about 39 to about 70 in FIG. 1A-1B (amino acidresidues 3 to 34 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 106 to about 120 in FIG. 1 (amino acid residues 70 to 84 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting o ⁇ amino acid residues from about 142 to about 189 in FIG.
  • 1A-1B amino acid residues 106 to 153 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting o£ amino acid residues from about 276 to about 283 in FIG. 1A-1B (amino acid residues 240 to 247 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 39 to about 70 in FIG. 4A-4C (amino acid residues 3 to 34 inSEQIDNO:5); amino acid residues from about 99 to about 136inFIG.4A-4C (amino acid residues 63 to 100 in SEQ ID NO:5); amino acid residues from about 171 to about 185 in FIG.
  • polypeptide fragments are antigenic regions of the TR2 receptors. Methods for determining other such epitope-bearing portions of the TR2 proteins are described in detail below. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • TR2 receptor polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to 64, 65 to 100, 101 to 150, 151 to 200,
  • RECTIFIED SHEET (RULE 91) ISA/EP 201 to 250, 225-265, 251 to 300, 301 to 350, 351 to 372, 373 to 450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, 701 to 750, 751 to 800, 801 to 850, 851 to 900, 901 to 950, 951 to 1000, 1001 to 1050, 1051 to 1100, 1070-1 1 13, 1 101 to 1150, 1 151 to 1200, 1201 to 1250, 1251 to 1300, 1301 to 1350, 1351 to 1400, 1401 to 1450, 1451 to 1500, 1501 to 1550, 1551 to 1600, or 1601 to 1670, of SEQ ID NO 1, the cDNA contained in the deposited identified as ATCC Deposit No 97059, or the complementary strand of any of these fragments
  • “about” includes the particularly recited ranges, larger or smaller by several (5, 4, 3, 2, or 1) nu
  • TR2 receptor polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 373 to 433, 373 to 450, 451 to 500, 501 to 550, 551 to 600, 601 to 650, 651 to 700, 701 to 750, 751 to 800, 801 to
  • cysteine repeat regions of the TR2 receptor disclosed in FIG 1 A- 1 B are important for interactions between the TR2 receptor and its ligands (e.g., AIM II (International Publication No WO 97/3491 1), Lymphotoxin- ⁇ , and the Herpes virus protein HSV1 glycoprotein D (gD))
  • ligands e.g., AIM II (International Publication No WO 97/3491 1), Lymphotoxin- ⁇ , and the Herpes virus protein HSV1 glycoprotein D (gD)
  • specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of cysteine repeat region A, B, C, or D disclosed in FIG. 16 and described in Example 6.
  • polynucleotides of the invention comprise, or alternatively consist of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polynucleotide sequence encoding one, two, or all three of the cysteine-rich motifs described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these nucleic acids and/or polynucleotide sequences are also encompassed by the invention.
  • the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under stringent hybridization conditions to one, two, or all three of the cysteine-rich motifs described above polynucleotides of the invention described above, or the complementary strand thereof.
  • stringent conditions as used herein is described infra.
  • the polynucleotide fragments of the invention encode a polypeptide which demonstrates one or more TR2 receptor functional activities.
  • a polypeptide demonstrating a TR2 receptor "functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) TR2 receptor protein.
  • Such functional activities include, but are not limited to, biological activity (e.g., inhibition of B cell proliferation), antigenicity, immunogenicity (ability to generate antibody which binds to a TR2 receptor polypeptide), the ability to bind (or compete with a TR2 receptor polypeptide for binding) to an anti-TR2 receptor antibody, the ability to form multimers with TR2 receptor polypeptides of the invention, and ability to bind to a receptor or ligand for a TR2 receptor polypeptide (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin- ⁇ , and the Herpes virus protein HSV1 glycoprotein D (gD)).
  • the functional activity of TR2 receptor 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.
  • competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric
  • 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.
  • Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • a TR2 receptor ligand e.g., AIM II (International Publication No WO 97/34911), Lymphotoxin- ⁇ , and the Herpes virus protein HSVl gD), or the ability of a polypeptide fragment, variant or derivative of the invention to multimerize is being evaluated, binding can be assayed, e.g.
  • TR2 receptor binding to its substrates can be assayed
  • assays described herein see, e g., Examples 6 and 8 and otherwise known in the art may routinely be applied to measure the ability of TR2 receptor polypeptides and fragments, variants derivatives and analogs thereof to elicit TR2 receptor related biological activity (e.g. , inhibition of B cell proliferation in vitro or m vivo)
  • TR2 receptor related biological activity e.g. , inhibition of B cell proliferation in vitro or m vivo
  • Other methods will be known to the skilled artisan and are within the scope of the invention
  • the invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, polynucleotides which hybridizes under stringent hybridization conditions to a portion of the polynucleotide of one of the nucleic acid molecules of the invention described above, for instance, the complement of a polynucleotide frgament described herein, or the cDNAs contained in ATCC Deposits 97059, 97058 and 97057
  • stringent hybridization conditions is intended overnight incubation at 42°C in a solution comprising, or alternatively consisting of 50% formamide, 5x SSC (750 mM NaCl, 75 mM 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 lx 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 nt of the reference polynucleotide.
  • nt nucleotides
  • the reference polynucleotide e.g., the deposited cDNAs or the nucleotide sequences as shown in SEQ ID NO:25, FIG. 1A-1B (SEQ ID NO:l), HG. 4A-4C (SEQ ID NO:4), or FIG. 7A-7C (SEQ ID NO:7).
  • nucleic acid molecules of the present invention which encode TR2 polypeptides may include, but are not limited to those encoding the amino acid sequences of the mature polypeptides, by itself; the coding sequence for the mature polypeptides and additional sequences, such as those encoding the about 36 amino acid leader or secretory sequences, such as pre-, or pro- or prepro- protein sequences; the coding sequence of the mature polypeptides, with or wimoutthe-iforementionedadditi ⁇ i ⁇ codingsequence ⁇ , together w 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;
  • the sequence encoding the polypeptides 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 etal, Proc. Natl. Acad. Sci.
  • 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 (1984)
  • other such fusion proteins include the TR2 receptors fused to IgG-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 TR2 receptors Variants may occur naturally, such as a natural allelic variant
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism Genes II, Lewin, B , ed , John Wiley & Sons, New York (1985)
  • 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.
  • 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.
  • silent substitutions, additions and deletions which do not alter the properties and activities of the TR2 receptors or portions thereof. Also especially preferred in this regard are conservative substitutions.
  • 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, and the production of TR2 polypeptides or fragments thereof by recombinant techniques.
  • the polynucleotides may be joined to a vector containing a selectable marker for propagation in a host.
  • a plasmid vector is introduced in a precipitate, such as a calcium phosphate precipitate, or in a complex with a charged lipid. 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 insert should be operatively linked to an appropriate promoter, such as the phage lambda PL promoter, the E. coli lac, trp and lac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • an appropriate promoter such as the phage lambda PL promoter, the E. coli lac, trp and lac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name a few.
  • Other suitable promoters will be known to the skilled artisan
  • the expression 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.
  • suitable heterologous hosts include, but are not limited to, bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and 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 pQ ⁇ 70, 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.
  • Other suitable vectors will be readily apparent to the skilled artisan.
  • 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
  • calcium phosphate transfection DEAE-dextran mediated transfection
  • cationic lipid-mediated transfection cationic lipid-mediated transfection
  • electroporation transduction
  • infection infection or other methods
  • methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals, but also additional heterologous functional regions. For instance, a region of additional amino acids, particularly charged amino acids, may be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification, or during subsequent handling and storage. Also, peptide moieties may be added to the polypeptide to facilitate purification. Such regions may be removed prior to final preparation of the polypeptide. The addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
  • a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins
  • EP-A-O 464 533 (Canadian counterpart 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin 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 (EP- A 0232262)
  • human proteins such as, human hIL-5 receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5 See, D Bennett e
  • TR2 receptors 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
  • 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, in some cases as
  • the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammali-m origin, that have been engineered to delete or replace endogenous genetic material (e.g., the TR2 receptor coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with TR2 receptor polynucleotides of the invention, and which activates, alters, and or amplifies endogenous TR2 receptor polynucleotides.
  • endogenous genetic material e.g., the TR2 receptor coding sequence
  • genetic material e.g., heterologous polynucleotide sequences
  • heterologous control regions e.g., promoter and/or enhancer
  • endogenous TR2 receptor polynucleotide sequences via homologous recombination
  • heterologous control regions e.g., promoter and/or enhancer
  • endogenous TR2 receptor polynucleotide sequences via homologous recombination
  • the invention further provides isolated TR2 polypeptides havingthe amino acid sequence encoded by the deposited cDNAs, or the amino acid sequence shown in SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), or FIG.7A-7C (SEQ ID NO:8), or apeptide or polypeptide comprising, or alternatively consisting of, a portion of the above polypeptides.
  • the polypeptides of this 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.
  • Soluble peptides are defined by amino acid sequence wherein the sequence comprises, or alternatively consists of, the polypeptide sequence lacking the transmembrane domain.
  • TR2-SV1 splice variant which has a secretory leader sequence but lacks both the intraceUular and transmembrane domains.
  • the TR2-SV1 receptor protein appears to be secreted in a soluble form from cells which express this protein-
  • 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.
  • FIG. 1 A-IB SEQ ID NO;2
  • TR2 receptor appears to be localized in the cytoplasmic membrane of cells which express this protein.
  • the polypeptides of the present invention also include the polypeptide encoded by the deposited cDNAs including the leader; the polypeptide encoded by the deposited the cDNAs minus the leader (le., the mature protein); the polypeptides of SEQ ID NO:26,FIG. 1A-1B (SEQIDNO:2)orFIG.4A-4C (SEQ ID NO:5) including the leader, the polypeptides of SEQ ID NO:26, FIG. 1A-1B (SEQ ID NO:2) or FIG.4A-4C (SEQ ID NO:5) including the leader butminus the N-te ⁇ ninal methionine; the polypeptides of SEQ ID NO:26, FIG. 1 A- IB (SEQ ID NO:2) or FIG. 4A-4C (SEQ ID NO.5) minus the leader; the polypeptide of FIG.
  • polypeptides which are at least 80% identical, more preferably at least 85%, 90%, 92% or 95% identical, still more preferably at least 96%, 97%, 98% or 99% identical to the polypeptides described above, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
  • polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TR2 polypeptide is
  • RECTIFIED SHEET (RULE 91) ISA/EP 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 a TR2 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 individuaUy 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%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:26, FIG. 1 A-IB (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), or FIG. 7A-7C (SEQ ID NO:8) or to the amino acid sequence encoded by one of the deposited cDNAs
  • the Bestfit program Wiconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer
  • 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.
  • RECTIFIED SHEET (RULE 91) ISA/EP
  • identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence, also referred to as a global sequence alignment, is determined using the FASTDB computer program based on the algorithm of Brutlag et al (Comp. App. Biosci.
  • the invention further includes variations of the TR2 receptors which show substantial TR2 receptor activity or which include regions of TR2 proteins such as the protein portions discussed below Such mutants include deletions, insertions, inversions, repeats, and type substitutions Guidance concerning which amino acid changes are likely to be phenotypically silent can be ⁇ 3-
  • the fragment, derivative or analog of the polypeptides of SEQ ID NO:26, HG. 1 A-IB (SEQ ID NO:2), FIG. 4A-4C (SEQ ID NO:5), and FIG. 7A- 7C (SEQ ID NO: 8), or that encoded by the deposited cDNAs may be (i) one in which one or more of the amino acid residues are substituted with a conserved or non-conserved amino acid residue (preferably a conserved amino acid residue) and such substituted amino acid residue may or may not be one encoded by the genetic code, or (ii) one in which one or more of the amino acid residues includes a substituent group, or (iii) 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 d e additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide
  • TR2 receptors 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 I).
  • Amino acids in the TR2 proteins of the present invention that are essential for function can be identified by methods known in the art, such as site-directed 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 for biological activity such as receptor binding or in vitro, or in vitro proliferative activity
  • Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al, J. Mol. Biol. 224 899-904 (1992) and de Vos et al. Science 255 306-312 (1992))
  • 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 contained within a recombinant host cell would be considered “isolated” for purposes of the present invention.
  • isolated polypeptide are polypeptides that have been purified, partially or substantially, from a recombinant host.
  • recombinantly produced versions of the TR2 receptors can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40
  • polypeptides of the present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • TR2 polypeptides of the invention can also inhibit mixed lymphocyte reactions (MLRs). As discussed below in Example 6, TR2 polypeptides inhibit three-way MLRs. An additional method for performing three-way MLRs is discussed in Harrop et al, Jour. Immunol 161: 1786-1794 (1998), which incorporated herein by reference.
  • MLRs mixed lymphocyte reactions
  • the present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the TR2 receptor polypeptide sequence set forth herein as n'-m 1 , n 2 -m 2 , n 3 -m ⁇ n 4 -m 4 , and/or n 5 -m 5 .
  • the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence of the specific TR2 receptor N- and C-terminal deletions recited herein.
  • TR2 receptor proteins of the invention comprise, or alternatively consist of, fusion proteins as described above wherein the TR2 receptor polypeptides are those described as n'-m 1 , n 2 -m 2 , n -m 3 , n 4 -m 4 , and/or n 5 -m 5 herein.
  • the application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%o identical to the nucleic acid sequences encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein Polynucleotides encoding these polypeptides are also encompassed by the invention As mentioned above, even if deletion of one or more amino acids from the
  • N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained
  • the ability of shortened TR2 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the 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 a TR2 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 TR2 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 TR2 amino acid sequence shown in FIG 1 A- IB (i.e., SEQ ID NO 2), up to the glycine residue at position number 278 and polynucleotides encoding such polypeptides
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-283 of FIG 1 A- IB (SEQ ID NO 2), where n 1 is an integer in the range of 2 to 278 Polynucleotides encoded by these polypeptides are also encompassed by the invention
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of E-2 to H-283, P-3 to H-283, P-4 to H-283, G-5 to H-283, D-6 to H-283, W-7 to H-283, G-8 to H-283, P-9 to H-283, P-10 to H-283, P-l l to H-283, W-12 to H-283, R-13 to H-283, S-14 to H-283, T-15 to H-283, P-16 to H-283, K-17 to H-283, T-18 to H-283, D-19 to H-283, N-20 to H-283, L-21 to H-283, R-22 to H-283, L-23 to H-283, N-24 to H-283, L-25 to H-283, Y-26 to H-283, L-27 to H-283, T-28 to H-283, F-29 to H-283, L-30 to H-283, G-31 to
  • H-283 G-174 to H-283, T-175 to H-283, L-176 to H-283, E-177 to H-283, E-178 to H-283, C-l 79 to H-283, Q-180 to H-283, H-181 to H-283, Q-182 to H-283, T-183 to H-283, K-184 to H-283, C-185 to H-283, S-186 to H-283, W-187 to H-283, L-188 to H-283, V-189 to H-283, T-190 to H-283, K-191 to H-283, A- 192 to H-283, G-193 to H-283, A- 194 to H-283, G-195 to H-283,
  • H-283 Q-242 to H-283, R-243 to H-283, K-244 to H-283, R-245 to H-283, Q-246 to H-283, E-247 to H-283, A-248 to H-283, E-249 to H-283, G-250 to H-283, E-251 to H-283, A-252 to H-283, T-253 to H-283, V-254 to H-283, 1-255 to H-283, E-256 to H-283, A-257 to H-283, L-258 to H-283, Q-259 to H-283, A-260 to H-283, P-261 to H-283, P-262 to H-283, D-263 to H-283,
  • 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 polypeptides described above
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence Polypeptides encoded
  • the ability of the shortened TR2 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus Whether a particular polypeptide lacking C-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 a TR2 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities In fact, peptides composed of as few as six TR2 amino acid residues may often evoke an immune response
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR2 polypeptide shown in FIG 1 A- 1 B (SEQ ID NO 2), up to the aspartic acid residue at position number 6, and polynucleotides encoding such polypeptides
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m 1 of FIG 1A-1B (i.e., SEQ ID NO.2), where m 1 is an integer in the range of 6 to 282 Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues M-1 to N-282, M-1 to P-281, M-1 to S-280, M-1 to R-279, M-1 to G-278, M-1 to T-277, M-1 to F-276, M-1 to S-275, M-1 to P-274, M-1 to 1-273, M- 1 to T-272, M- 1 to E-271 , M- 1 to E-270, M- 1 to N-269, M- 1 to A-268, M- 1 to N-267, M-1 to T-266, M-1 to T-265, M-1 to V-264, M-1 to D-263, M-1 to P-262, M-1 to P-261, M-1 to A-260, M-1 to Q-259, M-1 to L-258, M-1 to A-257, M-1 to E-256, M-1 to 1-255, M-1 to V-254, M-1 to T-253, M-1 to
  • M-1 to T-190 M-1 to V-189, M-1 to L-188, M-1 to W-187, M-1 to S-186, M-1 to C-185, M-1 to K-184, M-1 to T-183, M-1 to Q-182, M-1 to H-181, M-1 to Q-180, M-1 to C-179, M-1 to E-178, M-1 to E-177, M-1 to L-176, M-1 to T-175, M-1 to G-174, M-1 to ⁇ -173, M-1 to P-172, M-1 to S-171, M-l to F-170, M-l to T-169, M-l to G-168, M-l to P-167, M-l to P-166,
  • M-1 to C-l 19 M-1 to N-l 18, M-1 to A-1 17, M-1 to ⁇ -1 16, M-1 to E-1 15, M-1 to T-1 14, M-1 to R-113, M-1 to S-1 12, M-1 to C-l 1 1, M-1 to N-l 10, M-1 to R-109, M-1 to S-108, M-1 to A-107, M-1 to R-106, M-1 to L-105, M-1 to G-104, M-1 to M-103, M-1 to A-102, M-1 to P-101, M-1 to D-100, M-1 to C-99, M-1 to M-98, M-1 to Q-97, M-1 to C-96, M-1 to Q-95,
  • 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 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fiise to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR2 polypeptide, which may be described generally as having residues n'-m 1 of FIG. 1 A-IB (L e. , SEQ ID NO:2), where n l and m 1 are integers as described above. Polynucleotides encoded by these polypeptides are also encompassed by the invention. Also mentioned above, even if deletion of one or more amino acids from the N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other biological activities may still be retained.
  • the ability of shortened TR2-SV1 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the 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 a TR2-S VI 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 TR2-SV1 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 TR2-S V 1 amino acid sequence shown in FIG. 4A-4C (i. ⁇ . , SEQ ID NO:5), up to the threonine residue at position number 180 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n 2 -l 85 of FIG.4A-4C (SEQ ID NO:5)
  • n 2 is an integer in the range of 2 to 180, and 180 is the position of the first residue from the N-terminus of the complete TR2-SV1 polypeptide believed to be required for at least immunogenic activity of the TR2-SV1 polypeptide
  • Polynucleotides encoded by these polypeptides are also encompassed by the invention
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of E-2 to A-185, P-3 to A-185, P-4 to A-185, G-5 to A-185, D-6 to A-185, W-7 to A-185, G-8 to A-185, P-9 to A-185, P-10 to A-185, P-l l to
  • 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 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • RECTIFIED SHEET (RULE 91) ISA/EP complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-tenninal 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 a TR2-SV1 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or i munogenic activities. In fact, peptides composed of as few as six TR2-SV1 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the TR2-SV1 polypeptide shown in FIG.4A-4C (SEQ ID NO:5), up to the aspartic acid residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues I -m 2 of FIG. 4A-4C (i. e., SEQ ID NO:5), where m 2 is an integer in the range of
  • polypeptides 6 to 184.
  • Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues M-1 to R-184; M-1 to G-183; M-1 to G-182; M-1 to P-181; M-1 to
  • T-180 M-1 to G-179; M-1 to D-178; M-1 to A-177; M-1 to R-176 M-1 to D-175; M-1 to R-174; M-1 to L-173; M-1 to H-172; M-1 to L-171 M-1 to G-170; M-1 to A-169; M-1 to Q-168; M-1 to G-167; M-1 to C-166 M-1 to P-165; M-1 to A-164; M-1 to S-163; M-1 to A-162; M-1 to M-161 M-l to S-160; M-1 to L-159; M-I to R-158; M-1 to A-157; M-1 to H-156: M-1 to
  • M-1 to E-154 M-1 to L-153; M-1 to P-152; M-1 to V-151 M-1 to S-150; M-1 to G-149; M-1 to N-148; M-1 to A-147; M-1 to A-146 M-1 to H-145; M-1 to V-144; M-1 to P-143; M-1 to E-142; M-1 to A-141 M-1 to S-140; M-1 to L-139; M-1 to S-138; M-1 to E-137; M-1 to F-136 M- ⁇ to
  • RECTIFIED SHEET (RULE 91) ISA/EP A-135; M-1 to V-134; M-1 to E-133; M-1 to P-132; M-1 to D-13J; M-l to P-130; M-1 to E-129; M-1 to G-128; M-1 to K-127; M-1 to Q-126; M-1 to R-125; M-1 to G-124; M-1 to P-123; M-1 to S-122; M-1 to L-121; M-1 to H-120; M-1 to A-119; M-1 to G-118; M-1 to A-117; M-1 to E-116; M-1 to L-115; M-1 to H-J 14; M-1 to G-113; M-1 to R-112; M-1 to G-ll l; M-1 to
  • M-l to Y-26 M-l to L-25; M-l to V-24; M-l to L-23; M-l to R-22; M-l to L-21; M-l to V-20; M-l to D-19; M-l to T-18; M-l to R-17; M-l to P-16; M-l to T-15; M-l to S-14; M-l to R-13; M-l to W-12; M-l to P-l 1; M-l to P-10; M-l to P-9; M-l to G-8; M-l to W-7; and M-l to D-6 of the sequence of the TR2-S VI sequence shown in FIG.4 A-4C.
  • 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 polypeptides described above.
  • the present invention also encompasses the above
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR2-SV1 polypeptide, which may be described generally as having residues n 2 -m 2 of FIG. 4A-4C (i.e.,
  • n 2 and m 2 are integers as described above.
  • Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • TR2-S V2 mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities.
  • peptides composed of as few as six TR2-S V2 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 TR2-S V2 amino acid sequence shown in FIG. 7A-7C (be., SEQ ID NO: 8), up to the serine residue at position number 131 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting o ⁇ the amino acid sequence of residues n 3 -136 of FIG. 7A-7C (i.e., SEQ ID NO:8), where n 3 is an integer in the range of 2 to 131. Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of L-2 to K-136, G-3 to K-136, T-4 to K-136, S-5 to K-136, G-6 to K-136, H-7 to K-136, L-8 to K-136, N-9 to K-136, W-10 to K-136, L-1 1 to K-136, S-12 to K-136, Q-13 to K-136, G-14 to K-136, F-15 to K-136, S-16 to
  • 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 polypeptides described above,
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence oftheTR2-SV2 polypeptide shown in FIG.7A-7C (i.e., SEQIDNO.8), up to the glycine residue at position number 6, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1-m 1 of FIG. 7A-7C (i.e., SEQ ID NO:8), where m 3 is an integer in the range of 6 to 135. Polynucleotides encoded by these polypeptides are also encompassed
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues M-l to S-135; M-l to P-134; M-l to R-133; M-l to T-132; M-l to S-131; M-l to V-130; M-l to N-129; M-l to R-128; M-l to W-127; M-l to
  • RECTIFIED SHEET (RULE 91) ISA/EP of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a TR2-S V2 polypeptide, which may be described generally as having residues n 3 -ra 3 of FIG. 7A-7C (ie., SEQIDNO:8),wheren 3 andm 3 areintegersasdescribcd above. Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the predicted extracellular domain of the TR2 amino acid sequence shown in SEQ ID NO:2 (FIG. 1A-1B), up to the glycine residue at position number 159 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n 4 - 164 of SEQ ID NO :2, where n* is an integer in the range of 1 to 159. Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of P-l to H-164; A-2 to H-164; L-3 to H-164; P-4 to H-164; S-5 to H-164; C-6 to H-164; K-7 to H-164; E-8 to H-164; D-9 to H-164; E-10 to H-164; Y-ll to H-164; P-12 to H-164; V-13 to H-164; G-14 to H-164; S-15 to H-164;
  • RECTIFIED SHEET (RULE 91) ISA/EP H-164, G-36 to H-164, T-37 to H-164, N-38 to H-164, C-39 to H-164, E-40 to H-164, P-41 to H-164, C-42 to H-164, P-43 to H-164, P-44 to H-164, G-45 to H-164, T-46 to H-164, Y-47 to H-164, 1-48 to H-164, A-49 to H-164, H-50 to H-164, L-51 to H-164, ⁇ -52 to H-164, G-53 to H-164, L-54 to H-164, S-55 to H-164, K-56 to H-164, C-57 to H-164, L-58 to H-164, Q-59 to H-164, C-60 to
  • H- 164 Q-61 to H- 164, M-62 to H- 164, C-63 to H- 164, D-64 to H- 164, P-65 to H-164, A-66 to H- 164, M-67 to H- 164, G-68 to H- 164, L-69 to H- 164, R-70 to H-164, A-71 to H-164, S-72 to H-164, R-73 to H-164, N-74 to H-164, C-75 to H-164, S-76 to H-164, R-77 to H-164, T-78 to H-164, E-79 to H-164, N-80 to H-164, A-81 to H-164, V-82 to H-164, C-83 to H-164, G-84 to H-164, C-85 to
  • H-164 S-86 to H-164, P-87 to H-164, G-88 to H-164, H-89 to H-164, F-90 to H-164, C-91 to H-164, 1-92 to H-164, V-93 to H-164, Q-94 to H-164, D-95 to H-164, G-96 to H-164, D-97 to H-164, H-98 to H-164, C-99 to H-164, A- 100 to H-164, A-101 to H-164, C-102 to H-164, R-103 to H-164, A-104 to H-164, Y-105 to H-164, A-106 to H-164, T-107 to H-164, S-108 to H-164, S-109 to
  • 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 polypeptides described above
  • the present invention also encompasses the above polynucleotide sequences fused to a hetero
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence ofresidues P-l to H-164, P-l to S-163, P-l to S-162, P-l to S-161, P-l to T-160, P-l to G-159, P-l to A-158, P-l to G-157, P-l to A-156, P-l to K-155, P-l to T-154, P-l to V-153, P-l to L-152, P-l to W-151, P-l to S-150, P-l to C-149, P-l to K-148, P-l to T-147, P-l to Q-146, P-l to H-145, P-l to Q-144, P-l to C-143, P-l to E-142, P-l to E-141, P-l to L-140, P-l to T-139, P-l to G-138,
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a soluble TR2 polypeptide, which may be described generally as having residues n -m 4 of SEQ ID NO:
  • ED NO:2 (FIG. 1A-1B), where n* and m 4 are integers as described above. Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the predicted extracellular domain of the TR2-SV1 amino acid sequence shown in SEQ ID NO:5 (FIG. 4A-4C), up to the threonine residue at position number 144 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n 5 -149 of SEQ ID NO:5, where n 5 is an integer in the range of 1 to
  • Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of P-l to A-149; A-2 to A-149; L-3 to A-149; P-4 to A-149; S-5 to
  • A-149 P-129 to A-149; C-130 to A-149; G-13I to A-149; Q-132 to A-149;
  • A-149; R-138 to A-149; D-139 to A-149; R-140 to A-149; A-141 to A-149; D-142 to A-149; G-143 to A-l 49; and T-144 to A-149 of the TR2-SV1 amino acid sequence shown in SEQ ID NO:5 (which is identical to that shown in FIG. 4A-4C, with the exception that the amino acid residues in FIG. 4A-4C are numbered consecutively from 1 through 185 from the N-te ⁇ ninus to the C-terminus, while the amino acid residues in SEQ ID NO:5 are numbered consecutively from -36 through 149 to reflect the position of the predicted signal peptide).
  • 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 polypeptides described above.
  • the present invention also provides 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 polypeptides described above.
  • the present invention also provides 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 polypeptides described above.
  • the present invention
  • ISA/EP encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the predicted extracellular domain of the amino acid sequence of the TR2-SV1 shown in SEQ ED NO :5 (FIG. 4A-4C), up to the cysteine residue at position number 6 in SEQ ID NO:5, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1 -m 3 of SEQ ID NO:5 (FIG.4A-4C), where m 5 is an integer in the range of 6 to 149. Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues P-l to A-149; P-l to R-148; P-l to G-147; P-l to G-146; P-l to
  • 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 polypeptides described above.
  • the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotide sequences are also encompassed by the invention.
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a soluble TR2-SV1 polypeptide, which may be described generally as having residues n 5 -m s of SEQ ID NO:
  • n 5 and m 5 are integers as described above.
  • Polynucleotides encoded by these polypeptides are also encompassed by the invention.
  • ISA/EP of the invention e.g., arginine and lysine residues
  • ISA/EP of the invention may be deleted or substituted with another residue to eliminate undesired processing by proteases such as, for example, furins or kexins.
  • the invention further provides for the proteins containing polypeptide sequences encoded by the polynucleotides of the invention.
  • fragments of the invention are fragments characterized by structural or functional attributes of TR2 receptors of the invention.
  • Such fragments include amino acid residues that comprise, or alternatively consist of, 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”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (be., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson-Wolf program) of complete (i.e., full-length) TR2 receptor (SEQ ID NO:2).
  • Certain preferred regions are those set out in FIG. 3 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in FIG. 1A-1B (SEQ ID NO:2), such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn- regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Kyte-Doolittle predicted hydrophilic and hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson-Wolf high antigenic index regions, as predicted using the default parameters of these computer programs.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the polynucleotides of the invention encode functional attributes of TR2 receptors.
  • Preferred embodiments of the invention in this regard include fragments that comprise, or alternatively consist of, one, two, three, four or more of one or more of the following functional domains: 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”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TR2 receptors.
  • the data representing the structural or functional attributes of the TR2 receptors set forth in FIGs. 3, 6 and 9 and Tables II, II and IX were generated using the various identified modules and algorithms of the DNA*STAR set on default parameters.
  • the data presented in columns VIII, IX, XIII, and XIV of Table II can be used to determine regions of TR2 receptors which exhibit a high degree of potential for antigenicity. Regions of high antigenicity are determined from the data presented in columns VIII, IX,
  • XIII, and/or IV 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. Certain preferred regions in these regards are set out in FIGs. 3, 6 and 9, but may, as shown in Tables II, III and IV, respectively, be represented or identified by using tabular representations of the data presented in FIGs. 3, 6 and 9.
  • the DNA*STAR computer algorithm used to generate FIGs. 3, 6 and 9 (set on the original default parameters) was used to present the data in FIGs. 3, 6 and 9 in a tabular format. (See Tables II, III and IV, respectively).
  • FIGs. 3, 6 and 9 and in Tables II, III and IV include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in FIGs. 1, 4 and 7. As set out in FIGs.
  • such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (columns I, III, V, and VII in Tables II, III and IV), Chou-Fasman alpha-regions, beta-regions, and turn-regions (columns II, IV, and VI in Tables II, III and IV), Kyte-Doolittle hydrophilic regions (column VIII in Tables II, III and IV), Hopp-Woods hydrophobic regions (column IX in Tables II, III and IV), Eisenberg alpha- and beta-amphipathic regions (columns X and XI in Tables II, III and IV), Karplus-Schulz flexible regions (column XII in Tables II, III and IV), Jameson-Wolf regions of high antigenic index (column XIII in Tables II, III and IV), and Emini surface-forming regions
  • Trp 187 B I 3 0.04 0.69 k -0.60 0.15
  • Trp 36 1 T . -1.23 0.84 0.20 0.10
  • Trp 50 B 0.39 0.53 F -0.25 0.98
  • TR2 polypeptides of the present invention and epitope-bearing fragments thereof can be combined with heterologous 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, and any combination thereof, including both entire domains and portions thereof), resulting in chimeric polypeptides.
  • immunoglobulins IgA, IgE, IgG, IgM
  • CHI constant domain of immunoglobulins
  • CH2, CH3 any combination thereof, including both entire domains and portions thereof
  • the present invention is further directed to isolated polypeptides comprising, or alternatively consisting of, fragments of TR2, TR2-SV1, and TR2-SV2.
  • the invention provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids -36 to 24, -26 to 34, -16 to 44, -6 to 54, 1 to 60, 1 1 to 70, 21 to 80, 31 to 90, 41 to 100, 51 to 1 10, 61 to 120, 71 to 130, 81 to 140, 91 to 150, 101 to 160, 1 1 1 to 170, 121 to 180, 131 to 190, 141 to 200, 151 to 210, 161 to 220, 171 to 230, 181 to 240, and 191 to 247 of SEQ ID NO :2, as well as isolated polynucleotides which encode these polypeptides.
  • the invention further provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids -36 to 24, -26 to 34, -16 to 44, -6 to 54, 1 to 60, 11 to 70, 21 to 80, 31 to 90, 41 to 100, 51 to 110, 61 to 120, 71 to 130, 81 to 140, and
  • the invention also provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids 1 to 60, 1 1 to 70, 21 to 80, 31 to 90, 41 to 100, 51 to 1 10, 61 to 120, 71 to 130, and 81 to 136 of SEQ ID NO: 8, as well as isolated polynucleotides which encode these polypeptides.
  • the present invention is also directed to isolated polypeptides comprising, or alternatively consisting of, domains of TR2, TR2-SV1, and TR2-SV2.
  • the invention provides polypeptides comprising, or alternatively consisting of, beta-sheet regions of TR2, TR2-SV1, and TR2-SV2 set out in Tables II, III and IV.
  • These polypeptides include polypeptides comprising, or alternatively consisting of, amino acid sequences of a member selected from the group consisting of amino acid residues from about -19 to about -5, amino acid residues from about -18 to about -6, amino acid residues from about -2 to about
  • the invention is further directed to isolated polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode the beta-sheet regions set out in Tables II, III and IV, and isolated polypeptides comprising, or alternatively consisting of, amino acid sequences at least 80% identical, and more preferably at least 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to nucleic acid molecules encoding beta-sheet regions of the TR2, TR2-SV1, and TR2-SV2 proteins
  • the TR2 receptor 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 TR2 receptor 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 dimers,
  • Multimers encompassed by the invention may be homomers or heteromers
  • the term homomer refers to a multimer containing only TR2 receptor proteins of the invention (including TR2 receptor fragments, variants, and fusion proteins, as described herein) These homomers may contain TR2 receptor proteins having identical or different polypeptide sequences
  • a homomer of the invention is a multimer containing only TR2 receptor proteins having an identical polypeptide sequence
  • a homomer of the invention is a multimer containing TR2 receptor proteins having different polypeptide sequences
  • the multimer of the invention is a homodimer (e.g., containing TR2 receptor proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing TR2 receptor proteins having identical or different polypeptide sequences)
  • the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer
  • heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TR2 receptor gene) in addition to the TR2 receptor proteins of the invention
  • the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer
  • the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer
  • Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation
  • multimers of the invention such as, for example, homodimers or homotrimers
  • heteromultimers of the invention such as, for example, heterotrimers or heterotetramers
  • 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 TR2 receptor proteins of the invention
  • covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the TR2 receptor proteins (e.g. , the polypeptide sequence recited in SEQ ID NO 2, SEQ ID NO 5, SEQ ID NO 8, or SEQ ID NO 26, or the polypeptides encoded by the c
  • the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide
  • the covalent associations are the consequence of chemical or recombinant manipulation Alternatively, such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TR2 receptor fusion protein
  • covalent associations are between the heterologous sequence contained in a fusion protein of the invention (ee, e.g., U S Patent No 5,478,925)
  • the covalent associations are between the heterologous sequence contained in a TR2 receptor-Fc fusion protein of the invention (as described herein).
  • covalent associations of fusion proteins of the invention are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g., International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
  • two or more TR2 polypeptides of the invention are joined through synthetic linkers (e.g., peptide, carbohydrate or soluble polymer linkers). Examples include, but are not limited to, those peptide linkers described in U.S. Pat. No. 5,073,627 (hereby incorporated by reference). Proteins comprising multiple TR2 polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.
  • TR2 polypeptides of the invention involves use of TR2 polypeptides fused to a leucine zipper polypeptide sequence.
  • Leucine zipper domains are polypeptides that promote multimerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA-binding proteins (Landschulz et al. , Science 240: 1759, ( 1988)), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble multimeric TR2 proteins are those described in PCT application WO 94/10308, hereby incorporated by reference.
  • Recombinant fusion proteins comprising a soluble TR2 polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble multimeric TR2 is recovered from the culture supernatant using techniques known in the art.
  • trimeric TR2 may offer the advantage of enhanced biological activity
  • Preferred leucine zipper moieties are those that preferentially form trimers
  • SPD lung surfactant protein D
  • Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric TR2
  • proteins of the invention are associated by interactions between Flag® polypeptide sequence contained in Flag®-TR2 or Flag®-TR2 fusion proteins of the invention.
  • associations proteins of the invention are associated by interactions between heterologous polypeptide sequence contained in Flag®-TR2 or Flag®-TR2 fusion proteins of the invention and anti-Flag® antibody
  • the multimers of the invention may be generated using chemical techniques known in the art
  • proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g., U S Patent Number 5,478,925, which is herein incorporated by reference in its entirety)
  • multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., U S Patent Number 5,478,925, which is herein incorporated by reference in its entirety)
  • proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C-terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., U S Patent Number 5,478,925, which is herein incorporated
  • 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., U.S. 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., U.S. 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 polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., U.S. Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
  • 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, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in the deposited cDNA identified as ATCC Accession No. 97059, 97058 or 97057 or encoded by a polynucleotide that hybridizes to the complement of the polynucleotide sequence of SEQ ID NO: 1, SEQ ID NO:4, SEQ ID NO:7, or SEQ ID NO:25, or contained in the deposited cDNA identified as ATCC Accession No.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined herein.
  • polypeptide sequence of the invention such as, for example, the sequence disclosed in SEQ ID NO:2, SEQ ID NO:5, SEQ ID NO:8, or SEQ ID NO:26
  • polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined
  • 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, Proc.
  • antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically 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.
  • Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (/ ' . e. , immunogenic epitopes) nor to the amino or carboxyl terminals.
  • 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 57:767-778 (1984) at 777.
  • Antigenic epitopes of the invention 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
  • 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.
  • Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope. Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al, Cell 37:767-778 (1984); Sutcliffe et al, Science 219:660-666 (1983)).
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TR2 receptor-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 39 to about 70 in FIG. 1 (amino acid residues 3 to 34 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 106 to about 120 in FIG. 1A-1B (amino acid residues 70 to 84 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 142 to about 189 in FIG.
  • 1A-1B amino acid residues 106 to 153 in SEQ ID NO: 2
  • a polypeptide comprising, or alternatively consisting of, amino acid residues from about 276 to about 283 in FIG. 1 (amino acid residues 240 to 247 in SEQ ID NO:2)
  • a polypeptide comprising, or alternatively consisting of, amino acid residues from about 39 to about 70 in FIG.4A-4C (amino acid residues 3 to 34 in SEQ ID NO:5)
  • a polypeptide comprising, or alternatively cons-sting of, amino acid residues from about 99 to about 136 in FTG.
  • 4A-4C amino acid residues 63 to 100 in SEQ ID NO:5; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 171 to about 185 in FIG. 4A-4C (amino acid residues 135 to 149 in SEQ ID NO:5); a polypeptide comprising, or alternatively consisting of, amino acid residues from about 56 to about 68 in HG. 7A-7C (SEQ ID NO:8); and a polypeptide comprising, or alternatively consisting of, amino acid residues from about 93 to about 136 in FIG. 7A-7C (SEQ ID NO: 8).
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.
  • the inventors have determined that the above polypeptide fragments are antigenic regions of the TR2 receptor proteins.
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means. Houghten, . A. (1985) General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids, Proc. Nail. Acad Sci. USA 82:5131-5135. This "Simultaneous Multiple Peptide Synthesis
  • immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art (See, for instance, Sutcli fe et al., supra; Wilson et aL, supra; Chow et al. , Proc. Natl. Acad. Sci.
  • a preferred immunogenic epitope includes the secreted protein.
  • 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
  • RECTIFIED SHEET (RULE 91) ISA/EP system (such as, for example, 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 However, 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 ,
  • mice 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 as glutaraldehyde
  • Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intrape ⁇ toneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adju
  • 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) IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide 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.
  • 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
  • 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, 5,811,238, 5,830,721 , 5,834,252, and 5,837,458, and Patten et al. , Curr Opinion Biotechnol 8 724-33 (1997), Harayama, Trends Biotechnol
  • alteration of polynucleotides corresponding to SEQ ID NO 1 and the polypeptides encoded by these polynucleotides 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
  • Proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins' Structures and Molecular Principles, W H Freeman & Co , N Y , and Hunkapiller, M , et al.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids, 2,4-d ⁇ aminobutyric acid, ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, ⁇ -Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3 -amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic
  • 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 etal, Philos. Trans. R. Soc. London Ser A 317:415 (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
  • TR2 receptor polypeptides 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 4 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, 500, 1000, 1500, 2000, 2500, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 1 1,000, 1 1,500, 12,000, 12,500, 13,000, 13,500, 14,000,
  • the polyethylene glycol may have a branched structure. Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol.
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference
  • 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.
  • the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
  • Selective proteins chemically modified at the N- terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein Under the appropriate reaction conditions, substantially selective derivatization of the
  • 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 linker
  • Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al. , C . Rev. Thera. Drug Carrier Sys. 9 249-304 ( 1992), Francis et al. , Intern.
  • One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH 2 CF 3 )
  • MPEG monmethoxy polyethylene glycol
  • ClSO 2 CH 2 CF 3 tresylchloride
  • the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
  • Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
  • U.S. Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
  • Protein-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- succinimidylsuccinate, MPEG activated with 1 , 1 '-carbonyldiimidazole, MPEG- 2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various
  • MPEG-succinate derivatives A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope of the invention.
  • 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,
  • the present invention further relates to antibodies and T-cell antigen receptors (TCR) which specifically bind the polypeptides of the present invention.
  • the antibodies of the present invention include IgG (including IgGl, IgG2, IgG3, and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY
  • the term "antibody” (Ab) is meant to include whole antibodies, including single-chain whole antibodies, and antigen-binding fragments thereof
  • the antibodies are human antigen binding antibody fragments of the present invention 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 V L or V H domain
  • the antibodies may be from any animal origin including birds and mammals
  • the antibodies are human, murine, rabbit, goat, guinea
  • the antibodies of the present invention may be monospecific, bispecific, t ⁇ specific or of greater multi specificity 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 heterologous compositions, such as a heterologous polypeptide or solid support material See, .
  • Antibodies of the present invention may be described or specified in terms of the ep ⁇ tope(s) or port ⁇ on(s) of a polypeptide of the present invention which are recognized or specifically bound by the antibody
  • the ep ⁇ tope(s) or polypeptide port ⁇ on(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 the polypeptides of the present invention are included
  • 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. Further included in the present invention are antibodies which only bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions (as described herein) Antibodies of the present invention may also be described or specified in terms of their binding affinity Preferred binding affinities include those with a dissociation constant or Kd less than
  • 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 In preferred embodiments, the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, 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
  • 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 (i.e., signaling) may be determined by techniques described herein or otherwise known in the art For example, receptor activation can be determined by detecting the phosphorylation
  • antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, 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 Likewise, included in the invention are 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 Further included in the invention are antibodies which activate the receptor. These antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation.
  • 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
  • Antibodies of the present invention have uses that include, but are not limited to, methods known in the art 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 in the 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, or toxins. See, e.g., WO 92/08495, WO 91/14438, WO 89/12624, U S Patent Number 5,314,995, and EP 0 396 387
  • 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, phosphorylation, 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 prepared 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 For example, a polypeptide of the invention can be administered to
  • 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 ah, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981)
  • the term “monoclonal antibody” 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.
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant and phage display technology.
  • Hybridoma techniques include those known in the art and taught in Harlow et al, ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); and Hammerling, et al, in:
  • Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
  • antibodies of the present invention can be produced through the application of recombinant DNA and phage display technology or through synthetic chemistry using methods known in the art.
  • the antibodies of the present invention can be prepared using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them.
  • Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene NIII protein.
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman U. et al, J. Immunol. Methods 182:41-50 (1995); Ames, R.S. et al, J. Immunol. Methods 184: 177-1 ⁇ 6 (1995); Kettleborough, CA. etal., Eur. J. Immunol. 24:952-95% (1994); Persic, L.
  • it may be preferable to use chimeric, humanized, or human antibodies Methods for producing chimeric antibodies are known in the art See e.g., Morrison, Science 229 1202 (1985), Oi etal., B ⁇ oTechn ⁇ ques 4 214 (1986), Gillies, S D et al, J. Immunol.
  • antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide of the present invention may be specific for antigens other than polypeptides of the present invention
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors
  • Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art See e.g. , Harbor et al.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
  • the antibody portion fused to a polypeptide of the present invention may comprise the hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
  • the polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions. Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See e.g., U.S.
  • the invention further relates to antibodies which act as agonists or antagonists of the polypeptides of the present invention.
  • Antibodies which act as agonists or antagonists of the polypeptides of the present invention include, for example, antibodies which disrupt receptor/ligand interactions with the polypeptides of the invention either partially or fully.
  • the present invention includes antibodies which disrupt the ability of the proteins of the invention to multimerize.
  • the present invention includes antibodies which allow the proteins of the invention to multimerize, but disrupts the ability of the proteins of the invention to bind one or more TR2 receptor(s) or ligand(s) (e.g., AIM II (International Publication No.
  • the present invention includes antibodies which allow the proteins of the invention to multimerize, and bind TR2 receptor(s) or ligand(s) (e.g., AIM II (International Publication No. WO 97/34911), Lymphotoxin- ⁇ , and the Herpes virus protein
  • HSNl gD blocks biological activity associated with the TR2 receptor/ligand complex.
  • Antibodies which act as agonists or antagonists of the polypeptides of the present invention also include, both receptor-specific antibodies and ligand- specific antibodies. Included are receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation. Receptor activation (i.e. , signaling) may be determined by techniques described herein or otherwise known in the art. Also included are receptor-specific antibodies which both prevent ligand binding and receptor activation. Likewise, included are 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 Further included are antibodies which activate the receptor.
  • antibodies may act as agonists for either all or less than all of the biological activities affected by ligand-mediated receptor activation.
  • the antibodies may be specified as agonists or antagonists for biological activities comprising specific activities disclosed herein.
  • the above antibody agonists can be made using methods known in the art. See e.g. , WO 96/40281; U.S. Patent Number 5,811,097; Deng, B. et al, Blood 92: 1981-1988 (1998); Chen, Z. et al, Cancer Res. 5S:3668-3678 (1998); Harrop, J.A. et al., J. Immunol. 767: 1786-1794 (1998); Zhu, Z. et al, Cancer Res. 55:3209-3214
  • 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 isolated 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 that 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
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them
  • 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 Ml 3 binding domains expressed from phage with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene NIII protein
  • Examples of phage display methods that can be used to make the antibodies of the present invention include those disclosed in Brinkman etal
  • 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
  • techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in PCT publication WO 92/22324; Mullinax et al, BioTechniques 12(6) 864-869 (1992); and Sawai et al, AJRI 34:26-34 (1995); and Better et al, Science 240: 1041-1043 (1988) (said references incorporated by reference in their entireties).
  • 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.
  • 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.
  • 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.
  • veneering or resurfacing EP 592, 106, EP 519,596, Padlan, Molecular Immunology 28(4/5) 489-498 (1991), Studnicka et al, Protein Engineering 7(6) 805-814
  • Human antibodies are particularly desirable for therapeutic treatment of 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
  • 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
  • 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 that express human antibodies
  • the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptid
  • 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
  • is used to guide the selection of a completely human antibody recognizing the same epitope Jespers et al. , Bio/technology 12 899-903 (1988)
  • antibodies to the TR2 receptor proteins of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" TR2 receptors 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.
  • antibodies which bind to TR2 receptors and competitively inhibit TR2 receptor multimerization and/or binding to ligand can be used to generate anti-idiotypes that "mimic" TR2 receptor multimerization and/or binding domain and, as a consequence, bind to and neutralize TR2 receptors and/or their ligand(s)
  • Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize TR2 receptor ligand(s)
  • anti-idiotypic antibodies can be used to bind TR2 receptors, or to bind TR2 receptors or ligands, and thereby block TR2 receptor mediated inhibition of apoptosis
  • the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof
  • the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined herein, 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, SEQ ID NO 5, SEQ ID NO 8, or SEQ ID NO 26
  • the polynucleotides may be obtained, and the nucleotide sequence of the polynucleotides determined, by any method known in the art
  • a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g. , as described in Kutmeier et al, BioTechmques 77 242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR
  • a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence of the antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably polyA+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g., a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned into replicas the
  • nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc.
  • the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g., by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
  • CDRs complementarity determining regions
  • one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
  • the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et al. , J. Mol. Biol.
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the antibody to its antigen
  • such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds
  • Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art
  • a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine mAb and a human immunoglobulin constant region, e.g., humanized antibodies
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the Fv region via an amino acid bridge, resulting in a single chain polypeptide Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al, 1988, Science 242 1038-1041). B. Methods of Producing Antibodies
  • the antibodies of the invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques Recombinant expression of an antibody of the invention, or fragment, derivative or analog thereof, e.g.
  • a heavy or light chain of an antibody of the invention requires construction of an expression vector containing a polynucleotide that encodes the antibody
  • the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art
  • methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals
  • These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination
  • the invention thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the invention, or a
  • variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain
  • the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody of the invention
  • the invention includes host cells containing a polynucleotide encoding an antibody of the invention, or a heavy or light chain thereof, operably linked to a heterologous promoter
  • vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below
  • host-expression vector systems may be utilized to express the antibody molecules of the invention.
  • Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ
  • microorganisms such as bacteria (e.g., E. coli, B.
  • subtilis transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences, yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences, insect cell systems infected with recombinant virus expression vectors (e.g.
  • baculovirus containing antibody coding sequences
  • plant cell systems infected with recombinant virus expression vectors e.g , cauliflower mosaic virus, CaMV, tobacco mosaic virus, TMV
  • recombinant plasmid expression vectors e.g., Ti plasmid
  • mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 cells
  • promoters derived from the genome of mammalian cells
  • mammalian viruses e.g., the adenovirus late promoter, the vaccinia virus 7.5K promoter
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule
  • bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are
  • a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed. For example, when a large quantity of such a protein is to be produced, for the generation of pharmaceutical compositions of an antibody molecule, vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
  • vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al. , 1983,
  • pG ⁇ X vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
  • fusion proteins are soluble and can easily be purified from lysed cells by adsorption and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
  • the pG ⁇ X vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
  • Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
  • the virus grows in Spodoptera frugiperda cells.
  • the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • a number of viral-based expression systems may be utilized.
  • the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g., the late promoter and tripartite leader sequence
  • This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination Insertion in a non- essential region of the viral genome (e.g., region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts (e.g.
  • initiation signals may also be required for efficient translation of inserted antibody coding sequences These signals include the ATG initiation codon and adjacent sequences Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc (see Bittner et al , 1987, Methods in Enzymol
  • a host cell strain may be chosen which modulates the expression of the inserted sequences, or modifies and processes the gene product in the specific fashion desired Such modifications (e.g , glycosylation) and processing (e.g. , cleavage) of protein products may be important for the function of the protein
  • Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products
  • Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed
  • eukaryotichost cells which possess the cellular machinery for proper processing of the primary transcript, glycosylation, and phosphorylation of the gene product may be used
  • mammalian host cells include but are not limited to CHO, VERY, BHK, Hela, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL
  • cell lines which stably express the antibody molecule may be engineered.
  • host cells can be transformed with DNA controlled by appropriate expression control elements (e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
  • appropriate expression control elements e.g., promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
  • engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
  • the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
  • This method may advantageously be used to engineer cell lines which express the antibody molecule.
  • Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
  • a number of selection systems may be used, including but not limited to the herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 1 1 :223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192,
  • dhfr which confers resistance to methotrexate (Wigler et al, 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al, 1981, Proc.
  • the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol.3. (Academic Press, New York, 1987)).
  • a marker in the vector system expressing antibody is amplifiable
  • increase in the level of inhibitor present in culture of host cell will increase the number of copies of the marker gene. Since the amplified region is associated with the antibody gene, production of the antibody will also increase (Crouse et ⁇ /., 1983, Mol. Cell. Biol. 3 :257).
  • the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
  • the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
  • a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980, Proc. Natl. Acad. Sci. USA 77:2197).
  • the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
  • an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
  • centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
  • the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins.
  • the fusion does not necessarily need to be direct, but may occur through linker sequences.
  • the antibodies may be specific for antigens other than polypeptides (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention.
  • antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
  • Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art.
  • the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
  • the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof
  • the antibody portion fused to a polypeptide of the present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions
  • polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life of the polypeptides or for use in immunoassays using methods known in the art Further, the polypeptides of the present invention may be fused or conjugated to the above antibody portions to facilitate purification
  • 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 (EP 394,827, Traunecker et al, Nature 331 84-86 (1988)
  • the polypeptides of the present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone (Fountoulakis et al, J Biochem 270 3958-3964 (1995))
  • the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties (EP A 232,262)
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hIL-5 receptor
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5.
  • the antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification.
  • the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, CA, 91311), among others, many of which are commercially available.
  • hexa-histidine provides for convenient purification of the fusion protein.
  • peptide tags useful for purification include, but are not limited to, the "HA” tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al, Cell 37:767 (1984)) and the "flag" tag.
  • the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
  • the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No. 4,741,900 for metal ions which can be conjugated to antibodies for use as diagnostics according to the present invention.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, beta-galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelhferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin;
  • suitable radioactive material include 125 I, 131 I, ⁇ n In or "Tc.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
  • a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells. Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D,
  • Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and cis- dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
  • the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
  • the drug moiety may be a protein or polypeptide possessing a desired biological activity.
  • Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti- angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
  • a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
  • a protein such as tumor necrosis factor, a
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • an antibody can be conjugated to a second antibody to form an antibody hetero conjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
  • An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
  • the antibodies of the invention may be assayed for immunospecific binding by any method known in the art.
  • the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
  • Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH
  • a lysis buffer such as RIPA buffer (1% NP-40 or Triton X- 100, 1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate at pH
  • protein phosphatase and/or protease inhibitors e.g., EDTA, PMSF, aprotinin, sodium vanadate
  • adding the antibody of interest to the cell lysate incubating for a period of time (e.g., 1 -4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in SDS/sample buffer.
  • the ability of the antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g. , western blot analysis.
  • Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%- 20% SDS-PAGE depending on the molecular weight of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3%
  • washing the membrane in washing buffer e.g., PBS-Tween 20
  • primary antibody the antibody of interest
  • secondary antibody which recognizes the primary antibody, e.g., an anti-human antibody
  • an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
  • radioactive molecule e.g. , 32 P or 125 I
  • ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g. , horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence of the antigen.
  • a detectable compound such as an enzymatic substrate (e.g. , horseradish peroxidase or alkaline phosphatase)
  • the antibody of interest does not have to be conjugated to a detectable compound; instead, a second antibody (which recognizes the antibody of interest) conjugated to a detectable compound may be added to the well Further, instead of coating the well with the antigen, the antibody may be coated to the well In this case, a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well
  • a second antibody conjugated to a detectable compound may be added following the addition of the antigen of interest to the coated well.
  • the present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human, patient for treating one or more of the disclosed diseases, disorders, or conditions
  • Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies of the invention (including fragments, analogs and derivatives thereof and anti-idiotypic antibodies as described herein).
  • the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with aberrant expression and/or activity of a polypeptide of the invention, including, but not limited to, autoimmune diseases, disorders, or conditions associated with such diseases or disorders, including, but not limited to, autoimmune hemolytic anemia, autoimmune neonatal thrombocytopenia, idiopathic thrombocytopenia purpura, autoimmunocytopenia, hemolytic anemia, antiphospholipid syndrome, dermatitis, allergic encephalomyelitis, myocarditis, relapsing polychondritis, ulcerative colitis, dense deposit disease, rheumatic heart disease, glomerulonephritis (e.g., IgA nephropathy), pemphigus vulgaris, discoid lupus, Multiple Sclerosis, Neuritis, Uveitis Ophthalmia, Polyendocrinopathies, Purpura (e.g., Henloch-Scoenlein purpur
  • antibodies of the invention are used to treat, inhibit, prognose, diagnose or prevent systemic lupus erythematosis. Additionally, the antibodies of the invention can be used to treat, inhibit or prevent diseases, disorders or conditions associated with immunodeficiencies including, but not limited to, severe combined immunodeficiency (SCID)-X linked, SCID-autosomal, adenosine deaminase deficiency (ADA deficiency), X-linked agammaglobulinemia (XLA), Bruton's disease, congenital agammaglobulinemia, X-linked infantile agammaglobulinemia, acquired agammaglobulinemia, adult onset agammaglobulinemia, late-onset agammaglobulinemia, dysgammaglobulinemia, hyp o gammagl obulinemia, transient hypogammaglobulinemia of infancy, unspecified hypogammaglobulinemia, agamm
  • MHC Class II deficiency (Bare Lymphocyte Syndrome) and severe combined immunodeficiency.
  • Antibodies of the invention are used to prevent graft rejection and inflammation and for the treatment of arthritis.
  • the treatment and/or prevention of diseases and disorders associated with aberrant expression and/or activity of a polypeptide of the invention includes, but is not limited to, alleviating symptoms associated with those diseases and disorders
  • Antibodies of the invention may be provided in pharmaceutically acceptable compositions as known in the art or as described herein
  • a summary of the ways in which the antibodies of the present invention may be used therapeutically includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC) Some of these approaches are described in more detail below Armed with the teachings provided herein, one of ordinary skill in the art will know how to use the antibodies of the present invention for diagnostic, monitoring or therapeutic purposes without undue experimentation
  • the antibodies of this invention may be advantageously utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines or hematopoietic growth factors (such as, e.g., IL-2, IL-3 and IL-7), for example, which serve to increase the number or activity of effector cells which interact with the antibodies
  • the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g., radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents) Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that of the patient is preferred.
  • human antibodies, fragments derivatives, analogs, or nucleic acids are administered to a human patient for therapy or prophylaxis
  • polypeptides or polynucleotides of the present invention It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention.
  • Such antibodies, fragments, or regions will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof.
  • Preferred binding affinities include those with a dissociation constant orKd less than 5X10 '6 M, 10 "6 M, 5X10 “7 M, 10 “7 M, 5X10 '8 M, 10 “8 M, 5X10 "9 M, 10 "9 M, 5X10- 10 M, 10 "10 M, 5X10- ⁇ M, 10 ' ⁇ M, 5X10 "12 M, 10 "12 M,
  • the proteins 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. Any technique known in the art may be used to introduce the transgene
  • nucleic acids of the invention into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson etal, Appl. Microhiol. Biotechnol 40:691-698 (1994); Carver et al, Biotechnology) (NY) 77: 1263-1270 (1993); Wright et al, Biotechnology (NY) 9:830-834 (1991); and Hoppe et al, U.S. Patent Number
  • 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 355:810-813 (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. (Lasko et al, Proc. Natl. Acad. Sci. USA
  • 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, m 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 TR2 receptor polypeptides, studying conditions and/or disorders associated with aberrant TR2 receptor 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
  • 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.
  • 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.
  • 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.
  • 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.
  • TNF-family ligands induce various cellular responses by binding to TNF-family receptors, including the TR2 receptors of the present invention.
  • TNF- ⁇ a potent ligand of the TNF receptor proteins
  • TNF receptor proteins a potent ligand of the TNF receptor proteins
  • lymphocyte development tumor necrosis
  • induction of an antiviral state activation of polymorphonuclear leukocytes
  • induction of class I major histocompatibility complex antigens on endothelial cells induction of adhesion molecules on endothelium and growth hormone stimulation (Ruddle and Homer, Prog. Allergy, 40:162-182 (1988)).
  • TNF- ⁇ also a ligand of the TNF receptor proteins, has been reported to have a role in the rapid necrosis of tumors, immunostimulation, autoimmune disease, graft rejection, producing an anti-viral response, septic shock, cerebral malaria, cytotoxicity, protection against deleterious effects of ionizing radiation produced during a course of chemotherapy, such as denaturation of enzymes, lipid peroxidation and DNA damage (Nata et al, J. Immunol. 136(7) :2483 (1987);
  • TNF- ⁇ also triggers endothelial cells to secrete various factors, including PAI-1, IL-1, GM-CSF and IL-6 to promote cell proliferation.
  • TNF- ⁇ up-regulates various cell adhesion molecules such as E-Selectin, ICAM-1 and VCAM-1. TNF- ⁇ and the Fas ligand have also been shown to induce programmed cell death.
  • a cellular response to a TNF-family ligand is intended any genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand.
  • such cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased cell proliferation or the inhibition of increased cell proliferation, such as by the inhibition of apoptosis.
  • Apoptosis-programmed cell death-is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes (Ameisen, J.C.,- / S5: 1 197-1213 (1994); Krammer, P.H. etal, Curr. Opi . Immunol. 6:279-289 (1994)).
  • TR2 receptor protein and mRNA encoding TR2 receptor protein when compared to a corresponding "standard" mammal, i.e., a mammal of the same species not having the disease state. Further, since some forms of this protein are secreted, it is believed that enhanced levels of TR2 receptor protein can be detected in certain body fluids (e.g., sera, plasma, urine, and spinal fluid) from mammals with the disease state when compared to sera from mammals of the same species not having the disease state.
  • body fluids e.g., sera, plasma, urine, and spinal fluid
  • the invention provides a diagnostic method useful during diagnosis of disease states, which involves assaying the expression level of the gene encoding TR2 receptor protein in mammalian cells or body fluid and comparing the gene expression level with a standard TR2 receptor gene expression level, whereby an increase or decrease in the gene expression level over the standard is indicative of certain disease states associated with aberrant cell survival.
  • the present invention is useful as a prognostic indicator, whereby patients exhibiting significantly aberrant TR2 receptor gene expression will experience a worse clinical outcome relative to patients expressing the gene at a lower level.
  • test the expression level of the gene encoding TR2 receptor protein is intended qualitatively or quantitatively measuring or estimating the level of TR2, TR2-SV1 and/or TR2-SV2 receptor protein or the level of the mRNA encoding TR2, TR2-SV1 and/or TR2-SV2 receptor protein in a first biological sample either directly (e.g., by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to TR2, TR2-SV1 and/or TR2-SV2 receptor protein level or mRNA level in a second biological sample).
  • TR2 receptor protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard TR2 receptor protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disease state.
  • a standard TR2 receptor protein level or mRNA level is known, it can be used repeatedly as a standard for comparison.
  • biological sample is intended any biological sample obtained from an individual, cell line, tissue culture, or other source which contains TR2 receptor protein or mRNA.
  • Biological samples include mammalian body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain secreted mature TR2 receptor protein, and thymus, prostate, heart, placenta, muscle, liver, spleen, lung, kidney and other tissues. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
  • cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to, colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as systemic lupus erythematosus and immune-related glomerulonephritis rheumatoid arthritis) and viral infections (such as Herpes viruses, pox viruses and adenoviruses), information from Herpes viruses, pox viruses and adenoviruses, information from Her
  • AIDS Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration
  • myelodysplastic syndromes such as aplastic anemia
  • ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
  • toxin-induced liver disease such as that caused by alcohol
  • septic shock cachexia and anorexia.
  • TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention are used to treat or prevent autoimmune diseases and/or inhibit the growth, progression, and/or metastasis of cancers, including, but not limited to, those cancers disclosed herein, such as, for example, lymphocytic leukemias (including, for example, MLL and chronic lymphocytic leukemia (CLL)) and follicular lymphomas.
  • TR2-SV1 and/or TR2-SV2 polynucleotides or polypeptides of the invention are used to activate, differentiate or proliferate cancerous cells or tissue (e.g. , B cell lineage related cancers (e.g., CLL and MLL), lymphocytic leukemia, or lymphoma) and thereby render the cells more vulnerable to cancer therapy (e.g., chemotherapy or radiation therapy).
  • cancer therapy e.g., chemotherapy or radiation therapy
  • Assays available to detect levels of soluble receptors are well known to those of skill in the art, for example, radioimmunoassays, competitive-binding assays, Western blot analysis, and preferably an ELISA assay may be employed.
  • TR2 receptor-protein specific antibodies can be raised against intact TR2 receptor protein or an antigenic polypeptide fragment thereof, which may presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
  • a carrier protein such as an albumin
  • antibody or “monoclonal antibody” (mAb) is meant to include intact molecules as well as antibody fragments (such as, for example, Fab and F(ab') 2 fragments) which are capable of specifically binding to TR2 receptor protein.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
  • the antibodies of the present invention may be prepared by any of a variety of methods. For example, cells expressing TR2, TR2-SV1 and/or
  • TR2-SV2 receptor protein or an antigenic fragment thereof can be administered to an animal in order to induce the production of sera containing polyclonal antibodies.
  • a preparation of TR2, TR2-SV1 and/or TR2-SV2 receptor protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity.
  • the antibodies of the present invention are monoclonal antibodies (or TR2 receptor protein binding fragments thereof).
  • monoclonal antibodies can be prepared using hybridoma technology (Kohler et al. , Nature 256:495 (1975); Kohler et al, Eur. J. Immunol. 6:511 (1976); Kohler et al, Eur. J. Immunol. 6:292 (1976); Hammerling et al, In: Monoclonal Antibodies and T-Cell Hybridomas, Elsevier, N.Y., (1981) pp. 563-681).
  • such procedures involve immunizing an animal (preferably a mouse) with a TR2 receptor protein antigen or, more preferably, with a TR2 receptor protein-expressing cell.
  • Suitable cells can be recognized by their capacity to bind anti-TR2 receptor protein antibody.
  • Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56°C), and supplemented with about 10 g/1 of nonessential amino acids, about
  • mice 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
  • the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
  • Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP 2 O), available from the American Type Culture Collection, Rockville, Maryland.
  • SP 2 O parent myeloma cell line
  • the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 50:225-232 (1981)).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the TR2 receptor protein antigen.
  • the present invention is directed to a method for inhibiting a TR2 activity induced by a TNF-family ligand (e.g., cell proliferation, hematopoietic development), which involves administering to a cell which expresses a TR2 polypeptide an effective amount of a TR2 receptor ligand, analog or an antagonist capable of decreasing TR2 receptor mediated signaling.
  • TR2 receptor mediated signaling is increased to treat a disease wherein increased cell proliferation is exhibited.
  • An antagonist can include soluble forms of the TR2 receptors and antibodies directed against the TR2 polypeptides which block TR2 receptor mediated signaling
  • TR2 receptor mediated signaling is decreased to treat a disease
  • the present invention is directed to a method for increasing cell proliferation induced by a TNF-family ligand, which involves administering to a cell which expresses a TR2 polypeptide an effective amount of an agonist capable of increasing TR2 receptor mediated signaling
  • TR2 receptor mediated signaling is increased to treat a disease wherein decreased cell proliferation is exhibited
  • Agonists of the present invention include monoclonal antibodies directed against the TR2 polypeptides which stimulate TR2 receptor mediated signaling
  • TR2 receptor mediated signaling is increased to treat a disease
  • agonist is intended naturally occurring and synthetic compounds capable of enhancing cell proliferation and differentiation mediated by TR2 polypeptides
  • agonists include agents which increase expression of TR2 receptors or increase the sensitivity of the expressed receptor
  • antagonists include agents which decrease expression of TR2 receptors or decrease the sensitivity of the expressed receptor Whether any candidate "agonist” or “antagonist” of the present invention can enhance or inhibit cell proliferation and differentiation can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below
  • One such screening technique involves the use of cells which express the receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science 246 181-296 (October 1989)
  • compounds may be contacted with a cell which expresses the receptor polypeptide of the present invention and a second messenger response, e.g., signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the receptor.
  • Another such screening technique involves introducing RNA encoding the receptor into Xenopus oocytes to transiently express the receptor.
  • the receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation of the receptor.
  • Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof.
  • Such a method involves transfecting a eukaryotic cell with DNA encoding the receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand.
  • the ligand can be labeled, e.g., by radioactivity.
  • the amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors.
  • Soluble forms of the polypeptides of the present invention may be utilized in the ligand binding assay described above. These forms of the TR2 receptors are contacted with ligands in the extracellular medium after they are secreted. A determination is then made as to whether the secreted protein will bind to TR2 receptor ligands. Further screening assays for agonist and antagonist of the present invention are described in Tartaglia, L.A., and Goeddel, D.N., J. Biol. Chem. 2670:4304-4307(1992).
  • a screening method for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand.
  • the method involves contacting cells which express TR2 polypeptides with a candidate compound and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway.
  • assaying a cellular response is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and/or a TNF-family ligand (e.g. , determining or estimating an increase or decrease in T cell proliferation or tritiated thymidine labeling).
  • a cell expressing a TR2 polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.
  • a thymocyte proliferation assay may be employed to identify both ligands and potential drug candidates. For example, thymus cells are disaggregated from tissue and grown in culture medium.
  • Agonists according to the present invention include compounds such as, for example, TNF-family ligand peptide fragments, transforming growth factor ⁇ , and neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate)
  • Preferred agonist include polyclonal and monoclonal antibodies raised against a TR2 polypeptide, or a fragment thereof Such agonist antibodies raised against a T ⁇ F-family receptor are disclosed in Tartaglia, L A , et al, Proc. Natl. Acad.
  • chemotherapeutic drugs such as, for example, cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine Others include ethanol and ⁇ -amyloid peptide
  • Antagonist according to the present invention include soluble forms of the TR2 receptors (e.g., fragments of the TR2 receptor shown in FIG 1A-1B that include the ligand binding domain from the extracellular region of the full length receptor)
  • soluble forms of the receptor which may be naturally occurring or synthetic, antagonize TR2, TR2-SV1 or TR2-SV2 mediated signaling by competing with the cell surface bound forms of the receptor for binding to T ⁇ F- family ligands
  • Antagonists of the present invention also include antibodies specific for T ⁇ F-family ligands and TR2-Fc fusion proteins such as the one described below in Examples 5 and 6
  • T ⁇ F-family ligand is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the T ⁇ F receptor family and inducing the ligand/receptor signaling pathway
  • T ⁇ F ligand family include, but are not limited to, T ⁇ F- ⁇ , lymphotoxin- ⁇ (LT- ⁇ , also known as T ⁇ F- ⁇ ), LT- ⁇ (found in complex heterotrimer LT- ⁇ 2- ⁇ ), FasL,
  • CD40L CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor ( ⁇ GF)
  • TR2 receptors of the present invention are capable of inducing the proliferation of lymphocytes. Further, such proliferation can be inhibited by a TR2 protein fragment fused to an Fc antibody fragment.
  • TR2 receptox/Fc fusion proteins and nucleic acid molecules which encode such proteins. These fusion proteins include those having amino acid sequences of the extracellular domains of the TR2 proteins of the invention. Examples of portions of TR2 extracellular domains which are useful in the preparation of TR2 recept ⁇ r Fc fusion proteins include amino acids 1 to 192, 37 to 192, 50 to 192 and 100 to 192 in SEQ ID NO:2.
  • TNF ⁇ has been shown to protect mice firom infection with Herpes simplex virus type 1 (HSV-1). Rossol-Voth, R. etal, J .Gen. Virol. 72:143-147 (1991). The mechanism of the protective effect of TNF ⁇ is unknown but appears to involve neither interferons not NK cell killing. One member of the TNFR family has been shown to mediate HSV-1 entry into cells. Montgomery, R. et al., Eur.
  • TR2 receptors of the present invention include both TR2 amino acid sequences and antibodies capable of preventing TNFR mediated viral entry into cells. Such sequences and antibodies can function by either competing with cell surface localized TNFR for binding to virus or by directly blocking binding of virus to ceil surface receptors.
  • antibodies specific for the extracellular domain of the TR2 receptors of the invention can also block
  • HSV-1 entry into cells These antagonists are thus useful in the treatment and prevention of Herpes simplex infections.
  • Antibodies according to the present invention may be prepared by any of a variety of methods using TR2 receptor immunogcns of the present invention.
  • TR2 receptor immunogcns include the TR2 receptor protein shown in SEQ ID NO: 1
  • FIG. 1 A-IB SEQ ID NO:2
  • the TR2-SV1 FEG.4A-4C (SEQ ID NO:5)
  • TR2-SV2 FOG. 7A-7C (SEQ ID NO:8)
  • polypeptides any of which may or may not include a leader sequence
  • polypeptide fragments of the receptors comprising, or alternatively consisting of, the ligand binding
  • RECTIFIED SHEET (RULE 91) ISA/EP extracellular, transmembrane, the intraceUular domains of the TR2 receptors, or any combination thereof.
  • Polyclonal and monoclonal antibody agonist or antagonist according to the present invention can be raised according to the methods disclosed in Tartaglia and Goeddel, J. Biol Chem. 267(7):4304-4307(1992)); Tartaglia et al, Cell
  • antibody or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
  • Fab and F(ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24.316-325 (1983)).
  • antibodies according to the present invention are mAbs.
  • Such mAbs can be prepared using hybridoma technology (Kohler and Millstein, Nature 256:495-497 (1975) and U.S. Patent No. 4,376, 1 10; Harlow et al, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1988; Monoclonal Antibodies and Hybridomas: A New Dimension in Biological Analyses, Plenum Press, New York, NY, 1980, Campbell, "Monoclonal Antibody Technology," In. Laboratory Techniques in Biochemistry and Molecular Biology, Volume 13 (Burdon et al. , eds.), Elsevier,
  • Proteins and other compounds which bind the TR2 receptor domains are also candidate agonist and antagonist according to the present invention.
  • Such binding compounds can be "captured” using the yeast two-hybrid system (Fields and Song, Nature 340:245-246 (1989)).
  • a modified version of the yeast two- hybrid system has been described by Roger Brent and his colleagues (Gyuris, J. et al, Cell 75:791-803 (1993); Zervos, A.S. et al, Cell 72:223-232 (1993)).
  • the yeast two-hybrid system is used according to the present invention to capture compounds which bind to the ligand binding, extracellular, intraceUular, and transmembrane domains of the TR2 receptors.
  • Such compounds are good candidate agonist and antagonist of the present invention.
  • the intraceUular domain of the TR2 receptor may be used to identify cellular proteins which interact with the receptor in vivo.
  • Such an assay may also be used to identify ligands with potential agonistic or antagonistic activity of TR2 receptor function.
  • This screening assay has previously been used to identify protein which interact with the cytoplasmic domain of the murine TNF-RII and led to the identification of two receptor associated proteins. Rothe, M. et al, Cell 75:681 (1994).
  • proteins and amino acid sequences which bind to the cytoplasmic domain of the TR2 receptors are good candidate agonist and antagonist of the present invention.
  • screening techniques include the use of cells which express the polypeptide of the present invention (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science, 246: 181-296 (1989).
  • potential agonists or antagonists may be contacted with a cell which expresses the polypeptide of the present invention and a second messenger response, e.g. , signal transduction may be measured to determine whether the potential antagonist or agonist is effective.
  • the TR2 receptor agonists may be employed to stimulate ligand activities, such as inhibition of tumor growth and necrosis of certain transplantable tumors.
  • the agonists may also be employed to stimulate cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation.
  • Agonists to the TR2 receptor may also augment TR2's role in the host's defense against microorganisms and prevent related diseases (infections such as that from Listeria monocytogenes) and Chlamidiae.
  • the agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage
  • Agonists to the receptor polypeptides of the present invention may be used to augment TNF's role in host defenses against microorganisms and prevent related diseases
  • the agonists may also be employed to protect against the deleterious effects of ionizing radiation produced during a course of radiotherapy, such as denaturation of enzymes, lipid peroxidation, and DNA damage
  • the agonists may also be employed to mediate an anti-viral response, to regulate growth, to mediate the immune response and to treat immunodeficiencies related to diseases such as HIV by increasing the rate of lymphocyte proliferation and differentiation
  • the antagonists to the polypeptides of the present invention may be employed to inhibit ligand activities, such as stimulation of tumor growth and necrosis of certain transplantable tumors
  • the antagonists may also be employed to inhibit cellular differentiation, for example, T-cell, fibroblasts and hemopoietic cell differentiation
  • Antagonists may also be employed to treat autoimmune diseases, for example, graft versus host rejection and allograft rejection, and T-cell mediated autoimmune diseases such as AIDS It has been shown that T-cell proliferation is stimulated via a type 2 TNF receptor Accordingly, antagonizing the receptor may prevent the proliferation of T-cells and treat T-cell mediated autoimmune diseases
  • HIV-induced apoptotic cell death has been demonstrated not only in vitro but also, more importantly, in infected individuals (Ameisen, J C , AIDS 8 1 197- 1213 (1994) , Finkel, T H , and Banda, N K , Curr. Opm. Immunol. 6 605-615(1995), Muro-Cacho, C A et al, J. Immunol. 754 5555-5566 (1995)) Furthermore, apoptosis and CD4 + T-lymphocyte depletion is tightly correlated in different animal models of AIDS (Brunner, T., et al, Nature 373:441-444 (1995); Gougeon, M ., et al, AIDS Res. Hum.
  • the TNF-family ligand was detectable in uninfected macrophages and its expression was upregulated following HIV infection resulting in selective killing of uninfected CD4 + T-lymphocytes (Badley, A.D et al, J. Virol. 70: 199-206 (1996)).
  • the TR2 receptor shown in FIG. 1A-1B is expressed in CD4 + T-lymphocytes and is capable of inducing lymphocyte proliferation.
  • a method for treating HIV + individuals involves administering an agonist of the present invention to increase the rate of proliferation and differentiation of CD4 ⁇ T-lymphocytes
  • Such agonists include agents capable of inducing the expression of TR2 receptors (e.g. ,
  • TNF ⁇ TNF ⁇ , PMA and DMSO
  • Modes of administration and dosages are discussed in detail below.
  • Antagonists of the present invention are able to suppress the immune response to both allografts and xenografts by decreasing the rate of TR2 mediated lymphocyte proliferation and differentiation
  • antagonists include the TR2-Fc fusion protein described in Examples 5 and 6
  • the present invention further provides a method for suppression of immune responses
  • agonists and antagonists of the present invention may be useful in the treatment of autoimmune diseases such as type 1 diabetes
  • the role played by the TR2 receptors in cell proliferation and differentiation indicates that agonist or antagonist of the present invention may be used to treat disease states involving aberrant cellular expression of these receptors TR2 receptors may in some circumstances induce an inflammatory response, and antagonists may be useful reagents for blocking this response.
  • TR2 receptor antagonists e.g., soluble forms of the TR2 receptors, neutralizing antibodies
  • TR2 receptor antagonists may be useful for treating inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease
  • Antagonists to the TR2 receptor may also be employed to treat and/or prevent septic shock, which remains a critical clinical condition Septic shock results from an exaggerated host response, mediated by protein factors such as TNF and IL-1, rather than from a pathogen directly
  • Septic shock results from an exaggerated host response, mediated by protein factors such as TNF and IL-1, rather than from a pathogen directly
  • lipopolysaccharides have been shown to elicit the release of TNF leading to a strong and transient increase of its serum concentration TNF causes shock and tissue injury when administered in excessive amounts. Accordingly, it is believed that antagonists to the TR2 receptor will block the actions of TNF and treat/prevent septic shock.
  • These antagonists may also be employed to treat meningococcemia n children which correlates with high serum levels of TNF.
  • antagonists according to the present invention are nucleic acids corresponding to the sequences contained in SEQ ID NO.25, FIG. 1A-1B (SEQ ID NO:l), FIG. 4A-4C (SEQ ID NO:4) or FIG. 7A-7C (SEQ ID NO : 7) or the complementary strand thereof; and/or to the deposited nucleotide sequences of ATCC Deposit Numbers 97059, 97058 or 97057.
  • antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, e.g, O'Connor, J. Neurochem.56:560 (1991), and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, F (1988).
  • Antisense technology can be used to control gene expression through antisense DNA or RNA, or through triple-helix formation. Antisense techniques are discussed for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988). Triple helix formation is discussed in, for instance, Lee et al.
  • the 5' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an ant ense RNA oligonucleotide of from about 10 to 40 base pairs in length.

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EP00916583A 1999-03-22 2000-03-22 Menschlischer tumor necrosis faktor rezeptor änlicher rezeptor 2 Withdrawn EP1165185A2 (de)

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US12568399P 1999-03-22 1999-03-22
US125683P 1999-03-22
US12652299P 1999-03-26 1999-03-26
US126522P 1999-03-26
US13516999P 1999-05-20 1999-05-20
US135169P 1999-05-20
US14738399P 1999-08-06 1999-08-06
US147383P 1999-08-06
PCT/US2000/007521 WO2000056405A2 (en) 1999-03-22 2000-03-22 Human tumor necrosis factor receptor-like 2

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US20030068619A1 (en) * 2001-07-27 2003-04-10 Lal Preeti G. TNF receptor 2 related protein variant
WO2003011321A1 (en) * 2001-07-31 2003-02-13 Genset S.A. Agonists and antagonists of cobesin for the treatment of metabolic disorders
CA2605253C (en) * 2005-04-11 2017-06-13 Yale University Selective modulation of tumour necrosis factor receptors in therapy
US8895291B2 (en) 2010-10-08 2014-11-25 Terumo Bct, Inc. Methods and systems of growing and harvesting cells in a hollow fiber bioreactor system with control conditions
US9617506B2 (en) 2013-11-16 2017-04-11 Terumo Bct, Inc. Expanding cells in a bioreactor
EP3613841B1 (de) 2014-03-25 2022-04-20 Terumo BCT, Inc. Passives ersetzen von medien
CN106715676A (zh) 2014-09-26 2017-05-24 泰尔茂比司特公司 按计划供养
WO2016161415A2 (en) * 2015-04-02 2016-10-06 Memorial Sloan Kettering Cancer Center Tnfrsf14/ hvem proteins and methods of use thereof
WO2017004592A1 (en) 2015-07-02 2017-01-05 Terumo Bct, Inc. Cell growth with mechanical stimuli
US11965175B2 (en) 2016-05-25 2024-04-23 Terumo Bct, Inc. Cell expansion
US11104874B2 (en) 2016-06-07 2021-08-31 Terumo Bct, Inc. Coating a bioreactor
US11685883B2 (en) 2016-06-07 2023-06-27 Terumo Bct, Inc. Methods and systems for coating a cell growth surface
US11702634B2 (en) 2017-03-31 2023-07-18 Terumo Bct, Inc. Expanding cells in a bioreactor
US11624046B2 (en) 2017-03-31 2023-04-11 Terumo Bct, Inc. Cell expansion

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JP3443119B2 (ja) * 1989-08-07 2003-09-02 ペプテック リミテッド 腫瘍壊死因子結合リガンド
ATE234918T1 (de) * 1995-08-04 2003-04-15 Lek Tovarna Farmacevtskih Monoklonale antikörper gegen lösliche tnf-alpha rezeptoren p55 und p75 als auch gegen tnf-alpha und dessen analogen
EP1009431A4 (de) * 1997-05-12 2000-09-27 Smithkline Beckman Corp Antikörper gegen den menschlichen tumornekrosefaktor-rezeptor des typs 2 (tr2)

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WO2000056405A2 (en) 2000-09-28
WO2000056405A3 (en) 2001-01-04

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