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WO1999011790A1 - Recepteur ztnfr-6 du facteur de necrose des tumeurs - Google Patents

Recepteur ztnfr-6 du facteur de necrose des tumeurs Download PDF

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Publication number
WO1999011790A1
WO1999011790A1 PCT/US1998/018364 US9818364W WO9911790A1 WO 1999011790 A1 WO1999011790 A1 WO 1999011790A1 US 9818364 W US9818364 W US 9818364W WO 9911790 A1 WO9911790 A1 WO 9911790A1
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WIPO (PCT)
Prior art keywords
amino acid
seq
acid residues
sequence
polypeptide
Prior art date
Application number
PCT/US1998/018364
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English (en)
Inventor
Theresa M. Farrah
Jane A. Gross
Shellie M. Matthews
Original Assignee
Zymogenetics, Inc.
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Application filed by Zymogenetics, Inc. filed Critical Zymogenetics, Inc.
Priority to AU91305/98A priority Critical patent/AU9130598A/en
Publication of WO1999011790A1 publication Critical patent/WO1999011790A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • 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
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2875—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF/TNF superfamily, e.g. CD70, CD95L, CD153, CD154
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/30—Immunoglobulins specific features characterized by aspects of specificity or valency
    • C07K2317/34—Identification of a linear epitope shorter than 20 amino acid residues or of a conformational epitope defined by amino acid residues
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

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 47 ⁇ :545-54, 1989), p55 TNFR-I (Loetscher et al . , Cell 1:351-59, 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.
  • NGFR, TNFR- I, CD30, CD40, 4 -IBB, DR3 and OX40 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 , DR5 , CARl and low affinity NGFR) contains a death domain associated with apoptosis .
  • TNFR 1 Fas, DR3 , DR4 , DR5 , CARl and low affinity NGFR
  • TRID or DcRl Pan et al., Science 277 :815-8, 1997; Sheridan et al . , ibid.
  • 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.
  • the average homology is in the range of 25% to 30%.
  • cytoplasmic domain 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.
  • TNF receptor 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 X-ray crystallographic structures have been resolved for human TNF (Jones et al . , Nature 338 : 225-28 , 1989), LT- ⁇ (Eck et al . , J. Biol . Chem. 267:2119-122, 1992) and the LT- ⁇ /TNFR complex (Banner et al . , Cell 71:431-45, 1993) .
  • This complex features three receptor molecules bound symmetrically to one LT- ⁇ trimer.
  • a model of trimeric ligand binding through receptor oligomerization has been proposed to initiate signal transduction pathways.
  • the identification of biological activity of several TNF members has been facilitated through use of monoclonal antibodies specific for the corresponding receptor.
  • 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.
  • TNFR 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.
  • 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 present invention provides a novel human tumor necrosis factor receptor polypeptide and related compositions and methods.
  • the invention provides an isolated polynucleotide encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 66 to amino acid residue 145 of SEQ ID NOs:4 or 6; and (b) sequences of amino acid residues that are at least 80% identical to (a) ; the polypeptide having cysteine residues corresponding to amino acid residues 67, 82, 85, 99, 107, 109, 120, 126 and 144 of SEQ ID N0s:4 or 6.
  • polypeptide further comprises an extracellular region having cysteine residues corresponding to amino acid residues 43, 46, 56, 147, 162, 168 and 187 of SEQ ID NOs : 4 or 6.
  • extracellular region comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 37 to amino acid residue 188 of SEQ ID NOs : 4 or 6; (b) a sequence of amino acid residues from amino acid residue 18 to amino acid residue 188 of SEQ ID NOs: 4 or 6;
  • polypeptide further comprises a transmembrane domain.
  • the transmembrane domain comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 326 to amino acid residue 346 of SEQ ID NO : 4 and (b) sequences of amino acid residues that are at least 80% identical to (a) .
  • the polypeptide further comprises a death domain.
  • the death domain comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 390 to amino acid residue 471 of SEQ ID NO : 4 ; and (b) sequences of amino acid residues that are at least 80% identical to (a) .
  • polypeptide comprises amino acid residues 18 to 229 of SEQ ID NO : 6.
  • polypeptide comprises amino acid residues 18 to 631 of SEQ ID NO : 4.
  • polynucleotide is selected from the group consisting of : a) a sequence of nucleotides from nucleotide 284 to nucleotide 2225 of SEQ ID NO : 3 ; b) a sequence of nucleotides from nucleotide 284 to nucleotide 970 of SEQ ID NO : 5 ; c) a sequence of nucleotides from nucleotide 1 to nucleotide 1893 of SEQ ID NO: 18; and d) a sequence of nucleotides from nucleotide 1 to nucleotide 687 of SEQ ID NO: 19.
  • polypeptide further comprises an affinity tag.
  • an oligonucleotide probe or primer comprising at least 14 contiguous nucleotides of a polynucleotide selected from the group consisting of: a) the nucleotide sequence of SEQ ID NO: 18; b) the nucleotide sequence of SEQ ID NO: 19; or c) nucleotide sequences complementary to a) or b) .
  • an isolated polynucleotide encoding a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond, the first portion consisting essentially of a polypeptide selected from the group consisting of: a) a polypeptide comprising amino acid residue 18 to amino acid residue 188 of SEQ ID NOs:4 or 6 and having cysteine residues corresponding to amino acid residues 43, 46, 56, 64, 67, 82, 85, 89, 99, 107, 109, 120, 126, 147, 162, 168 and 187 of SEQ ID NOs:4 or 6; and b) sequences of amino acid residues that are at least 80% identical to a) ; and the second portion consisting essentially of another protein.
  • the second portion is an affinity tag.
  • the affinity tag is an immunoglobulin heavy chain constant region.
  • the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from residue 66 to residue 145 of SEQ ID NOs: 4 or 6; and (b) sequences of amino acid residues that are at least 80% identical to (a) ; the polypeptide having cysteine residues corresponding to amino acid residues 67, 82, 85, 99, 107, 109, 120, 126 and 144 of SEQ ID NOs : 4 or 6; and a transcription terminator.
  • a transcription promoter a DNA segment encoding a polypeptide comprising a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from residue 66 to residue 145 of SEQ ID NOs: 4 or 6; and (b) sequences of amino acid residues that are at least 80% identical to (a)
  • the DNA encodes a polypeptide further comprising an extracellular region having cysteine residues corresponding to amino acid residues 43, 46, 56, 147, 162, 168 and 187 of SEQ ID NOs : 4 or 6.
  • the extracellular region comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from residue 37 to residue 188 of SEQ ID NOs : 4 or 6; (b) a sequence of amino acid residues from residue 18 to residue 188 of SEQ ID NOs: 4 or 6; (c) a sequence of amino acid residues from residue 18 to residue 325 of SEQ ID NO: 4; (d) a sequence of amino acid residues from residue 18 to residue 229 of SEQ ID NO: 4; and (e) sequences of amino acid residues that are at least 80% identical to (a) , (b) , (c) or (d) .
  • the DNA encodes a polypeptide further comprising a transmembran
  • the DNA encodes a polypeptide further comprising a death domain.
  • the death domain comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 390 to amino acid residue 471 of SEQ ID NO : 4 ; and (b) sequences of amino acid residues that are at least 80% identical to (a) .
  • the invention provides a cultured eukaryotic cell into which has been introduced an expression vector as described above, wherein the cell expresses a tumor necrosis factor receptor polypeptide encoded by the DNA segment.
  • the invention provides an expression vector comprising the following operably linked elements: a transcription promoter; a DNA segment encoding a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond, the first portion consisting essentially of a polypeptide selected from the group consisting of: a) a polypeptide comprising amino acid residue 18 to amino acid residue 188 of SEQ ID NOs: 4 or 6 and having cysteine residues corresponding to amino acid residues 43, 46, 56, 64, 67, 82, 85, 89, 99, 107, 109, 120, 126, 147, 162, 168 and 187 of SEQ ID NOs: 4 or 6; and b) sequences of amino acid residues that are at least 80% identical to a) ; and the second portion consisting essentially of another protein; and a transcription terminator.
  • the invention also provides a cultured eukaryotic cell into which has been introduced an expression vector as described above, wherein said cell expresses the chimeric polypeptide encoded by the DNA segment.
  • an isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of : (a) a sequence of amino acid residues from amino acid residue 66 to amino acid residue 145 of SEQ ID N0s:4 or 6 ; and (b) sequences of amino acid residues that are at least 80% identical to (a) ; the polypeptide having cysteine residues corresponding to amino acid residues 67, 82, 85, 99, 107, 109, 120, 126 and 144 of SEQ ID NOs : 4 or 6.
  • the polypeptide further comprises an extracellular region having cysteine residues corresponding to amino acid residues 43, 46, 56, 147, 162, 168 and 187 of SEQ ID NOs: 4 or 6.
  • the extracellular region comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 37 to amino acid residue 188 of SEQ ID NOs : 4 or 6; (b) a sequence of amino acid residues from amino acid residue 18 to amino acid residue 188 of SEQ ID NOs : 4 or 6 ; (c) a sequence of amino acid residues from amino acid residue 18 to amino acid residue 325 of SEQ ID NO : 4 ; (d) a sequence of amino acid residues from amino acid residue 18 to amino acid residue 229 of SEQ ID NO: 6; and (e) sequences of amino acid residues that are at least 80% identical to (a) , (b) , (c) or (d) .
  • the polypeptide further comprises a transmembrane domain.
  • the transmembrane domain comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 326 to amino acid residue 346 of SEQ ID NO : 4 and (b) sequences of amino acid residues that are at least 80% identical to (a) .
  • the polypeptide further comprises a death domain.
  • the death domain comprises a sequence of amino acid residues selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 390 to amino acid residue 471 of SEQ ID NO : 4 ; and (b) sequences of amino acid residues that are at least 80% identical to (a) .
  • the invention provides an isolated polypeptide comprising a sequence of amino acid residues selected from the group consisting of : a) a sequence of amino acid residues from amino acid residue 18 to amino acid residue 188 of SEQ ID NO : 4 ; and b) a sequence of amino acid residues from amino acid 18 to amino acid 229 of SEQ ID NO: 6; wherein the polypeptide is substantially free of transmembrane and intracellular domains ordinarily associated with cell surface receptors.
  • the polypeptide further comprises affinity tag.
  • a chimeric polypeptide consisting essentially of a first portion and a second portion joined by a peptide bond
  • the first portion consisting essentially of a polypeptide selected from the group consisting of: a) a polypeptide comprising amino acid residue 18 to amino acid residue 188 of SEQ ID NOs : 4 or 6 and having cysteine residues corresponding to amino acid residues 43, 46, 56, 64, 67, 82, 85, 89, 99, 107, 109, 120, 126, 147, 162, 168 and 187 of SEQ ID NOs : 4 or 6; and b) sequences of amino acid residues that are at least 80% identical to a) ; and the second portion consisting essentially of another protein.
  • the second portion is an affinity tag.
  • the affinity tag is an immunoglobulin heavy chain constant region.
  • an antibody that specifically binds to a polypeptide as described above .
  • composition comprising a polypeptide as described above in combination with a pharmaceutically acceptable vehicle.
  • a method for detecting a genetic abnormality in a patient comprising: obtaining a genetic sample from a patient; incubating the genetic sample with a polynucleotide comprising at least 14 contiguous nucleotides of SEQ ID NOs: 3 or 5 or the complement of SEQ ID NOs : 3 or 5, under conditions wherein the polynucleotide will hybridize to complementary polynucleotide sequence, to produce a first reaction product; comparing the first reaction product to a control reaction product, wherein a difference between the first reaction product and the control reaction product is indicative of a genetic abnormality in the patient.
  • Figure 2 shows a comparison of the death domains of ZTNFR-6 (SEQ ID NO : 4 ) and murine FADD (SEQ ID NO: 16) .
  • 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 (Nilsson et al . , EMBO J . 4:1075, 1985; Nilsson et al . , Methods Enzvmol . 198 : 3 , 1991), glutathione S transferase
  • 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.
  • 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 ' .
  • Conticf Denotes a polynucleotide, a segment of which is equivalent in nucleotide sequence to a segment of another polynucleotide sequence.
  • a "contig assembly” denotes a collection of EST contigs that define a larger polynucleotide segment containing an open reading frame encoding a full-length or partial polypeptide.
  • Degenerate As applied to a nucleotide sequence such as a probe or primer, 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 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. In a preferred form, the isolated polypeptide is substantially free of other polypeptides, particularly other polypeptides of animal origin.
  • polypeptides in a highly purified form, i.e. greater than 95% pure, more preferably greater than 99% pure.
  • 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
  • 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 DNA sequences that provide for the binding of RNA polymerase and initiation of transcription. Promoter i 6 y
  • sequences are commonly, but not always, found in the 5' 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- 6 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.
  • Splice variant As used herein to denote alternative forms of RNA transcribed from a gene. Splice variation arises naturally through use of alternative splicing sites within a transcribed RNA molecule, or less commonly between separately transcribed RNA molecules, and may result in several mRNAs transcribed from the same gene.
  • Splice variants may encode polypeptides having altered amino acid sequence.
  • the term splice variant is also used herein to denote a protein encoded by a splice variant of an mRNA transcribed from a gene.
  • the present invention is based in part upon the discovery of a novel 2686 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-6.
  • a correctly spliced polypetide sequence is disclosed in SEQ ID NO : 4 and the corresponding polynucleotide sequence in SEQ ID NO : 3.
  • the 153 amino acid residue correctly spliced sequence of SEQ ID NO : 4 begins at amino acid residue 479 of SEQ ID NO : 4 and replaces amino acid residues 479 to 508 of SEQ ID NO : 2.
  • the newly identified segment of cytoplasmic sequence does not alter the extracellular ligand binding domain, transmembrane domain or death domain which remain as originally disclosed in SEQ ID NOs : 1 and 2.
  • Novel ZTNFR-6 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 assembly of 5 ESTs was constructed.
  • a 2646 bp ZTNFR-6 receptor (SEQ ID NO: 5) was identified which, like osteoprotegerin (OPG), was soluble.
  • OPG osteoprotegerin
  • the insertion of a 40 bp exon (SEQ ID NO: 7) is needed to put the sequence in frame for the transmembrane and cytoplasmic domains, resulting in a 3440 bp membrane-bound form of ZTNFR-6 as disclosed in SEQ ID NO : 3 (exon sequence inserted between nucleotides 959-999 of SEQ ID NO : 3 ) .
  • the 5' and 3' ends of the 40 bp insert implicitly define two splice junctions.
  • Sequence analysis of a deduced amino acid sequence of the soluble form as represented in SEQ ID NO: 6 and the membrane-bound form as represented in SEQ ID NO: 4 indicates the presence of 17 amino acid residues of a signal peptide (residues 1-17 of SEQ ID NOs : 4 and 6 nucleotides 284-334 of SEQ ID NOs : 3 and 5), four extracellular, cysteine-rich repeats (residues 37-65, 66- 107, 108-145 and 146-188 of SEQ ID NOs : 4 and 6, nucleotides 392-478, 479-604, 605-718 and 719-789 of SEQ ID NOs : 3 and 5) .
  • the soluble form ends with 41 amino acid residues (189-229 of SEQ ID NO : 6 , nucleotides 848-970 of SEQ ID NO: 5) of the linker sequence resulting in a mature soluble protein comprising amino acid residues 18-229 of SEQ ID NO:6, nucleotides 285-970 of SEQ ID NO:5).
  • the membrane- bound form further consists of a linker region (residues 189-325 of SEQ ID NO : 4 , nucleotides 790-1306 of SEQ ID NO:3), a transmembrane domain (residues 326-346 of SEQ ID NO:4, nucleotides 791-1369 of SEQ ID NO : 3 ) and a cytoplasmic domain (residues 347-631 of SEQ ID NO : 4 , nucleotides 1370-2225 of SEQ ID NO:3) containing a death domain (residues 390-471 of SEQ ID NO : 4 , nucleotides 1499- 1696 of SEQ ID NO : 3 ) .
  • the mature membrane-bound ZTNFR-6 protein comprises amino acid residue 18-631 of SEQ ID NO: 4, nucleotides 285-2225 of SEQ ID NO : 3.
  • SEQ ID NO: 4 nucleotides 285-2225 of SEQ ID NO : 3.
  • these domain boundaries are approximate, and are based on alignments with known proteins and predictions of protein folding.
  • 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,
  • Sequence motifs for the four pseudo-repeat sequence motifs are shown below. Within in each 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. Alternative sequences are indicated with asterisks.
  • Pseudo repeat #1 motif (SEQ ID NO: 8) X-C-X ⁇ l0-14 ⁇ -C-C-X-X-C-X ⁇ 5-9 ⁇ -C-X ⁇ 6-8 ⁇ -C-X
  • Pseudo-repeat #1 of ZTNFR-6 (amino acid residues 37 to 65 of SEQ ID NOs : 4 and 6) is missing the first three cysteines of this motif.
  • TNF receptors such as OX-40, osteoprotegerin and CD-27
  • Pseudo-repeat #3 of ZTNFR-6 amino acid residues 108 to 145 SEQ ID NOs: 4 and 6) most closely resembles that in osteoprotegerin and is best described by the alternative motif (SEQ ID NO:12) C-X ⁇ 10 ⁇ -C-X ⁇ 5 ⁇ -C-X ⁇ 17 ⁇ -C .
  • Pseudo-repeat #4 of ZTNFR-6 (amino acid residues 146-188 of SEQ ID NOs : 4 and 6) is encompassed within the alternative motif #4 sequence (SEQ ID NO: 14) .
  • the region corresponding to pseudo-repeat #1 has conserved cysteine residues at amino acid residues 43, 46, 56 and 64 of SEQ ID NOs : 4 and 6.
  • the region corresponding to pseudo-repeat #2 has conserved cysteine residues at amino acid residues 67, 82, 85, 89, 99 and 107 of SEQ ID NOs : 4 and 6.
  • the region corresponding to pseudo-repeat #3 has conserved cysteine residues at amino acid residues 109, 120, 126 and 144 of SEQ ID NOs : 4 and 6.
  • the region corresponding to pseudo-repeat #4 has conserved cysteine residues at amino acid residues 147, 162, 168 and 187 of SEQ ID NOs: 4 and 6.
  • cysteine-rich pseudo-repeats #2 and #3 are most likely essential for binding. Crystal structure of the LT- ⁇ receptor- ligand complex (Banner et al . , ibid.) show that only repeats #2 and #3 had significant contact with the ligand. Based on the homology of ZTNFR-6 to other members of the TNFR family, amino acid residues involved in ligand binding are likely to be found within residues 66- 145 of SEQ ID NOs : 4 and 6) . The four cysteine-rich domains (pseudo-repeats) of the extracellular ligand binding region of ZTNFR-6 are similar to several other members of the TNF receptor family.
  • ZTNFR-6 is most homologous to OPG, sharing a 35% amino acid identity within the four cysteine-rich repeats. Although the percent homology is only slightly above that typical within the TNF family, the general structure of the extracellular domains of ZTNFR-6 and OPG, in particular truncation of the first two cysteines in cysteine-rich pseudo-repeat #1, is the same between the two proteins and the divergence of pseudo-repeat #3 ( Figure 1) . Both ZTNFR- 6 and OPG exist in soluble forms. OPG is a soluble molecule which appears to bind an as yet unidentified ligand reducing the ligand concentration in circulation and preventing it from binding and activating the ligand' s true membrane-bound receptor.
  • This "true membrane-bound receptor” has not yet been identified and may be an alternatively spliced version of OPG or a different receptor altogether.
  • Two ZTNFR-6 receptors are disclosed herein.
  • the ligand for ZTNFR-6 is most likely a maturation factor, it may be like the proposed OPG ligand and act as an osteoclast maturation factor, however it is more likely to be homologous to TNF or NGF, or perhaps have a completely different structure from known TNF ligands.
  • the cytoplasmic death domain of ZTNFR-6 most resembles that of murine FADD death domain (Hsu et al . ,
  • ZTNFR-6 shares 28% homology with the murine FADD death domain ( Figure 2) .
  • Northern blot analysis of various human tissues was performed using a 404 bp DNA probe (SEQ ID NO: 17).
  • a 4.4 kb transcript was detected corresponding to ZTNFR-6.
  • a high level of transcription was detected in heart, brain, placenta, kidney, pancreas, colon, spinal cord and trachea.
  • a lower level of transcription was detected in most other tissues.
  • a second 3.0 kb transcript was detected in testis.
  • the transcript distribution represents a mixture of the soluble and membrane-bound forms of ZTNFR-6.
  • TNF receptor family Several members, TNRFR-I, TNRFR-II, TNRFR-III and DR4 , are found in most human tissues. OPG was detected in lung, heart, kidney, placenta, and to a lesser degree, in hematopoietic and immune organs (Simmonet et al., ibid. ) . Four of the ESTs which made up the ZTNFR-6 contig assembly were from tumor libraries. Expression in tumor cells is consistent with other members of the TNFR family that are associated with growth regulation, differentiation and tumorigenesis .
  • Chromosomal localization of ZTNFR-6 to 6p21.1 was determined using radiation hybrid chimeras.
  • the present invention further provides polynucleotide molecules, including DNA and RNA molecules, encoding ZTNFR-6 proteins.
  • the polynucleotides of the present invention include the sense strand; the anti-sense strand; and the DNA as double-stranded, having both the sense and anti-sense strand annealed together by their respective hydrogen bonds.
  • Representative DNA sequences encoding ZTNFR-6 proteins are set forth in SEQ ID NOs : 3 and 5.
  • DNA sequences encoding other ZTNFR-6 proteins can be readily generated by those of ordinary skill in the art based on the genetic code.
  • Counterpart RNA sequences can be generated by substitution of U (uracil) for T (thymine) .
  • SEQ ID NO: 18 is a degenerate DNA sequence that encompasses all DNAs that encode the ZTNFR-6 polypeptide of SEQ ID NO:4.
  • SEQ ID NO: 19 is a degenerate DNA sequence that encompasses all DNAs that encode the ZTNFR-6 polypeptide of SEQ ID NO : 6.
  • the degenerate sequences of SEQ ID NOs: 18 and 19 also provide all RNA sequences encoding SEQ ID NOs: 4 and 6 by substituting U for T.
  • the ZTNFR-6 polypeptide-encoding polynucleotides comprising nucleotide 1 to nucleotide 3440 of SEQ ID NO : 4 , nucleotide 1 to 2646 of SEQ ID NO: 6, and their RNA equivalents are contemplated by the present invention.
  • Table 1 sets forth the one- letter codes used within SEQ ID NOs: 18 and 19 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
  • its complement R denotes A (adenine) or G (guanine) , A being complementary to T, and G being complementary to C.
  • 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) .
  • 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 sequences of SEQ ID NOs:4 and 6. Variant sequences can be readily tested for functionality as described herein.
  • 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) .
  • the 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.
  • 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. Therefore, the degenerate codon sequences disclosed in SEQ ID NOs: 18 and 19 serve 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.
  • isolated polynucleotides will hybridize to similar sized regions of SEQ ID NOs : 3 and 5, or to sequences 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. Methods for isolating DNA and RNA are well known in the art. It is generally preferred to isolate RNA from human heart tissue, although 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 18:52-94, 1979).
  • Poly (A) + RNA is prepared from total RNA using the method of Aviv and Leder (Proc .
  • cDNA is prepared from poly (A) + RNA using known methods.
  • Polynucleotides encoding ZTNFR-6 polypeptides are then identified and isolated by, for example, hybridization or PCR.
  • sequences disclosed in SEQ ID NOs : 3 and 5 represent single alleles of the human gene and that allelic variation and alternative splicing is expected to occur.
  • Allelic variants of the DNA sequences shown in SEQ ID NOs : 3 and 5, 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 NOs : 4 and 6.
  • cDNAs generated from alternatively spliced mRNAs, which retain the properties of the ZTNFR-6 polypeptide are included within the scope of the present invention, as are polypeptides encoded by such cDNAs and mRNAs.
  • the 40 bp exon aligns the soluble sequence in frame to encode a transmembrane and cytoplasmic domain.
  • the 5' and 3' ends of the 40 bp insert (SEQ ID NO: 7) implicitly define two splice junctions. 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.
  • species would include, but are not limited to, mammalian, avian, amphibian, reptile, fish, insect and other vertebrate and invertebrate species.
  • ZTNFR-6 receptors from other mammalian species, including murine, porcine, ovine, bovine, canine, feline, equine, and other primate receptors. Using the human ZTNFR-6 sequences disclosed herein, the partial sequence of a murine ortholog was discovered.
  • the resulting 764 bp sequence extends the EST by 394 bp and encodes a C- terminal portion of the death domain and nearly all of the cytoplasmic C-terminal tail of a mouse ZTNFR-6 ortholog (SEQ ID NO: 20) .
  • the mouse sequence corresponds to amino acid residues 374 to 626 of SEQ ID NO : 4. Alignment with the human ZTNFR-6 amino acid sequence (SEQ ID NO : 4 ) shows 93% identity at the amino acid level.
  • Species orthologs of the human ZTNFR-6 receptor can be cloned using information and compositions provided by the present invention in combination with conventional cloning techniques.
  • 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.
  • Orthologs can also be identified by searching various electronic database collections of ESTs from other organisms.
  • the present invention also provides isolated receptor polypeptides that are substantially homologous to the receptor polypeptides of SEQ ID NOs : 4 and 6 and their species orthologs.
  • isolated is meant a protein or polypeptide that is found in a condition other than its native environment, such as apart from blood and animal tissue.
  • the isolated protein or polypeptide is substantially free of other proteins or polypeptides, particularly other proteins or polypeptides of animal origin. 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 80% sequence identity to the sequences shown in SEQ ID NOs : 4 and 6, or their species orthologs. Such proteins or polypeptides will more preferably be at least 90% identical, and most preferably 95% or more identical to SEQ ID NOs : 4 and 6 or their species 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 a small extension that facilitates purification, an affinity tag.
  • Polypeptides comprising affinity tags can further comprise a proteolytic cleavage site between the ZTNFR-6 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, trans-4-hydroxyproline, N-methyl -glycine , allo-threonine, methylthreonine, hydroxyethyl-cysteine, hydroxyethylhomocysteine, nitroglutamine, homo-glutamine, pipecolic acid, thiazolidine carboxylic acid, dehydroproline, 3- and 4-methylproline, 3 , 3 -dimethyl - proline, tert- leucine, norvaline, 2-azaphenylalanine, 3- azaphenylalanine, 4-azaphenylalanine, and 4-fluoro- phenylalanine .
  • 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. 33 : 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. 1:395-403, 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-6 amino acid residues.
  • Essential amino acids in the polypeptides of the present invention can be identified according to procedures known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science
  • Sites of ligand-receptor interaction can also be determined by physical analysis of structure, as determined by such techniques as nuclear magnetic resonance, crystallography, electron diffraction or photoaffinity labeling, in conjunction with mutation of putative contact site amino acids. See, for example, de Vos et al., Science 255:306-12, 1992; Smith et al . , J. Mol .
  • variants of the disclosed ZTNFR-6 DNA and polypeptide sequences can be generated through DNA shuffling as disclosed by Stemmer, Nature 370 :389-91, 1994, Stemmer, Proc. Natl. Acad. Sci. USA £:10747-51, 1994 and WIPO Publication WO 97/20078. Briefly, variant DNAs are generated by in vi tro homologous recombination by random fragmentation of a parent DNA followed by reassembly using PCR, resulting in randomly introduced point mutations. This technique can be modified by using a family of parent DNAs, such as allelic variants or DNAs from different species, to introduce additional variability into the process.
  • 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) can be recovered from the host cells and rapidly sequenced using modern equipment. 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 18-188 of SEQ ID NOs: 4 and 6 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-6 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. Selection of promoters, terminators, selectable markers, vectors and other elements is a matter of routine design within the level of ordinary skill in the art. Many such elements are described in the literature and are available through commercial suppliers.
  • 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-6 polypeptide, or may be derived from another secreted protein (e.g., t-PA) or synthesized de novo .
  • the secretory signal sequence is joined to the ZTNFR-6 DNA sequence in the correct reading frame.
  • 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.
  • the secretory signal sequence contained in the polypeptides of the present invention is used to direct other polypeptides into the secretory pathway.
  • the present invention provides for such fusion polypeptides.
  • a signal fusion polypeptide can be made wherein a secretory signal sequence derived from amino acid residues 1-17 of SEQ ID NO : 4 or 6 is be operably linked to another polypeptide using methods known in the art and disclosed herein.
  • the secretory signal sequence contained in the fusion polypeptides of the present invention is preferably fused amino-terminally to an additional peptide to direct the additional peptide into the secretory pathway.
  • these novel secretory signal sequence fusion constructs can direct the secretion of an active component of a normally non-secreted protein, such as another receptor.
  • Such fusions may be used in vivo ox in vi tro to direct peptides through the secretory pathway.
  • Cultured mammalian cells are suitable hosts within the present invention.
  • Methods for introducing exogenous DNA into mammalian host cells include calcium phosphate-mediated transfection (Wigler et al . , Cell 14:725, 1978; Corsaro and Pearson, Somatic Cell Genetics 2:603, 1981; Graham and Van der Eb, Virology £2:456, 1973), electroporation (Neumann et al . , EMBO J . 1:841-45, 1982), DEAE-dextran mediated transfection (Ausubel et al . , ibid . ) , and liposome-mediated transfection (Hawley-Nelson et al .
  • 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. CRL 1573; Graham et al . , J. Gen. Virol. 3_£:59-72, 1977) and Chinese hamster ovary (e.g., CHO-K1; ATCC No. CCL 61) cell lines. Additional suitable cell lines are known in the art and available from public depositories such as the American Type Culture Collection, Rockville, Maryland. In general, 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) H:47-58, 1987. Transformation of insect cells and production of foreign polypeptides therein is disclosed by Guarino et al .
  • Insect cells can be infected with recombinant baculovirus, commonly derived from Autographa calif ornica nuclear polyhedrosis virus (AcNPV) .
  • DNA encoding the ZTNFR-6 polypeptide is inserted into the baculoviral genome in place of the AcNPV polyhedrin gene coding sequence by one of two methods. The first is the traditional method of homologous DNA recombination between wild-type AcNPV and a transfer vector containing the ZTNFR-6 flanked by AcNPV sequences.
  • Suitable insect cells e.g.
  • SF9 cells are infected with wild-type AcNPV and transfected with a transfer vector comprising a ZTNFR-6 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • a transfer vector comprising a ZTNFR-6 polynucleotide operably linked to an AcNPV polyhedrin gene promoter, terminator, and flanking sequences.
  • the second method of making recombinant baculovirus utilizes a transposon-based system described by Luckow (Luckow et al . , J. Virol. £7:4566-79, 1993). This system is sold in the Bac-to-Bac kit (Life Technologies, Rockville, MD) . This system utilizes a transfer vector, pFastBaclTM (Life Technologies) containing a Tn7 transposon to move the DNA encoding the ZTNFR-6 polypeptide into a baculovirus genome maintained in E.
  • the pFastBaclTM transfer vector utilizes the AcNPV polyhedrin promoter to drive the expression of the gene of interest, in this case ZTNFR-6.
  • pFastBaclTM can be modified to a considerable degree .
  • the polyhedrin promoter can be removed and substituted with the baculovirus basic protein promoter (also known as Pcor, p6.9 or MP promoter) which is expressed earlier in the baculovirus infection, and has been shown to be advantageous for expressing secreted proteins. See, Hill-Perkins and Possee, J. Gen. Virol. 21:971-6, 1990; Bonning et al . , J. Gen. Virol.
  • transfer vector constructs a short or long version of the basic protein promoter can be used.
  • transfer vectors can be constructed which replace the native ZTNFR-6 secretory signal sequences with secretory signal sequences derived from insect proteins.
  • a secretory signal sequence from Ecdysteroid Glucosyltransferase (EGT) , honey bee Melittin (Invitrogen, Carlsbad, CA) , or baculovirus gp67 (PharMingen, San Diego, CA) can be used in constructs to replace the native secretory signal sequence.
  • transfer vectors can include an in- frame fusion with DNA encoding an epitope tag at the C- or N-terminus of the expressed ZTNFR-6 polypeptide, for example, a Glu-Glu epitope tag
  • ZTNFR-6 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. Sf9 cells.
  • Recombinant virus that expresses ZTNFR-6 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
  • 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 (Expression Systems) for the Sf9 cells; and Ex-cellO405TM (JRH Biosciences, Lenexa, KS) or Express FiveOTM (Life Technologies) for the T. ni cells.
  • the cells are grown up from an inoculation density of approximately 2-5 x 10 s cells to a density of 1-2 x 10 6 cells at which time a recombinant viral stock is added at a multiplicity of infection (MOD of 0.1 to 10, more typically near 3.
  • MOD multiplicity of infection
  • the recombinant virus-infected cells typically produce the recombinant ZTNFR-6 polypeptide at 12-72 hours post- infection and secrete it with varying efficiency into the medium.
  • the culture is usually harvested 48 hours post- infection. Centrifugation is used to separate the cells from the medium (supernatant) .
  • the supernatant containing the ZTNFR-6 polypeptide is filtered through micropore filters, usually 0.45 ⁇ m pore size. Procedures used are generally described in available laboratory manuals (King and Possee, ibid. ; O'Reilly et al . , ibid. ; Richardson, C. D., ibid. ) .
  • 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 .
  • Methods for transforming S . cerevisiae cells with exogenous DNA and producing recombinant polypeptides therefrom are disclosed by, for example, Kawasaki, U.S. Patent No. 4,599,311; Kawasaki et al . , U.S. Patent No. 4,931,373; Brake, U.S. Patent No. 4,870,008; Welch et al .
  • 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.
  • Kluyveromyces fragilis , Ustilago maydis , Pi chia pastoris , 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 Sumino et al . , U.S. Patent No.
  • Pichia methanolica as host for the production of recombinant proteins is disclosed in WIPO Publications WO 97/17450, WO 97/17451, WO 98/02536, and WO 98/02565.
  • 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) .
  • DHAS dihydroxyacetone synthase
  • FMD formate dehydrogenase
  • CAT catalase
  • a preferred selectable marker for use in 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 .
  • host cells For large-scale, industrial processes where it is desirable to minimize the use of methanol, it is preferred to use host cells in which both methanol utilization genes (AUG1 and AUG2) are deleted. • For production of secreted proteins, 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 , 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-6 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
  • 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 soluble ZTNFR-6 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, Montgomeryville, PA), Octyl-Sepharose (Pharmacia) 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.
  • 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 or proteins having histidine 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.
  • Other methods of purification include purification of glycosylated proteins by lectin affinity chromatography and ion exchange chromatography (Methods in Enzymol . , Vol. 182, "Guide to Protein Purification", M. Deutscher, (ed.), Acad. Press, San Diego, 1990, pp.529-39) .
  • a fusion of the polypeptide of interest and an affinity tag may be constructed to facilitate purification.
  • an affinity tag e.g., Glu-Glu, FLAG, maltose-binding protein, an immunoglobulin domain
  • One such method for purifying a ZTNFR-6 polypeptide having an affinity tag comprises adsorbing ZTNFR-6 affinity-tagged proteins to a matrix, Sepharose coupled to an anti-affinity tag antibody, and eluting by competitive elution using a competing peptide such as the FLAG peptide (Eastman Kodak, Rochester, NY) , N- AspTyrLysAspAspAspLys-C (SEQ ID NO: 22) or a Glu-Glu peptide (AnaSpec, San Jose, CA) , Glu-Tyr-Met-Pro-Val-Asp,
  • 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-6 polypeptides or fragments thereof may also be prepared through chemical synthesis.
  • ZTNFR-6 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-6 receptors, used to form fusion or chimeric proteins with human Ig, as His-tagged proteins, or FLAGTM-tagged proteins.
  • a soluble ZTNFR-6 protein is represented in SEQ ID NO : 6.
  • Soluble ZTNFR-6 or ZTNFR-6-Ig chimeric proteins are used, for example, to identify the ZTNFR-6 ligands, including the natural ligand, as well as agonists and antagonists of the natural ligand.
  • cells expressing the ligand are identified by fluorescence immunocytometry or immunohistochemistry .
  • the soluble fusion proteins or soluble Ig fusion protein is useful in studying the distribution of the ligand on tissues or specific cell lineages, and to provide insight into receptor/ligand biology.
  • 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 (Hopp et al., Biotechnology £: 1204-10, 1988; available from Eastman Kodak Co., New Haven, CT) or another polypeptide or protein for which an antibody or other specific binding agent is available, can be fused to the receptor polypeptide .
  • a soluble ZTNFR-6 receptor extracellular ligand-binding region can be expressed as a chimera 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-6-Ig fusion protein chimera
  • 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 .
  • the invention provides a method for detecting a ligand within a test sample, comprising contacting a test sample with a polypeptide comprising a segment selected from the group consisting of: (a) a sequence of amino acid residues from amino acid residue 18 to amino acid residue 188 of SEQ ID NOs : 4 or 6 having cysteine residues corresponding to amino acid residues 43, 46, 56, 64, 67, 82, 85, 89, 99, 107, 109, 120, 126, 147,
  • the polypeptide is membrane bound within a cultured cell, and the detecting step comprises measuring a biological response in the cultured cell.
  • the biological response is cell proliferation or activation of transcription of a reporter gene.
  • the tumor necrosis factor receptor polypeptide is immobilized on a solid support.
  • 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-6 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. 65 : 55-63, 1983).
  • 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.
  • 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 ££: 563-72, 1989) .
  • a preferred such reporter gene is a luciferase gene (de Wet et al . , Mol . Cell. Biol. 2:725, 1987) . Expression of the luciferase gene is detected by luminescence using methods known in the art (e.g., Baumgartner et al . , J. Biol. Chem.
  • 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 .
  • 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 .
  • 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.
  • Methods for linking polypeptides to solid supports 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 soluble ZTNFR-6 is useful in studying the distribution of ligands on tissues or specific cell lineages, and to provide insight into receptor/ligand biology. Using labeled soluble ZTNFR-6, cells expressing the ligand are identified by fluorescence immunocytometry or immunohistochemistry. Application may also be made of the specificity of TNF receptors for their ligands. Soluble receptors or antibodies to the receptor may be directly or indirectly conjugated to drugs, toxins, radionuclides and the like, and these conjugates used for in vivo diagnostic or therapeutic applications. For instance, polypeptides or antibodies of the present invention may used to identify or treat tissues or organs that express a corresponding anti- complementary molecule (receptor or antigen, respectively, for instance) .
  • ZTNFR-6 polypeptides or anti-ZTNR-6 antibodies, or bioactive fragments or portions thereof can be coupled to detectable or cytotoxic molecules and delivered to a mammal having cells, tissues or organs that express the anti -complementary molecule.
  • Suitable detectable molecules may be directly or indirectly attached to the polypeptide or antibody, and include radionuclides, enzymes, substrates, cofactors, inhibitors, fluorescent markers, chemiluminescent markers, magnetic particles and the like.
  • Suitable cytotoxic molecules may be directly or indirectly attached to the polypeptide or antibody, and include bacterial or plant toxins (for instance, diphtheria toxin, Pseudomonas exotoxin, ricin, abrin and the like) , as well as therapeutic radionuclides, such as iodine-131, rhenium-188 or yttrium-90 (either directly attached to the polypeptide or antibody, or indirectly attached through means of a chelating moiety, for instance) .
  • Polypeptides or antibodies may also be conjugated to cytotoxic drugs, such as adriamycin.
  • cytotoxic drugs such as adriamycin.
  • the detectable or cytotoxic molecule may be conjugated with a member of a complementary/ anticomplementary pair, where the other member is bound to the polypeptide or antibody portion.
  • biotin/streptavidin is an exemplary complementary/ anticomplementary pair.
  • Such polypeptide-toxin fusion proteins or antibody/fragment-toxin fusion proteins may be used for targeted cell or tissue inhibition or ablation (for instance, to treat cancer cells or tissues) .
  • a fusion protein including only the targeting domain may be suitable for directing a detectable molecule, a cytotoxic molecule or a complementary molecule to a cell or tissue type of interest.
  • the anticomplementary molecule may be conjugated to a detectable or cytotoxic molecule.
  • Such domain-complementary molecule fusion proteins thus represent a generic targeting vehicle for cell/tissue-specific delivery of generic anti- complementary-detectable/cytotoxic molecule conjugates.
  • the bioactive polypeptide or antibody conjugates described herein can be delivered intravenously, intraarterially or intraductally, or may be introduced locally at the intended site of action.
  • ZTNFR-6 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-6 polypeptides will also find use in mediating metabolic or physiological processes in vivo .
  • the effects of a compound on proliferation and differentiation can be measured in vi tro using cultured cells.
  • Bioassays and ELISAs are available to measure cellular response to ZTNFR-6, 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) .
  • Assays to measure other cellular responses including antibody isotype, monocyte activation, NK cell formation, antigen presenting cell function, apoptos
  • soluble ZTNFR-6 transfected expression host cells may be embedded in an alginate environment and injected (implanted) into recipient animals.
  • Alginate-poly-L-lysine micro- encapsulation, 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 .
  • 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.
  • 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 (i.e., the human 293 cell line) .
  • the host cell i.e., the human 293 cell line
  • 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.
  • the host's tissue i.e., liver
  • the host's tissue i.e., liver
  • the host's tissue 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 By culturing adenovirus- infected non-293 cells under conditions where the cells are not rapidly dividing, the cells can produce proteins for extended periods of time. For instance, 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 A. Garnier et al., Cvtotechnol. 15:145-55, 1994). With either protocol, 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-6 shares homology with OPG, a soluble TNF receptor involved in the regulation of bone density (Simonet et al . , ibid. ) .
  • Well established animal models are available to test the in vivo efficacy of ZTNFR- ⁇ polypeptides for certain disease states, such as bone- related disorders.
  • the hypocalcemic rat model can be used to determine the effect of ZTNFR-6 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-6 polypeptides can also be used to prepare antibodies that specifically bind to ZTNFR-6 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: Techniques 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 immunogenicity of a ZTNFR-6 polypeptide may be increased through the use of an adjuvant, such as alum
  • the ZTNFR-6 polypeptide or a fragment thereof serves as an antigen (immunogen) to inoculate an animal or elicit an immune response.
  • Suitable antigens would include the ZTNFR-6 polypeptide encoded by SEQ ID NOs : 4 or 6 from amino acid residues 37 to 65, 66 to 107, 108 to 145, 146 to
  • Polypeptides useful for immunization also include fusion polypeptides, such as fusions of ZTNFR-6 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. If the 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.
  • KLH keyhole limpet hemocyanin
  • BSA bovine serum albumin
  • antibodies includes polyclonal antibodies, affinity-purified polyclonal antibodies, monoclonal antibodies, and antigen-binding fragments thereof, such as F(ab')2 and Fab 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 human- like 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-6 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-6 protein or peptide, and selection of antibody display libraries in phage or similar vectors (for instance, through use of immobilized or labeled ZTNFR-6 protein or peptide) .
  • ZTNFR-6 polypeptides can also be used to prepare antibodies that specifically bind to ZTNFR-6 epitopes, peptides or polypeptides.
  • Antibodies are determined to be specifically binding if: 1) they exhibit a threshold level of binding activity, and/or 2) they do not significantly cross-react with related polypeptide molecules.
  • Antibodies herein specifically bind if they bind to a ZTNFR-6 polypeptide, peptide or epitope with a binding affinity
  • K a 10 6 M 1 or greater, preferably 107 M-1 or greater, more preferably 10 M " or greater, and most preferably 10 M "1 or greater.
  • the binding affinity of an antibody can be readily determined by one of ordinary skill in the art, for example, by Scatchard analysis (Scatchard, G. , Ann. NY Acad. Sci. 51 : 660-72, 1949).
  • Antibodies specifically bind if they do not significantly cross-react with related polypeptides. Antibodies do not significantly cross-react with related polypeptide molecules, for example, if they detect ZTNFR-6 but not known related polypeptides using a standard Western blot analysis (Ausubel et al . , ibid. ) .
  • known related polypeptides are orthologs, proteins from the same species that are members of a protein family such as other known human tumor necrosis factor receptors (e.g. DR4 ) , mutant ZTNFR-6 polypeptides, and non-human tumor necrosis factor receptors.
  • antibodies may be "screened against" known related polypeptides to isolate a population that specifically binds ZTNFR-6 polypeptides. For example, antibodies raised to ZTNFR-6 are adsorbed to related polypeptides adhered to insoluble matrix; antibodies specific to ZTNFR-6 will flow through the matrix under the proper buffer conditions.
  • assays known to those skilled in the art can be utilized to detect antibodies which specifically bind to ZTNFR-6 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-6 protein or peptide.
  • ELISA enzyme-linked immunosorbent assay
  • Antibodies to ZTNFR-6 may be used for immunohistochemical tagging of cells that express human ZTNFR-6, for example, to use in a diagnostic assays; for isolating ZTNFR-6 by affinity purification; for screening expression libraries; for generating anti-idiotypic antibodies; and as neutralizing antibodies or as antagonists to block ZTNFR-6 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-6 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-6 can be used in analysis of tissue distribution of ZTNFR-6 by immunohistochemistry on human or primate tissue.
  • These soluble ZTNFR-6 polypeptides can also be used to immunize mice in order to produce monoclonal antibodies to a soluble human ZTNFR-6 polypeptide.
  • Monoclonal antibodies to a soluble human ZTNFR-6 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-6 can be used to determine the distribution, regulation and biological interaction of the ZTNFR-6/ZTNFR- 6 -ligand pair on specific cell lineages identified by tissue distribution studies.
  • Genes encoding polypeptides having potential ZTNFR-6 polypeptide binding domains can be obtained by screening random peptide libraries displayed on phage (phage display) or on bacteria, such as E. coli .
  • Nucleotide sequences encoding the polypeptides can be obtained in a number of ways, such as through random mutagenesis and random polynucleotide synthesis.
  • These random peptide display libraries can be used to screen for peptides which interact with a known target which can be a protein or polypeptide, such as a ligand or receptor, a biological or synthetic macromolecule, or organic or inorganic substances.
  • Techniques for creating and screening such random peptide display libraries are known in the art (Ladner et al . , US Patent NO : 5 , 223 , 409 ; Ladner et al., US Patent NO : 4 , 946 , 778 ; Ladner et al .
  • Random peptide display libraries can be screened using the ZTNFR-6 sequences disclosed herein to identify proteins which bind to ZTNFR-6.
  • binding proteins which interact with ZTNFR-6 polypeptides may be used for tagging cells; for isolating homolog polypeptides by affinity purification; they can be directly or indirectly conjugated to drugs, toxins, radionuclides and the like. These binding proteins can also be used in analytical methods such as for screening expression libraries and neutralizing activity.
  • binding proteins can also be used for diagnostic assays for determining circulating levels of polypeptides; for detecting or quantitating soluble polypeptides as marker of underlying pathology or disease. These binding proteins can also act as ZTNFR-6 "antagonists" to block ZTNFR-6 receptor binding and signal transduction in vi tro and in vivo .
  • the invention also provides isolated and purified
  • ZTNFR-6 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
  • the synthetic oligonucleotides of the present invention have at least 80% identity to a representative ZTNFR-6 DNA sequence (SEQ ID NOs: 4 or 6) or its complements.
  • the invention also provides oligonucleotide probes or primers comprising at least 14 contiguous nucleotides of a polynucleotide of SEQ ID NOs : 21 or 22 or a sequence complementary to SEQ ID NOs : 21 or 22.
  • 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 .
  • the molecules 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.
  • 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.
  • Such probes can also be used in hybridizations to detect the presence or quantify the amount of ZTNFR-6 gene or mRNA transcript in a sample.
  • ZTNFR-6 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-6-like proteins.
  • ZTNFR-6 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 polynucleotide probes could be used to hybridize to counterpart sequences on individual chromosomes.
  • Chromosomal identification and/or mapping of the ZTNFR-6 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) .
  • FISH fluorescence in si tu hybridization
  • 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 . Chromosomal localization of ZTNFR-6 to 6p21.1 was determined using radiation hybrid chimeras. The present invention also provides reagents which will find use in diagnostic applications.
  • dbSTS Database of Sequence Tagged Sites
  • GenBank National Center for Biological Information, National Institutes of Health, Bethesda, MD http://www.ncbi.nlm.nih.gov
  • the ZTNFR-6 gene, a probe comprising ZTNFR-6 DNA or RNA, or a subsequence thereof can be used to determine if the ZTNFR-6 gene is present on chromosome 6 or if a mutation has occurred.
  • Detectable chromosomal aberrations at the ZTNFR-6 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.
  • Such aberrations can be detected using polynucleotides of the present invention by employing molecular genetic techniques, such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid.; Ausubel, et . al . , ibid. ; Marian, A.J., Chest , 108: 255-265, 1995) .
  • molecular genetic techniques such as restriction fragment length polymorphism (RFLP) analysis, short tandem repeat (STR) analysis employing PCR techniques, and other genetic linkage analysis techniques known in the art (Sambrook et al . , ibid.; Ausubel, et . al . , ibid. ; Marian, A.J., Chest , 108: 255-265, 1995) .
  • RFLP restriction fragment length polymorphism
  • STR short tandem repeat
  • 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 NOs : 3 or 5, the complement of SEQ ID NOs : 3 or 5 , 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 (Sambrook et al . , ibid. ; Ausubel et . al . , ibid.
  • Ribonuclease protection assays comprise the hybridization of an RNA probe to a patient RNA sample, after which the reaction product (RNA-RNA hybrid) is exposed to RNase . 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-6 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- 6 -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- 6 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- 6 -bearing cells in culture .
  • the invention also provides antagonists, which either bind to ZTNFR-6 polypeptides or, alternatively, to a ligand to which ZTNFR- 6 polypeptides bind, thereby inhibiting or eliminating the function of ZTNFR-6.
  • ZTNFR-6 antagonists would include antibodies; polypeptides which bind either to the ZTNFR- 6 polypeptide or to its ligand; natural or synthetic analogs of ZTNFR-6 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- 6 polypeptides and prevent signaling are also contemplated as antagonists.
  • ZTNFR-6 receptors are also contemplated.
  • ZTNFR-6 antagonists would be useful as therapeutics for treating certain disorders where blocking signal from either a ZTNFR- 6 receptor or ligand would be beneficial.
  • Antagonists are useful as research reagents for characterizing ligand-receptor interaction.
  • ZTNFR-6 polypeptides may be used within diagnostic systems to detect the presence of ligand polypeptides.
  • Antibodies or other agents that specifically bind to ZTNFR- 6 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- 6 antibodies can be used to detect ZTNFR- 6 receptor and/or ligands in tissue samples.
  • ZTNFR-6 levels can also be monitored by such methods as RT-PCR, where ZTNFR- 6 mRNA can be detected and quantified. The information derived from such detection methods would provide insight into the significance of ZTNFR-6 polypeptides in various diseases, and as such would serve as diagnostic tools for diseases for which altered levels of ZTNFR- 6 are significant.
  • Altered levels of ZTNFR- 6 receptor polypeptides may be indicative of pathological conditions including cancer, autoimmune disorders, bone disorders, " inflammation and immunodeficiencies .
  • ZTNFR- 6 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.
  • the invention also provides nucleic acid-based therapeutic treatment. If a mammal lacks or has a mutated
  • the ZTNFR- 6 gene can be introduced into the cells of the mammal.
  • a gene encoding a ZTNFR- 6 polypeptide is introduced in vivo in a viral vector.
  • viral vectors include an attenuated or defective
  • DNA virus such as but not limited to herpes simplex virus
  • HSV herpes virus 1
  • EBV Epstein Barr virus
  • AAV adeno-associated virus
  • HSV herpes virus 1
  • adenovirus vector such as the vector described by Stratford-Perricaudet et al . (J. Clin. Invest. £0:626-30, 1992), and a defective adeno- associated virus vector (Samulski et al . , J. Virol. £1:3096-101, 1987; Samulski et al . , J. Virol. £:3822-8,
  • the gene can be introduced in a retroviral vector, e . g. , as described in Anderson et al., U.S. Patent No. 5,399,346; Mann et al . , Cell 33 :153, 1983; Temin et al . , U.S. Patent No. 4,650,764; Temin et al . , U.S. Patent No. 4,980,289; Markowitz et al . , J. Virol.
  • the vector can be introduced by lipofection in vivo using liposomes.
  • Synthetic cationic lipids can be used to prepare liposomes for in vivo transfection of a gene encoding a marker (Feigner et al . , Proc. Natl. Acad. Sci. USA £4:7413-7, 1987; and Mackey et al., Proc. Natl. Acad. Sci.
  • lipofection to introduce exogenous genes into specific organs in vivo has certain practical advantages.
  • Molecular targeting of liposomes to specific cells represents one area of benefit. It is clear that directing transfection to particular cells represents one area of benefit. It is clear that directing transfection to particular cell types would be particularly advantageous in a tissue with cellular heterogeneity, such as the pancreas, liver, kidney, and brain.
  • Lipids may be chemically coupled to other molecules for the purpose of targeting.
  • Targeted peptides e.g., hormones or neurotransmitters, and proteins such as antibodies, or non-peptide molecules could be coupled to liposomes chemically.
  • DNA vector for gene therapy can be introduced into the desired host cells by methods known in the art, e.g., transfection, electroporation, microinjection, transduction, cell fusion, DEAE dextran, calcium phosphate precipitation, use of a gene gun or use of a DNA vector transporter (see, for example, Wu et al . , J. Biol. Chem. 267 :963-7, 1992; Wu et al., J. Biol. Chem. 263.-14621-4, 1988).
  • antisense polynucleotide compositions that are complementary to a segment of the polynucleotides set forth in SEQ ID NOs : 3 or 5.
  • Such synthetic antisense oligonucleotides are designed to bind to mRNA encoding ZTNFR- 6 polypeptides and to inhibit translation of such mRNA.
  • Such antisense oligonucleotides are used to inhibit expression of ZTNFR- 6 polypeptide-encoding genes in cell culture or in a subject.
  • mice engineered to express the ZTNFR- 6 gene, and mice that exhibit a complete absence of ZTNFR- 6 gene function, referred to as "knockout mice” (Snouwaert et al . , Science 257 : 1083 , 1992), may also be generated (Lowell et al., Nature 366 :740-2, 1993). These mice may be employed to study the ZTNFR- 6 gene and the protein encoded thereby in an in vivo system.
  • ZTNFR- 6 polypeptides or ZTNFR-6 antagonists of the present invention can be formulated with pharmaceutically acceptable carriers for parenteral, oral, nasal, rectal, topical, transdermal administration or the like, according to conventional methods.
  • Formulations may further include one or more diluents, fillers, emulsifiers, preservatives, buffers, excipients, and the like, and may be provided in such forms as liquids, powders, emulsions, suppositories, liposomes, transdermal patches and tablets, for example.
  • Slow or extended-release delivery systems including any of a number of biopolymers (biological-based systems) , systems employing liposomes, and polymeric delivery systems, can also be utilized with the compositions described herein to provide a continuous or long-term source of the ZTNFR-6 polypeptide or antagonist.
  • Such slow release systems are applicable to formulations, for example, for oral, topical and parenteral use.
  • pharmaceutically acceptable carrier refers to a carrier medium which does not interfere with the effectiveness of the biological activity of the active ingredients and which is not toxic to the host or patient.
  • One skilled in the art may formulate the compounds of the present invention in an appropriate manner, and in accordance with accepted practices, such as those disclosed in Remington: The Science and Practice of Pharmacy, Gennaro, ed. , Mack Publishing Co., Easton PA, 19 th ed., 1995.
  • a "pharmaceutically effective amount" of a ZTNFR- 6 polypeptide, agonists or antagonist is an amount sufficient to induce a desired biological result.
  • the result can be alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.
  • an effective amount of a pharmaceutical agent for example, an effective amount of a pharmaceutical agent, or any other suitable alteration of a biological system.
  • ZTNFR-6 polypeptide is that which provides either subjective relief of symptoms or an objectively identifiable improvement as noted by the clinician or other qualified observer.
  • an effective amount of a ZTNFR- 6 polypeptide results in a decrease in glucose levels in patients with IDDM, prevention or significant delay of onset of disease or loss of islet infiltration in
  • ZTNFR- 6 polypeptides can vary widely depending on the disease or symptom to be treated.
  • the amount of the polypeptide to be administered and its concentration in the formulations depends upon the vehicle selected, route of administration, the potency of the particular polypeptide, the clinical condition of the patient, the side effects and the stability of the compound in the formulation.
  • the clinician will employ the appropriate preparation containing the appropriate concentration in the formulation, as well as the amount of formulation administered, depending upon clinical experience with the patient in question or with similar patients.
  • a dose will be in the range of 0.1-100 mg/kg of subject.
  • Doses for specific compounds may be determined from in vi tro or ex vivo studies in combination with studies on experimental animals. Concentrations of compounds found to be effective m vi tro or ex vivo provide guidance for animal studies, wherein doses are calculated to provide similar concentrations at the site of action.
  • the invention is further illustrated by the following non-limiting examples.
  • Novel ZTNFR- 6 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 receptor family. Using this information, 5 independent ESTs were identified and aligned to produce a novel human contig assembly. 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.
  • 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:24) and ZC5939 (SEQ ID NO: 25) to the vector were used as sequencing primers.
  • ZC14189 (SEQ ID NO:30), ZC15020 (SEQ ID NO:31), ZC14415
  • SEQ ID NO:32 and ZC14873 (SEQ ID NO:33) 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 2646 bp sequence is disclosed in SEQ ID NO: 5 and encodes a soluble ZTNFR- 6 receptor.
  • a second clone considered likely to contain a full length ZTNFR- 6 polynucleotide was sequenced as described above using oligonucleotides ZC694 (SEQ ID NO:24), ZC695 (SEQ ID NO:34), ZC15826 (SEQ ID NO:35), ZC15827 (SEQ ID NO:36), ZC15955 (SEQ ID NO:37) and ZC15956
  • SEQ ID NO: 38 The resulting 2646 bp sequence is disclosed in SEQ ID NO: 3 and encodes a full length, membrane bound ZTNFR-6 receptor.
  • the MarathonTM-ready cDNA libraries were prepared according to manufacturer's instructions (MarathonTM cDNA Amplification Kit; Clontech, Palo Alto, CA) using human heart and uterus poly A+ RNA (Clontech) and MCF7 and CaC02 poly A+ RNA prepared using a guanidine isothiocyanate, phenol, chloroform mixture according to Cheomczynski and Sacchi (Anal. Biochem. 162 :156-9, 1987) and isolated using oligo d(T) cellulose chromatography (Aviv and Leder, Proc. Natl. Acad. Sci. USA £9:1408-12, 1972) .
  • the probe was amplified in a polymerase chain reaction as follows: 94°C for 1.5 minutes followed by 35 cycles of 94°C for 10 seconds, 64°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 1.5% agarose gel (GIBCO BRL, Gaithersburg, MD) , the fragment was purified using the QIAEXTM 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 (Clontech) solution was used for prehybridization and as a hybridizing solution for the Northern blots. Hybridization took place overnight at 55°C using 2.2 x 10 6 cpm/ml of labeled probe. The blots were then washed at 65°C in 0. IX SSC, 0.1% SDS.
  • a transcript of approximately 4.4 kb was detected heart, brain, placenta, lung, kidney, pancreas, colon, stomach, spinal cord and trachea. A lesser transcript was detected in liver, skeletal muscle, spleen, thymus, prostate, small intestine, thyroid, lymph node, adrenal gland and bone marrow.
  • a second transcript of 3 kb was detected in testis.
  • HOS cells a human osteogenic sarcoma clone, ATCC CRL-1547
  • MG-63 cells a human osteosarcoma, ATCC CRL-1427
  • Saos2 human osteogenic sarcoma, ATCC HTB-85
  • U205 cells human primary osteogenic sarcoma ATCC HTB-96
  • RNA was denatured in 2.2 M formaldehyde/phosphate buffer (50 mM Na 2 HP0 4 , 50 mM NaH 2 P0 4 , 50 mM NaOAc , 1 mM EDTA and 2.2 M formaldehyde) and separated by 1.5% agarose mini gel electrophoresis in formaldehyde/phosphate buffer.
  • the RNA was blotted overnight onto a nytran filter (Schleicher & Schuell, Keene, NH) , and the filter was UV crosslinked (1,200 ⁇ Joules) in a STRATALINKER® UV crosslinker (Stratagene Cloning Systems) .
  • Hybridization took place overnight at 55°C using 2.2 x 10 cpm/ml of labeled probe. The blots were then washed at 50°C in 0. IX SSC, 0.1% SDS and exposed to film for two days. Transcript signals were detected in all four cell lines.
  • ZTNFR-6 was mapped to chromosome 6 using the commercially available GeneBridge 4 Radiation Hybrid Panel
  • Radiation Hybrid Panel contains PCRable DNAs from each of 93 radiation hybrid clones, plus two control DNAs (the HFL donor and the A23 recipient) .
  • a publicly available WWW server http : //www-genome . wi .mit . edu/cgi-bin/contig/ rhmapper.pl
  • WICGR Whitehead Institute/MIT Center for Genome Research's radiation hybrid map of the human genome
  • Each of the 95 PCR reactions consisted of 2 ⁇ l 10X KlenTaq PCR reaction buffer (Clontech Laboratories, Inc., Palo Alto, CA) , 1.6 ⁇ l dNTPs mix (2.5 mM each, Perkin-Elmer, Foster City, CA) , 1 ⁇ l sense primer, ZC 14,861 (SEQ ID NO:41), 1 ⁇ l antisense primer, ZC 14,851 (SEQ ID N0:42), 2 ⁇ l .RediLoad (Research Genetics, Inc.), 0.4 ⁇ l 50X Advantage
  • KlenTaq Polymerase Mix (Clontech) , 25 ng of DNA from an individual hybrid clone or control and ddH 2 0 for a total volume of 20 ⁇ l .
  • the reactions were overlaid with an equal amount of mineral oil and sealed.
  • the PCR cycler conditions were as follows : an initial 1 cycle 5 minute denaturation at 95°C, 40 cycles of a 1 minute denaturation at 95°C, 1 minute annealing at 64°C and 1.5 minute extension at 72°C, followed by a final 1 cycle extension of 7 minutes at 72oC.
  • the reactions were separated by electrophoresis on a 2% agarose gel (Life Technologies, Gaithersburg, MD) .
  • ZTNFR- 6 maps 271.75 cR_3000 from the top of the human chromosome 6 linkage group on the WICGR radiation hybrid map.
  • Proximal and distal framework markers were D6S452 and CHLC.GATA85F06 , respectively.
  • the use of surrounding markers positions ZTNFR-6 in the 6p21.1 region on the integrated LDB chromosome 6 map (The Genetic Location Database, University of Southampton, WWW server: http : //cedar . genetics . soton . ac . uk/public_html/ ) .
  • the Fc region of human IgGl (the hinge region and the CH2 and CH3 domains) was modified.
  • the Fc region was isolated from a human fetal liver library (Clontech) by PCR using oligo primers ZC10,134 (SEQ ID NO: 43) and ZC10,135 (SEQ ID NO: 44) .
  • 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: 45) according to Baum et al. (EMBO J.
  • Oligonucleotide primers ZC15,345 (SEQ ID NO:46) and ZC15,347 (SEQ ID NO:47) were used to introduce the mutation.
  • the reaction products were electrophoresed and the band corresponding to the predicted size of ⁇ 676 bp was so y detected.
  • the band was excised from the gel and recovered using a QIAGEN QIAquickTM 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: 45) and Pro to Ser (amino acid residue 135 of SEQ ID NO: 45) 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.
  • the second reaction contained 2 ⁇ l of 20 mM each of oligonucleotide primers ZC15,518 (SEQ ID NO:50), a 5' primer beginning at nucleotide 388 of SEQ ID NO: 45 and ZC15,347 (SEQ ID NO:47), 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 QIAquickTM 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: 51) , a 5' primer beginning at nucleotide 1 of SEQ ID NO: , introducing a Bam HI restriction site, and 20 mM ZC15,347 (SEQ ID NO: 47) 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.
  • the remainder of the IgFc PCR fragment and the baculovirus expression vector pFBL2 (pFASTBacTM (Gibco BRL) with baculo basic protein promoter added in place of existing promoter) were digested with the restriction enzymes Bam HI and Xba I .
  • the IgFc fragment was ligated into the vector at a molar ratio of insert to vector of approximately 4 to 1.
  • the IgFc containing pFBL2 vector was then used to transform competent E. coli DH10B cells (Life).
  • 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. One half microliter of Luria Broth (LB) was added to the transformation reaction and it was then plated on LB plus ampicillin (100 mg/ml) plates and incubated overnight as 37°C. To screen for positive transformants, 5 colonies were cultured overnight in LB containing 100 mg/ml ampicillin. DNA was prepared from each culture using a QIAGEN QIAprep Spin Miniprep Kit (QIAGEN) according the manufacturer's instruction.
  • QIAGEN QIAprep Spin Miniprep Kit
  • 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 .
  • col i 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:52), a sequence from the pHZ200 expression vector, and ZC15,530 (SEQ ID NO:49).
  • a soluble ZTNFR- 6 fragment was prepared using the clone described in Example 1.
  • Oligonucleotide primer ZC15,825 (SEQ ID NO: 53) adds a Bam HI restriction site to the 5' side of nucleotide 284 in SEQ ID NO : 3 and oligonucleotide primer ZC15,824 (SEQ ID NO: 21) adds sequence encoding for amino acid residues Glu and Pro and a
  • the reaction was heated to 94°C for 1 minute.
  • Pfu polymerase 2.5 units, Stratagene
  • the reaction products were electrophoresed and the band corresponding to the predicted size of ⁇ 713 bp was detected.
  • the remainder of the PCR product was digested with restriction enzymes Bam HI and Bgl II to release a fragment containing the ZTNFR- 6 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, dephosphorylated IgFc4/pHZ200 vector as described above at a 10 to 1 molar ratio of insert to vector.
  • Competent DH10B cells (Life Technologies) were transformed with the ligation product, plated and a clone containing the correct insert was identified by restriction enzyme mapping analysis and designated ZTNFR6Fc4/pHZ200.
  • a Bam HI -Xba I fragment containing the ZTNFR6-IgFc fusion protein coding region from ZTNFR6Fc4/pHZ200 was restriction digested from ZTNFR6Fc4/pHZ200 and was ligated into a Bam HI -Xba I digested pFBL2 vector as described above at a 4 to 1 molar ration of insert to vector.
  • E. coli TOP10 competent cells (Invitrogen Inc., San Diego, CA) 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 ZTNFR6Fc4/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%
  • the cells were then transfected with the plasmid ztnfr6xlFc4/pHZ200 , using LipofectamineTM (Gibco BRL) , in serum free (SF) media formulation (DMEM, 10 ⁇ g/ml transferrin, 5 ⁇ g/ml insulin, 2 ng/ml selenium, 10 ⁇ g/ml fetuin, 1% L-glutamine and 1% sodium pyruvate) .
  • DMEM serum free
  • the cells were incubated at 37°C for five hours, then 6.4 ml of DMEM/10% FBS, 1% PSN media was added to each plate. The plates were incubated at 37°C overnight and the DNA: LipofectamineTM mixture was replaced with fresh FBS/DMEM media the next day. On day 2 post-transfection, 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.
  • DMEM/FBS media from above with the addition of 1 ⁇ M methotrexate (Sigma Chemical Co., St. Louis, Mo.)
  • 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/
  • a sterile Teflon mesh (Spectrum Medical Industries, Los Angeles, CA) pre-soaked in serum- free ESTEP 2 was then placed over the cells.
  • a sterile nitrocellulose filter -0 pre-soaked in serum-free ESTEP 2 was then placed over the mesh. Orientation marks on the nitrocellulose were transferred to the culture dish. The plates were then incubated for 5 hours in a 37°C, 5% C0 2 incubator. Following incubation, the filter was removed, and the media 5 aspirated and replaced with DMEM/5% FBS, IX PSN (Gibco BRL) media.
  • 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- 0 human IgG-HRP antibody (Southern Biotechnology Associates, Inc., Birmingham, AL) at a 1:4000 dilution 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) for 1 hour at room temperature on a rotating shaker.
  • the filter 5 was then washed three times at room temperature in PBS plus 0.1% Tween 20, 5-15 minutes per wash.
  • the filter was developed with ECL reagent (Amersham Corp., Arlington Heights, IL) according the manufacturer's directions and exposed to film (Hyperfilm ECL, Amersham) for approximately 0 5 minutes.
  • the film was aligned with the plate containing the colonies. Using the film as a guide, 12 suitable colonies were selected. Sterile, 3 mm coloning discs (PGC Scientific Corp., Frederick, MD) were soaked in trypsin, 5 and placed on the colonies. Twelve colonies were transferred into 200 ⁇ l of selection medium in a 96 well plate. A series of seven, two-fold dilutions were carried out for each colony The cells were grown for one week at 37°C at which time the wells which received the lowest dilution of cells which are now at the optimum density were selected, trypsmized and transferred to a 12 well plate containing selection media. When confluent the cells were transferred into 2 T-75 flasks, one flask switched to serum free media, harvested and subjected to Western analysis.
  • 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 m 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 w th 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 was aspirated from the plate of cells and the lipid-DNA mixture, to which 0.8 ml of Sf-900 II SFM was added, was distributed over the plate.
  • the cells were incubated at 27°C for 5 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 4 days 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 3 days after which time the virus was harvested, titer 1.2 x 10 8 pfu/ml .
  • Beckman JLA-10.5 rotor (Beckman Instruments, Palo Alto, CA) in a Beckman Avanti J25I centrifuge (Beckman Instruments) to remove cell debris.
  • a 50.0 ml sample of protein G-Sepharose (Pharmacia Biotech, Piscataway, NJ) and the mixture was gently agitated on a Wheaton (Millville, NJ) roller culture apparatus for 18.0 h at 4°C.
  • the mixture was poured into a 5.0 x 20.0 cm Econo-Column (Bio-Rad, Laboratories, Hercules, CA) and the gel was washed with 10 column volumes of phosphate buffered saline at pH 7.2 (PBS) . The unretained flow-through fraction was discarded. Once the absorbance of the effluent at 280 nM was less than 0.05, flow through the column was reduced to zero and the Protein G Sepharose gel was washed with 2.0 column volumes of 0.2 M glycine, pH 3.0. Flow was resumed and the eluted protein was collected.
  • PBS phosphate buffered saline at pH 7.2
  • the pH of the glycine-eluted fraction was adjusted to 7.2 by the addition of a small volume of 2.0 M Tris-HCl.
  • the glycine elution was concentrated to 5.0 ml using a 5,000 molecular weight cutoff membrane concentrator (Millipore, Bedford, MA) according to the manufacturer's instructions.
  • the concentrated sample was further purified and buffer exchanged by chromatography on a 1.6 x 94 cm Sephacryl S-100 (Pharmacia, Piscataway, NJ) column equilibrated in PBS at a flow rate of 1.5 ml/min using a BioCad Sprint HPLC (PerSeptive BioSystems, Framingham, MA) .
  • the peptides were reduced then conjugated to maleimide activated keyhole limpet hemocyanin (KLH) carrier protein.
  • KLH keyhole limpet hemocyanin
  • the rabbits were each given an initial intraperitoneal (ip) injection of 200 ⁇ g of peptide in Complete Freund ' s Adjuvant followed by booster ip injections of 100 ⁇ g peptide in Incomplete Freund' s Adjuvant every three weeks. Seven to ten days after the administration of the third booster injection, the animals were bled and the serum was collected. The animals were then boosted and bled every three weeks .
  • Human ZTNFR-6 peptide-specific antibodies designated Rb ZTNFR6-l and Rb ZTNFR6-2 (corresponding to the peptides disclosed in SEQ ID NOs: 54 and 55) were affinity purified from the serum using an CNBr-SEPHAROSE 4B peptide column (Pharmacia LKB) that was prepared using 10 mg of the respective peptides per gram CNBr-SEPHAROSE, followed by dialysis in PBS overnight.
  • Rabbit specific- ZTNFR-6 antibodies were characterized by an ELISA titer check using 5 ⁇ g/ml of the peptides (SEQ ID NOs : 54 and 55) used to make the antibodies as the antibody target.
  • LLD Rb ⁇ ZTNFR6-l , 0.5 ng/ml and Rb ⁇ ZTNFR6-2 0.5 ng/ml .

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Abstract

L'invention se rapporte à de nouveaux polypeptides de récepteurs du facteur de nécrose des tumeurs, sécrétés et à liaison membranaire, aux polynucléotides codant ces polypeptides et à des compositions et procédés associés. Ces polypeptides comportent quatre répétitions riches en cystéine qui sont homologues d'autres récepteurs du facteur de nécrose des tumeurs, en particulier de l'ostéoprotégérine soluble, sécrétée, réceptrice du facteur de nécrose des tumeurs. Ces polypeptides peuvent servir à la détection de ligands, d'agonistes et d'antagonistes. Ils peuvent également être utilisés dans des procédés qui favorisent la maturation cellulaire et la régulation des cellules osseuses.
PCT/US1998/018364 1997-09-04 1998-09-03 Recepteur ztnfr-6 du facteur de necrose des tumeurs WO1999011790A1 (fr)

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US5760897P 1997-09-04 1997-09-04
US60/057,608 1997-09-04
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Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1999066039A1 (fr) * 1998-06-12 1999-12-23 Astrazeneca Ab Proteine du type du recepteur du facteur de necrose tumorale et comportant un domaine d'apoptose
US6194151B1 (en) 1997-09-26 2001-02-27 Millenium Pharmaceuticals, Inc. Molecules of the TNF receptor superfamily and uses therefor
US6358508B1 (en) 1997-06-11 2002-03-19 Human Genome Sciences, Inc. Antibodies to human tumor necrosis factor receptor TR9
EP0988371A4 (fr) * 1997-06-11 2002-11-04 Human Genome Sciences Inc Recepteur humain tr9 du facteur de necrose tumorale
US6667390B2 (en) 1997-06-11 2003-12-23 Human Genome Sciences, Inc. Human tumor necrosis factor receptor TR9
US6916907B1 (en) 1998-10-23 2005-07-12 Curagen Corporation Nucleic acids encoding osteoprotegern-like proteins and methods of using same
WO2005037865A3 (fr) * 2003-10-16 2005-08-04 Zymogenetics Inc Ztnfr14, recepteur du facteur de necrose tumorale
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

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US5447851A (en) * 1992-04-02 1995-09-05 Board Of Regents, The University Of Texas System DNA encoding a chimeric polypeptide comprising the extracellular domain of TNF receptor fused to IgG, vectors, and host cells
WO1997023614A1 (fr) * 1995-12-22 1997-07-03 Amgen Inc. Osteoprotegerine
EP0869179A1 (fr) * 1997-04-02 1998-10-07 Smithkline Beecham Corporation Récepteur en rapport avec la nécrose des tumeurs, TR7

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* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5447851A (en) * 1992-04-02 1995-09-05 Board Of Regents, The University Of Texas System DNA encoding a chimeric polypeptide comprising the extracellular domain of TNF receptor fused to IgG, vectors, and host cells
US5447851B1 (en) * 1992-04-02 1999-07-06 Univ Texas System Board Of Dna encoding a chimeric polypeptide comprising the extracellular domain of tnf receptor fused to igg vectors and host cells
WO1997023614A1 (fr) * 1995-12-22 1997-07-03 Amgen Inc. Osteoprotegerine
EP0869179A1 (fr) * 1997-04-02 1998-10-07 Smithkline Beecham Corporation Récepteur en rapport avec la nécrose des tumeurs, TR7

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Title
DATABASE GENBANK 14 February 1996 (1996-02-14), HILLIER ET AL.: "H. sapiens cDNA clone IMAGE 280262, EST.", XP002085987 *
DATABASE GENBANK 8 January 1997 (1997-01-08), HILLIER ET AL.: "H. sapiens cDNA 625115, EST", XP002085986 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6358508B1 (en) 1997-06-11 2002-03-19 Human Genome Sciences, Inc. Antibodies to human tumor necrosis factor receptor TR9
EP0988371A4 (fr) * 1997-06-11 2002-11-04 Human Genome Sciences Inc Recepteur humain tr9 du facteur de necrose tumorale
US6667390B2 (en) 1997-06-11 2003-12-23 Human Genome Sciences, Inc. Human tumor necrosis factor receptor TR9
US6919078B2 (en) 1997-06-11 2005-07-19 Human Genome Sciences, Inc. Antibodies to human tumor necrosis factor receptor TR9
US6949358B1 (en) 1997-06-11 2005-09-27 Human Genome Sciences, Inc. Human tumor necrosis factor receptor TR9
US7776560B2 (en) 1997-06-11 2010-08-17 Human Genome Scienes, Inc. Human tumor necrosis factor receptor TR9 antibody
US6194151B1 (en) 1997-09-26 2001-02-27 Millenium Pharmaceuticals, Inc. Molecules of the TNF receptor superfamily and uses therefor
WO1999066039A1 (fr) * 1998-06-12 1999-12-23 Astrazeneca Ab Proteine du type du recepteur du facteur de necrose tumorale et comportant un domaine d'apoptose
GB2355461A (en) * 1998-06-12 2001-04-25 Astrazeneca Ab TNFR-like protein with death domain
US6916907B1 (en) 1998-10-23 2005-07-12 Curagen Corporation Nucleic acids encoding osteoprotegern-like proteins and methods of using same
WO2005037865A3 (fr) * 2003-10-16 2005-08-04 Zymogenetics Inc Ztnfr14, recepteur du facteur de necrose tumorale
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

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