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WO1999004001A1 - Recepteur ztnfr-5 du facteur de necrose tumorale - Google Patents

Recepteur ztnfr-5 du facteur de necrose tumorale Download PDF

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Publication number
WO1999004001A1
WO1999004001A1 PCT/US1998/015072 US9815072W WO9904001A1 WO 1999004001 A1 WO1999004001 A1 WO 1999004001A1 US 9815072 W US9815072 W US 9815072W WO 9904001 A1 WO9904001 A1 WO 9904001A1
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Prior art keywords
amino acid
seq
polypeptide
sequence
acid residue
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PCT/US1998/015072
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English (en)
Inventor
Theresa M. Farrah
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Zymogenetics, Inc.
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Priority to AU90139/98A priority Critical patent/AU9013998A/en
Publication of WO1999004001A1 publication Critical patent/WO1999004001A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70503—Immunoglobulin superfamily
    • C07K14/70535—Fc-receptors, e.g. CD16, CD32, CD64 (CD2314/705F)
    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
    • 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

Definitions

  • TNFR tumor necrosis factor receptor
  • the TNFR family consists of a number of integral membrane glycoprotein receptors many of which, in conjunction with their respective ligands, regulate interactions between different hematopoietic cell lineages (Smith et al . , The TNF Receptor Superfamily of Cellular and Viral Proteins : Activation, Costimulation and Death, 7_6: 959-62, 1994; Cosman, Stem Cells 12:440-55, 1994) .
  • the TNF receptor family is composed of a number of type I integral membrane glycoproteins which exhibit sequence homology, particularly with respect to cysteine- rich repeats in their extracellular domains.
  • the TNF receptor family includes p75 NGFR (Johnson et al . , Cell 4 . 7:545-54, 1989), p55 TNFR-I (Loetscher et al . , Cell £:351-59, 1990), p75 TNFR-II (Schall et al . , Cell 61:361- 70, 1990), TNFR-RP/TNFR-III (Crowe et al . Science 264:707- 10, 1994), CD27 (Camerini et al .. J. Immunol.
  • NGFR, TNFR- I, CD30, CD40, 4 -IBB, DR3 and 0X40 are mainly restricted to cells of the lymphoid/hematopoietic system.
  • TNRFR-I, TNRFR-II, TNRFR-III and DR4 are found in most human tissues.
  • TNF receptor family are characterized by a multi-domain structure comprising an extracellular region, a transmembrane domain, a spacer region between the extracellular ligand-binding region and the transmembrane domain and a cytoplasmic domain, which in several members of this family (TNFR 1, Fas, DR3 , DR4 , CAR1 and low affinity NGFR) contains a death domain associated with apoptosis.
  • the extracellular ligand-binding region is characterized by the presence of one to six cysteine-rich motifs each containing about six cysteines and approximately 40 amino acids, although variation in the size and number of these motifs occurs among members of this family.
  • the cysteine-rich regions provide the motif for binding to shared structures in the ligands.
  • the highest degree of homology among the TNFR family members is within this extracellular cysteine-rich region.
  • human TNFRs the average homology is in the range of 25% to 30%.
  • Between the last cysteine-rich repeat and the transmembrane domain is a small spacer region of between 8 to 70 amino acid residues.
  • Cell surface TNF receptors are anchored in the cell membrane by a transmembrane domain characterized by a sequence of hydrophobic amino acid residues.
  • the cytoplasmic domain On the opposite end of the protein from the extracellular ligand-binding region and separated from it by the transmembrane domain is the cytoplasmic domain.
  • the cytoplasmic domains of TNFR family members are small, from 46 to 221 amino acid residues, which suggests possible differences in the signaling mechanisms among family members.
  • activation is triggered by the aggregation of cytoplasmic domains of three receptors when their corresponding extracellular domains bind to trimeric ligand.
  • One member of the TNF receptor family, osteoprotegerin (Simonet et al . , ibid) , is unique in that it is a secreted protein.
  • Soluble forms of other TNF receptors have been described for TNFR- I, TNFR- II, low- affinity NGFR, FAS, CD27, CD30, CD40 and 4-1BB, but these were generated either by cleaving from the cell membrane or secreted by alternatively spliced mRNA.
  • OPG inhibits osteoclast maturation and it is thought that it might serve to regulate bone density by modulating osteoclast differentiation from hematopoietic precursors.
  • OPG provided protection from normal osteoclast remodeling and ovariectomy-associated bone loss.
  • TNF TNF-binding protein
  • the members of the TNF ligand family share approximately 20% sequence homology in the extracellular ligand-binding regions, and exist mainly as type II membrane glycoproteins, biologically active as trimeric or multimeric complexes.
  • TNF TNF-alpha converting enzyme
  • TACE TNF-alpha converting enzyme
  • receptor crosslinking is a prerequisite for signal transduction in this receptor family.
  • the use of receptor-specific monoclonal antibodies or soluble receptors in the form of multimeric Ig fusion proteins has been useful in determining biological function in vi tro and in vivo for several family members. Soluble receptor- Ig fusion proteins have been used successfully in the cloning of the cell surface ligands corresponding to the CD40, CD30, CD27,
  • the members of the tumor necrosis factor ligand family mediate interactions between different hematopoietic cells, such as T cell/B cell, T cell/monocyte and T cell/T cell interactions.
  • the result of this two-way communication can be stimulatory or inhibitory, depending on the target cell or the activation state.
  • TNF proteins are involved in regulation of cell proliferation, activation and differentiation, including control of cell survival or death by apoptosis or cytotoxicity .
  • One member of this family, OX-40 is restricted to T cells where it acts as a costimulatory receptor.
  • TNF receptor ligand family members there are differences in distribution, kinetics of induction and requirements for induction, which support a defined role for each of the ligands in T cell- mediated immune responses .
  • the demonstrated in vi tro and in vivo activities of these TNF receptor ligand family members illustrate the enormous clinical potential of, and need for, other TNF receptors, TNF ligands, TNFR agonists, and TNFR antagonists.
  • the present invention addresses this need by providing a novel TNF receptor and related compositions and methods .
  • the invention provides an isolated polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2.
  • the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2.
  • the polypeptide comprises the region between amino acid residue 1 and amino acid residue 300 of SEQ ID NO : 2.
  • the polypeptide further comprises an affinity tag.
  • the invention provides a fusion protein consisting essentially of a first portion and a second portion joined by a peptide bond, said first portion comprising a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2; and the second portion comprising another polypeptide.
  • the second portion is an IgG Fc region.
  • a fusion protein comprising a secretory signal sequence having the amino acid sequence of amino acid residues 1-23 of SEQ ID NO : 2 , wherein said secretory signal sequence is operably linked to an additional polypeptide.
  • a pharmaceutical composition comprising an isolated polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine- rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2; in combination with a pharmaceutically acceptable vehicle .
  • an isolated polynucleotide encoding an isolated polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo- repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO : 2.
  • the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO : 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2.
  • the polypeptide comprises the region between amino acid residue 1 and amino acid residue 194 of SEQ ID NO: 2.
  • the polypeptide further comprises an affinity tag.
  • Another aspect provided by the invention is an isolated polynucleotide according to claim 11, selected from the group consisting of, a) a polynucleotide having a sequence of nucleotides from nucleotide 252 to nucleotide 764 of SEQ ID NO : 1 ; b) a polynucleotide having a sequence of nucleotides from nucleotide 252 to nucleotide 1082 of SEQ ID NO : 1 ; c) a polynucleotide having a sequence of nucleotides from nucleotide 183 to nucleotide 764 of SEQ ID NO:l; d) a polynucleotide having a sequence of nucleotides from nucleotide 183 to nucleotide 1082 of SEQ ID NO:l; e) a polynucleotide having a sequence of nucleotides from nucleotide 1 to nucleotide 1205 of
  • an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine- rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2; and a transcription terminator.
  • the DNA segment encodes a polypeptide comprising an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO : 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo- repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO : 2.
  • the DNA segment encodes a polypeptide comprising residues 24-194 of SEQ ID NO : 2.
  • the DNA segment encodes a polypeptide covalently linked amino terminally or carboxy terminally to an affinity tag.
  • the secretory signal sequence comprises residues 1-23 of SEQ ID NO: 2 or SEQ ID NO: 44.
  • a cultured cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine- rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2; and a transcription terminator, wherein said cell expresses said polypeptide encoded by said DNA segment
  • Also provided is a method of producing a polypeptide comprising: culturing a cell into which has been introduced an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO : 2 from amino acid residue 24 to amino acid residue 194, wherein the polypeptide has four extracellular, cysteine-rich pseudo- repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO : 2 ; and a transcription terminator; whereby said cell expresses said polypeptide encoded by said DNA segment; and recovering said expressed polypeptide.
  • an antibody that specifically binds to an epitope of a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO : 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2.
  • binding protein that specifically binds to an epitope of a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence of SEQ ID NO: 2 from amino acid residue 24 to amino acid residue 194, wherein said polypeptide has four extracellular, cysteine-rich pseudo-repeats having cysteine residues corresponding to amino acid residues 49, 52, 62, 70, 73, 88, 91, 95, 105, 113, 115, 126, 132, 150, 153, 168, 174 and 193 of SEQ ID NO: 2.
  • Another aspect provided is an isolated polynucleotide comprising the sequence of nucleotide 1 to nucleotide 900 of SEQ ID NO: 14.
  • an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NO: 14 or a sequence complementary to SEQ ID NO: 14.
  • the Figure shows a comparison of the deduced amino acid sequence of ZTNFR-5 (SEQ ID NO: 2) with the deduced amino acid sequence of osteoprotegerin (SEQ ID NO: 3) .
  • the predicted cleavage sites for ZTNFR-5 and OPG are marked with double carets, and cysteine-rich repeats #1, #2, #3 and #4 are indicated.
  • Affinity tag is used herein to denote a polypeptide segment that can be attached to a second polypeptide to provide for purification or detection of the second polypeptide or provide sites for attachment of the second polypeptide to a substrate.
  • any peptide or protein for which an antibody or other specific binding agent is available can be used as an affinity tag.
  • Affinity tags include a poly-histidine tract, protein A
  • Allelic variant Any of two or more alternative forms of a gene occupying the same chromosomal locus . Allelic variation arises naturally through mutation, and may result in phenotypic polymorphism within populations.
  • Gene mutations can be silent (i.e., no change in the encoded polypeptide) , or may encode polypeptides having altered amino acid sequence.
  • allelic variant is also used herein to denote a protein encoded by an allelic variant of a gene. Also included are the same protein from the same species which differs from a reference amino acid sequence due to allelic variation. Allelic variation refers to naturally occurring differences among individuals in genes encoding a given protein.
  • Amino-terminal and carboxyl-terminal are used herein to denote positions within polypeptides and proteins. Where the context allows, these terms are used with reference to a particular sequence or portion of a polypeptide or protein to denote proximity or relative position. For example, a certain sequence positioned carboxyl-terminal to a reference sequence within a protein is located proximal to the carboxyl terminus of the reference sequence, but is not necessarily at the carboxyl terminus of the complete protein.
  • Complement/anti-complement pair Denotes non- identical moieties that form a non-covalently associated, stable pair under appropriate conditions. For instance, biotin and avidin (or streptavidin) are prototypical members of a complement/anti-complement pair.
  • Other exemplary complement/anti-complement pairs include receptor/ligand pairs, antibody/antigen (or hapten or epitope) pairs, sense/antisense polynucleotide pairs, and the like. Where subsequent dissociation of the complement/anti-complement pair is desirable, the complement/anti-complement pair preferably has a binding affinity of ⁇ 10 -9 M.
  • Contig Denotes a polynucleotide that has a contiguous stretch of identical or complementary sequence to another polynucleotide. Contiguous sequences are said to "overlap" a given stretch of polynucleotide sequence either in their entirety or along a partial stretch of the polynucleotide. For example, representative contigs to the polynucleotide sequence 5 ' -ATGGCTTAGCTT-3 ' are 5'- TAGCTTgagtct-3 ' and 3 ' -gtcgacTACCGA-5 ' .
  • Complements of polynucleotide molecules Denotes polynucleotide molecules having a complementary base sequence and reverse orientation as compared to a reference sequence. For example, the sequence 5' ATGCACGGG 3' is complementary to 5 ' CCCGTGCAT 3 ' .
  • Degenerate Nucleotide Sequence or Degenerate Sequence Denotes a sequence of nucleotides that includes one or more degenerate codons (as compared to a reference polynucleotide molecule that encodes a polypeptide) .
  • Degenerate codons contain different triplets of nucleotides, but encode the same amino acid residue (i.e., GAU and GAC triplets each encode Asp) .
  • Expression vector A DNA molecule, linear or circular, that comprises a segment encoding a polypeptide of interest operably linked to additional segments that provide for its transcription. Such additional segments may include promoter and terminator sequences, and optionally one or more origins of replication, one or more selectable markers, an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or- viral DNA, or may contain elements of both. Isolated: when applied to a polynucleotide, denotes that the polynucleotide has been removed from its natural genetic milieu and is thus free of other extraneous or unwanted coding sequences, and is in a form suitable for use within genetically engineered protein production systems.
  • isolated molecules are those that are separated from their natural environment and include cDNA and genomic clones.
  • isolated DNA molecules of the present invention are free of other genes with which they are ordinarily associated, but may include naturally occurring 5' and 3' untranslated regions such as promoters and terminators. The identification of associated regions will be evident to one of ordinary skill in the art (see for example, Dynan and Tijan, Nature 316 :774-78, 1985).
  • Isolated polypeptide or protein is a polypeptide or protein that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin. It is preferred to provide the polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term "isolated” does not exclude the presence of the same polypeptide in alternative physical forms, such as dimers or alternatively glycosylated or derivatized forms.
  • operably linked indicates that the segments are arranged so that they function in concert for their intended purposes, e.g., transcription initiates in the promoter and proceeds through the coding segment to the terminator.
  • Ortholog Denotes a polypeptide or protein obtained from one species that is the functional counterpart of a polypeptide or protein from a different species. Sequence differences among orthologs are the result of speciation.
  • Paralogs are distinct but structurally related proteins made by an organism. Paralogs are believed to arise through gene duplication. For example, ⁇ -globin, ⁇ - globin, and myoglobin are paralogs of each other.
  • Polynucleotide denotes a single- or double- stranded polymer of deoxyribonucleotide or ribonucleotide bases read from the 5' to the 3' end. Polynucleotides include RNA and DNA, and may be isolated from natural sources, synthesized in vi tro, or prepared from a combination of natural and synthetic molecules.
  • bp base pairs
  • nt nucleotides
  • kb kilobases
  • the two strands of a double-stranded polynucleotide may differ slightly in length and that the ends thereof may be staggered as a result of enzymatic cleavage; thus all nucleotides within a double-stranded polynucleotide molecule may not be paired. Such unpaired ends will in general not exceed 20 nt in length.
  • Polypeptide Is a polymer of amino acid residues joined by peptide bonds, whether produced naturally or synthetically. Polypeptides of less than about 10 amino acid residues are commonly referred to as “peptides" .
  • Promoter Denotes a portion of a gene containing
  • Promoter sequences are commonly, but not always, found in the 5 1 non-coding regions of genes.
  • Protein is a macromolecule comprising one or more polypeptide chains.
  • a protein may also comprise non- peptidic components, such as carbohydrate groups. Carbohydrates and other non-peptidic substituents may be added to a protein by the cell in which the protein is produced, and will vary with the type of cell. Proteins are defined herein in terms of their amino acid backbone structures; substituents such as carbohydrate groups are generally not specified, but may be present nonetheless.
  • Receptor A cell-associated protein, or a polypeptide subunit of such protein, that binds to a bioactive molecule (the "ligand") and mediates the effect of the ligand on the cell. Binding of ligand to receptor results in a change in the receptor (and, in some cases, receptor multimerization, i.e., association of identical or different receptor subunits) that causes interactions between the effector domain (s) of the receptor and other molecule (s) in the cell. These interactions in turn lead to alterations in the metabolism of the cell.
  • ligand a bioactive molecule
  • Metabolic events that are linked to receptor-ligand interactions include gene transcription, phosphorylation, dephosphorylation, cell proliferation, increases in cyclic AMP production, mobilization of cellular calcium, mobilization of membrane lipids, cell adhesion, hydrolysis of inositol lipids and hydrolysis of phospholipids .
  • ZTNFR- 5 has characteristics of TNF receptors, as discussed in more detail below.
  • Secretory signal sequence A DNA sequence that encodes a polypeptide (a "secretory peptide") that, as a component of a larger polypeptide, directs the larger polypeptide through a secretory pathway of a cell in which it is synthesized.
  • the larger polypeptide is commonly cleaved to remove the secretory peptide during transit through the secretory pathway.
  • Soluble receptor A receptor polypeptide that is not bound to a cell membrane. Soluble receptors are most commonly ligand-binding receptor polypeptides that lack transmembrane and cytoplasmic domains . Soluble receptors can comprise additional amino acid residues, such as affinity tags that provide for purification of the polypeptide or provide sites for attachment of the polypeptide to a substrate. Many cell -surface receptors have naturally occurring, soluble counterparts that are produced by proteolysis or translated from alternatively spliced mRNAs. Receptor polypeptides are said to be substantially free of transmembrane and intracellular polypeptide segments when they lack sufficient portions of these segments to provide membrane anchoring or signal transduction, respectively.
  • the present invention is based in part upon the discovery of a novel 1205 bp DNA sequence (SEQ ID N0:1) and corresponding polypeptide sequence (SEQ ID NO: 2) which have homology to members of the tumor necrosis factor receptor family.
  • the receptor has been designated ZTNFR-5.
  • Novel ZTNFR-5 receptor-encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST (Expressed Sequence Tag) database for sequences homologous to conserved motifs within the TNF receptor family. Based on this search a contig of 16 ESTs was constructed.
  • EST expressed Sequence Tag
  • the TNF receptor family is characterized by an extracellular portion composed of several modules called, historically, "cysteine-rich pseudo-repeats".
  • a prototypical family member has four of these pseudo-repeats, each about 29-43 residues long, one right after the other.
  • a typical pseudo-repeat has 6 cysteine residues. They are called pseudo-repeats because, although they appear to originate from a common ancestral module, they do not repeat exactly: pseudo-repeats #1, #2, #3 and #4 have characteristic sequence features which distinguish them from one another.
  • the crystal structure of the p55 TNF receptor revealed that each pseudo-repeat corresponds to one folding domain, and that all four pseudo-repeats fold into the same tertiary structure, held together internally by disulfide bonds.
  • Sequence motifs for the four sequence motifs are shown below. Within in the motif, X represents an amino acid residue, the numbers contained within curly brackets are multipliers for the preceding residue and residues in brackets are optional and alternative sequences are indicated with asterisks.
  • Pseudo repeat #1 motif (SEQ ID NO: 28) X-C-X ⁇ 10-14 ⁇ -C-C-X-X-C-X ⁇ 5-9 ⁇ -C-X ⁇ 6-8 ⁇ -C-X 5 Pseudo-repeat #1 of ZTNFR-5 (amino acid residues 49-71 of
  • SEQ ID NO: 2 is missing the first two cysteines of this motif .
  • Pseudo-repeat #2 motif (SEQ ID NO: 29) X-C-X ⁇ l3-15 ⁇ -C-X-X-C-X ⁇ 2-3 ⁇ -C-X ⁇ 8-ll ⁇ -C-X ⁇ 7 ⁇ -C 10 Pseudo-repeat #2 of ZTNFR-5 (amino acid residues 72-113 of
  • SEQ ID NO: 2 match this motif.
  • Pseudo-repeat #3 motif (SEQ ID NO: 30) X-C-X ⁇ 5-6 ⁇ -X-X ⁇ 4-9 ⁇ -C-X-X-C-X ⁇ 2-7 ⁇ -C-X ⁇ 8-9 ⁇ -C-X ⁇ 7 ⁇ -C- (X)
  • Pseudo-repeat #3 of ZTNFR-5 (amino acid residues 114-151 SEQ ID NO:2) is encompassed within the alternative sequences .
  • Pseudo-repeat #4 motif (SEQ ID NO: 32) X-C-X ⁇ l0-14 ⁇ -C-X-X-C-X-X-C-X ⁇ 4-10 ⁇ -C-X ⁇ 3-7 ⁇ -C-X
  • Pseudo-repeat #4 of ZTNFR-5 (amino acid residues 151-194 of SEQ ID NO : 2 ) is encompassed within the alternative sequences.
  • cysteine-rich pseudo-repeats (residues 49-71, 72-113, 114- 151 and 152-194 of SEQ ID NO : 2 ) .
  • a typical pseudo-repeat has six cysteines.
  • Pseudo-repeat #1 has conserved cysteine residues as amino acid residues 49, 52, 62 and 70 of SEQ ID NO : 2.
  • Pseudo-repeat #2 has conserved cysteine residues at amino acid residues 73, 88, 91, 95, 105 and 113 of SEQ ID NO: 2.
  • Pseudo-repeat has conserved cysteine residues at amino acid residues 115, 126, 132 and 150 of SEQ ID NO : 2.
  • Pseudo-repeat #4 has conserved cysteine residues at amino acid residues 153, 168, 174 and 193 of SEQ ID NO: 2. Those skilled in the art will recognize that these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding. These features indicate that the receptor encoded by the DNA sequence of SEQ ID NO : 1 is a member of the TNF receptor family.
  • ZTNFR-5 The four cysteine-rich domains of the extracellular ligand binding region of ZTNFR-5 are similar to several other members of the TNF receptor family, in particular, OPA and TNFR-2. ZTNFR-5 shares an overall 31% amino acid identity with OPG. The greatest homology, 42%, is within the four cysteine-rich repeats. Like OPG, ZTNFR- 5 is a soluble, secreted TNF receptor, however it may also exist, as do other TNFRs, as a membrane bound receptor having a transmembrane domain and a cytoplasmic domain.
  • OPG was detected in lung, heart, kidney, placenta, and to a lesser degree, in hematopoietic and immune organs (Simmonet et al . , ibid. ) .
  • Several ESTs which made up the ZTNFR-5 contig were from tumor libraries, prostate, colon and breast. Expression in tumor cells is consistent with other members of the TNFR family that are associated with growth regulation, differentiation and tu origenesis .
  • SEQ ID NO : 14 is a degenerate DNA sequence that encompasses all DNAs that encode the ZTNFR-5 polypeptide of SEQ ID NO: 2. Those skilled in the art will recognize that the degenerate sequence of SEQ ID NO: 14 also provides all RNA sequences encoding SEQ ID NO : 2 by substituting U (uracil) for T (thymine) .
  • ZTNFR-5 polypeptide- encoding polynucleotides comprising nucleotide 1 to nucleotide 900 of SEQ ID NO: 14 and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one-letter codes used within SEQ ID NO: 14 to denote degenerate nucleotide positions. "Resolutions” are the nucleotides denoted by a code letter. "Complement” indicates the code for the complementary nucleotide (s) .
  • the code Y denotes either C (cytosine) or T
  • R denotes A (adenine) or G (guanine) , A being complementary to T, and G being complementary to C.
  • degenerate codons used in SEQ ID NO: 14, encompassing all possible codons for a given amino acid, are set forth in Table 2.
  • any X NNN One of ordinary skill in the art will appreciate that some ambiguity is introduced in determining a degenerate codon, representative of all possible codons encoding each amino acid.
  • the degenerate codon for serine can, in some circumstances, encode arginine (AGR)
  • the degenerate codon for arginine (MGN) can, in some circumstances, encode serine (AGY) .
  • some polynucleotides encompassed by the degenerate sequence may encode variant amino acid sequences, but one of ordinary skill in the art can easily identify such variant sequences by reference to the amino acid sequence of SEQ ID NO : 2.
  • preferential codon usage or “preferential codons” is a term of art referring to protein translation codons that are most frequently used in cells of a certain species, thus favoring one or a few representatives of the possible codons encoding each amino acid (See Table 2) .
  • amino acid threonine amino acid threonine
  • Thr may be encoded by ACA, ACC, ACG, or ACT, but in mammalian cells ACC is the most commonly used codon; in other species, for example, insect cells, yeast, viruses or bacteria, different Thr codons may be preferential.
  • Preferential codons for a particular species can be introduced into the polynucleotides of the present invention by a variety of methods known in the art. Introduction of preferential codon sequences into recombinant DNA can, for example, enhance production of the protein by making protein translation more efficient within a particular cell type or species.
  • the degenerate codon sequence disclosed in SEQ ID NO: 17 serves as a template for optimizing expression of polynucleotides in various cell types and species commonly used in the art and disclosed herein. Sequences containing preferential codons can be tested and optimized for expression in various species, and tested for functionality as disclosed herein.
  • RT-PCR reverse transcription-polymerase chain reaction
  • highly degenerate primers designed from the ZTNFR-5 sequences are useful for this purpose.
  • isolated polynucleotides will hybridize to similar sized regions of SEQ ID NO:l, or to a sequence complementary thereto, under stringent conditions.
  • stringent conditions are selected to be about 5°C lower than the thermal melting point (T m ) for the specific sequence at a defined ionic strength and pH.
  • T m is the temperature (under defined ionic strength and pH) at which 50% of the target sequence hybridizes to a perfectly matched probe.
  • Typical stringent conditions are those in which the salt concentration is up to about 0.03 M at pH 7 and the temperature is at least about 60°C.
  • the isolated polynucleotides of the present invention include DNA and RNA.
  • RNA DNA from lung or lymphoid tissue
  • DNA can also be prepared using RNA from other tissues or isolated as genomic DNA.
  • Total RNA can be prepared using guanidine HCl extraction followed by isolation by centrifugation in a CsCl gradient (Chirgwin et al., Biochemistry 11:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc. Natl. Acad. Sci. USA £1:1408-12, 1972) .
  • Complementary DNA (cDNA) is prepared from poly (A) + RNA using known methods. Polynucleotides encoding ZTNFR-5 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • SEQ ID NO:l represents a single allele of the human gene, and that allelic variation and alternative splicing is expected to occur.
  • Allelic variants of the DNA sequence shown in SEQ ID NO:l including those containing silent mutations and those in which mutations result in amino acid sequence changes, are within the scope of the present invention, as are proteins which are allelic variants of SEQ ID NO: 2.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ZTNFR-5 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • Allelic variants and splice variants of these sequences can be cloned by probing cDNA or genomic libraries from different individuals or tissues according to standard procedures known in the art .
  • the present invention further provides counterpart receptors and polynucleotides from other species (orthologs) .
  • species include, but are not limited to mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species .
  • ZTNFR-5 receptors from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate receptors.
  • Orthologs of the human ZTNFR-5 receptor can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques. For example, a cDNA can be cloned using mRNA obtained from a tissue or cell type that expresses the receptor.
  • Suitable sources of mRNA can be identified by probing Northern blots with probes designed from the sequences disclosed herein.
  • a library is then prepared from mRNA of a positive tissue or cell line.
  • a receptor- encoding cDNA can then be isolated by a variety of methods, such as by probing with a complete or partial human cDNA or with one or more sets of degenerate probes based on the disclosed sequence.
  • a cDNA can also be cloned using PCR, using primers designed from the sequences disclosed herein.
  • the cDNA library can be used to transform or transfect host cells, and expression of the cDNA of interest can be detected with an antibody to the receptor. Similar techniques can also be applied to the isolation of genomic clones.
  • the present invention also provides isolated receptor polypeptides that are substantially homologous to the receptor polypeptide of SEQ ID NO : 2 and its orthologs. It is preferred to provide the proteins or polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • the term "substantially homologous” is used herein to denote proteins or polypeptides having 50%, preferably 60%, more preferably at least 80%, sequence identity to the sequence shown in SEQ ID NO: 2 or its orthologs. Such proteins or polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NO : 2 or its orthologs. Percent sequence identity is determined by conventional methods.
  • Sequence identity of polynucleotide molecules is determined by similar methods using a ratio as disclosed above .
  • Substantially homologous proteins and polypeptides are characterized as having one or more amino acid substitutions, deletions or additions. These changes are preferably of a minor nature, that is conservative amino acid substitutions (see Table 4) and other substitutions that do not significantly affect the folding or activity of the protein or polypeptide; small deletions, typically of one to about 30 amino acids; and small amino- or carboxyl-terminal extensions, such as an amino-terminal methionine residue, a small linker peptide of up to about 20-25 residues, or an affinity tag.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the ZTNFR-5 polypeptide and the affinity tag. Preferred such sites include thrombin cleavage sites and factor Xa cleavage sites .
  • Aromatic phenylalanine tryptophan tyrosine
  • the proteins of the present invention can also comprise non-naturally occurring amino acid residues.
  • Non-naturally occurring amino acids include, without limitation, trans-3-methylproline, 2 , 4-methanoproline, cis-4-hydroxyproline, tra ⁇ s-4-hydroxyproline, N- methylglycine, allo-threonine, methylthreonine, hydroxyethylcysteine, hydroxyethylhomocysteine, nitroglutamine, homoglutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4- methylproline, 3 , 3-dimethylproline, tert-leucine, norvaline, 2-azaphenylalanine, 3-azaphenylalanine, 4- azaphenylalanine, and 4-fluorophenylalanine .
  • an in vi tro system can be employed wherein nonsense mutations are suppressed using chemically aminoacylated suppressor tRNAs.
  • Methods for synthesizing amino acids and aminoacylating tRNA are known in the art . Transcription and translation of plasmids containing nonsense mutations is carried out in a cell-free system comprising an E. coli S30 extract and commercially available enzymes and other reagents. Proteins are purified by chromatography. See, for example, Robertson et al., J . Am . Chem . Soc . 113 :2722, 1991; Ellman et al .
  • E. coli cells are cultured in the absence of a natural amino acid that is to be replaced (e.g., phenylalanine) and in the presence of the desired non-naturally occurring amino acid(s) (e.g., 2- azaphenylalanine, 3-azaphenylalanine, 4-azaphenylalanine, or 4-fluorophenylalanine) .
  • the non-naturally occurring amino acid is incorporated into the protein in place of its natural counterpart. See, Koide et al . , Biochem. 11:7470-6, 1994.
  • Naturally occurring amino acid residues can be converted to non-naturally occurring species by in vi tro chemical modification. Chemical modification can be combined with site-directed mutagenesis to further expand the range of substitutions (Wynn and Richards, Protein Sci. 2:395-403, 1993).
  • non-conservative amino acids amino acids that are not encoded by the genetic code, non-naturally occurring amino acids, and unnatural amino acids may be substituted for ZTNFR-5 amino acid residues.
  • Mutagenesis methods as disclosed above can be combined with high-throughput screening methods to detect activity of cloned, mutagenized receptors in host cells.
  • Mutagenized DNA molecules that encode active receptors or portions thereof e.g., ligand-binding fragments
  • These methods allow the rapid determination of the importance of individual amino acid residues in a polypeptide of interest, and can be applied to polypeptides of unknown structure.
  • polypeptides that are substantially homologous to residues 24-194 of SEQ ID NO: 2 or allelic variants thereof and retain the ligand-binding properties of the wild-type protein.
  • polypeptides may include additional amino acids from affinity tags and the like.
  • polypeptides may also include additional polypeptide segments as generally disclosed above.
  • the receptor polypeptides of the present invention can be produced in genetically engineered host cells according to conventional techniques.
  • Suitable host cells are those cell types that can be transformed or transfected with exogenous DNA and grown in culture, and include bacteria, fungal cells, and cultured higher eukaryotic cells. Eukaryotic cells, particularly cultured cells of multicellular organisms, are preferred. Techniques for manipulating cloned DNA molecules and introducing exogenous DNA into a variety of host cells are disclosed by Sambrook et al .
  • a DNA sequence encoding a ZTNFR-5 polypeptide is operably linked to other genetic elements required for its expression, generally including a transcription promoter and terminator, within an expression vector.
  • the vector will also commonly contain one or more selectable markers and one or more origins of replication, although those skilled in the art will recognize that within certain systems selectable markers may be provided on separate vectors, and replication of the exogenous DNA may be provided by integration into the host cell genome.
  • a secretory signal sequence (also known as a leader sequence, prepro sequence or pre sequence) is provided in the expression vector.
  • the secretory signal sequence may be that of the ZTNFR-5 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized e novo .
  • the secretory signal sequence is joined to the ZTNFR-5 DNA sequence in the correct reading frame and positioned to direct the newly synthesized polypeptide into the secretory pathway of the host cell.
  • Secretory signal sequences are commonly positioned 5' to the DNA sequence encoding the polypeptide of interest, although certain signal sequences may be positioned elsewhere in the DNA sequence of interest (see, e.g., Welch et al . , U.S. Patent No. 5,037,743; Holland et al . , U.S. Patent No. 5,143,830).
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Suitable cultured mammalian cells include the COS-1 (ATCC No. CRL 1650), COS-7 (ATCC No. CRL 1651), BHK (ATCC No. CRL 1632), BHK 570 (ATCC No. CRL 10314), 293 (ATCC No.
  • Chinese hamster ovary e.g., CH0-K1; ATCC No. CCL 61
  • Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland.
  • strong transcription promoters are preferred, such as promoters from SV-40 or cytomegalovirus. See, e.g., U.S. Patent No. 4,956,288.
  • Other suitable promoters include those from metallothionein genes (U.S. Patent Nos. 4,579,821 and 4,601,978 and the adenovirus major late promoter .
  • Drug selection is generally used to select for cultured mammalian cells into which foreign DNA has been inserted. Such cells are commonly referred to as “transfectants” . Cells that have been cultured in the presence of the selective agent and are able to pass the gene of interest to their progeny are referred to as “stable transfectants . " A preferred selectable marker is a gene encoding resistance to the antibiotic neomycin. Selection is carried out in the presence of a neomycin- type drug, such as G-418 or the like.
  • Selection systems may also be used to increase the expression level of the gene of interest, a process referred to as "amplification.” Amplification is carried out by culturing transfectants in the presence of a low level of the selective agent and then increasing the amount of selective agent to select for cells that produce high levels of the products of the introduced genes.
  • a preferred amplifiable selectable marker is dihydrofolate reductase, which confers resistance to methotrexate.
  • Other drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • drug resistance genes e.g., hygromycin resistance, multi-drug resistance, puromycin acetyltransferase
  • Alternative markers that introduce an altered phenotype such as green fluorescent protein, or cell surface proteins such as CD4 , CD8 , Class I MHC, placental alkaline phosphatase may be used to sort transfected cells from untransfected cells by such means as FACS sorting or magnetic bead separation technology.
  • Other higher eukaryotic cells can also be used as hosts, including plant cells, insect cells and avian cells.
  • Agrobacterium rhizogenes as a vector for expressing genes in plant cells has been reviewed by Sinkar et al . , J. Biosci. (Bangalore; 11:47-58, 1987.
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa californica nuclear polyhedrosis virus (AcNPV) . See, King and Possee, The Baculovirus Expression System: A Laboratory Guide,
  • transfer vectors can include an in-frame fusion with DNA encoding an epitope tag at the C- or N- terminus of the expressed ZTNFR-5 polypeptide, for example, a Glu-Glu epitope tag (Grussenmeyer et al . , Proc . Natl. Acad. Sci. 12:7952-4, 1985) .
  • a transfer vector containing ZTNFR-5 is transformed into E. coli , and screened for bacmids which contain an interrupted lacZ gene indicative of recombinant baculovirus .
  • the bacmid DNA containing the recombinant baculovirus genome is isolated, using common techniques, and used to transfect Spodoptera frugiperda cells, e.g.
  • Recombinant virus that expresses ZTNFR-5 is subsequently produced.
  • Recombinant viral stocks are made by methods commonly used the art .
  • the recombinant virus is used to infect host cells, typically a cell line derived from the fall armyworm, Spodoptera frugiperda . See, in general, Glick and Pasternak, Molecular Biotechnology: Principles and Applications of Recombinant DNA, ASM Press, Washington, D.C., 1994.
  • host cells typically a cell line derived from the fall armyworm, Spodoptera frugiperda .
  • Another suitable cell line is the High FiveOTM cell line (Invitrogen) derived from Trichoplusia ni (U.S.
  • Patent #5,300,435) Commercially available serum-free media are used to grow and maintain the cells. Suitable media are Sf900 IITM (Life Technologies) or ESF 921TM
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 5 cells to a density of 1-2 x 10 s cells at which time a recombinant viral stock is added at a multiplicity of infection (MOI) of 0.1 to 10, more typically near 3.
  • MOI multiplicity of infection
  • Fungal cells including yeast cells, can also be used within the present invention.
  • Yeast species of particular interest in this regard include Saccharomyces cerevisiae, Pichia pastoris , and Pichia methanolica .
  • Transformed cells are selected by phenotype determined by the selectable marker, commonly drug resistance or the ability to grow in the absence of a particular nutrient (e.g., leucine) .
  • a preferred vector system for use in Saccharomyces cerevisiae is the POT1 vector system disclosed by Kawasaki et al . (U.S. Patent No. 4,931,373), which allows transformed cells to be selected by growth in glucose-containing media.
  • Suitable promoters and terminators for use in yeast include those from glycolytic enzyme genes (see, e.g., Kawasaki, U.S. Patent No. 4,599,311; Kingsman et al . , U.S. Patent No. 4,615,974; and Bitter, U.S. Patent No. 4,977,092) and alcohol dehydrogenase genes. See also U.S. Patents Nos. 4,990,446; 5,063,154; 5,139,936 and 4,661,454. Transformation systems for other yeasts, including Hansenula polymorpha , Schizosaccharomyces pombe ,
  • Kl uyveromyces lactis, Kl uyveromyces fragilis , Ustilago maydis , Pichia pas toris , Pichia methanolica , Pichia guillermondii and Candida mal tosa are known in the art. See, for example, Gleeson et al . , J. Gen. Microbiol . 132:3459-65, 1986 and Cregg, U.S. Patent No. 4,882,279. Aspergillus cells may be utilized according to the methods of McKnight et al . , U.S. Patent No. 4,935,349. Methods for transforming Acremonium chrysogenum are disclosed by
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO
  • DNA molecules for use in transforming P . methanolica will commonly be prepared as double-stranded, circular plasmids, which are preferably linearized prior to transformation.
  • the promoter and terminator in the plasmid be that of a P. methanolica gene, such as a P. methanolica alcohol utilization gene
  • ⁇ AUG1 or AUG2 ⁇ AUG1 or AUG2
  • Other useful promoters include those of the dihydroxyacetone synthase (DHAS) , formate dehydrogenase (FMD) , and catalase (CAT) genes.
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • Pichia methanolica is a P. methanolica ADE2 gene, which encodes phosphoribosyl-5-aminoimidazole carboxylase (AIRC; EC 4.1.1.21), which allows ade2 host cells to grow in the absence of adenine .
  • EPA phosphoribosyl-5-aminoimidazole carboxylase
  • host cells deficient in vacuolar protease genes ⁇ PEP4 and PRB1 are preferred.
  • Electroporation is used to facilitate the introduction of a plasmid containing DNA encoding a polypeptide of interest into P. methanolica cells. It is preferred to transform P. methanolica cells by electroporation using an exponentially decaying, pulsed electric field having a field strength of from 2.5 to 4.5 kV/cm, preferably about 3.75 kV/cm, and a time constant ( ⁇ ) of from 1 to 40 milliseconds, most preferably about 20 milliseconds.
  • Prokaryotic host cells including strains of the bacteria Escherichia coli , Bacillus and other genera are also useful host cells within the present invention. Techniques for transforming these hosts and expressing foreign DNA sequences cloned therein are well known in the art (see, e.g., Sambrook et al . , ibid. ) .
  • the polypeptide When expressing a ZTNFR-5 polypeptide in bacteria such as E . coli , the polypeptide may be retained in the cytoplasm, typically as insoluble granules, or may be directed to the periplasmic space by a bacterial secretion sequence.
  • the cells are lysed, and the granules are recovered and denatured using, for example, guanidine isothiocyanate or urea.
  • the denatured polypeptide can then be refolded and dimerized by diluting the denaturant , such as by dialysis against a solution of urea and a combination of reduced and oxidized glutathione, followed by dialysis against a buffered saline solution.
  • the polypeptide can be recovered from the periplasmic space in a soluble and functional form by disrupting the cells (by, for example, sonication or osmotic shock) to release the contents of the periplasmic space and recovering the protein, thereby obviating the need for denaturation and refolding.
  • Transformed or transfected host cells are cultured according to conventional procedures in a culture medium containing nutrients and other components required for the growth of the chosen host cells.
  • suitable media including defined media and complex media, are known in the art and generally include a carbon source, a nitrogen source, essential amino acids, vitamins and minerals. Media may also contain such components as growth factors or serum, as required.
  • the growth medium will generally select for cells containing the exogenously added DNA by, for example, drug selection or deficiency in an essential nutrient which is complemented by the selectable marker carried on the expression vector or co- transfected into the host cell.
  • P. methanolica cells are cultured in a medium comprising adequate sources of carbon, nitrogen and trace nutrients at a temperature of about 25°C to 35°C.
  • Liquid cultures are provided with sufficient aeration by conventional means, such as shaking of small flasks or sparging of fermentors .
  • a preferred culture medium for P. methanolica is YEPD (2% D-glucose, 2% BactoTM Peptone (Difco Laboratories, Detroit, MI) , 1% BactoTM yeast extract (Difco Laboratories), 0.004% adenine and 0.006% L-leucine) .
  • Expressed recombinant ZTNFR-5 polypeptides (or chimeric ZTNFR-5 polypeptides) can be purified using fractionation and/or conventional purification methods and media. Ammonium sulfate precipitation and acid or chaotrope extraction may be used for fractionation of samples.
  • Exemplary purification steps may include hydroxyapatite, size exclusion, FPLC and reverse-phase high performance liquid chromatography.
  • Suitable anion exchange media include derivatized dextrans, agarose, cellulose, polyacrylamide, specialty silicas, and the like. PEI, DEAE, QAE and Q derivatives are preferred, with DEAE Fast-Flow Sepharose (Pharmacia, Piscataway, NJ) being particularly preferred.
  • Exemplary chromatographic media include those media derivatized with phenyl, butyl, or octyl groups, such as Phenyl-Sepharose FF (Pharmacia) , Toyopearl butyl 650 (Toso Haas, Montgomeryvilie, PA) ,
  • Octyl -Sepharose and the like; or polyacrylic resins, such as Amberchrom CG 71 (Toso Haas) and the like.
  • Suitable solid supports include glass beads, silica-based resins, cellulosic resins, agarose beads, cross-linked agarose beads, polystyrene beads, cross-linked polyacrylamide resins and the like that are insoluble under the conditions in which they are to be used. These supports may be modified with reactive groups that allow attachment of proteins by amino groups, carboxyl groups, sulfhydryl groups, hydroxyl groups and/or carbohydrate moieties.
  • Examples of coupling chemistries include cyanogen bromide activation, N-hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, hydrazide activation, and carboxyl and amino derivatives for carbodiimide coupling chemistries. These and other solid media are well known and widely used in the art, and are available from commercial suppliers. Methods for binding receptor polypeptides to support media are well known in the art . Selection of a particular method is a matter of routine design and is determined in part by the properties of the chosen support. See, for example, Affinity Chromatography: Principles & Methods, Pharmacia LKB Biotechnology, Uppsala, Sweden, 1988.
  • the polypeptides of the present invention can be isolated by exploitation of their physical properties.
  • immobilized metal ion adsorption (IMAC) chromatography can be used to purify histidine-rich proteins including those comprising polyhistidine tags. Briefly, a gel is first charged with divalent metal ions to form a chelate (Sulkowski, Trends in Biochem. 1:1-7, 1985) . Histidine-rich proteins will be adsorbed to this matrix with differing affinities, depending upon the metal ion used, and will be eluted by competitive elution, lowering the pH, or use of strong chelating agents.
  • IMAC immobilized metal ion adsorption
  • Protein refolding (and optionally reoxidation) procedures may be advantageously used. It is preferred to purify the protein to >80% purity, more preferably to >90% purity, even more preferably >95%, and particularly preferred is a pharmaceutically pure state, that is greater than 99.9% pure with respect to contaminating macromolecules, particularly other proteins and nucleic acids, and free of infectious and pyrogenic agents. Preferably, a purified protein is substantially free of other proteins, particularly other proteins of animal origin.
  • ZTNFR-5 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • ZTNFR-5 polypeptides may be monomers or multimers; glycosylated or non-glycosylated; pegylated or non-pegylated; and may or may not include an initial methionine amino acid residue.
  • the invention also provides soluble ZTNFR-5 receptors used to form fusion proteins with human Ig, to form His-tagged proteins, or FLAGTM-tagged proteins.
  • Soluble ZTNFR-5 or ZTNFR-5-Ig fusion proteins are used, for example, to identify the ZTNFR-5 ligands, including the natural ligand, as well as agonists and antagonists of the natural ligand.
  • soluble ZTNFR-5 cells expressing the ligand are identified by fluorescence immunocytometry or immunohistochemistry .
  • the soluble fusion proteins or soluble Ig fusion proteins are useful in studying the distribution of the ligand on tissues or specific cell lineages, and to provide insight into receptor/ligand biology.
  • Immunoglobulin-ZTNFR-5 polypeptide fusions can be expressed in genetically engineered cells to produce a variety of multimeric ZTNFR-5 analogs.
  • Auxiliary domains can be fused to ZTNFR-5 polypeptides to target them to specific cells, tissues, or macromolecules .
  • a ZTNFR-5 polypeptide or protein could be targeted to a predetermined cell type by fusing a ZTNFR-5 polypeptide to a ligand that specifically binds to a receptor on the surface of the target cell. In this way, polypeptides and proteins can be targeted for therapeutic or diagnostic purposes.
  • a ZTNFR-5 polypeptide can be fused to two or more moieties, such as an affinity tag for purification and a targeting domain.
  • Polypeptides containing an amino acid sequence associated with ligand-binding can be used for purification of ligand.
  • the receptor polypeptide is immobilized on a solid support, such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • a solid support such as beads of agarose, cross-linked agarose, glass, cellulosic resins, silica-based resins, polystyrene, cross-linked polyacrylamide, or like materials that are stable under the conditions of use.
  • Methods for linking polypeptides to solid supports are known in the art, and include amine chemistry, cyanogen bromide activation, N- hydroxysuccinimide activation, epoxide activation, sulfhydryl activation, and hydrazide activation.
  • the resulting media will generally be configured in the form of a column, and fluids containing ligand are passed through the column one or more times to allow ligand to bind to the receptor polypeptide.
  • the ligand is then eluted using changes in salt concentration, chaotropic agents (MnCl2) , or pH to disrupt ligand-receptor binding.
  • the receptor DNA is linked to a second DNA segment encoding a secretory peptide, such as a t-PA secretory peptide.
  • a secretory peptide such as a t-PA secretory peptide.
  • an N- or C-terminal extension such as a poly-histidine tag, substance P, FLAGTM peptide
  • a soluble ZTNFR-5 receptor extracellular ligand-binding region can be expressed as a fusion with immunoglobulin heavy chain constant regions, typically an F c fragment, which contains two constant region domains and a hinge region, but lacks the variable region.
  • immunoglobulin heavy chain constant regions typically an F c fragment, which contains two constant region domains and a hinge region, but lacks the variable region.
  • Such fusions are typically secreted as multimeric molecules, wherein the Fc portions are disulfide bonded to each other and two receptor polypeptides are arrayed in close proximity to each other. Fusions of this type can be used to affinity purify the cognate ligand from solution, as an in vi tro assay tool, to block signals in vi tro by specifically titrating out ligand, and as antagonists in vivo by administering them to block ligand stimulation.
  • a ZTNFR-5- Ig fusion protein (chimera) is added to a sample containing the ligand under conditions that facilitate receptor-ligand binding (typically near-physiological temperature, pH, and ionic strength) .
  • the chimera-ligand complex is then separated by the mixture using protein A, which is immobilized on a solid support (e.g., insoluble resin beads) .
  • the ligand is then eluted using conventional chemical techniques, such as with a salt or pH gradient.
  • the chimera itself can be bound to a solid support, with binding and elution carried out as above.
  • the chimeras are bound to a support via the F c region and used in an ELISA format .
  • Cells expressing functional receptor are used within screening assays .
  • a variety of suitable assays are known in the art. These assays are based on the detection of a biological response in a target cell . An increase in metabolism above a control value indicates a test compound that modulates ZTNFR- 5 mediated metabolism.
  • One such assay is a cell proliferation assay. Cells are cultured in the presence or absence of a test compound, and cell proliferation is detected by, for example, measuring incorporation of tritiated thymidine or by colorimetric assay based on the metabolic breakdown of 3- (4, 5- dimethylthiazol-2-yl) -2 , 5-diphenyl tetrazolium bromide (MTT) (Mosman, J. Immunol. Meth.
  • An alternative assay format uses cells that are further engineered to express a reporter gene.
  • the reporter gene is linked to a promoter element that is responsive to the receptor- linked pathway, and the assay detects activation of transcription of the reporter gene.
  • Numerous reporter genes that are easily assayed for in cell extracts are known in the art, for example, the E. coli lacZ , chloroamphenicol acetyl transferase (CAT) and serum response element (SRE) (see, e.g., Shaw et al . , Cell 5_£: 563-72, 1989) .
  • a preferred such reporter gene is a luciferase gene (de Wet et al . , Mol . Cell. Biol.
  • Luciferase activity assay kits are commercially available from, for example, Promega Corp., Madison, WI .
  • Target cell lines of this type can be used to screen libraries of chemicals, cell-conditioned culture media, fungal broths, soil samples, water samples, and the like. For example, a bank of cell-conditioned media samples can be assayed on a target cell to identify cells that produce ligand.
  • Positive cells are then used to produce a cDNA library in a mammalian expression vector, which is divided into pools, transfected into host cells, and expressed. Media samples from the transfected cells are then assayed, with subsequent division of pools, re-transfection, subculturing, and re-assay of positive cells to isolate a cloned cDNA encoding the ligand.
  • An assay system that uses a ligand-binding receptor (or an antibody, one member of a complement/anti- complement pair) or a binding fragment thereof, and a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ) may also may be advantageously employed.
  • a ligand-binding receptor or an antibody, one member of a complement/anti- complement pair
  • a commercially available biosensor instrument (BIAcoreTM, Pharmacia Biosensor, Piscataway, NJ)
  • Such receptor, antibody, member of a complement/anti-complement pair or fragment is immobilized onto the surface of a receptor chip.
  • Use of this instrument is disclosed by Karlsson, J. Immunol. Methods 145 :229-40 , 1991 and Cunningham and Wells, J. Mol . Biol . 234 : 554-63 , 1993.
  • a receptor, antibody, member or fragment is covalently attached, using amine or sulfhydryl chemistry, to dextran fibers that are attached to gold film within the flow cell.
  • a test sample is passed through the cell. If a ligand, epitope, or opposite member of the complement/anti-complement pair is present in the sample, it will bind to the immobilized receptor, antibody or member, respectively, causing a change in the refractive index of the medium, which is detected as a change in surface plasmon resonance of the gold film.
  • This system allows the determination of on- and off-rates, from which binding affinity can be calculated, and assessment of stoichiometry of binding.
  • Ligand-binding receptor polypeptides can also be used within other assay systems known in the art . Such systems include Scatchard analysis for determination of binding affinity (see, Scatchard, Ann. NY Acad. Sci. 51 : 660-72, 1949) and calorimetric assays (Cunningham et al . , Science 253 : 545- 48, 1991; Cunningham et al . , Science 245:821-25, 1991).
  • the soluble ZTNFR- 5 is useful in studying the distribution of ligands on tissues or specific cell lineages, and to provide insight into receptor/ligand biology. Application may also be made of the specificity of TNF receptors for their ligands as a mechanism by which to destroy ligand-bearing target cells. For example, toxic compounds may be coupled to ZTNFR-5 receptors, in particular to soluble receptors.
  • Examples of toxic compounds would include radiopharmaceuticals that inactivate target cells; chemotherapeutic agents such as doxorubicin, daunorubicin, methotrexate, and cytoxan; toxins, such as ricin, diphtheria, Pseudomonas exotoxin A and abrin; and antibodies to cytotoxic T-cell surface molecules.
  • ZTNFR-5 polynucleotides and/or polypeptides may be useful for regulating the maturation of TNF ligand- bearing cells, such as T cells, B cells, lymphocytes, peripheral blood mononuclear cells, polymorphonuclear leukocytes, fibroblasts and hematopoietic cells.
  • ZTNFR-5 polypeptides will also find use in mediating metabolic or physiological processes in vivo . Proliferation and differentiation can be measured in vi tro using cultured cells. Bioassays and ELISAs are available to measure cellular response to ZTNFR-5, in particular are those which measure changes in cytokine production as a measure of cellular response (see for example, Current Protocols in Immunology ed. John E. Coligan et al . , NIH, 1996) .
  • soluble ZTNFR-5 transfected expression host cells may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine microencapsulation, permselective membrane encapsulation and diffusion chambers have been described as a means to entrap transfected mammalian cells or primary mammalian cells.
  • non-immunogenic "encapsulations” or microenvironments permit the transfer of nutrients into the microenvironment, and also permit the diffusion of proteins and other macromolecules secreted or released by the captured cells across the environmental barrier to the recipient animal. Most importantly, the capsules or microenvironments mask and shield the foreign, embedded cells from the recipient animal's immune response. Such microenvironments can extend the life of the injected cells from a few hours or days (naked cells) to several weeks (embedded cells) .
  • Alginate threads provide a simple and quick means for generating embedded cells.
  • the materials needed to generate the alginate threads are readily available and relatively inexpensive. Once made, the alginate threads are relatively strong and durable, both in vi tro and, based on data obtained using the threads, in vivo .
  • the alginate threads are easily manipulable and the methodology is scalable for preparation of numerous threads.
  • 3% alginate is prepared in sterile H2O, and sterile filtered. Just prior to preparation of alginate threads, the alginate solution is again filtered. An approximately 50% cell suspension (containing about 5 x 10 5 to about 5 x 10 7 cells/ml) is mixed with the 3% alginate solution.
  • One ml of the alginate/cell suspension is extruded into a 100 mM sterile filtered CaCl 2 solution over a time period of ⁇ 15 min, forming a "thread" .
  • the extruded thread is then transferred into a solution of 50 mM CaCl 2 , and then into a solution of 25 mM CaCl 2 .
  • the thread is then rinsed with deionized water before coating the thread by incubating in a 0.01% solution of poly-L-lysine .
  • the thread is rinsed with Lactated Ringer's Solution and drawn from solution into a syringe barrel (without needle attached) .
  • a large bore needle is then attached to the syringe, and the thread is intraperitoneally injected into a recipient in a minimal volume of the Lactated Ringer's Solution.
  • viruses for this purpose include adenovirus, herpesvirus, vaccinia virus and adeno- associated virus (AAV) .
  • Adenovirus a double-stranded DNA virus, is currently the best studied gene transfer vector for delivery of heterologous nucleic acid (for a review, see Becker et al . , Meth. Cell Biol. 41:161-89, 1994; and Douglas and Curiel, Science & Medicine 4_:44-53, 1997).
  • adenovirus can (i) accommodate relatively large DNA inserts; (ii) be grown to high-titer; (iii) infect a broad range of mammalian cell types; and (iv) be used with a large number of available vectors containing different promoters. Also, because adenoviruses are stable in the bloodstream, they can be administered by intravenous injection. By deleting portions of the adenovirus genome, larger inserts (up to 7 kb) of heterologous DNA can be accommodated. These inserts may be incorporated into the viral DNA by direct ligation or by homologous recombination with a co-transfected plasmid.
  • the essential El gene has been deleted from the viral vector, and the virus will not replicate unless the El gene is provided by the host cell (the human 293 cell line is exemplary) .
  • adenovirus When intravenously administered to intact animals, adenovirus primarily targets the liver. If the adenoviral delivery system has an El gene deletion, the virus cannot replicate in the host cells. However, the host's tissue (e.g., liver) will express and process (and, if a signal sequence is present, secrete) the heterologous protein. Secreted proteins will enter the circulation in the highly vascularized liver, and effects on the infected animal can be determined.
  • the adenovirus system can also be used for protein production in vi tro .
  • the cells can produce proteins for extended periods of time.
  • BHK cells are grown to confluence in cell factories, then exposed to the adenoviral vector encoding the secreted protein of interest.
  • the cells are then grown under serum-free conditions, which allows infected cells to survive for several weeks without significant cell division.
  • adenovirus vector infected 293S cells can be grown in suspension culture at relatively high cell density to produce significant amounts of protein (see Gamier et al . , Cvtotechnol . 15 . : 145-55, 1994).
  • an expressed, secreted heterologous protein can be repeatedly isolated from the cell culture supernatant. Within the infected 293S cell production protocol, non-secreted proteins may also be effectively obtained.
  • ZTNFR-5 shares homology with OPG, a soluble TNF receptor involved in the regulation of bone density
  • the hypocalcemic rat model can be used to determine the effect of ZTNFR-5 on serum calcium
  • the ovariectomized rat or mouse can be used as a model system for osteoporosis . Bone changes seen in these models and in humans during the early stages of estrogen deficiency are qualitatively similar.
  • ZTNFR-5 polypeptides can also be used to prepare antibodies that specifically bind to ZTNFR- 5 epitopes, peptides or polypeptides.
  • Methods for preparing polyclonal and monoclonal antibodies are well known in the art (see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual , Second Edition, Cold Spring Harbor, NY, 1989; and Hurrell, J. G. R. , Ed., Monoclonal Hybridoma Antibodies: Technigues and Applications, CRC Press, Inc., Boca Raton, FL, 1982) .
  • polyclonal antibodies can be generated from a variety of warm-blooded animals such as horses, cows, goats, sheep, dogs, chickens, rabbits, mice, hamsters, guinea pigs and rats as well as transgenic animals such as transgenic sheep, cows, goats or pigs.
  • Antibodies may also be expressed in yeast and fungi in modified forms as well as in mammalian and insect cells.
  • the ZTNFR-5 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal or elicit an immune response.
  • Suitable antigens would include the ZTFNR-5 polypeptide encoded by SEQ ID NO : 2 from amino acid residue 24-300 of SEQ ID NO:2, or a contiguous 9-300 amino acid residue fragment thereof.
  • the immunogenicity of a ZTNFR-5 polypeptide may be increased through the use of an adjuvant, such as alum (aluminum hydroxide) or Freund's complete or incomplete adjuvant.
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZTNFR-5 or a portion thereof with an immunoglobulin polypeptide or with maltose binding protein.
  • the polypeptide immunogen may be a full- length molecule or a portion thereof.
  • polypeptide portion is "hapten-like"
  • such portion may be advantageously joined or linked to a macromolecular carrier (such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid) for immunization.
  • a macromolecular carrier such as keyhole limpet hemocyanin (KLH) , bovine serum albumin (BSA) or tetanus toxoid
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof, such as F(ab')2 an ⁇ ⁇ Fa ⁇ proteolytic fragments. Genetically engineered intact antibodies or fragments, such as chimeric antibodies, Fv fragments, single chain antibodies and the like, as well as synthetic antigen-binding peptides and polypeptides, are also included.
  • Non-human antibodies may be humanized by grafting only non-human CDRs onto human framework and constant regions, or by incorporating the entire non-human variable domains (optionally "cloaking" them with a humanlike surface by replacement of exposed residues, wherein the result is a "veneered” antibody) .
  • humanized antibodies may retain non-human residues within the human variable region framework domains to enhance proper binding characteristics. Through humanizing antibodies, biological half-life may be increased, and the potential for adverse immune reactions upon administration to humans is reduced.
  • Humanized monoclonal antibodies directed against ZTNFR- 5 polypeptides could be used as a protein therapeutic, in particular for use as an immunotherapy.
  • Alternative techniques for generating or selecting antibodies useful herein include in vi tro exposure of lymphocytes to ZTNFR-5 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZTNFR-5 protein or peptide) .
  • Antibodies are defined to be specifically binding if they bind to a ZTNFR-5 polypeptide with a binding affinity (K a ) of 10 M “1 or greater, preferably 10 7 M “1 or greater, more preferably 10 8 M “1 or greater, and most preferably 10 9 M- 1 or greater.
  • K a binding affinity
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art (for example, by Scatchard analysis) .
  • assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to ZTNFR-5 proteins or peptides. Exemplary assays are described in detail in Antibodies: A Laboratory Manual , Harlow and Lane (Eds.), Cold Spring Harbor Laboratory Press, 1988. Representative examples of such assays include: concurrent immunoelectrophoresis, radioimmunoassay, radioimmuno-precipitation, enzyme-linked immunosorbent assay (ELISA) , dot blot or Western blot assay, inhibition or competition assay, and sandwich assay. In addition, antibodies can be screened for binding to wild-type versus mutant ZTNFR-5 protein or peptide.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies to ZTNFR-5 may be used for immunohistochemical tagging of cells that express human ZTNFR-5, for example, to use in a diagnostic assays; for isolating ZTNFR-5 by affinity purification; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZTNFR-5 in vi tro and in vivo .
  • Suitable direct tags or labels include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like; indirect tags or labels may feature use of biotin-avidin or other complement/anti-complement pairs as intermediates.
  • Antibodies herein may also be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications.
  • Antibodies can be made to soluble, ZTNFR-5 polypeptides which are His or FLAGTM tagged. Alternatively, such polypeptides form a fusion protein with Human Ig.
  • antiserum containing polypeptide antibodies to His-tagged, or FLAGTM-tagged soluble ZTNFR-5 can be used in analysis of tissue distribution of ZTNFR-5 by immunohistochemistry on human or primate tissue.
  • These soluble ZTNFR-5 polypeptides can also be used to immunize mice in order to produce monoclonal antibodies to a soluble human ZTNFR-5 polypeptide.
  • Monoclonal antibodies to a soluble human ZTNFR-5 polypeptide can also be used to mimic ligand/receptor coupling, resulting in activation or inactivation of the ligand/receptor pair. For instance, it has been demonstrated that cross-linking anti-soluble CD40 monoclonal antibodies provides a stimulatory signal to B cells that have been sub-optimally activated with anti-IgM or LPS, and results in proliferation and immunoglobulin production. These same monoclonal antibodies act as antagonists when used in solution by blocking activation of the receptor. Monoclonal antibodies to ZTNFR-5 can be used to determine the distribution, regulation and biological interaction of the ZTNFR-5/ZTNFR-5-ligand pair on specific cell lineages identified by tissue distribution studies.
  • the invention also provides isolated and purified ZTNFR-5 polynucleotide probes or primers.
  • polynucleotide probes can be RNA or DNA.
  • DNA can be either cDNA or genomic DNA.
  • Polynucleotide probes are single or double-stranded DNA or RNA, generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences and will generally comprise at least 16 nucleotides, more often from 17 nucleotides to 25 or more nucleotides, sometimes 40 to 60 nucleotides, and in some instances a substantial portion, domain or even the entire ZTNFR-5 gene or cDNA.
  • Probes and primers are generally synthetic oligonucleotides, but may be generated from cloned cDNA or genomic sequences or its complements. Analytical probes will generally be at least 20 nucleotides in length, although somewhat shorter probes
  • PCR primers are at least
  • a polynucleotide probe may comprise an entire exon or more. Probes can be labeled to provide a detectable signal, such as with an enzyme, biotin, a radionuclide, fluorophore, chemiluminescer, paramagnetic particle and the like, which are commercially available from many sources, such as Molecular Probes, Inc., Eugene, OR, and Amersham Corp., Arlington Heights, IL, using techniques that are well known in the art.
  • the synthetic oligonucleotides of the present invention have at least 80% identity to a representative ZTNFR-5 DNA sequence (SEQ ID NO:l) or its complements.
  • Preferred regions from which to construct probes include the 5 ' and/or 3' coding sequences, ligand binding regions, and signal sequences, and the like.
  • Techniques for developing polynucleotide probes and hybridization techniques are known in the art, see for example, Ausubel et al . , eds., Current Protocols in Molecular Biology, John Wiley and Sons, Inc., NY, 1991.
  • Such probes can also be used in hybridizations to detect the presence or quantify the amount of ZTNFR-5 gene or mRNA transcript in a sample.
  • ZTNFR-5 polynucleotide probes could be used to hybridize to DNA or RNA targets for diagnostic purposes, using such techniques such as fluorescent in si tu hybridization (FISH) or immunohistochemistry .
  • Polynucleotide probes can be used to identify genes encoding ZTNFR-5-like proteins.
  • ZTNFR- 5 polynucleotides can be used as primers and/or templates in PCR reactions to identify other novel members of the TNFR family.
  • Such probes can also be used to screen libraries for related sequences encoding novel tumor necrosis factor receptors .
  • Such screening would be carried out under conditions of low stringency which would allow identification of sequences which are substantially homologous, but not requiring complete homology to the probe sequence.
  • Such methods and conditions are well known in the art, see, for example, Sambrook et al . , Molecular Cloning: A Laboratory Manual, Second Edition, Cold Spring Harbor, NY, 1989.
  • Such low stringency conditions could include hybridization temperatures less than 42°C, formamide concentrations of less than 50% and moderate to low concentrations of salt .
  • Libraries may be made of genomic DNA or cDNA.
  • Polynucleotide probes are also useful for Southern, Northern, or slot blots, colony and plaque hybridization and in si tu hybridization. Mixtures of different ZTNFR-5 polynucleotide probes can be prepared which would increase sensitivity or the detection of low copy number targets, in screening systems. In addition, such polynucleotide probes could be used to hybridize to counterpart sequences on individual chromosomes. Chromosomal identification and/or mapping of the ZTNFR-5 gene could provide useful information about gene function and disease association. Many mapping techniques are available to one skilled in the art, for example, mapping somatic cell hybrids, and fluorescence in si tu hybridization (FISH) . A preferred method is radiation hybrid mapping.
  • Radiation hybrid mapping is a somatic cell genetic technique developed for constructing high-resolution, contiguous maps of mammalian chromosomes (Cox et al., Science 250 :245-50, 1990). Partial or full knowledge of a gene's sequence allows the designing of PCR primers suitable for use with chromosomal radiation hybrid mapping panels.
  • Commercially available radiation hybrid mapping panels which cover the entire human genome, such as the Stanford G3 RH Panel and the GeneBridge 4 RH Panel (Research Genetics, Inc., Huntsville, AL) , are available.
  • These panels enable rapid, PCR based, chromosomal localizations and ordering of genes, sequence-tagged sites (STSs) , and other non-polymorphic- and polymorphic markers within a region of interest. This includes establishing directly proportional physical distances between newly discovered genes of interest and previously mapped markers.
  • the precise knowledge of a gene's position can be useful in a number of ways including: 1) determining if a sequence is part of an existing contig and obtaining additional surrounding genetic sequences in various forms such as YAC-, BAC- or cDNA clones, 2) providing a possible candidate gene for an inheritable disease which shows linkage to the same chromosomal region, and 3) for cross-referencing model organisms such as mouse which may be beneficial in helping to determine what function a particular gene might have.
  • Chromosomal localization can also be done using STSs.
  • An STS is a DNA sequence that is unique in the human genome and can be used as a reference point for a particular chromosome or region of a chromosome.
  • An STS can be defined by a pair of oligonucleotide primers that can be used in a polymerase chain reaction to specifically detect this site in the presence of all other genomic sequences.
  • STSs are based solely on DNA sequence they can be completely described within a database, for example, Database of Sequence Tagged Sites (dbSTS) , GenBank, (National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov), they can be searched with a gene sequence of interest for the mapping data contained within these short genomic landmark STS sequences .
  • dbSTS Database of Sequence Tagged Sites
  • the present invention also provides reagents for use in diagnostic applications.
  • the ZTNFR- 5 gene, a probe comprising ZTNFR- 5 DNA or RNA, or a subsequence thereof can be used to determine if the ZTNFR- 5 gene is present on a particular chromosome or if a mutation has occurred.
  • Detectable chromosomal aberrations at the ZTNFR-5 gene locus include, but are not limited to, aneuploidy, gene copy number changes, insertions, deletions, restriction site changes and rearrangements. These aberrations can occur within the coding sequence, within introns, or within flanking sequences, including upstream promoter and regulatory regions, and may be manifested as physical alterations within a coding sequence or changes in gene expression level.
  • these diagnostic methods comprise the steps of (a) obtaining a genetic sample from a patient; (b) incubating the genetic sample with a polynucleotide probe or primer as disclosed above, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; and (iii) comparing the first reaction product to a control reaction product. A difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
  • Genetic samples for use within the present invention include genomic DNA, cDNA, and RNA.
  • the polynucleotide probe or primer can be RNA or DNA, and will comprise a portion of SEQ ID NO:l, the complement of SEQ ID NO:l, or an RNA equivalent thereof.
  • Suitable assay methods in this regard include molecular genetic techniques known to those in the art, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, ligation chain reaction (Barany, PCR Methods and Applications 1:5- 16, 1991), ribonuclease protection assays, and other genetic linkage analysis techniques known in the art
  • RNA-RNA hybrid RNA-RNA hybrid
  • RNase RNase-activated DNA cleavage reaction
  • Hybridized regions of the RNA are protected from digestion.
  • PCR assays a patient's genetic sample is incubated with a pair of polynucleotide primers, and the region between the primers is amplified and recovered. Changes in size or amount of recovered product are indicative of mutations in the patient.
  • Another PCR-based technique that can be employed is single strand conformational polymorphism (SSCP) analysis (Hayashi, PCR Methods and Applications 1:34-8, 1991) .
  • SSCP single strand conformational polymorphism
  • ZTNFR-5 agonists are useful for modifying the proliferation and development of target cells in vi tro and in vivo .
  • agonist compounds are useful alone or in combination with other cytokines and hormones as components of defined cell culture media.
  • Agonists are thus useful in specifically mediating the growth and/or development of ZTNFR-5 -bearing T lymphocytes cells in culture.
  • Agonists and antagonists may also prove useful in the study of effector functions of T lymphocytes, in particular T lymphocyte activation and differentiation.
  • Antagonists are useful as research reagents for characterizing ligand-receptor interaction.
  • ZTNFR-5 agonists are useful for modifying the proliferation and development of target cells in vi tro and in vivo .
  • agonist compounds are useful alone or in combination with other cytokines and hormones as components of defined cell culture media. Agonists are thus useful in specifically mediating the maturation of ZTNFR-5 -bearing cells in culture.
  • the invention also provides antagonists, which either bind to ZTNFR-5 polypeptides or, alternatively, to a ligand to which ZTNFR-5 polypeptides bind, thereby inhibiting or eliminating the function of ZTNFR-5.
  • ZTNFR-5 antagonists would include antibodies; oligonucleotides which bind either to the ZTNFR-5 polypeptide or to its ligand; natural or synthetic analogs of ZTNFR-5 ligands which retain the ability to bind the receptor but do not result in either ligand or receptor signaling. Such analogs could be peptides or peptide-like compounds. Natural or synthetic small molecules which bind to ZTNFR-5 polypeptides and prevent signaling are also contemplated as antagonists. As such, ZTNFR-5 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a ZTNFR-5 receptor or ligand would be beneficial. Antagonists are useful as research reagents for characterizing ligand-receptor interaction.
  • ZTNFR-5 polypeptides may be used within diagnostic systems to detect the presence of ligand polypeptides.
  • Antibodies or other agents that specifically bind to ZTNFR-5 may also be used to detect the presence of circulating receptor or ligand polypeptides.
  • detection methods are well known in the art and include, for example, enzyme-linked immunosorbent assay (ELISA) and radioimmunoassay.
  • ELISA enzyme-linked immunosorbent assay
  • Immunohistochemically labeled ZTNFR- 5 antibodies can be used to detect ZTNFR-5 receptor and/or ligands in tissue samples.
  • ZTNFR-5 levels can also be monitored by such methods as RT-PCR, where ZTNFR-5 mRNA can be detected and quantified.
  • ZTNFR-5 polypeptides in various diseases, and as a would serve as diagnostic tools for diseases for which altered levels of ZTNFR-5 are significant.
  • Altered levels of ZTNFR-5 receptor polypeptides may be indicative of pathological conditions including cancer, autoimmune disorders, bone disorders, inflammation and immunodeficiencies .
  • ZTNFR-5 polynucleotides and/or polypeptides, agonists and antagonists disclosed herein can be useful as therapeutics to modulate one or more biological processes in cells, tissues and/or biological fluids.
  • Novel ZTNFR-5 encoding polynucleotides and polypeptides of the present invention were initially identified by querying an EST database for sequences homologous to conserved motifs within the TNF ligand family. Using this information, 16 independent ESTs were identified and aligned to produce a novel human contig of about 1181 bp . To identify the corresponding cDNA, a clone considered likely to contain the entire sequence was used for sequencing. Using a QIAwell 8 plasmid kit (Qiagen, Inc., Chatsworth, CA) according to manufacturer's instructions, a 5 ml overnight culture in LB + 50 ⁇ g/ml ampicillin was prepared.
  • QIAwell 8 plasmid kit Qiagen, Inc., Chatsworth, CA
  • the template was sequenced on an Applied BiosystemsTM model 377 DNA sequencer (Perkin-Elmer Cetus, Norwalk, Ct . ) using the ABI PRISMTM Dye Terminator Cycle Sequencing Ready Reaction Kit (Perkin-Elmer Corp.) according to the manufacturer's instructions. Oligonucleotides ZC694 (SEQ ID NO: 4) and ZC695 (SEQ ID NO: 5) to the T7 and SP6 promoters on the vector were used as sequencing primers.
  • Oligonucleotides ZC14153 (SEQ ID NO:6), ZC14343 (SEQ ID NO:7), ZC14344 (SEQ ID NO : 8 ) , ZC14331 (SEQ ID NO:9) and ZC14467 (SEQ ID NO:10) were used to complete the sequence from the clone. Sequencing reactions were carried out in a Hybaid OmniGene Temperature Cycling System (National Labnet Co., Woodbridge, NY). SequencherTM 3.0 sequence analysis software (Gene Codes Corporation, Ann Arbor, MI) was used for data analysis. The resulting 1205 bp sequence is disclosed in SEQ ID NO : 1.
  • the MarathonTM-ready cDNA libraries were prepared according to manufacturer's instructions (MarathonTM cDNA Amplification Kit; Clontech, Palo Alto, CA) using human heart, uterus, K562 and ⁇ MCF7 poly A+ RNA (Clontech) .
  • the probe was amplified in a polymerase chain reaction as follows: 1 cycle at 94°C for 1 minute 30 seconds; 35 cycles of 94°C for 10 seconds, 62°C for 20 seconds, and 72°C for 30 seconds, followed by 1 cycle at 72°C for 10 minutes.
  • the resulting DNA fragment was electrophoresed on a 2% low melt agarose gel (SEA PLAQUE GTG low melt agarose, FMC Corp., Rockland, ME), the fragment was purified using the QIAquickTM method (Qiagen, Chatsworth, CA) , and the sequence was confirmed by sequence analysis.
  • the probe was radioactively labeled using the random priming MULTIPRIME DNA labeling system (Amersham, Arlington Heights, IL) , according to the manufacturer's specifications.
  • the probe was purified using a NUCTRAP push column (Stratagene, La Jolla, CA) .
  • ExpressHybTM ExpressHybTM
  • Hybridization took place overnight at 55°C using 2.9 x 10 6 cpm/ml of labeled probe. The blots were then washed at 65°C in 0.1X SSC, 0.1% SDS. Signal intensity was highest in lung, spinal cord, stomach, lymph node, spleen, colon and trachea. Less intense signals were present in kidney, thymus, heart, pancreas, prostate, small intestine, placenta, thyroid, and bone marrow tissue. The transcript size was approximately 1.2 kb. In the case of lung a strongly hybridizing 2.4 kb transcript was also seen.
  • the Fc region of human IgGl was modified.
  • the Fc region was isolated from a human fetal liver library (Clontech) by PCR using oligo primers ZC10,134 (SEQ ID NO: 15) and ZC10,135 (SEQ ID NO: 16) .
  • PCR was used to introduce mutations within the Fc region to reduce FcgRI binding.
  • the FcgRI binding site (Leu-Leu-gly-Gly) was mutated to Ala-Glu-gly-Ala (amino acid residues 38-41 of SEQ ID NO: 17) according to Baum et al. (EMBO J. 11:3992-4001, 1994), to reduce FcRl binding (Duncan et al . , Nature 112:563-4, 1988).
  • Oligonucleotide primers ZC15,345 (SEQ ID NO: 18) and ZC15,347 (SEQ ID NO: 19) were used to introduce the mutation.
  • reaction products were electrophoresed and the band corresponding to the predicted size of ⁇ 676 bp was detected.
  • the band was excised from the gel and recovered using a QIAGEN QIAquick ⁇ Gel Extraction Kit (Qiagen) according to the manufacturers instructions.
  • PCR was also used to introduce a mutation of Ala to Ser (amino acid residue 134 of SEQ ID NO: 17) and Pro to Ser (amino acid residue 135 of SEQ ID NO: 17) to reduce complement Clq binding and/or complement fixation (Duncan and Winter, Nature 332 : 788, 1988) and add a 5' Bam HI restriction site, a signal sequence for secretion, a 3' Xba I restriction site as well as the stop codon TAA.
  • Two first round reactions were done using the FcgRI binding site mutated IgFc sequence as a template.
  • 1 ⁇ l FcgRI binding site mutated IgFc template 5 ⁇ l 10X Pfu Reaction Buffer (Stratagene) , 8 ⁇ l 1.25 mM dNTPs, 31 ⁇ l dH 2 0, 2 ⁇ l 20 mM ZC15,517 (SEQ ID NO:20), a 5' primer beginning at nucleotide 36 of SEQ ID NO:17 and 2 ⁇ l 20 mM ZC15,530 (SEQ ID NO:21), a 3' primer beginning at the complement of nucleotide 405 of SEQ ID NO: 17.
  • the second reaction contained 2 ⁇ l of 20 mM each of oligonucleotide primers ZC15,518 (SEQ ID NO:22), a 5' primer beginning at nucleotide 388 of SEQ ID NO: 17 and ZC15,347 (SEQ ID NO:19), a 3' primer, to introduce the Ala to Ser mutation, Xba I restriction site and stop codon.
  • An equal volume of mineral oil was added and the reactions were heated to 94 °C for 1 minute.
  • Pfu polymerase 2.5 units, Stratagene was added followed by 25 cycles at 94 °C for 30 seconds, 55°C for 30 seconds, 72°C for 2 minutes followed by a 7 minute extension at 72 °C.
  • the reaction products were electrophoresed and bands corresponding to the predicted sizes, ⁇ 370 and ⁇ 395 bp respectfully, were detected.
  • the bands were excised from the gel and extracted using a QIAGEN QIAquick ⁇ J Gel Extraction Kit (Qiagen) according to the manufacturers instructions.
  • a second round reaction was done to join the above fragments and add the 5' Bam HI restriction site.
  • To a 50 ⁇ l final volume was added 30 ⁇ l dH 2 0, 8 ⁇ l 1.25 mM dNTPs, 5 ⁇ l 10X Pfu polymerase reaction buffer (Stratagene) and 1 ⁇ l each of the two first round PCR products .
  • An equal volume of mineral oil was added and the reaction was heated to 94 °C for 1 minute.
  • Pfu polymerase (2.5 units, Stratagene) was added followed by 5 cycles at 94°C for 30 seconds, 55°C for 30 seconds, and 72 °C for 2 minutes. The temperature was again brought to 94 °C and 2 ⁇ l each of 20 mM ZC15,516 (SEQ ID NO: 23), a 5' primer beginning at nucleotide 1 of SEQ ID NO: 17, introducing a Bam HI restriction site, and 20 mM ZC15,347 (SEQ ID NO: 19) were added followed by 25 cycles at 94 °C for 30 seconds, 55°C for 30 seconds and 72°C for 2 minutes, and a final 7 minute extension at 72°C. A portion of the reaction was visualized using gel electrophoresis. A 789 bp band corresponding the predicted size was detected. The remainder of the IgFc
  • the transformation reaction consisted of 1 ⁇ l of the ligation reaction in 100 ⁇ l competent cells. The transformation reaction was incubated for 30 minutes on ice, heat shocked at 42 °C for 40 seconds, and incubated on ice for 2 minutes.
  • pHZ200 A second expression vector, mammalian vector pHZ200, was digested with restriction enzymes Bam HI and Xba I and ligated to the IgFc fragment as described above.
  • pHZ200 is an expression vector that may be used to express protein in mammalian cells or in a frog oocyte translation system from mRNAs that have been transcribed in vi tro .
  • the pHZ200 expression unit comprises the mouse metallothionein-1 promoter, the bacteriophage T7 promoter flanked by multiple cloning banks containing unique restriction sites for insertion of coding sequences, the human growth hormone terminator and the bacteriophage T7 terminator.
  • pHZ200 contains an E. coli origin of replication; a bacterial beta lactamase gene; a mammalian selectable marker expression unit comprising the SV40 promoter and origin, a dihydrofolate reductase gene and the SV40 transcription terminator.
  • Colonies were screened by PCR using primers, ZC6583 (SEQ ID NO:24), a sequence from the pHZ200 expression vector, and ZC15,530 (SEQ ID NO: 21) .
  • a sample of each colony in 5 ⁇ l LB was added to 27 ⁇ l d ⁇ O, 5 ⁇ l 10X Advantage cDNA Polymerase Mix Buffer, 8 ⁇ l 1.25 mM dNTP, 2 ⁇ l each 20 mM ZC6583 (SEQ ID NO:24) and ZC15,530 (SEQ ID NO:21), and 1 ⁇ l Advantage cDNA Polymerase Mix (Clontech) .
  • the reactions were heated at 94°C for 1 minute followed by 25 cycles at 94°C for 30 seconds, 55°C for 30 seconds, 68°C for 2 minutes followed by a 7 minute extension at 68 °C.
  • Positive clones were identified by the presence of a band at 486 bp .
  • the insert sequence of a positive clone, designated IgFc4/pHZ200 was verified by sequence analysis.
  • a soluble ZTNFR-5 fragment was prepared using PCR, truncating the transmembrane and cytoplasmic domains.
  • Oligonucleotide primer ZC15,334 (SEQ ID NO: 25) adds a Bam HI restriction site to the 5' side of nucleotide 181 in SEQ ID NO:l and oligonucleotide primer ZC15,519 (SEQ ID NO: 26) adds sequence encoding for amino acid residues Glu and Pro and a Bgl II site just 3 ' to nucleotide 1892 in SEQ ID NO:l.
  • the reaction products were electrophoresed and the band corresponding to the predicted size of ⁇ 926 bp was detected.
  • the band was excised from the gel and recovered using a QIAGEN QIAquick ⁇ Gel Extraction Kit (Qiagen) according to the manufacturers instructions.
  • the soluble ZTNFR5 PCR fragment was ligated in pCR ⁇ -Blunt (Clontech) following the manufacturer's recommended protocol for ligation and transformation of E. coli TOP10 competent cells. Positive clones were selected by PCR screening using pCR -Blunt oligonucleotide primers ZC447 (SEQ ID NO:27) and ZC694 (SEQ ID NO:4) .
  • Ztnfr5/pCR-Blunt was digested with restriction enzymes Bam HI and Bgl II to release a fragment containing the ztnfr5 signal sequence and extracellular domain fragment .
  • the fragment was isolated by gel electrophoresis as described above and ligated into a Bam HI and Bgl II digested IgFc4/pHZ200 vector as described above at a 4 to 1 molar ratio of insert to vector.
  • Competent DH10B cells were transformed with the ligation product, plated and a clone containing the correct insert was identified by restriction enzyme mapping analysis and designated ZTNFR5Fc4/pHZ200.
  • the Bam HI -Xba I fragment containing the ZTNFR5-IgFc fusion protein coding region from ZTNFR5Fc4/pHZ200 was ligated into Bam HI -Xba I digested pFBL2 vector as described above at a 4 to 1 molar ration of insert to vector.
  • DH10B competent cells (Life Technologies) were transformed with the ligation product according to manufacturer's instructions and a clone containing the correct insert was identified by restriction enzyme mapping analysis and designated ZTNFR5Fc4/pFBL2.
  • BHK 570 cells (ATCC NO: CRL-10314) were plated in 10 cm tissue culture dishes and allowed to grow to approximately 50 to 70% confluency overnight at 37°C, 5% C0 2 , in DMEM/FBS media (DMEM, Gibco/BRL High Glucose,
  • LipofectamineTM mixture was replaced with fresh FBS/DMEM media the next day.
  • the cells were split into the selection media (DMEM/FBS media from above with the addition of 1 ⁇ M methotrexate (Sigma Chemical Co., St. Louis, Mo.)) in 150 mm plates at 1:50, 1:100 and 1:200. The plates were refed at day 5 post-transfection with fresh selection media. Screening colonies
  • 10 ml serum-free ESTEP 2 media (668.7g/50L DMEM (Gibco), 5.5 g/50L pyruvic acid, sodium salt 96% (Mallinckrodt) , 185.0 g/50L NaHC0 3 (Mallinkrodt) , 5.0 mg/m
  • a sterile Teflon mesh (Spectrum Medical Industries, Los Angeles, CA) pre-soaked in serum- free ESTEP 2 was then placed over the cells.
  • the filters were blocked in 2.5% nonfat dry milk/Western A buffer (Western A: 50mM Tris pH 7.4, 5 mM EDTA, 0.05% NP-40, 150 mM NaCl and 0.25% gelatin) overnight at 4°C.
  • the filter was then incubated with a goat anti -human IgG-HRP antibody (Southern Biotechnology Associates, Inc., Birmingham, AL) at a
  • DHlOBac Max Efficiency competent cells Gibco-BRL, Gaithersburg, MD
  • the transformants were then diluted in 980 ⁇ l SOC media and plated on to Luria Agar plates containing 50 ⁇ g/ml kanamycin, 7 ⁇ g/ml gentamicin, 10 ⁇ g/ml tetracycline, IPTG and Bluo Gal.
  • the cells were incubated for 48 hours at 37°C.
  • a color selection was used to identify those cells having virus that had incorporated into the plasmid (referred to as a "bacmid”) . Those colonies, which were white in color, were picked for analysis. Bacmid DNA was isolated from positive colonies and used to transfect Spodoptera frugiperda (Sf9) cells.
  • Sf9 cells were seeded at 5 x 10 6 cells per 35 mm plate and allowed to attach for 1 hour at 27°C.
  • Five microliters of bacmid DNA was diluted with 100 ⁇ l Sf-900 II SFM.
  • Six microliters of CellFECTIN Reagent (Life Technologies) was diluted with 100 ⁇ l Sf-900 II SMF .
  • the bacmid DNA and lipid solutions were gently mixed and incubated 30-45 minutes at room temperature. The media the plate of cells was aspirated, and the lipid-DNA mixture to which 0.8 ml of Sf-900 II SFM was added.
  • the cells were incubated at 27°C for 4 hours, then 2 ml of Sf- 900 II media containing penicillin/streptomycin was added to each plate.
  • the plates were incubated at 27°C, 90% humidity, for 48 hours after which the virus was harvested.
  • Sf9 cells were grown in 50 ml Sf-900 II SFM in a shake flask to an approximate density of 0.50 x 10 6 cells/ml. They were then transfected with 50 ⁇ l of the virus stock from above and incubated at 27°C for 2 days after which time the virus was harvested, titer 2.2 x 10 7 pfu/ml . To scale up, Sf9 cells were grown to a density of 1.7 x 10 6 SF9 cells/ml in 5 liter batches. The cells were then transfected with the harvested virus (MOI 3) and incubated as above for 46 hours followed by harvest.
  • MOI 3 harvested virus

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Abstract

L'invention concerne de nouveaux polypeptides solubles sécrétés récepteurs du facteur de nécrose tumorale (TNRF), des polynucléotides codant ces polypeptides, et les compositions et les procédés associés. Les polypeptides comprennent quatre séquences répétées riches en cystéine, qui sont homologues à d'autres récepteurs du facteur de nécrose tumorale, en particulier au récepteur du facteur de nécrose tumorale soluble sécrété, l'ostéoprotégérine Ces polypeptides peuvent être utilisés pour la détection de ligands, d'agonistes et d'antagonistes. Ils peuvent également être utilisés dans des procédés favorisant la maturation des cellules et la régulation des cellules osseuses.
PCT/US1998/015072 1997-07-21 1998-07-21 Recepteur ztnfr-5 du facteur de necrose tumorale WO1999004001A1 (fr)

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AU90139/98A AU9013998A (en) 1997-07-21 1998-07-21 Tumor necrosis factor receptor ztnfr-5

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WO1999026977A1 (fr) * 1997-11-24 1999-06-03 Biogen, Inc. Recepteurs 'opg-2'
WO1999031128A2 (fr) * 1997-12-16 1999-06-24 Incyte Pharmaceuticals, Inc. Proteines du type facteur humain de necrose tumorale r2
WO1999046376A1 (fr) * 1998-03-09 1999-09-16 Basf Aktiengesellschaft Recepteur de la superfamille des recepteurs du facteur de necrose tumorale issu du poumon humain
WO1999050413A2 (fr) * 1998-03-30 1999-10-07 Eli Lilly And Company APPLICATIONS THERAPEUTIQUES DE POLYPEPTIDES mFLINT MURS, OU DE OPG3 EN TANT QUE MEMBRE DE LA SUPERFAMILLE DU RECEPTEUR DU TNF
WO2000037094A2 (fr) * 1998-12-22 2000-06-29 Eli Lilly And Company Applications therapeutiques de polypeptides flint
EP1020521A1 (fr) * 1998-12-09 2000-07-19 Eli Lilly And Company Compositions qui contiennent des poteines FLINT ( Fas ligand inhibitor ) et leur utilisations
WO2000046247A1 (fr) * 1999-02-05 2000-08-10 Merck & Co., Inc. Procedes d'utilisation de molecules d'adn codant m68, proteine apparentee au recepteur de tnf
WO2000053755A2 (fr) * 1999-03-08 2000-09-14 Genentech, Inc. Compositions et procedes pour le traitement de tumeur
WO2000058465A2 (fr) * 1999-03-30 2000-10-05 Eli Lilly And Company Analogues polypeptidiques de flint
WO2000075316A1 (fr) * 1999-06-02 2000-12-14 Genentech, Inc. Procedes et compositions pour l'inhibition de la croissance de cellules neoplastiques
WO2001010908A1 (fr) * 1999-08-04 2001-02-15 Amgen Inc. Ntr3, membre de la famille de supergenes recepteurs du tnf
EP1159286A1 (fr) * 1999-03-04 2001-12-05 Human Genome Sciences, Inc. Recepteurs du facteur de necrose tumorale 6 alpha et 6 beta
US6521422B1 (en) 1999-08-04 2003-02-18 Amgen Inc. Fhm, a novel member of the TNF ligand supergene family
US6627199B1 (en) 1999-07-09 2003-09-30 Amgen Inc Isolation, identification and characterization of tmst2, a novel member of the TNF-receptor supergene family
US6764679B2 (en) 1997-09-18 2004-07-20 Genentech, Inc. Antibodies to DcR3 Polypeptide, a TNFR Homolog
US6835814B1 (en) 1999-03-30 2004-12-28 Eli Lilly And Company Protease resistant flint analogs
WO2005037865A2 (fr) * 2003-10-16 2005-04-28 Zymogenetics, Inc. Ztnfr14, recepteur du facteur de necrose tumorale
US6965012B1 (en) 1999-03-30 2005-11-15 Eli Lilly And Company Flint polypeptide analogs
EP1666052A1 (fr) 2000-02-16 2006-06-07 Genentech, Inc. Utilisation d'agonistes ou d'antagonistes pour moduler l'activite de molecules associees au tnf
US7112421B2 (en) 2000-09-18 2006-09-26 Biogen Idec Ma Inc. Nucleic acids encoding BAFF receptor, chimeric proteins and methods and compositions related thereto
US7186800B1 (en) 1997-01-14 2007-03-06 Human Genome Sciences, Inc. Tumor necrosis factor 6α and 6β
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US7285267B2 (en) 1997-01-14 2007-10-23 Human Genome Sciences, Inc. Tumor necrosis factor receptors 6α & 6β
JP2008500804A (ja) * 2003-10-16 2008-01-17 ザイモジェネティクス，インコーポレイティド ｚｔｎｆｒ１４、腫瘍壊死因子レセプター
EP2014303A2 (fr) 2000-07-27 2009-01-14 Genentech, Inc. Agoniste de récepteur APO-2L et synergie CPT-11
US7501497B2 (en) * 2001-05-24 2009-03-10 Zymogenetics, Inc. TACI-immunoglobulin fusion proteins
US7700317B2 (en) 2003-03-28 2010-04-20 Biogen Idec Ma Inc. Truncated baff receptors
US7722886B2 (en) 2003-05-20 2010-05-25 Wyeth Compositions and methods for treatment of severe acute respiratory syndrome (SARS)
US7772365B2 (en) 1999-01-07 2010-08-10 Zymogenetics, Inc. Soluble receptor BR43x2
EP2241328A1 (fr) 2000-05-12 2010-10-20 Immunex Corporation Inhibiteurs d'interleukine 1 dans le traitement de maladies
US7842292B2 (en) 2005-08-09 2010-11-30 Ares Trading S.A. Methods for treating B-cell malignancies using a TACI-Ig fusion molecule
US8722859B2 (en) 2000-04-11 2014-05-13 Genentech, Inc. Multivalent antibodies and uses therefor
US8784812B2 (en) 2006-05-15 2014-07-22 Zymogenetics, Inc. Methods for treating autoimmune diseases using a TACI-Ig fusion molecule
US8808696B2 (en) 2005-08-09 2014-08-19 Ares Trading S.A. Methods for the treatment and prevention of abnormal cell proliferation using TACI-fusion molecules
WO2014136117A1 (fr) 2013-03-06 2014-09-12 Protalix Ltd. Utilisation de cellules végétales exprimant un inhibiteur du polypeptide tnf alpha en thérapie
WO2014136113A1 (fr) 2013-03-06 2014-09-12 Protalix Ltd. Polypeptides chimériques, polynucléotides codant pour ceux-ci, cellules exprimant ceux-ci et leurs procédés de production
US9545086B2 (en) 1999-01-25 2017-01-17 Biogen Ma Inc. BAFF, inhibitors thereof and their use in the modulation of B-cell response and treatment of autoimmune disorders
US9726673B2 (en) 2005-11-23 2017-08-08 Genentech, Inc. Methods and compositions related to B cell assays

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WO1999031128A2 (fr) * 1997-12-16 1999-06-24 Incyte Pharmaceuticals, Inc. Proteines du type facteur humain de necrose tumorale r2
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WO1999050413A3 (fr) * 1998-03-30 1999-12-02 Lilly Co Eli APPLICATIONS THERAPEUTIQUES DE POLYPEPTIDES mFLINT MURS, OU DE OPG3 EN TANT QUE MEMBRE DE LA SUPERFAMILLE DU RECEPTEUR DU TNF
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US7772365B2 (en) 1999-01-07 2010-08-10 Zymogenetics, Inc. Soluble receptor BR43x2
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US7320796B2 (en) 1999-07-09 2008-01-22 Amgen Inc. Isolation, identification and characterization of tmst2, a novel member of the TNF-receptor supergene family
US6627199B1 (en) 1999-07-09 2003-09-30 Amgen Inc Isolation, identification and characterization of tmst2, a novel member of the TNF-receptor supergene family
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US7118863B2 (en) 1999-08-04 2006-10-10 Amgen, Inc. Methods for detecting NTR3 nucleic acids by hybridization
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