EP1053351A1 - Neue moleküle der tnf-rezeptorsuperfamilie und verwendungen dafür - Google Patents

Neue moleküle der tnf-rezeptorsuperfamilie und verwendungen dafür

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Publication number
EP1053351A1
EP1053351A1 EP99904304A EP99904304A EP1053351A1 EP 1053351 A1 EP1053351 A1 EP 1053351A1 EP 99904304 A EP99904304 A EP 99904304A EP 99904304 A EP99904304 A EP 99904304A EP 1053351 A1 EP1053351 A1 EP 1053351A1
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EP
European Patent Office
Prior art keywords
strife2
strifel
protein
seq
nucleic acid
Prior art date
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Application number
EP99904304A
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English (en)
French (fr)
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EP1053351A4 (de
Inventor
Samantha J. Busfield
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Millennium Pharmaceuticals Inc
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Millennium Pharmaceuticals Inc
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Publication of EP1053351A1 publication Critical patent/EP1053351A1/de
Publication of EP1053351A4 publication Critical patent/EP1053351A4/de
Withdrawn legal-status Critical Current

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Classifications

    • 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
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/06—Antianaemics
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01K—ANIMAL HUSBANDRY; AVICULTURE; APICULTURE; PISCICULTURE; FISHING; REARING OR BREEDING ANIMALS, NOT OTHERWISE PROVIDED FOR; NEW BREEDS OF ANIMALS
    • A01K2217/00—Genetically modified animals
    • A01K2217/05—Animals comprising random inserted nucleic acids (transgenic)
    • 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
    • C07K2319/00—Fusion polypeptide

Definitions

  • TNFR tumor necrosis factor receptor
  • a TNFR superfamily member is typically a membrane-bound, trimeric or multimeric complex which is stabilized via intracysteine disulfide bonds that are formed between the cysteine-rich domains of individual subunit members (Banner et al. (1993) Cell
  • the proteins themselves do not have intrinsic catalytic activity, rather they function via association with other proteins to transduce cellular signals.
  • a functional TNFR superfamily protein can also exist in a soluble form. Soluble versions ofthe superfamily bind cognate ligands and influence bioavailability. For instance, the osteoprotegerin protein family exists as a soluble protein. (Simonet et al. (1997) Cell 89:309-319.) Many soluble forms of the TNFR have been identified. Certain soluble TNFRs are elevated in disease states such as lupus and rheumatoid arthritis. (Gabay et al. (1997) J. Rheumatol.
  • the soluble superfamily members lack the transmembrane domain characteristic ofthe majority of superfamily members due to either proteolytic cleavage or, at least in one instance, to alternative splicing (Grass et al. (1995) Blood 85, 3378-3404.)
  • the present invention is based, at least in part, on the discovery of novel molecules ofthe TNF receptor superfamily, referred to herein as "STRIFE” nucleic acid and protein molecules.
  • STRIFE novel molecules ofthe TNF receptor superfamily
  • Two splice forms ofthe "STRIFE” nucleic acid molecule have been identified and are referred to herein as the "STRIFEl” and “STRIFE2” nucleic acid and protein molecules.
  • the STRIFEl and STRIFE2 molecules ofthe present invention are useful as modulating agents in regulating a variety of cellular processes. Accordingly, in one aspect, this invention provides isolated nucleic acid molecules encoding STRIFEl and STRIFE2 proteins or biologically active portions thereof, as - 2 -
  • nucleic acid fragments suitable as primers or hybridization probes for the detection of STRIFEl and STRIFE2-encoding nucleic acids.
  • a STRIFEl nucleic acid molecule is at least about 60%, 65%, 70%, 71%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence shown in SEQ ID NO: 1 , SEQ ID NO:3, SEQ ID NO:4, or a complement thereof.
  • a STRIFE2 nucleic acid molecule is at least about 60%, 65%, 70%, 71%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide sequence shown in SEQ ID NO:5, SEQ ID NO:7, SEQ ID NO:8, or a complement thereof.
  • an isolated STRIFEl nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:3, or a complement thereof.
  • a STRIFEl nucleic acid molecule further comprises nucleotides 1-106 of SEQ ID NO:l.
  • a STRIFEl nucleic acid molecule further comprises nucleotides 751-981 of SEQ ID NO:l.
  • an isolated STRIFEl nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO: 1.
  • an isolated STRIFE2 nucleic acid molecule has the nucleotide sequence shown SEQ ID NO:7, or a complement thereof. In another embodiment, a STRIFE2 nucleic acid molecule further comprises nucleotides 1-109 of SEQ ID NO:5. In yet another preferred embodiment, a STRIFE2 nucleic acid molecule further comprises nucleotides 562-655 of SEQ ID NO:5. In another preferred embodiment, an isolated STRIFE2 nucleic acid molecule has the nucleotide sequence shown in SEQ ID NO:5.
  • a STRIFEl or a STRIFE2 nucleic acid molecule include a nucleotide sequence encoding a protein having an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:6, respectively.
  • a STRIFEl or a STRIFE2 nucleic acid molecule include a nucleotide sequence encoding a protein having an amino acid sequence 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% or more homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:6, respectively.
  • an isolated nucleic acid molecule ofthe present invention encodes a STRIFEl protein which includes a cysteine-rich domain, optionally a signal sequence, and is membrane bound.
  • an isolated nucleic acid molecule ofthe present invention encodes a STRIFEl protein which includes a signal sequence and a cysteine-rich domain, wherein the cysteine-rich domain comprises at least one module, and is membrane bound.
  • a STRIFEl nucleic acid molecule encodes a STRIFEl protein and is a naturally occurring nucleotide sequence. - 3 -
  • an isolated nucleic acid molecule ofthe present invention encodes a STRIFE2 protein which includes a cysteine-rich domain, optionally a signal sequence, and is secreted.
  • an isolated nucleic acid molecule ofthe present invention encodes a STRIFE2 protein which includes a signal sequence and a cysteine-rich domain, wherein the cysteine-rich domain comprises at least one module, and is secreted.
  • a STRIFE2 nucleic acid molecule encodes a STRIFE2 protein and is a naturally occurring nucleotide sequence.
  • STRIFEl or STRIFE2 nucleic acid molecules which specifically detect STRIFEl or STRIFE2 nucleic acid molecules, respectively, relative to nucleic acid molecules encoding non-STRIFEl or non-STRIFE2 proteins.
  • a STRIFEl or STRIFE2 nucleic acid molecule hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotides 107-751, 1-16, 413-602, or 711-981 ofthe nucleotide sequence shown in SEQ ID NO: 1, or to nucleotides 110-562, 1-16, 416-489, or 519-655 of nucleotide sequence shown in SEQ ID NO:5, respectively.
  • the STRIFEl or STRIFE2 nucleic acid molecule is at least 450 nucleotides in length and hybridizes under stringent conditions to a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO:l, or SEQ ID NO:5, respectively, or a complement thereof.
  • Another embodiment ofthe invention provides an isolated nucleic acid molecule which is antisense to the coding strand of a STRIFEl or a STRIFE2 nucleic acid molecule.
  • Another aspect ofthe invention provides a vector comprising a STRIFEl or a STRIFE2 nucleic acid molecule.
  • the vector is a recombinant expression vector.
  • the invention provides a host cell containing a vector ofthe invention.
  • the invention also provides a method for producing a STRIFEl or a STRIFE2 protein by culturing in a suitable medium, a host cell ofthe invention containing a recombinant expression vector such that a STRIFEl or a STRIFE2 protein, respectively, is produced.
  • Another aspect of this invention features isolated or recombinant STRIFEl or
  • an isolated STRIFEl protein has a cysteine-rich domain, optionally a signal sequence, and is membrane bound.
  • an isolated STRIFE2 protein has a cysteine-rich domain, optionally a signal sequence, and is secreted.
  • an isolated STRIFEl or STRIFE2 protein has an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:6, respectively.
  • a STRIFEl protein has an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% or more homologous to the amino acid sequence of SEQ ID NO:2.
  • a STRIFE2 protein has an amino acid sequence at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98%) or more homologous to the amino acid sequence of SEQ ID NO:6.
  • a STRIFEl or a STRIFE2 protein has the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:6, respectively.
  • Another embodiment ofthe invention features an isolated STRIFEl protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 60% homologous to a nucleotide sequence of SEQ ID NO:l, or a complement thereof.
  • Another embodiment ofthe invention features an isolated STRIFE2 protein which is encoded by a nucleic acid molecule having a nucleotide sequence at least about 60% homologous to a nucleotide sequence of SEQ ID NO:5, or a complement thereof.
  • This invention further features an isolated STRIFEl or STRIFE2 protein which is encoded by a nucleic acid molecule having a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising the nucleotide sequence of SEQ ID NO:l, or SEQ ID NO:5, respectively, or a complement thereof.
  • the STRIFEl and STRIFE2 proteins ofthe present invention can be operatively linked to a non-STRIFEl and a non-STRIFE2 polypeptide to form STRIFEl and STRIFE2 fusion proteins.
  • the invention further features antibodies that specifically bind STRIFEl and STRIFE2 proteins, such as monoclonal or polyclonal antibodies.
  • the STRIFEl and STRIFE2 proteins or biologically active portions thereof can be incorporated into pharmaceutical compositions, which optionally include pharmaceutically acceptable carriers.
  • the present invention provides a method for detecting STRIFEl and STRIFE2 expression in a biological sample by contacting the biological sample with an agent capable of detecting a STRIFEl and a STRIFE2 nucleic acid molecule, protein or polypeptide such that the presence of a STRIFEl and a STRIFE2 nucleic acid molecule, protein or polypeptide is detected in the biological sample.
  • the present invention provides a method for detecting the presence of STRIFEl and STRIFE2 activity in a biological sample by contacting the biological sample with an agent capable of detecting an indicator of STRIFEl and STRIFE2 activity such that the presence of STRIFEl and STRIFE2 activity is detected in the biological sample.
  • the invention provides a method for modulating STRIFEl and STRIFE2 activity comprising contacting the cell with an agent that modulates STRIFEl and/or STRIFE2 activity such that STRIFEl and/or STRIFE2 activity in the cell is modulated.
  • the agent inhibits STRIFEl and/or STRIFE2 activity.
  • the agent stimulates STRIFEl and/or STRIFE2 activity.
  • the agent is an antibody that specifically binds to a STRIFEl and/or a STRIFE2 protein.
  • the agent modulates expression of STRIFEl and STRIFE2 by modulating transcription of a STRIFEl and a STRIFE2 gene or translation of a STRIFEl and a STRIFE2 mRNA.
  • the agent is a nucleic acid molecule having a nucleotide sequence that is antisense to the coding strand of a STRIFEl and a STRIFE2 mRNA or a STRIFE gene.
  • the methods ofthe present invention are used to treat a subject having a disorder characterized by aberrant STRIFEl and/or STRIFE2 protein or nucleic acid expression or activity by administering an agent which is a STRIFEl and/or STRIFE2 modulator to the subject.
  • the STRIFEl and STRIFE2 modulator is a STRIFEl and a STRIFE2 protein, respectively.
  • the STRIFEl or STRIFE2 modulator is a STRIFEl or a STRIFE2 nucleic acid molecule, respectively.
  • the STRIFEl and the STRIFE2 modulator is a peptide, peptidomimetic, or other small molecule.
  • the disorder characterized by aberrant STRIFEl and/or STRIFE2 protein or nucleic acid expression is a developmental, differentiative, or proliferative disorder.
  • the present invention also provides a diagnostic assay for identifying the presence or absence of a genetic alteration characterized by at least one of (i) aberrant modification or mutation of a gene encoding a STRIFEl and/or a STRIFE2 protein; (ii) mis-regulation of said gene; and (iii) aberrant post-translational modification of a STRIFEl and/or a STRIFE2 protein, wherein a wild-type form of said gene encodes an protein with a STRIFEl and a STRIFE2 activity, respectively.
  • the invention provides a method for identifying a compound that binds to or modulates the activity of a STRIFEl or a STRIFE2 protein, by providing an indicator composition comprising a STRIFEl and/or STRIFE2 protein having STRIFEl and/or STRIFE2 activity, respectively, contacting the indicator composition with a test compound, and determining the effect ofthe test compound on STRIFEl or STRIFE2 activity in the indicator composition to identify a compound that modulates the activity of a STRIFEl or a STRIFE2 protein, respectively.
  • Figure 1 depicts the cDNA sequence and predicted amino acid sequence of murine STRIFEl.
  • the nucleotide sequence corresponds to nucleic acids 1 to 981 of SEQ ID NO:l.
  • the amino acid sequence corresponds to amino acids 1 to 214 of SEQ ID NO:2.
  • Figure 2 depicts the cDNA sequence and predicted amino acid sequence of murine STRIFE2.
  • the nucleotide sequence corresponds to nucleic acids 1 to 655 of SEQ ID NO:5.
  • the amino acid sequence corresponds to amino acids 1 to 150 of SEQ ID NO:6.
  • Figure 3 depicts an alignment ofthe amino acid sequences of murine STRIFEl
  • FIG. 4 depicts the results from a FASTA search using the amino acid sequence of STRIFEl as a query.
  • Figure 5 depicts the results froma FASTA search using the nucleotide sequence of STRIFEl as a query.
  • TNF receptors are typically membrane-bound, trimeric or multimeric complexes which are stabilized via intracysteine disulfide bonds that are formed between the cysteine-rich domains of individual subunit members (Banner et al. (1993) Cell 73 :431 -445).
  • Functional TNF receptors can also exist in a soluble form. Soluble members ofthe superfamily bind cognate ligands and influence bioavailability.
  • the soluble superfamily members lack the transmembrane domain characteristic ofthe majority of superfamily members due to either proteolytic cleavage or, at least in one instance, to alternative splicing (Grass et al. (1995) Blood 85, 3378-3404).
  • TNF receptors are the sole mediators of Tumor Necrosis Factor (TNF) signaling.
  • TNF Tumor Necrosis Factor
  • TNF is a cytokine that is capable of acting independently or in conjunction with other factors to affect various different body functions.
  • TNF has diverse biological effects, including killing of transformed cells, stimulation of granulocytes and fibroblasts, damage to endothelial cells, and anti-parasitic effects.
  • TNF plays a key role as an endogenous mediator of inflammatory, immune, and host defense functions.
  • TNF plays a role in various neoplastic disease states. - 7 -
  • the STRIFEl and STRIFE2 molecules ofthe present invention having homology to the TNF receptors may also be TNF receptors involved in TNF signaling.
  • the STRIFEl and STRIFE2 molecules ofthe present invention may play a role in mediating inflammatory, immune, and host defense functions.
  • the STRIFEl and STRIFE2 molecules ofthe present invention may play a role in various neoplastic disease states.
  • the STRIFEl and STRIFE2 molecules may be useful as targets for developing novel diagnostic and therapeutic agents to treat TNF-associated disorders and TNF receptor-associated disorders.
  • TNF-associated disorder and “TNF receptor-associated disorder” include any disorder, disease, or condition which is associated with an abnormal or undesired TNF or TNF receptor function or an abnormal or undesired TNF or TNF receptor level, e.g., plasma, tissue, or cellular levels or concentration.
  • TNF-associated and TNF receptor-associated disorders include, but are not limited to, sepsis syndrome, including cachexia; circulatory collapse and shock resulting from acute or chronic bacterial infection; acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections; acute and chronic immune and autoimmune pathologies, such as systemic lupus erythematosus and rheumatoid arthritis; alcohol-induced hepatitis; chronic inflammatory pathologies such as sarcoidosis and Crohn's pathology; vascular inflammatory pathologies such as disseminated intravascular coagulation; graft-versus-host pathology; Rawasaki's pathology; malignant pathologies involving TNF-secreting tumors; cerebral malaria; and multiple sclerosis.
  • sepsis syndrome including cachexia; circulatory collapse and shock resulting from acute or chronic bacterial infection; acute and chronic parasitic or infectious processes, including bacterial, viral and fungal infections; acute and chronic immune and autoimmune pathologies, such as systemic
  • family when referring to the protein and nucleic acid molecules of the invention is intended to mean two or more proteins or nucleic acid molecules having a common structural domain and having sufficient amino acid or nucleotide sequence homology as defined herein.
  • family members can be naturally occurring and can be from either the same or different species.
  • a family can contain a first protein of human origin, as well as other, distinct proteins of human origin or alternatively, can contain homologues of non-human origin.
  • Members of a family may also have common functional characteristics.
  • a STRIFEl and a STRIFE2 family member is identified based on the presence of at least one "cysteine-rich domain" in the protein or corresponding nucleic acid molecule.
  • the term "cysteine-rich domain” refers to a protein domain of about 110-160 amino acid residues in length, preferably about 100-150 amino acid residues in length, more preferably about 90-140 amino acid residues in length, and even more preferably at least about 80-130 amino acid residues in length, of which at least about 10-30, preferably about 10-20, and more preferably about - 8 -
  • cysteine residues 12, 13, 14, or 15 amino acid residues are cysteine residues.
  • a cysteine-rich domain is located in the N-terminal region of a STRIFEl and STRIFE2 protein and includes about amino acid residues 34 through 114 of SEQ ID NO:2 and SEQ ID NO:6, respectively.
  • Preferred cysteine rich domains contain at least about two, three, or four modules or motifs, wherein each module is a region of about 20-60 amino acid residues in length, preferably 30-50 amino acid residues in length, more preferably 40 amino acid residues in length and includes about 3-10 cysteines, preferably 5-7 cysteines, and more preferably 6 cysteines.
  • the module has the following motif: C-Xaal(4-14)-C-Xaa2(0-2)-C-Xaa3(2-4)-C-Xaa4(6-12)-C-Xaa5(6-10)-C(SEQ ID NO:
  • Xaal is between 4-6, 6-8, 8-10, 10-12, or 12-14 amino acid residues; Xaa4 is between 6-8, 8-10, or 10-12 amino acid residues; and Xaa5 is between 6-8 or 8-10 amino acid residues.
  • Xaal is 4-6 amino acid residues, of which at least one is the amino acid phenylalanine, at least one is the amino acid tyrosine, and/or at least one is the amino acid histidine.
  • Xaa5 is 6-10 amino acid residues, of which at least one is the amino acid aspartic acid, at least one is the amino acid asparagine, at least one is the amino acid glutamic acid, at least one is the amino acid glutamine, at least one is the amino acid serine, at least one is the amino acid lysine, and/or at least one is the amino acid proline.
  • the module has the following motif:
  • a STRIFEl protein contains a cysteine-rich domain including a first module containing about amino acids 34-72 of SEQ ID NO:2 (shown separately as SEQ ID NO:l 1) having 6 cysteine residues at positions indicated by the aforementioned motifs, and a second module containing about amino acids 75-114 of SEQ ID NO:2 (shown separately as SEQ ID NO: 12) having 6 cysteine residues at positions indicated by the aforementioned motifs.
  • a STRIFE2 protein contains a cysteine rich domain including a first module containing about amino acids 34-72 of SEQ ID NO:6 (shown separately as SEQ ID NO: 14) having 6 cysteine residues at positions indicated by the aforementioned motifs, and a second module containing about amino acids 75-114 of SEQ ID NO:6 (shown separately as SEQ ID NO: 15) having 6 cysteine residues at positions indicated by the aforementioned motifs.
  • a STRIFEl and STRIFE2 protein has at least one cysteine-rich domain and a signal sequence.
  • a signal sequence refers to a peptide containing about 20 amino acids which occurs at the N- terminus of secretory and integral membrane proteins and which contains a large number of hydrophobic amino acid residues.
  • a signal sequence contains at least about 14-28 amino acid residues, preferably about 16-26 amino acid residues, more preferably about 18-24 amino acid residues, and more preferably about 20-22 amino acid residues, and has at least about 40-70%, preferably about 50-65%, and more preferably about 55-60% hydrophobic amino acid residues (e.g., Alanine.
  • a signal sequence serves to direct a protein containing such a sequence to a lipid bilayer.
  • a STRIFEl protein contains a signal sequence of about amino acids 1-29 of SEQ ID NO:2 (shown separately as SEQ ID NO:9).
  • a STRIFE2 protein contains a signal sequence of about amino acids 1 -29 of SEQ ID NO: 6 (shown separately as SEQ ID NO: 13).
  • one embodiment ofthe invention features a STRIFEl or a STRIFE2 protein having at least one cysteine-rich domain.
  • Another embodiment features a STRIFEl or a STRIFE2 protein having at least one cysteine-rich domain, wherein the cysteine-rich domain includes at least one module having the predicted motif of SEQ ID NO: 16.
  • Another embodiment features a STRIFEl or a STRIFE2 protein having at least one cysteine-rich domain, wherein the cysteine-rich domain includes at least two modules.
  • Another embodiment features a protein having 20, 30, 40, 50, 60, 70, 80, 90, 95, or 99% homology to a cysteine-rich domain of a STRIFEl or a STRIFE2 protein of the invention.
  • Yet another embodiment ofthe invention features a STRIFEl or a STRIFE2 protein having at least one cysteine-rich domain and a signal peptide.
  • Another embodiment features a STRIFEl or a STRIFE2 protein having at least one cysteine-rich domain and a signal peptide, wherein the cysteine-rich domain includes at least one module having the predicted motif of SEQ ID NO: 16.
  • a STRIFEl protein has a transmembrane domain.
  • transmembrane domain refers to a structural amino acid motif which includes a hydrophobic helix that spans the plasma membrane.
  • a transmembrane domain preferably includes a series of hydrophobic residues, such as leucine, valine, and tyrosine residues.
  • a STRIFEl protein contains a transmembrane domain containing amino acids 169-193 of SEQ ID NO:2 (shown seperately as SEQ ID NO: 10). - 10 -
  • Preferred STRIFEl or STRIFE2 molecules ofthe present invention have an amino acid sequence sufficiently homologous to the amino acid sequence of SEQ ID NO:2, or SEQ ID NO:6, respectively.
  • the term "sufficiently homologous" refers to a first amino acid or nucleotide sequence which contains a sufficient or minimum number of identical or equivalent (e.g., an amino acid residue which has a similar side chain) amino acid residues or nucleotides to a second amino acid or nucleotide sequence such that the first and second amino acid or nucleotide sequences share common structural domains and/or a common functional activity.
  • amino acid or nucleotide sequences which share common structural domains have at least about 40% homology, preferably 50%) homology, more preferably 60%-70% homology across the amino acid sequences of the domains and contain at least one, preferably two, more preferably three, and even more preferably four, five or six structural domains, are defined herein as sufficiently homologous.
  • amino acid or nucleotide sequences which share at least 40%, preferably 50%, more preferably 60, 70, or 80% homology and share a common functional activity are defined herein as sufficiently homologous.
  • a "STRIFEl and a STRIFE2 activity” refers to an activity exerted by a STRIFEl and a STRIFE2 protein, polypeptide or nucleic acid molecule on a STRIFEl or a STRIFE2 responsive cell as determined in vivo, or in vitro, according to standard techniques.
  • a STRIFEl and a STRIFE2 activity is a direct activity, such as an association with a STRIFEl or a STRIFE2 -target molecule.
  • a "target molecule” is a molecule with which a STRIFEl and a STRIFE2 protein binds or interacts in nature, such that STRIFEl or STRIFE2 -mediated function is achieved.
  • a STRIFEl or a STRIFE2 target molecule can be a non-STRIFEl and a non-STRIFE2 molecule or a STRIFEl or STRIFE2 protein or polypeptide ofthe present invention.
  • a STRIFE2 target molecule is a membrane-bound protein (e.g., a "STRIFE2 receptor") or a modified form of such a protein which has been altered such that the protein is soluble (e.g., recombinantly produced such that the protein does not express a membrane-binding domain).
  • STRIFE2 target is a second soluble protein molecule (e.g., a "STRIFEl or a STRIFE2 binding partner" or a "STRIFEl and STRIFE2 substrate").
  • a STRIFEl and a STRIFE2 binding partner can be a soluble non-STRIFEl and non-STRIFE2 protein or a second STRIFEl and a STRIFE2 protein molecule ofthe present invention.
  • a STRIFEl and a STRIFE2 activity is an indirect activity, such as a cellular signaling activity mediated by interaction ofthe STRIFEl and - 11 -
  • the STRIFE2 protein with a second protein (e.g., a STRIFEl ligand or a STRIFE2 receptor).
  • a second protein e.g., a STRIFEl ligand or a STRIFE2 receptor
  • a STRIFEl activity is at least one or more ofthe following activities: (i) interaction of a STRIFEl protein on the cell surface with a second non-STRIFEl protein molecule on the surface ofthe same cell; (ii) interaction of a STRIFEl protein on the cell surface with a second non-STRIFEl protein molecule on the surface of a different cell; (iii) complex formation between a membrane-bound STRIFEl protein and a cytokine, e.g., TNF; (iv) interaction of a STRIFEl protein with an intracellular protein including SH2 domain-containing proteins or cytoskeletal proteins; (v) formation of a homogeneous multimeric signaling complex with STRIFE 1- like proteins; and (vi) formation of a heterogeneous multimeric signaling complex with other TNFR superfamily proteins.
  • a STRIFE2 activity is at least one or more of the following activities: (i) interaction of a STRIFE2 protein with a membrane-bound STRIFE2 receptor; (ii) interaction of a STRIFE2 protein with a soluble form of a
  • STRIFE2 receptor interaction of a STRIFE2 protein with an intracellular protein via a membrane-bound STRIFE2 receptor; (iv) complex formation between a soluble STRIFE2 protein and a second soluble STRIFE2 binding partner; (v) complex formation between a soluble STRIFE2 protein and a second soluble STRIFE2 binding partner, wherein the STRIFE2 binding partner is a non-STRIFE2 protein molecule; and (vi) complex formation between a soluble STRIFE2 protein and a second soluble STRIFE2 binding partner, wherein the STRIFE2 binding partner is a second STRIFE2 protein molecule.
  • a STRIFEl or a STRIFE2 activity is at least one or more ofthe following activities: (i) modulation of cellular signal transduction, either in vitro or in vivo; (ii) regulation of gene transcription in a cell involved in development or differentiation, either in vitro or in vivo; (iii) modulation of cellular signal transduction; (iv) regulation of cellular proliferation; (v) regulation of cellular differentiation; and (vi) regulation of cell survival.
  • another embodiment ofthe invention features isolated STRIFEl and STRIFE2 proteins and polypeptides having a STRIFEl and/or STRIFE2 activity, respectively.
  • STRIFEl and STRIFE2 proteins have at least one cysteine-rich domain and a STRIFEl and/or a STRIFE2 activity.
  • the STRIFEl and STRIFE2 protein has at least one cysteine-rich domain, wherein the cysteine-rich domain comprises at least one module, and a STRIFEl and STRIFE2 activity, respectively.
  • the STRIFEl and STRIFE2 protein has at least one cysteine-rich domain, wherein the cysteine-rich domain - 12 -
  • a STRIFEl and STRIFE2 protein further comprises a signal sequence.
  • a STRIFEl and a STRIFE2 protein has a cysteine-rich domain, a STRIFEl and a STRIFE2 activity, and an amino acid sequence sufficiently homologous to an amino acid sequence of SEQ ID NO:2, or SEQ ID NO:6, respectively.
  • the murine STRIFEl cDNA which is approximately 981 nucleotides in length, encodes a protein which is approximately 214 amino acid residues in length.
  • the murine STRIFEl protein contains an N-terminal signal sequence and a cysteine-rich domain comprising two modules.
  • a STRIFEl cysteine-rich domain can be found at least, for example, from about amino acids 34-114 of SEQ ID NO:2.
  • the STRIFEl cysteine-rich domain comprises a first module from about amino acids 34-72 of SEQ ID NO:2 (shown separately as SEQ ID NO:l 1) and a second module from about amino acids 75-114 of SEQ ID NO:2 (shown separately as SEQ ID NO: 12).
  • the murine STRIFEl protein is a membrane bound protein which contains a transmembrane domain at about amino acids 169-193 of SEQ ID NO:2 (shown seperately as SEQ ID NO: 10) and a signal sequence at about amino acids 1-29 of SEQ ID NO:2 (shown separately as SEQ ID NO:9).
  • SIGNALP Computer algorithm
  • the murine STRIFE2 cDNA which is approximately 655 nucleotides in length, encodes a protein which is approximately 150 amino acid residues in length.
  • the murine STRIFE2 protein contains an N-terminal signal sequence and a cysteine-rich domain comprising two modules.
  • a STRIFE2 cysteine-rich domain can be found at least, for example, from about amino acids 34-114 of SEQ ID NO:6.
  • the STRIFE2 cysteine-rich domain comprises a first module from about amino acids 34-72 of SEQ ID NO: 6 (shown separately as SEQ ID NO: 14) and a second module from about amino acids 75-114 of SEQ ID NO:6 (shown separately as SEQ ID NO: 15).
  • the murine STRIFE2 protein is a secreted protein which further contains a signal sequence at about amino acids 1 -29 of SEQ ID NO:6 (shown separately as SEQ ID NO: 13).
  • I. Isolated Nucleic Acid Molecules One aspect ofthe invention pertains to isolated nucleic acid molecules which encode STRIFEl and STRIFE2 proteins or biologically active portions thereof, as well as nucleic acid fragments sufficient for use as hybridization probes to identify STRIFEl - 13 -
  • nucleic acid molecule is intended to include DNA molecules (e.g., cDNA or genomic DNA) and RNA molecules (e.g., mRNA) and analogs ofthe DNA or RNA generated using nucleotide analogs.
  • the nucleic acid molecule can be single-stranded or double-stranded, but preferably is double-stranded DNA.
  • an “isolated” nucleic acid molecule is one which is separated from other nucleic acid molecules which are present in the natural source ofthe nucleic acid.
  • an “isolated” nucleic acid is free of sequences which naturally flank the nucleic acid (i.e., sequences located at the 5' and 3' ends ofthe nucleic acid) in the genomic DNA ofthe organism from which the nucleic acid is derived.
  • the isolated STRIFEl and STRIFE2 nucleic acid molecule can contain less than about 5 kb, 4kb, 3kb, 2kb, 1 kb, 0.5 kb or 0.1 kb of nucleotide sequences which naturally flank the nucleic acid molecule in genomic DNA ofthe cell from which the nucleic acid is derived.
  • an "isolated" nucleic acid molecule such as a cDNA molecule, can be substantially free of other cellular material, or culture medium when produced by recombinant techniques, or substantially free of chemical precursors or other chemicals when chemically synthesized.
  • a nucleic acid molecule ofthe present invention e.g., a nucleic acid molecule having the nucleotide sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 or a portion thereof, can be isolated using standard molecular biology techniques and the sequence information provided herein.
  • STRIFEl and STRIFE2 nucleic acid molecules can be isolated using standard hybridization and cloning techniques (e.g., as described in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
  • nucleic acid molecule encompassing all or a portion of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 can be isolated by the polymerase chain reaction (PCR) using synthetic oligonucleotide primers designed based upon the sequence of SEQ ID NO:l, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8.
  • PCR polymerase chain reaction
  • a nucleic acid ofthe invention can be amplified using cDNA, mRNA or alternatively, genomic DNA, as a template and appropriate oligonucleotide primers according to standard PCR amplification techniques.
  • the nucleic acid so amplified can be cloned into an appropriate vector and characterized by DNA sequence analysis.
  • oligonucleotides corresponding to STRIFEl and STRIFE2 nucleotide sequences can be prepared by standard synthetic techniques, e.g., using an automated DNA synthesizer.
  • an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:l .
  • the sequence of SEQ ID NO:l corresponds to the murine STRIFEl cDNA.
  • This cDNA comprises sequences encoding the murine STRIFEl protein (i.e., "the coding region", from nucleotides 107- 751), as well as 5' untranslated sequences (nucleotides 1 to 106) and 3' untranslated sequences (nucleotides 752-981).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO:l (e.g., nucleotides 107-751, corresponding to SEQ ID NO:3).
  • an isolated nucleic acid molecule ofthe invention comprises the nucleotide sequence shown in SEQ ID NO:5.
  • the sequence of SEQ ID NO: 5 corresponds to the murine STRIFE2 cDNA.
  • This cDNA comprises sequences encoding the murine STRIFE2 protein (i.e., "the coding region", from nucleotides 110-562), as well as 5' untranslated sequences (nucleotides 1-109) and 3' untranslated sequences (nucleotides 563-655).
  • the nucleic acid molecule can comprise only the coding region of SEQ ID NO:5 (e.g., nucleotides 110-562, corresponding to SEQ ID NO:7).
  • an isolated nucleic acid molecule of the invention comprises a nucleic acid molecule which is a complement ofthe nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID , or a portion of either of these nucleotide sequences.
  • a nucleic acid molecule which is complementary to the nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 is one which is sufficiently complementary to the nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 such that it can hybridize to the nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, thereby forming a stable duplex.
  • an isolated nucleic acid molecule ofthe present invention comprises a nucleotide sequence which is at least about 60%, 65%, 70%, 71%, 75%, 80%, 85%, 90%, 95%, 98% or more homologous to the nucleotide - 15 -
  • sequences show in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, or a portion of either of these nucleotide sequences larger than 450 bp.
  • the nucleic acid molecule ofthe invention can comprise only a portion ofthe nucleic acid sequence of SEQ ID NOT, or SEQ ID NO:5, for example a fragment which can be used as a probe or primer or a fragment encoding a biologically active portion of a STRIFEl or a STRIFE2 protein.
  • the nucleotide sequence determined from the cloning ofthe murine STRIFEl and STRIFE2 genes allows for the generation of probes and primers designed for use in identifying and/or cloning STRIFE homologues in other cell types, e.g., from other tissues, as well as STRIFE homologues from other mammals including humans.
  • the probe/primer typically comprises a substantially purified oligonucleotide.
  • the oligonucleotide typically comprises a region of nucleotide sequence that hybridizes under stringent conditions to at least about 12, preferably about 25, more preferably about 40, 50 or 75 consecutive nucleotides of a sense sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, of an antisense sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, or of a naturally occurring mutant of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8.
  • a nucleic acid molecule ofthe present invention comprises a nucleotide sequence which hybridizes under stringent hybridization conditions to a nucleic acid molecule comprising nucleotides 1-16, 413-602, or 711-981 of SEQ ID NOT or to a nucleic acid molecule comprising nucleotides 1-16, 416-489, or 519-655 of SEQ ID NO:5.
  • Probes based on the murine STRIFEl and STRIFE2 nucleotide sequence can be used to detect transcripts or genomic sequences encoding the same or homologous proteins.
  • the probe further comprises a label group attached thereto, e.g., the label group can be a radioisotope, a fluorescent compound, an enzyme, or an enzyme co-factor.
  • Such probes can be used as a part of a diagnostic test kit for identifying cells or tissue which misexpress a STRIFEl or a STRIFE2 protein, such as by measuring a level of a STRIFEl or a STRIFE2-encoding nucleic acid in a sample of cells from a subject e.g., detecting STRIFEl or STRIFE2 mRNA levels or determining whether a genomic STRIFEl or STRIFE2 gene has been mutated or deleted.
  • a nucleic acid fragment encoding a "biologically active portion of a STRIFEl or a STRIFE2 protein" can be prepared by isolating a portion of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 which encodes a polypeptide having a STRIFEl or a STRIFE2 biological activity (the biological activities ofthe STRIFEl and STRIFE2 proteins include biological activities attributed - 16 -
  • the TNFR super-family of proteins expressing the encoded portion ofthe STRIFEl or the STRIFE2 protein (e.g., by recombinant expression in vitro) and assessing the activity ofthe encoded portion ofthe STRIFEl or STRIFE2 protein.
  • the invention further encompasses nucleic acid molecules that differ from the nucleotide sequence shown in SEQ ID NO , SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 due to degeneracy ofthe genetic code and thus encode the same STRIFEl or STRIFE2 proteins as those encoded by the nucleotide sequence shown in SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8, respectively.
  • an isolated nucleic acid molecule of the invention has a nucleotide sequence encoding a protein having an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:6.
  • the terms "gene” and “recombinant gene” refer to nucleic acid molecules comprising an open reading frame encoding a STRIFEl or STRIFE2 protein, preferably a mammalian STRIFEl or STRIFE2 protein.
  • Such natural allelic variations can typically result in 1-5% variance in the nucleotide sequence of a STRIFEl or a STRIFE2 gene. Any and all such nucleotide variations and resulting amino acid polymorphisms in STRIFEl or STRIFE2 genes that are the result of natural allelic variation and that do not alter the functional activity of a STRIFEl or STRIFE2 protein are intended to be within the scope ofthe invention.
  • nucleic acid molecules encoding STRIFEl and STRIFE2 proteins from other species and thus which have a nucleotide sequence which differs from the murine sequence of SEQ ID NO: 1 and SEQ ID NO: 5 are intended to be within the scope ofthe invention.
  • Nucleic acid molecules corresponding to natural allelic variants and homologues ofthe STRIFEl or STRIFE2 cDNAs ofthe invention can be isolated based on their homology to the murine STRIFEl or STRIFE2 nucleic acids disclosed herein using the murine cDNA, or a portion thereof, as a hybridization probe according to standard hybridization techniques under stringent hybridization conditions.
  • an isolated nucleic acid molecule ofthe invention is at least 15 nucleotides in length and hybridizes under stringent conditions to the nucleic acid molecule comprising the nucleotide sequence of SEQ ID NOT, SEQ ID - 17 -
  • the nucleic acid is at least 30, 50, 100, 250 or 500 nucleotides in length.
  • hybridizes under stringent conditions is intended to describe conditions for hybridization and washing under which nucleotide sequences at least 60% homologous to each other typically remain hybridized to each other.
  • the conditions are such that sequences at least about 70%, more preferably at least about 80%), even more preferably at least about 85% or 90% homologous to each other typically remain hybridized to each other.
  • stringent hybridization conditions are known to those skilled in the art and can be found in Current Protocols in Molecular Biology, John Wiley & Sons, N.Y. (1989), 6.3.1-6.3.6.
  • a preferred, non-limiting example of stringent hybridization conditions are hybridization in 6X sodium chloride/sodium citrate (SSC) at about 45°C, followed by one or more washes in 0.2 X SSC, 0.1% SDS at 50-65°C.
  • an isolated nucleic acid molecule ofthe invention that hybridizes under stringent conditions to the sequence of SEQ ID NO: 1 corresponds to a naturally- occurring nucleic acid molecule.
  • a "naturally-occurring" nucleic acid molecule refers to an RNA or DNA molecule having a nucleotide sequence that occurs in nature (e.g., encodes a natural protein).
  • allelic variants ofthe STRIFEl or STRIFE2 sequences that may exist in the population, the skilled artisan will further appreciate that changes can be introduced by mutation into the nucleotide sequences of SEQ ID NO , or SEQ ID NO:5, thereby leading to changes in the amino acid sequence ofthe encoded STRIFEl or STRIFE2 proteins, without altering the functional ability ofthe STRIFEl or STRIFE2 proteins.
  • nucleotide substitutions leading to amino acid substitutions at "non-essential" amino acid residues can be made in the sequence of SEQ ID NO , or SEQ ID NO:5.
  • non-essential amino acid residue is a residue that can be altered from the wild-type sequence of STRIFEl or STRIFE2 (e.g., the sequence of SEQ ID NO:2 or SEQ ID NO:6) without altering the biological activity, whereas an "essential" amino acid residue is required for biological activity.
  • amino acid residues that are conserved among the STRIFEl or STRIFE2 proteins ofthe present invention are predicted to be particularly unamenable to alteration.
  • amino acid residues that are conserved between STRIFEl or STRIFE2 protein and other proteins having cysteine-rich domains are not likely to be amenable to alteration.
  • nucleic acid molecules encoding STRIFEl or STRIFE2 proteins that contain changes in amino acid residues that are not essential for activity.
  • STRIFEl or STRIFE2 proteins differ in amino acid sequence from SEQ ID NO:2 or SEQ ID NO:6 yet retain biological activity.
  • the isolated nucleic acid molecule comprises a nucleotide sequence - 18 -
  • the protein comprises an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO.10, SEQ ID NOT 1, SEQ ID NO.12, SEQ ID NO:13, SEQ ID NO: 14, or SEQ ID NO: 15.
  • the protein encoded by the nucleic acid molecule is at least about 60%, 65%, 70%, 75%, 80%, 85%, 86%, 90%, 95%, 98% or more homologous to SEQ ID NO:2, SEQ ID NO:6, SEQ ID NO:9, SEQ ID NO: 10, SEQ ID NOT 1, SEQ ID NOT2, SEQ ID NOT3, SEQ ID NO.14, or SEQ ID NOT5.
  • An isolated nucleic acid molecule encoding a STRIFEl or STRIFE2 protein homologous to the protein of SEQ ID NO:2 or SEQ ID NO:6, respectively, can be created by introducing one or more nucleotide substitutions, additions or deletions into the nucleotide sequence of SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO: 7, such that one or more amino acid substitutions, additions or deletions are introduced into the encoded protein. Mutations can be introduced into SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:5, or SEQ ID NO:7 by standard techniques, such as site- directed mutagenesis and PCR-mediated mutagenesis.
  • conservative amino acid substitutions are made at one or more predicted non-essential amino acid residues.
  • a "conservative amino acid substitution” is one in which the amino acid residue is replaced with an amino acid residue having a similar side chain. Families of amino acid residues having similar side chains have been defined in the art.
  • amino acids with basic side chains e.g., lysine, arginine, histidine
  • acidic side chains e.g., aspartic acid, glutamic acid
  • uncharged polar side chains e.g., glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine
  • nonpolar side chains e.g., alanine, valine, leucine, isoleucine, proline, phenylalanine, methionine, tryptophan
  • beta-branched side chains e.g., threonine, valine, isoleucine
  • aromatic side chains e.g., tyrosine, phenylalanine, tryptophan, histidine
  • a predicted nonessential amino acid residue in a STRIFEl or STRIFE2 protein is preferably replaced with another amino acid residue from the same side chain family.
  • mutations can be introduced randomly along all or part of a STRIFEl or STRIFE2 coding sequence, such as by saturation mutagenesis, and the resultant mutants can be screened for STRIFEl or STRIFE2 biological activity to identify mutants that retain activity.
  • SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8 the encoded protein can be expressed recombinantly and the activity ofthe protein can be determined.
  • a mutant STRIFEl or STRIFE2 protein can be assayed for the ability to (i) modulate cellular signal transduction, either in vitro or in vivo; (ii) regulate gene transcription in a cell involved in development or differentiation, either in vitro or in vivo; (iii) modulate cellular signal transduction; (iv) regulate cellular - 19 -
  • an antisense nucleic acid comprises a nucleotide sequence which is complementary to a "sense" nucleic acid encoding a protein, e.g., complementary to the coding strand of a double-stranded cDNA molecule or complementary to an mRNA sequence. Accordingly, an antisense nucleic acid can hydrogen bond to a sense nucleic acid.
  • the antisense nucleic acid can be complementary to an entire STRIFEl or STRIFE2 coding strand, or to only a portion thereof.
  • an antisense nucleic acid molecule is antisense to a "coding region" ofthe coding strand of a nucleotide sequence encoding STRIFEl or STRIFE2.
  • the term "coding region” refers to the region ofthe nucleotide sequence comprising codons which are translated into amino acid residues (e.g., the coding region of murine STRIFEl corresponds to SEQ ID NO: 3 and the coding region of murine STRIFE2 corresponds to SEQ ID NO:7).
  • the antisense nucleic acid molecule is antisense to a "noncoding region" ofthe coding strand of a nucleotide sequence encoding STRIFEl or STRIFE2.
  • the term “noncoding region” refers to 5' and 3' sequences which flank the coding region that are not translated into amino acids (i.e., also referred to as 5' and 3' untranslated regions).
  • antisense nucleic acids ofthe invention can be designed according to the rales of Watson and Crick base pairing.
  • the antisense nucleic acid molecule can be complementary to the entire coding region of STRIFEl or STRIFE2 mRNA, but more preferably is an oligonucleotide which is antisense to only a portion ofthe coding or noncoding region of STRIFEl or STRIFE2 mRNA.
  • the antisense oligonucleotide can be complementary to the region surrounding the translation start site of STRIFEl or STRIFE2 mRNA.
  • An antisense oligonucleotide can be, for example, about 5, 10, 15, 20, 25, 30, 35, 40, 45 or 50 nucleotides in length.
  • An antisense nucleic acid ofthe invention can be constructed using chemical synthesis and enzymatic ligation reactions using procedures known in the art.
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • an antisense nucleic acid e.g., an antisense oligonucleotide
  • modified nucleotides which can be used to generate the antisense nucleic acid include 5- - 20 -
  • fluorouracil 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4- acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-2- thiouridine, 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5- methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5- methoxyaminomethyl-2-thiouracil, beta-D-mannosylqueosine, 5'- methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6
  • the antisense nucleic acid can be produced biologically using an expression vector into which a nucleic acid has been subcloned in an antisense orientation (i.e., RNA transcribed from the inserted nucleic acid will be of an antisense orientation to a target nucleic acid of interest, described further in the following subsection).
  • the antisense nucleic acid molecules ofthe invention are typically administered to a subject or generated in situ such that they hybridize with or bind to cellular mRNA and/or genomic DNA encoding a STRIFEl or STRIFE2 protein to thereby inhibit expression ofthe protein, e.g., by inhibiting transcription and/or translation.
  • the hybridization can be by conventional nucleotide complementarity to form a stable duplex, or, for example, in the case of an antisense nucleic acid molecule which binds to DNA duplexes, through specific interactions in the major groove ofthe double helix.
  • An example of a route of administration of antisense nucleic acid molecules ofthe invention include direct injection at a tissue site.
  • antisense nucleic acid molecules can be modified to target selected cells and then administered systemically.
  • antisense molecules can be modified such that they specifically bind to receptors or antigens expressed on a selected cell surface, e.g., by linking the antisense nucleic acid molecules to peptides or antibodies which bind to cell surface receptors or antigens.
  • the antisense nucleic acid molecules can also be delivered to cells using the vectors described herein. To achieve sufficient intracellular concentrations ofthe antisense molecules, vector constructs in which the antisense nucleic acid molecule is placed under the control of a strong pol II or pol III promoter are preferred.
  • the antisense nucleic acid molecule ofthe invention is an ⁇ -anomeric nucleic acid molecule.
  • An ⁇ -anomeric nucleic acid molecule forms specific double-stranded hybrids with complementary RNA in which, contrary to the - 21 -
  • an antisense nucleic acid ofthe invention is a ribozyme.
  • Ribozymes are catalytic RNA molecules with ribonuclease activity which are capable of cleaving a single-stranded nucleic acid, such as an mRNA, to which they have a complementary region.
  • ribozymes e.g., hammerhead ribozymes (described in Haselhoff and Gerlach (1988) Nature 334:585-591)
  • a ribozyme having specificity for a STRIFEl or STRIFE2-encoding nucleic acid can be designed based upon the nucleotide sequence of a STRIFEl or STRIFE2 cDNA disclosed herein (i.e., SEQ ID NOT, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8).
  • SEQ ID NOT SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:7, or SEQ ID NO:8
  • a derivative of a Tetrahymena L- 19 IVS RNA can be constracted in which the nucleotide sequence ofthe active site is complementary to the nucleotide sequence to be cleaved in a STRIFEl or STRIFE2-encoding mRNA. See, e.g., Cech et al.
  • STRIFEl or STRIFE2 mRNA can be used to select a catalytic RNA having a specific ribonuclease activity from a pool of RNA molecules. See, e.g., Bartel, D. and Szostak, J.W. (1993) Science 261 :1411-1418.
  • STRIFEl or STRIFE2 gene expression can be inhibited by targeting nucleotide sequences complementary to the regulatory region ofthe STRIFEl or STRIFE2 (e.g., the STRIFEl or STRIFE2 promoter and/or enhancers) to form triple helical structures that prevent transcription ofthe STRIFEl or STRIFE2 gene in target cells.
  • nucleotide sequences complementary to the regulatory region ofthe STRIFEl or STRIFE2 e.g., the STRIFEl or STRIFE2 promoter and/or enhancers
  • the STRIFEl or STRIFE2 nucleic acid molecules of the present invention can be modified at the base moiety, sugar moiety or phosphate backbone to improve, e.g., the stability, hybridization, or solubility ofthe molecule.
  • the deoxyribose phosphate backbone ofthe nucleic acid molecules can be modified to generate peptide nucleic acids (see Hyrup B. et al. (1996) Bioorganic & Medicinal Chemistry 4 (1): 5-23).
  • peptide nucleic acids or "PNAs” refer to nucleic acid mimics, e.g., DNA mimics, in which the deoxyribose phosphate backbone is replaced by a pseudopeptide backbone and only the four natural nucleobases are retained.
  • the neutral backbone of PNAs has been shown to allow for - 22 -
  • PNA oligomers can be synthesized using standard solid phase peptide synthesis protocols as described in Hyrup B. et al. (1996) supra; Perry-O'Keefe et al. PNAS 93: 14670-675.
  • PNAs of STRIFEl or STRIFE2 nucleic acid molecules can be used for therapeutic and diagnostic applications.
  • PNAs can be used as antisense or antigene agents for sequence-specific modulation of gene expression by, for example, inducing transcription or translation arrest or inhibiting replication.
  • PNAs of STRIFEl or STRIFE2 nucleic acid molecules can also be used in the analysis of single base pair mutations in a gene, (e.g., by PNA-directed PCR clamping); as 'artificial restriction enzymes' when used in combination with other enzymes, (e.g., SI nucleases (Hyrup B. (1996) supra)); or as probes or primers for DNA sequencing or hybridization (Hyrup B. et al. (1996) supra; Perry-O'Keefe supra).
  • PNAs of STRIFEl or STRIFE2 can be modified, (e.g., to enhance their stability or cellular uptake), by attaching lipophilic or other helper groups to PNA, by the formation of PNA-DNA chimeras, or by the use of liposomes or other techniques of drug delivery known in the art.
  • PNA-DNA chimeras of STRIFEl or STRIFE2 nucleic acid molecules can be generated which may combine the advantageous properties of PNA and DNA.
  • Such chimeras allow DNA recognition enzymes, (e.g., RNAse H and DNA polymerases), to interact with the DNA portion while the PNA portion would provide high binding affinity and specificity.
  • PNA-DNA chimeras can be linked using linkers of appropriate lengths selected in terms of base stacking, number of bonds between the nucleobases, and orientation (Hyrup B. (1996) supra).
  • the synthesis of PNA-DNA chimeras can be performed as described in Hyrup B. (1996) supra and Finn P.J. et al. (1996) Nucleic Acids Res. 24 (17): 3357-63.
  • a DNA chain can be synthesized on a solid support using standard phosphoramidite coupling chemistry and modified nucleoside analogs, e.g., 5'-(4- methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite, can be used as a between the PNA and the 5' end of DNA (Mag, M. et al. (1989) Nucleic Acid Res. 17: 5973-88). PNA monomers are then coupled in a stepwise manner to produce a chimeric molecule with a 5' PNA segment and a 3' DNA segment (Finn P.J. et al. (1996) supra).
  • modified nucleoside analogs e.g., 5'-(4- methoxytrityl)amino-5'-deoxy-thymidine phosphoramidite
  • chimeric moleclues can be synthesized with a 5' DNA segment and a 3' PNA segment (Peterser, K.H. et al. (1975) Bioorganic Med. Chem. Lett. 5: 1119-11124).
  • the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g., Letsinger et al. (1989) Proc. Natl. Acad. Sci. US. 86:6553-6556; Lemaitre et al. (1987) Proc. Natl. Acad. Sci. USA 84:648-652; - 23 -
  • oligonucleotides can be modified with hybridization-triggered cleavage agents (See, e.g., Krol et al. (1988) BioTechniques 6:958-976) or intercalating agents. (See, e.g., Zon (1988) Pharm. Res. 5:539-549).
  • the oligonucleotide may be conjugated to another molecule, (e.g., a peptide, hybridization triggered cross-linking agent, transport agent, or hybridization-triggered cleavage agent).
  • One aspect ofthe invention pertains to isolated STRIFEl and STRIFE2 proteins, and biologically active portions thereof, as well as polypeptide fragments suitable for use as immunogens to raise anti-STRIFEl and STRIFE2 antibodies.
  • native STRIFEl or STRIFE2 proteins can be isolated from cells or tissue sources by an appropriate purification scheme using standard protein purification techniques.
  • STRIFEl or STRIFE2 proteins are produced by recombinant DNA techniques.
  • a STRIFEl or STRIFE2 protein or polypeptide can be synthesized chemically using standard peptide synthesis techniques.
  • an “isolated” or “purified” protein or biologically active portion thereof is substantially free of cellular material or other contaminating proteins from the cell or tissue source from which the STRIFEl or STRIFE2 protein is derived, or substantially free from chemical precursors or other chemicals when chemically synthesized.
  • the language “substantially free of cellular material” includes preparations of STRIFEl or STRIFE2 protein in which the protein is separated from cellular components ofthe cells from which it is isolated or recombinantly produced.
  • the language "substantially free of cellular material” includes preparations of STRIFEl or STRIFE2 protein having less than about 30% (by dry weight) of non-STRIFEl or STRIFE2 protein (also referred to herein as a "contaminating protein"), more preferably less than about 20% of non-STRIFEl or non-STRIFE2 protein, still more preferably less than about 10% of non-STRIFEl or non-STRIFE2 protein, and most preferably less than about 5% non-STRIFEl or non-STRIFE2 protein.
  • STRIFEl or STRIFE2 protein or biologically active portion thereof is recombinantly produced, it is also preferably substantially free of culture medium, i.e., culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume ofthe protein preparation.
  • culture medium represents less than about 20%, more preferably less than about 10%, and most preferably less than about 5% ofthe volume ofthe protein preparation.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of STRIFEl or STRIFE2 protein in which the protein is separated from chemical precursors or other chemicals which are involved in the synthesis ofthe protein.
  • the language “substantially free of chemical precursors or other chemicals” includes preparations of STRIFEl or STRIFE2 protein having less than about 30% (by dry weight) of chemical precursors or non-STRIFEl or non-STRIFE2 chemicals, more preferably less than about 20% chemical precursors or non-STRIFEl or non-STRIFE2 chemicals, still more preferably less than about 10% chemical precursors or non-STRIFEl or non-STRIFE2 chemicals, and most preferably less than about 5% chemical precursors or non-STRIFEl or non-STRIFE2 chemicals.
  • Biologically active portions of a STRIFEl or STRIFE2 protein include peptides comprising amino acid sequences sufficiently homologous to or derived from the amino acid sequence ofthe STRIFEl or STRIFE2 protein, e.g., the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:6, which include less amino acids than the full length STRIFE 1 or STRIFE2 proteins, and exhibit at least one activity of a STRIFE 1 or
  • biologically active portions comprise a domain or motif with at least one activity ofthe STRIFEl or STRIFE2 protein.
  • a biologically active portion of a STRIFEl or STRIFE2 protein can be a polypeptide which is, for example, 10, 25, 50, 100 or more amino acids in length.
  • a biologically active portion of a STRIFEl or STRIFE2 protein comprises at least a cysteine-rich domain.
  • a biologically active portion of a STRIFEl or STRIFE2 protein comprises at least a cysteine-rich domain, wherein the cysteine-domain includes at least one module.
  • a biologically active portion of a STRIFEl or STRIFE2 protein comprises at least a signal sequence. In yet a further embodiment, a biologically active portion of a STRIFEl or STRIFE2 protein comprises at least a cysteine-rich domain and a signal sequence.
  • a biologically active portion of a STRIFEl or STRIFE2 protein comprises a STRIFEl or STRIFE2 amino acid sequence lacking a signal sequence.
  • a biologically active portion of a STRIFEl or STRIFE2 protein comprises a STRIFEl or STRIFE2 amino acid sequence lacking a cysteine-rich domain.
  • a preferred biologically active portion of a STRIFEl or STRIFE2 protein ofthe present invention may contain at least one ofthe above- identified structural domains.
  • Another preferred biologically active portion of a STRIFEl or STRIFE2 protein ofthe present invention may contain at least one ofthe above- identified structural domains.
  • STRIFEl or STRIFE2 protein may contain at least two ofthe above-identified structural domains. Another more preferred biologically active portion of a STRIFEl or - 25 -
  • STRIFE2 protein may contain at least three or more ofthe above-identified structural domains.
  • biologically active portions in which other regions ofthe protein are deleted, can be prepared by recombinant techniques and evaluated for one or more ofthe functional activities of a native STRIFEl or STRIFE2 protein.
  • the STRIFEl or STRIFE2 protein has an amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:6, respectively.
  • the STRIFEl or STRIFE2 protein is substantially homologous to SEQ ID NO:2 or SEQ ID NO:6, and retains the functional activity ofthe protein of SEQ ID NO:2 or SEQ ID NO:6, respectively, yet differs in amino acid sequence due to natural allelic variation or mutagenesis, as described in detail in subsection I above.
  • the STRIFEl or STRIFE2 protein is a protein which comprises an amino acid sequence at least about 60% homologous to the amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6 and preferably retains a functional activity ofthe STRIFEl or STRIFE2 protein of SEQ ID NO:2 or SEQ ID NO:6, respectively.
  • the protein is at least about 70% homologous to SEQ ID NO:2 or SEQ ID NO:6, more preferably at least about 80% homologous to SEQ ID NO:2 or SEQ ID NO:6, even more preferably at least about 90% homologous to SEQ ID NO:2 or SEQ ID NO:6, and most preferably at least about 95% or more homologous to SEQ ID NO:2 or SEQ ID NO:6.
  • the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleic acid sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes).
  • the length of a reference sequence aligned for comparison purposes is at least 30%, preferably at least 40%, more preferably at least 50%, even more preferably at least 60%, and even more preferably at least 70%, 80%, or 90% ofthe length ofthe reference sequence (e.g., when aligning a second sequence to the STRIFEl and STRIFE2 amino acid sequence of SEQ ID NO:2 or SEQ ID NO:6 having 177 amino acid residues, at least 80, preferably at least 100, more preferably at least 120, even more preferably at least 140, and even more preferably at least 150, 160 or 170 amino acid residues are aligned).
  • the amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein amino acid or nucleic acid
  • identity is equivalent to amino acid or nucleic acid “homology”).
  • percent identity is a function ofthe number of identical positions shared by - 26 -
  • sequences taking into account the number of gaps, and the length of each gap, which need to be introduced for optimal alignment ofthe two sequences.
  • the comparison of sequences and determination of percent identity between two sequences can be accomplished using a mathematical algorithm.
  • the percent identity between two amino acid sequences is determined using the Needleman and Wunsch (J. Mol. Biol. (48):444-453 (1970)) algorithm which has been inco ⁇ orated into the GAP program in the GCG software package (available at http://www.gcg.com), using either a Blossom 62 matrix or a PAM250 matrix, and a gap weight of 16, 14, 12, 10, 8, 6, or 4 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two nucleotide sequences is determined using the GAP program in the GCG software package (available at http://www.gcg.com), using a NWSgapdna.CMP matrix and a gap weight of 40, 50, 60, 70, or 80 and a length weight of 1, 2, 3, 4, 5, or 6.
  • the percent identity between two amino acid or nucleotide sequences is determined using the algorithm of E. Meyers and W. Miller (CABIOS, 4:11-17 (1989)) which has been inco ⁇ orated into the ALIGN program (version 2.0), using a PAM120 weight residue table, a gap length penalty of 12 and a gap penalty of 4.
  • the nucleic acid and protein sequences ofthe present invention can further be used as a "query sequence" to perform a search against public databases to, for example, identify other family members or related sequences.
  • search can be performed using the NBLAST and XBLAST programs (version 2.0) of Altschul, et al. (1990) J Mol. Biol. 215:403-10.
  • Gapped BLAST can be utilized as described in Altschul et al., (1997) Nucleic Acids Res. 25(17):3389-3402.
  • the default parameters ofthe respective programs e.g., XBLAST and NBLAST
  • a STRIFEl or STRIFE2 "chimeric protein" or “fusion protein” comprises a STRIFEl or STRIFE2 polypeptide operatively linked to a non-STRIFEl or non-STRIFE2 polypeptide.
  • a "STRIFEl or STRIFE2 polypeptide” refers to a polypeptide having an amino acid sequence corresponding to STRIFEl or STRIFE2
  • a “non-STRIFEl or non-STRIFE2 polypeptide” refers to a polypeptide having - 27 -
  • a STRIFEl or STRIFE2 fusion protein comprises at least one biologically active portion of a STRIFEl or STRIFE2 protein.
  • a STRIFEl or STRIFE2 fusion protein comprises at least two biologically active portions of a STRIFEl or STRIFE2 protein. In another preferred embodiment, a STRIFEl or STRIFE2 fusion protein comprises at least three biologically active portions of a STRIFEl or STRIFE2 protein.
  • the term "operatively linked" is intended to indicate that the STRIFEl or STRIFE2 polypeptide and the non-STRIFEl or non-STRIFE2 polypeptide are fused in-frame to each other.
  • the non-STRIFEl or non-STRIFE2 polypeptide can be fused to the N-terminus or C- terminus of the STRIFEl or STRIFE2 polypeptide.
  • the fusion protein is a GST-STRIFE 1 or STRIFE2 fusion protein in which the STRIFEl or STRIFE2 sequences are fused to the C-terminus ofthe GST sequences.
  • Such fusion proteins can facilitate the purification of recombinant STRIFEl or STRIFE2.
  • the fusion protein is a STRIFEl or STRIFE2 protein containing a heterologous signal sequence at its N- terminus.
  • the native STRIFEl or STRIFE2 signal sequence i.e, about amino acids 1-29 of SEQ ID NO:2 or SEQ ID NO:6 can be removed and replaced with a signal sequence from another protein.
  • expression and/or secretion of STRIFEl or STRIFE2 can be increased through use of a heterologous signal sequence.
  • the fusion protein is a STRIFEl or STRIFE2- immunoglobulin fusion protein in which the STRIFEl or STRIFE2 sequences comprising primarily the STRIFEl or STRIFE2 cysteine-rich domains are fused to sequences derived from a member ofthe immunoglobulin protein family.
  • Soluble derivatives have also been made of cell surface glycoproteins in the immunoglobulin gene superfamily consisting of an extracellular domain ofthe cell surface glycoprotein fused to an immunoglobulin constant (Fc) region (see e.g., Capon, D.J. et al. (1989) Nature 337:525-531 and Capon U.S.
  • Patents 5,116,964 and 5,428,130 [CD4-IgGl constructs]; Linsley, P.S. et al. (1991) J Exp. Med. 173:721-730 [a CD28-IgGl construct and a B7-l-IgGl construct]; and Linsley, P.S. et al. (1991) J. Exp. Med. 174:561-569 and U.S. Patent 5,434,131[a CTLA4-IgGl]).
  • Such fusion proteins have proven useful for modulating receptor-ligand interactions. Soluble derivatives of cell - 28 -
  • TNFR tumor necrosis factor receptor
  • the STRIFEl or STRIFE2-immunoglobulin fusion proteins ofthe invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a STRIFEl ligand and a STRIFEl receptor on the surface of a cell, or between a STRIFE2 receptor and the STRIFE2 ligand, to thereby suppress STRIFEl or STRIFE2 -mediated signal transduction in vivo.
  • the STRIFEl or STRIFE2-immunoglobulin fusion proteins ofthe invention can be inco ⁇ orated into pharmaceutical compositions and administered to a subject to inhibit an interaction between a STRIFEl ligand and a STRIFEl receptor on the surface of a cell, or between a STRIFE2 receptor and the STRIFE2 ligand, to thereby suppress STRIFEl or STRIFE2 -mediated signal transduction in vivo.
  • the STRIFEl or STRIFE2-immunoglobulin fusion proteins ofthe invention can
  • STRIFE2-immunoglobulin fusion proteins can be used to affect the bioavailability of a STRIFEl or STRIFE2 cognate receptor. Inhibition ofthe STRIFEl or STRIFE2 ligand/STRIFEl or STRIFE2 interaction may be useful therapeutically for the treatment of TNF-associated disorders, e.g., inflammatory, immune, or neoplastic disorders.
  • TNF-associated disorders e.g., inflammatory, immune, or neoplastic disorders.
  • the STRIFEl or STRIFE2-immunoglobulin fusion proteins ofthe invention can be used as immunogens to produce anti-STRIFEl or STRIFE2 antibodies in a subject, to purify STRIFEl or STRIFE2 ligands and in screening assays to identify molecules which inhibit the interaction of STRIFEl or STRIFE2 with a STRIFEl or STRIFE2 ligand.
  • a STRIFEl or STRIFE2 chimeric or fusion protein ofthe invention is produced by standard recombinant DNA techniques.
  • DNA fragments coding for the different polypeptide sequences are ligated together in-frame in accordance with conventional techniques, for example by employing blunt-ended or stagger-ended termini for ligation, restriction enzyme digestion to provide for appropriate termini, filling-in of cohesive ends as appropriate, alkaline phosphatase treatment to avoid undesirable joining, and enzymatic ligation.
  • the fusion gene can be synthesized by conventional techniques including automated DNA synthesizers.
  • PCR amplification of gene fragments can be carried out using anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence (see, for example, Current Protocols in Molecular Biology, eds. Ausubel et al. John Wiley & Sons: 1992).
  • anchor primers which give rise to complementary overhangs between two consecutive gene fragments which can subsequently be annealed and reamplified to generate a chimeric gene sequence
  • many expression vectors are commercially available that already encode a fusion moiety (e.g., a GST polypeptide).
  • a STRIFEl or STRIFE2-encoding nucleic acid can be cloned into such an expression vector such that the fusion moiety is linked in-frame to the STRIFEl or STRIFE2 protein.
  • the present invention also pertains to variants of the STRIFEl or STRIFE2 proteins which function as either STRIFEl or STRIFE2 agonists (mimetics) or as STRIFEl or STRIFE2 antagonists.
  • Variants ofthe STRIFEl or STRIFE2 proteins can be generated by mutagenesis, e.g., discrete point mutation or trancation of a STRIFEl or STRIFE2 protein.
  • An agonist ofthe STRIFEl or STRIFE2 proteins can retain substantially the same, or a subset, ofthe biological activities ofthe naturally occurring form of a STRIFEl or STRIFE2 protein.
  • An antagonist of a STRIFEl or STRIFE2 protein can inhibit one or more ofthe activities ofthe naturally occurring form ofthe STRIFEl or STRIFE2 protein by, for example, competitively binding to a downstream or upstream member of a cellular signaling cascade which includes the STRIFEl or STRIFE2 protein.
  • specific biological effects can be elicited by treatment with a variant of limited function.
  • treatment of a subject with a variant having a subset ofthe biological activities ofthe naturally occurring form ofthe protein has fewer side effects in a subject relative to treatment with the naturally occurring form ofthe STRIFEl or STRIFE2 protein.
  • variants of a STRIFEl or STRIFE2 protein which function as either STRIFEl or STRIFE2 agonists (mimetics) or as STRIFEl or STRIFE2 antagonists can be identified by screening combinatorial libraries of mutants, e.g., trancation mutants, of a STRIFEl or STRIFE2 protein for STRIFEl or STRIFE2 protein agonist or antagonist activity.
  • a variegated library of STRIFEl or STRIFE2 variants is generated by combinatorial mutagenesis at the nucleic acid level and is encoded by a variegated gene library.
  • a variegated library of STRIFEl or STRIFE2 variants can be produced by, for example, enzymatically ligating a mixture of synthetic oligonucleotides into gene sequences such that a degenerate set of potential STRIFEl or STRIFE2 sequences is expressible as individual polypeptides, or alternatively, as a set of larger fusion proteins (e.g., for phage display) containing the set of STRIFEl or STRIFE2 sequences therein.
  • libraries of fragments of a STRIFEl or STRIFE2 protein coding sequence can be used to generate a variegated population of STRIFEl or STRIFE2 fragments for screening and subsequent selection of variants of a STRIFEl or STRIFE2 protein.
  • a library of coding sequence fragments can be generated by treating a double stranded PCR fragment of a STRIFEl or STRIFE2 coding sequence with a nuclease under conditions wherein nicking occurs only about once per molecule, denaturing the double stranded DNA, renaturing the DNA to form double stranded DNA which can include sense/antisense pairs from different nicked products, removing single stranded portions from reformed duplexes by treatment with S 1 nuclease, and ligating the resulting fragment library into an expression vector.
  • an expression library can be derived which encodes N-terminal, C-terminal and internal fragments of various sizes ofthe STRIFEl or STRIFE2 protein.
  • Recrusive ensemble mutagenesis (REM), a new technique which enhances the frequency of functional mutants in the libraries, can be used in combination with the screening assays to identify STRIFEl or STRIFE2 variants (Arkin and Yourvan (1992) PNAS 59:7811-7815; Delgrave et al. (1993) Protein Engineering 6(3):327-331).
  • cell based assays can be exploited to analyze a variegated STRIFEl or STRIFE2 library.
  • a library of expression vectors can be transfected into a cell line which ordinarily responds to a particular ligand, e.g., a cytokine, in a STRIFEl or STRIFE2-dependent manner.
  • the transfected cells are then contacted with the ligand and the effect of expression ofthe mutant on signaling by the ligand can be detected, e.g., by measuring NF- ⁇ B activity or cell survival.
  • Plasmid DNA can then be recovered from the cells which score for inhibition, or alternatively, potentiation of cytokine induction, and the individual clones further characterized.
  • STRIFEl or STRIFE2 protein can be used as an immunogen to generate antibodies that bind STRIFEl or STRIFE2 using standard techniques for polyclonal and monoclonal antibody preparation.
  • STRIFE1 or STRIFE2 protein can be used or, alternatively, the invention provides antigenic peptide fragments of STRIFEl or STRIFE2 for use as immunogens.
  • the antigenic peptide of STRIFEl or STRIFE2 comprises at least 8 amino acid residues of the amino acid sequence shown in SEQ ID NO:2 or SEQ ID NO:6 and encompasses an epitope of STRIFEl or STRIFE2 such that an antibody raised against the peptide forms a specific immune complex with STRIFEl or STRIFE2.
  • the antigenic peptide comprises at least 10 amino acid residues, more preferably at least 15 amino acid residues, even more preferably at least 20 amino acid residues, and most preferably at least 30 amino acid residues.
  • Preferred epitopes encompassed by the antigenic peptide are regions of STRIFEl or STRIFE2 that are located on the surface ofthe protein, e.g., hydrophilic regions.
  • a STRIFEl or STRIFE2 immunogen typically is used to prepare antibodies by immunizing a suitable subject, (e.g., rabbit, goat, mouse or other mammal) with the immunogen.
  • An appropriate immunogenic preparation can contain, for example, recombinantly expressed STRIFEl or STRIFE2 protein or a chemically synthesized STRIFEl or STRIFE2 polypeptide.
  • the preparation can further include an adjuvant, such as Freund's complete or incomplete adjuvant, or similar immunostimulatory agent. Immunization of a suitable subject with an immunogenic STRIFEl or STRIFE2 preparation induces a polyclonal anti-STRIFEl or STRIFE2 antibody response. Accordingly, another aspect ofthe invention pertains to anti-STRIFEl or
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site which specifically binds (immunoreacts with) an antigen, such as STRIFEl or STRIFE2.
  • immunologically active portions of immunoglobulin molecules include F(ab) and F(ab')2 fragments which can be generated by treating the antibody with an enzyme such as pepsin.
  • the invention provides polyclonal and monoclonal antibodies that bind STRIFEl or STRIFE2.
  • monoclonal antibody or “monoclonal antibody composition”, as used herein, refers to a population of antibody molecules that contain only one species of an antigen binding site capable of immunoreacting with a particular epitope of STRIFEl or
  • a monoclonal antibody composition thus typically displays a single binding affinity for a particular STRIFEl or STRIFE2 protein with which it immunoreacts.
  • Polyclonal anti-STRIFEl or STRIFE2 antibodies can be prepared as described above by immunizing a suitable subject with a STRIFEl or STRIFE2 immunogen.
  • the anti-STRIFEl or STRIFE2 antibody titer in the immunized subject can be monitored over time by standard techniques, such as with an enzyme linked immunosorbent assay (ELISA) using immobilized STRIFEl or STRIFE2. If desired, the antibody molecules - 32 -
  • STRIFEl or STRIFE2 directed against STRIFEl or STRIFE2 can be isolated from the mammal (e.g., from the blood) and further purified by well known techniques, such as protein A chromatography to obtain the IgG fraction.
  • antibody-producing cells can be obtained from the subject and used to prepare monoclonal antibodies by standard techniques, such as the hybridoma technique originally described by Kohler and Milstein (1975) Nature 256:495-497) (see also, Brown et al. (1981) J. Immunol. 127:539-46; Brown et al. (1980) J. Biol.
  • an immortal cell line typically a myeloma
  • lymphocytes typically splenocytes
  • STRIFEl or STRIFE2 immunogen as described above, and the culture supernatants ofthe resulting hybridoma cells are screened to identify a hybridoma producing a monoclonal antibody that binds STRIFEl or STRIFE2.
  • the immortal cell line (e.g., a myeloma cell line) is derived from the same mammalian species as the lymphocytes.
  • murine hybridomas can be made by fusing lymphocytes from a mouse immunized with an immunogenic preparation ofthe present invention with an immortalized mouse cell line.
  • Preferred immortal cell lines are mouse myeloma cell lines that are sensitive to culture medium containing hypoxanthine, aminopterin and thymidine ("HAT medium").
  • myeloma cell lines can be used as a fusion partner according to standard techniques, e.g., the P3-NSl/l-Ag4-l, P3-x63-Ag8.653 or Sp2/O-Agl4 myeloma lines. These myeloma lines are available from ATCC. Typically, HAT-sensitive mouse myeloma cells are fused to mouse splenocytes using polyethylene glycol ("PEG"). - 33 -
  • Hybridoma cells resulting from the fusion are then selected using HAT medium, which kills unfused and unproductively fused myeloma cells (unfused splenocytes die after several days because they are not transformed).
  • Hybridoma cells producing a monoclonal antibody ofthe invention are detected by screening the hybridoma culture supernatants for antibodies that bind STRIFEl or STRIFE2, e.g., using a standard ELISA assay.
  • a monoclonal anti-STRIFEl or STRIFE2 antibody can be identified and isolated by screening a recombinant combinatorial immunoglobulin library (e.g., an antibody phage display library) with STRIFEl or STRIFE2 to thereby isolate immunoglobulin library members that bind STRIFEl or STRIFE2.
  • Kits for generating and screening phage display libraries are commercially available (e.g., the Pharmacia Recombinant Phage Antibody System, Catalog No. 27-9400-01; and the Stratagene SurfZAPTM Phage Display Kit, Catalog No. 240612).
  • examples of methods and reagents particularly amenable for use in generating and screening antibody display library can be found in, for example, Ladner et al. U.S. Patent No. 5,223,409; Kang et al. PCT International Publication No. WO 92/18619; Dower et al. PCT International Publication No. WO 91/17271; Winter et al. PCT International Publication WO 92/20791; Markland et al. PCT International Publication No. WO 92/15679; Breitling et al. PCT International Publication WO 93/01288; McCafferty et al. PCT International Publication No.
  • recombinant anti-STRIFEl or STRIFE2 antibodies such as chimeric and humanized monoclonal antibodies, comprising both human and non- human portions, which can be made using standard recombinant DNA techniques, are within the scope ofthe invention.
  • Such chimeric and humanized monoclonal antibodies can be produced by recombinant DNA techniques known in the art, for example using methods described in Robinson et al. International Application No. PCT/US86/02269; Akira, et al. European Patent Application 184,187; Taniguchi, M., European Patent Application 171,496; Morrison et al. European Patent Application 173,494; Neuberger et al. PCT International Publication No. WO 86/01533; Cabilly et al. U.S. Patent No. - 34 -
  • An anti-STRIFEl or STRIFE2 antibody (e.g., monoclonal antibody) can be used to isolate STRIFEl or STRIFE2 by standard techniques, such as affinity chromatography or immunoprecipitation.
  • An anti-STRIFEl or STRIFE2 antibody can facilitate the purification of natural STRIFEl or STRIFE2 from cells and of recombinantly produced STRIFEl or STRIFE2 expressed in host cells.
  • an anti-STRIFEl or STRIFE2 antibody can be used to detect STRIFEl or STRIFE2 protein (e.g., in a cellular lysate or cell supernatant) in order to evaluate the abundance and pattern of expression ofthe STRIFEl or STRIFE2 protein.
  • Anti-STRIFEl or STRIFE2 antibodies can be used diagnostically to monitor protein levels in tissue as part of a clinical testing procedure, e.g., to, for example, determine the efficacy of a given treatment regimen. Detection can be facilitated by coupling (i.e., physically linking) the antibody to a detectable substance.
  • detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, and radioactive materials.
  • suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
  • suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
  • suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
  • an example of a luminescent material includes luminol;
  • examples of bioluminescent materials include luciferase, luciferin, and aequorin, and examples of suitable radioactive material include 125 1, 131 I, 35 S or 3 H.
  • vectors preferably expression vectors, containing a nucleic acid encoding STRIFEl or STRIFE2 (or a portion thereof).
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • vector refers to a nucleic acid molecule capable of transporting another nucleic acid to which it has been linked.
  • plasmid refers to a circular double stranded DNA loop into which additional - 35 -
  • DNA segments can be ligated.
  • Another type of vector is a viral vector, wherein additional DNA segments can be ligated into the viral genome.
  • Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g., bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome.
  • certain vectors are capable of directing the expression of genes to which they are operatively linked. Such vectors are referred to herein as "expression vectors”.
  • expression vectors of utility in recombinant DNA techniques are often in the form of plasmids.
  • plasmid and “vector” can be used interchangeably as the plasmid is the most commonly used form of vector.
  • the invention is intended to include such other forms of expression vectors, such as viral vectors (e.g., replication defective retrovirases, adenovirases and adeno-associated viruses), which serve equivalent functions.
  • viral vectors e.g., replication defective retrovirases, adenovirases and adeno-associated viruses
  • the recombinant expression vectors ofthe invention comprise a nucleic acid of the invention in a form suitable for expression ofthe nucleic acid in a host cell, which means that the recombinant expression vectors include one or more regulatory sequences, selected on the basis ofthe host cells to be used for expression, which is operatively linked to the nucleic acid sequence to be expressed.
  • "operably linked" is intended to mean that the nucleotide sequence of interest is linked to the regulatory sequence(s) in a manner which allows for expression ofthe nucleotide sequence (e.g., in an in vitro transcription/translation system or in a host cell when the vector is introduced into the host cell).
  • regulatory sequence is intended to includes promoters, enhancers and other expression control elements (e.g., polyadenylation signals). Such regulatory sequences are described, for example, in Goeddel; Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990). Regulatory sequences include those which direct constitutive expression of a nucleotide sequence in many types of host cell and those which direct expression ofthe nucleotide sequence only in certain host cells (e.g., tissue-specific regulatory sequences). It will be appreciated by those skilled in the art that the design ofthe expression vector can depend on such factors as the choice ofthe host cell to be transformed, the level of expression of protein desired, etc.
  • the expression vectors ofthe invention can be introduced into host cells to thereby produce proteins or peptides, including fusion proteins or peptides, encoded by nucleic acids as described herein (e.g., STRIFEl or STRIFE2 proteins, mutant forms of STRIFEl or STRIFE2, fusion proteins, etc.).
  • STRIFEl or STRIFE2 proteins e.g., STRIFEl or STRIFE2 proteins, mutant forms of STRIFEl or STRIFE2, fusion proteins, etc.
  • the recombinant expression vectors ofthe invention can be designed for expression of STRIFEl or STRIFE2 in prokaryotic or eukaryotic cells.
  • STRIFEl or STRIFE2 can be expressed in bacterial cells such as E. coli, insect cells (using baculovirus expression vectors) yeast cells or mammalian cells. Suitable host cells are discussed further in Goeddel, Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, CA (1990).
  • the recombinant expression vector can be transcribed and translated in vitro, for example using T7 promoter regulatory sequences and T7 polymerase.
  • Fusion vectors add a number of amino acids to a protein encoded therein, usually to the amino terminus ofthe recombinant protein.
  • Such fusion vectors typically serve three pu ⁇ oses: 1) to increase expression of recombinant protein; 2) to increase the solubility ofthe recombinant protein; and 3) to aid in the purification ofthe recombinant protein by acting as a ligand in affinity purification.
  • a proteolytic cleavage site is introduced at the junction ofthe fusion moiety and the recombinant protein to enable separation ofthe recombinant protein from the fusion moiety subsequent to purification ofthe fusion protein.
  • enzymes, and their cognate recognition sequences include Factor Xa, thrombin and enterokinase.
  • Typical fusion expression vectors include pGEX (Pharmacia Biotech Inc; Smith, D.B. and Johnson, K.S.
  • fusion proteins can be utilized in STRIFEl or STRIFE2 activity assays, in STRIFEl or STRIFE2 ligand binding (e.g., direct assays or competitive assays described in detail below), to generate antibodies specific for STRIFEl or STRIFE2 proteins, as examples.
  • a STRIFEl or STRIFE2 fusion expressed in a retroviral expression vector ofthe present invention can be utilized to infect bone marrow cells which are subsequently transplanted into irradiated recipients. The pathology ofthe subject recipient is then examined after sufficient time has passed (e.g six (6) weeks).
  • Suitable inducible non-fusion E. coli expression vectors include pTrc (Amann et al., (1988) Gene 69:301-315) and pET l id (Studier et al., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 60-89).
  • Target gene expression from the pTrc vector relies on host RNA polymerase transcription from a hybrid t ⁇ -lac fusion promoter.
  • Target gene expression from the pET l id vector relies on transcription from a T7 gnlO-lac fusion - 37 -
  • T7 gnl coexpressed viral RNA polymerase
  • This viral polymerase is supplied by host strains BL21(DE3) or HMS174(DE3) from a resident ⁇ prophage harboring a T7 gnl gene under the transcriptional control ofthe lacUV 5 promoter.
  • One strategy to maximize recombinant protein expression in E. coli is to express the protein in a host bacteria with an impaired capacity to proteolytically cleave the recombinant protein (Gottesman, S., Gene Expression Technology: Methods in Enzymology 185, Academic Press, San Diego, California (1990) 119-128).
  • nucleic acid sequence ofthe nucleic acid is altered by standard DNA synthesis techniques.
  • the STRIFEl or STRIFE2 expression vector is a yeast expression vector.
  • yeast expression vectors for expression in yeast S. cerivisae include pYepSecl (Baldari, et al., (1987) Embo J 6:229-234), pMFa (Kurjan and Herskowitz, (1982) Cell 30:933-943), pJRY88 (Schultz et al., (1987) Gene 54:113-123), pYES2 (Invitrogen Co ⁇ oration, San Diego, CA), and picZ (InVitrogen Co ⁇ , San Diego, CA).
  • STRIFEl or STRIFE2 can be expressed in insect cells using baculoviras expression vectors.
  • Baculoviras vectors available for expression of proteins in cultured insect cells include the pAc series (Smith et al. (1983) Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow and Summers (1989) Virology 170:31-39).
  • a nucleic acid ofthe invention is expressed in mammalian cells using a mammalian expression vector.
  • mammalian expression vectors include pCDM8 (Seed, B. (1987) N ⁇ twre 329:840) and pMT2PC (Kaufman et al. (1987) EMBO J 6:187-195).
  • the expression vector's control functions are often provided by viral regulatory elements.
  • commonly used promoters are derived from polyoma, Adenoviras 2, cytomegaloviras and Simian Virus 40.
  • the recombinant mammalian expression vector is capable of directing expression ofthe nucleic acid preferentially in a particular cell type (e.g., tissue-specific regulatory elements are used to express the nucleic acid).
  • tissue-specific promoters include the albumin promoter (liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277), lymphoid-specific promoters (Calame and Eaton (1988) Adv. Immunol. 43:235-275), in particular promoters of T cell receptors (Winoto and Baltimore (1989) EMBO J 8:729-733) and immunoglobulins (Banerji et al.
  • albumin promoter liver-specific; Pinkert et al. (1987) Genes Dev. 1 :268-277
  • lymphoid-specific promoters Calame and Eaton (1988) Adv. Immunol. 43:235-275
  • promoters of T cell receptors Winoto and Baltimore (1989) EMBO J 8:729-733
  • immunoglobulins Bonerji et al.
  • Neuron-specific promoters e.g., the neurofilament promoter; Byrne and Ruddle (1989) RN4S 86:5473-5477
  • pancreas-specific promoters e.g., milk whey promoter; U.S. Patent No. 4,873,316 and European Application Publication No. 264, 166.
  • promoters are also encompassed, for example the murine hox promoters (Kessel and Grass (1990) Science 249:374-379) and the -fetoprotein promoter (Campes and Tilghman (1989) Genes Dev. 3:537-546).
  • the invention further provides a recombinant expression vector comprising a DNA molecule ofthe invention cloned into the expression vector in an antisense orientation. That is, the DNA molecule is operatively linked to a regulatory sequence in a manner which allows for expression (by transcription ofthe DNA molecule) of an RNA molecule which is antisense to STRIFEl or STRIFE2 mRNA. Regulatory sequences operatively linked to a nucleic acid cloned in the antisense orientation can be chosen which direct the continuous expression ofthe antisense RNA molecule in a variety of cell types, for instance viral promoters and/or enhancers, or regulatory sequences can be chosen which direct constitutive, tissue specific or cell type specific expression of antisense RNA.
  • the antisense expression vector can be in the form of a recombinant plasmid, phagemid or attenuated virus in which antisense nucleic acids are produced under the control of a high efficiency regulatory region, the activity of which can be determined by the cell type into which the vector is introduced.
  • a high efficiency regulatory region the activity of which can be determined by the cell type into which the vector is introduced.
  • a host cell can be any prokaryotic or eukaryotic cell.
  • STRIFEl or STRIFE2 protein can be expressed in bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • bacterial cells such as E. coli, insect cells, yeast or mammalian cells (such as Chinese hamster ovary cells (CHO) or COS cells).
  • CHO Chinese hamster ovary cells
  • COS cells Chinese hamster ovary cells
  • Vector DNA can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques.
  • transformation and transfection are intended to refer to a variety of art-recognized techniques for introducing foreign nucleic acid (e.g., DNA) into a host cell, including calcium phosphate or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, or electroporation. Suitable methods for transforming or transfecting host cells can be found in Sambrook, et al. (Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), and other laboratory manuals.
  • a gene that encodes a selectable marker (e.g., resistance to antibiotics) is generally introduced into the host cells along with the gene of interest.
  • selectable markers include those which confer resistance to drags, such as G418, hygromycin and methotrexate.
  • Nucleic acid encoding a selectable marker can be introduced into a host cell on the same vector as that encoding STRIFEl or STRIFE2 or can be introduced on a separate vector.
  • Cells stably transfected with the introduced nucleic acid can be identified by drag selection (e.g., cells that have inco ⁇ orated the selectable marker gene will survive, while the other cells die).
  • a host cell ofthe invention such as a prokaryotic or eukaryotic host cell in culture, can be used to produce (i.e., express) STRIFEl or STRIFE2 protein.
  • the invention further provides methods for producing STRIFEl or STRIFE2 protein using the host cells ofthe invention.
  • the method comprises culturing the host cell of invention (into which a recombinant expression vector encoding STRIFEl or STRIFE2 has been introduced) in a suitable medium such that STRIFEl or STRIFE2 protein is produced.
  • the method further comprises isolating STRIFEl or STRIFE2 from the medium or the host cell.
  • the host cells ofthe invention can also be used to produce nonhuman transgenic animals.
  • a host cell ofthe invention is a fertilized oocyte or an embryonic stem cell into which STRIFEl or STRIFE2-coding sequences have been introduced. Such host cells can then be used to create non-human transgenic animals in which exogenous STRIFEl or STRIFE2 sequences have been introduced into - 40 -
  • transgenic animal is a non-human animal, preferably a mammal, more preferably a rodent such as a rat or mouse, in which one or more ofthe cells ofthe animal includes a transgene.
  • transgenic animals include non-human primates, sheep, dogs, cows, goats, chickens, amphibians, etc.
  • a transgene is exogenous DNA which is integrated into the genome of a cell from which a transgenic animal develops and which remains in the genome ofthe mature animal, thereby directing the expression of an encoded gene product in one or more cell types or tissues ofthe transgenic animal.
  • a "homologous recombinant animal” is a non-human animal, preferably a mammal, more preferably a mouse, in which an endogenous STRIFEl or STRIFE2 gene has been altered by homologous recombination between the endogenous gene and an exogenous DNA molecule introduced into a cell ofthe animal, e.g., an embryonic cell ofthe animal, prior to development ofthe animal.
  • a transgenic animal ofthe invention can be created by introducing STRIFEl or STRIFE2-encoding nucleic acid into the male pronuclei of a fertilized oocyte, e.g., by microinjection, retroviral infection, and allowing the oocyte to develop in a pseudopregnant female foster animal.
  • the murine STRIFEl or STRIFE2 cDNA sequence of SEQ ID NO: 1 or SEQ ID NO:4 can be introduced as a transgene into the genome of a non-human animal.
  • a nonmurine homologue of a murine STRIFEl or STRIFE2 gene such as a human STRIFEl or STRIFE2 gene
  • a transgene can be isolated based on hybridization to the murine STRIFEl or STRIFE2 cDNA (described further in subsection I above) and used as a transgene.
  • Intronic sequences and polyadenylation signals can also be included in the transgene to increase the efficiency of expression ofthe transgene.
  • a tissue-specific regulatory sequence(s) can be operably linked to the STRIFEl or STRIFE2 transgene to direct expression of STRIFEl or STRIFE2 protein to particular cells.
  • transgenic founder animal can be identified based upon the presence ofthe STRIFEl or STRIFE2 transgene in its genome and/or expression of STRIFEl or STRIFE2 mRNA in tissues or cells ofthe animals.
  • transgenic animals carrying a transgene encoding STRIFEl or STRIFE2 can further be bred to other transgenic animals carrying other transgenes.
  • a vector is prepared which contains at least a portion of a STRIFEl or STRIFE2 gene into which a deletion, addition or substitution has been introduced to thereby alter, e.g., functionally disrupt, the STRIFEl or STRIFE2 gene.
  • the STRIFEl or STRIFE2 gene can be a murine gene (e.g., the cDNA of SEQ ID NO:3 or SEQ ID NO:7), but can also be a non-murine homologue of a murine STRIFEl or STRIFE2 gene.
  • a human STRIFEl or STRIFE2 gene can be used to construct a homologous recombination vector suitable for altering an endogenous STRIFEl or STRIFE2 gene in the mouse genome.
  • the vector is designed such that, upon homologous recombination, the endogenous STRIFEl or STRIFE2 gene is functionally disrupted (i.e., no longer encodes a functional protein; also referred to as a "knock out" vector).
  • the vector can be designed such that, upon homologous recombination, the endogenous STRIFEl or STRIFE2 gene is mutated or otherwise altered but still encodes functional protein (e.g., the upstream regulatory region can be altered to thereby alter the expression ofthe endogenous STRIFEl or STRIFE2 protein).
  • the altered portion ofthe STRIFEl or STRIFE2 gene is flanked at its 5' and 3' ends by additional nucleic acid ofthe STRIFEl or STRIFE2 gene to allow for homologous recombination to occur between the exogenous STRIFEl or STRIFE2 gene carried by the vector and an endogenous STRIFEl or STRIFE2 gene in an embryonic stem cell.
  • the additional flanking STRIFEl or STRIFE2 nucleic acid is of sufficient length for successful homologous recombination with the endogenous gene.
  • flanking DNA both at the 5' and 3' ends
  • flanking DNA are included in the vector (see e.g., Thomas, K.R.
  • the vector is introduced into an embryonic stem cell line (e.g., by electroporation) and cells in which the introduced STRIFEl or STRIFE2 gene has homologously recombined with the endogenous STRIFEl or STRIFE2 gene are selected (see e.g., Li, E. et al. (1992) Cell 69:915).
  • the selected cells are then injected into a blastocyst of an animal (e.g., a mouse) to form aggregation chimeras (see e.g., Bradley, A.
  • a chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embryo brought to term.
  • Progeny harboring the homologously recombined DNA in their germ cells can be used to breed animals in which all cells of the animal contain the homologously recombined DNA by germline transmission ofthe - 42 -
  • transgenic non-humans animals can be produced which contain selected systems which allow for regulated expression ofthe transgene.
  • a system is the cre/loxP recombinase system of bacteriophage PI .
  • cre/loxP recombinase system of bacteriophage PI .
  • the cre/loxP recombinase system see, e.g., Lakso et al. (1992) PNAS 89:6232-6236.
  • Another example of a recombinase system is the FLP recombinase system of Saccharomyces cerevisiae (O'Gorman et al. (1991) Science 251 :1351-1355.
  • mice containing transgenes encoding both the Cre recombinase and a selected protein are required.
  • Such animals can be provided through the construction of "double" transgenic animals, e.g., by mating two transgenic animals, one containing a transgene encoding a selected protein and the other containing a transgene encoding a recombinase.
  • Clones ofthe non-human transgenic animals described herein can also be produced according to the methods described in Wil ut, I. et al. (1997) Nature 385:810- 813 and PCT International Publication Nos. WO 97/07668 and WO 97/07669.
  • a cell e.g., a somatic cell
  • the quiescent cell can then be fused, e.g., through the use of electrical pulses, to an enucleated oocyte from an animal ofthe same species from which the quiescent cell is isolated.
  • the recontructed oocyte is then cultured such that it develops to morala or blastocyte and then transferred to pseudopregnant female foster animal.
  • the offspring borne of this female foster animal will be a clone ofthe animal from which the cell, e.g., the somatic cell, is isolated.
  • compositions suitable for administration can be inco ⁇ orated into pharmaceutical compositions suitable for administration.
  • compositions typically comprise the nucleic acid molecule, protein, or antibody and a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier is intended to include any and all solvents, dispersion media, coatings, antibacterial and antifungal agents, isotonic and abso ⁇ tion delaying agents, and the like, compatible with pharmaceutical administration. The use of such media and agents for pharmaceutically active substances is well known - 43 -
  • a pharmaceutical composition ofthe invention is formulated to be compatible with its intended route of administration.
  • routes of administration include parenteral, e.g., intravenous, intradermal, subcutaneous, oral (e.g., inhalation), transdermal (topical), transmucosal, and rectal administration.
  • Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerine, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide.
  • the parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.
  • compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersion.
  • suitable carriers include physiological saline, bacteriostatic water, Cremophor ELTM (BASF, Parsippany, NJ) or phosphate buffered saline (PBS).
  • the composition must be sterile and should be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, and liquid polyetheylene glycol, and the like), and suitable mixtures thereof.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use of surfactants.
  • Prevention ofthe action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like.
  • isotonic agents for example, sugars, polyalcohols such as manitol, sorbitol, sodium chloride in the composition.
  • Prolonged abso ⁇ tion ofthe injectable compositions can be brought about by including in the composition an agent which delays abso ⁇ tion, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions can be prepared by inco ⁇ orating the active compound (e.g., a STRIFEl or STRIFE2 protein or anti-STRIFEl or STRIFE2 antibody) in the required amount in an appropriate solvent with one or a combination of ingredients enumerated above, as required, followed by filtered sterilization.
  • the active compound e.g., a STRIFEl or STRIFE2 protein or anti-STRIFEl or STRIFE2 antibody
  • dispersions are prepared by inco ⁇ orating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and freeze-drying which yields a powder ofthe active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.
  • Oral compositions generally include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the pu ⁇ ose of oral therapeutic administration, the active compound can be inco ⁇ orated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Pharmaceutically compatible binding agents, and/or adjuvant materials can be included as part ofthe composition.
  • the tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or corn starch; a lubricant such as magnesium stearate or Sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.
  • a suitable propellant e.g., a gas such as carbon dioxide, or a nebulizer.
  • Systemic administration can also be by transmucosal or transdermal means.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives.
  • Transmucosal administration can be accomplished through the use of nasal sprays or suppositories.
  • the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art. - 45 -
  • the compounds can also be prepared in the form of suppositories (e.g., with conventional suppository bases such as cocoa butter and other glycerides) or retention enemas for rectal delivery.
  • suppositories e.g., with conventional suppository bases such as cocoa butter and other glycerides
  • retention enemas for rectal delivery.
  • the active compounds are prepared with carriers that will protect the compound against rapid elimination from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems.
  • a controlled release formulation including implants and microencapsulated delivery systems.
  • Biodegradable, biocompatible polymers can be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, and polylactic acid. Methods for preparation of such formulations will be apparent to those skilled in the art. The materials can also be obtained commercially from Alza Co ⁇ oration and Nova
  • Liposomal suspensions can also be used as pharmaceutically acceptable carriers. These can be prepared according to methods known to those skilled in the art, for example, as described in U.S. Patent No. 4,522,811. It is especially advantageous to formulate oral or parenteral compositions in dosage unit form for ease of administration and uniformity of dosage.
  • Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subject to be treated; each unit containing a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.
  • the specification for the dosage unit forms ofthe invention are dictated by and directly dependent on the unique characteristics ofthe active compound and the particular therapeutic effect to be achieved, and the limitations inherent in the art of compounding such an active compound for the treatment of individuals.
  • Toxicity and therapeutic efficacy of such compounds can be determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., for determining the LD50 (the dose lethal to 50% ofthe population) and the ED50 (the dose therapeutically effective in 50% ofthe population).
  • the dose ratio between toxic and therapeutic effects is the therapeutic index and it can be expressed as the ratio LD50/ED50.
  • Compounds which exhibit large therapeutic indices are preferred. While compounds that exhibit toxic side effects may be used, care should be taken to design a delivery system that targets such compounds to the site of affected tissue in order to minimize potential damage to uninfected cells and, thereby, reduce side effects.
  • the data obtained from the cell culture assays and animal studies can be used in formulating a range of dosage for use in humans.
  • the dosage of such compounds lies preferably within a range of circulating concentrations that include the ED50 with little or no toxicity.
  • the dosage may vary within this range depending upon the dosage form employed and the route of administration utilized. For any compound used in the - 46 -
  • the therapeutically effective dose can be estimated initially from cell culture assays.
  • a dose may be formulated in animal models to achieve a circulating plasma concentration range that includes the IC50 (i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms) as determined in cell culture.
  • IC50 i.e., the concentration of the test compound which achieves a half-maximal inhibition of symptoms
  • levels in plasma may be measured, for example, by high performance liquid chromatography.
  • the nucleic acid molecules ofthe invention can be inserted into vectors and used as gene therapy vectors.
  • Gene therapy vectors can be delivered to a subject by, for example, intravenous injection, local administration (see U.S. Patent 5,328,470) or by stereotactic injection (see e.g., Chen et al. (1994) PNAS 91:3054-3057).
  • the pharmaceutical preparation ofthe gene therapy vector can include the gene therapy vector in an acceptable diluent, or can comprise a slow release matrix in which the gene delivery vehicle is imbedded.
  • the pharmaceutical preparation can include one or more cells which produce the gene delivery system.
  • compositions can be included in a container, pack, or dispenser together with instructions for administration.
  • nucleic acid molecules, proteins, protein homologues, and antibodies described herein can be used in one or more ofthe following methods: a) screening assays; b) detecting assays (e.g., chromosome mapping, tissue typing, and forensic biology); c) predictive medicine (e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics); and d) methods of treatment (e.g., therapeutic and prophylactic methods as well as such methods in the context of pharmacogenomics).
  • assays e.g., chromosome mapping, tissue typing, and forensic biology
  • predictive medicine e.g., diagnostic assays, prognostic assays, monitoring clinical trials, and pharmacogenetics
  • methods of treatment e.g., therapeutic and prophylactic methods as well as such methods in the context of pharmacogenomics.
  • a STRIFEl protein ofthe invention has one or more ofthe following activities: (i) interaction of a STRIFEl protein on the cell surface with a second non- STRIFEl protein molecule on the surface ofthe same cell; (ii) interaction of a STRIFEl protein on the cell surface with a second non-STRIFEl protein molecule on the surface of a different cell; (iii) complex formation between a membrane-bound STRIFEl protein and a cytokine, e.g., TNF; (iv) interaction of a STRIFEl protein with an intracellular protein including SH2 domain-containing proteins or cytoskeletal proteins; (v) formation of a homogeneous multimeric signaling complex with like STRIFEl proteins; and (vi) formation of a heterogeneous multimeric signaling complex with other TNFR superfamily proteins.
  • STRIFE2 protein ofthe invention has one or - 47 -
  • the STRIFEl and STRIFE2 proteins ofthe invention can thus be used in, for example, (1) modulation of cellular signal transduction, either in vitro or in vivo; (2) regulation of gene transcription in a cell involved in development or differentiation, either in vitro or in vivo; (3) regulation of gene transcription in a cell involved in in development or differentiation, wherein at least one gene encodes a differentiation- specific protein; (4) regulation of gene transcription in a cell involved in in development or differentaition, wherein at least one gene encodes a second secreted protein; (5) regulation of gene transcription in a cell involved in development or differentiation, wherein at least one gene encodes a signal transduction molecule; and (6) regulation of cellular proliferation, either in vitro or in vivo.
  • the isolated nucleic acid molecules of the invention can be used, for example, to express STRIFEl or STRIFE2 protein (e.g., via a recombinant expression vector in a host cell in gene therapy applications), to detect STRIFEl or STRIFE2 mRNA (e.g., in a biological sample) or a genetic alteration in a STRIFEl or STRIFE2 gene, and to modulate STRIFEl or STRIFE2 activity, as described further below.
  • the STRIFEl or STRIFE2 proteins can be used to screen drugs or compounds which modulate the STRIFEl or STRIFE2 activity as well as to treat disorders characterized by insufficient or excessive production of STRIFEl or STRIFE2 protein or production of STRIFEl or STRIFE2 protein forms which have decreased or aberrant activity compared to STRIFEl or STRIFE2 wild type protein (e.g., developmental disorders or proliferative diseases such as cancer).
  • the anti- STRIFEl or STRIFE2 antibodies ofthe invention can be used to detect and isolate STRIFEl or STRIFE2 proteins, regulate the bioavailability of STRIFEl or STRIFE2 proteins, and modulate STRIFEl or STRIFE2 activity.
  • the invention provides a method (also referred to herein as a "screening assay") for identifying modulators, i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drags) which bind to STRIFEl or STRIFE2 - 48 -
  • modulators i.e., candidate or test compounds or agents (e.g., peptides, peptidomimetics, small molecules or other drags) which bind to STRIFEl or STRIFE2 - 48 -
  • STRIFEl or STRIFE2 expression or STRIFEl or STRIFE2 activity proteins or have a stimulatory or inhibitory effect on, for example, STRIFEl or STRIFE2 expression or STRIFEl or STRIFE2 activity.
  • assays that can be used to identify candidate or test compounds or agents which have a stimulatory or inhibitory effect on, for example, STRIFEl or STRIFE2 expression or STRIFEl or STRIFE2 activity.
  • assays based on the effects of TNF on some cells can be used to evaluate the modulatory activity of test compounds on STRIFEl or STRIFE2 expression or STRIFEl or STRIFE2 activity.
  • Known effects of TNF on fibroblast cells include effects on mitogenesis, IL-6 secretion and HLA class II antigen induction.
  • Known effects of TNF on monocytes include effects on secretion of cytokines such as GM-CSF, IL-6, and IL-8.
  • TNF is known to be cytotoxic to some cells, such as WEHI- 164 murine fibrosarcoma cells (described in Espevik et al. (1986) J. Immunol. Methods 95:99-105). TNF is also known to have effects on cytokine secretion by endothelial cells, as well as effect induction of adhesion molecules such as ICAM-1, E-selectin, VCAM, and tissue factor production in endothelial cells. Thus, these cells and the detectable phenotypic changes resulting from the effect of TNF in the presence or absence of a test compound can be used to evaluate the modulatory activity ofthe test compound on STRIFEl or STRIFE2 expression or STRIFEl or STRIFE2 activity.
  • TNF is known to modulate neutrophil responses. Comparisons can be made between TNF effects on neutrophils in the presence or absence of a test compound using cellular activation, priming, degranulation, and/or superoxide production as detectable endpoints for evaluation of STRIFEl or STRIFE2 modulatory activity. These and other related assays are well known to those having ordinary skill in the art.
  • the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a STRIFEl or STRIFE2 protein or polypeptide or biologically active portion thereof. In another embodiment, the invention provides assays for screening candidate or test compounds which bind to or modulate the activity of a STRIFEl receptor.
  • the test compounds ofthe present invention can be obtained using any ofthe numerous approaches in combinatorial library methods known in the art, including: biological libraries; spatially addressable parallel solid phase or solution phase libraries; synthetic library methods requiring deconvolution; the 'one- bead one-compound' library method; and synthetic library methods using affinity chromatography selection.
  • an assay is a cell-based assay in which a cell which expresses a STRIFEl or STRIFE2 receptor on the cell surface is contacted with a test compound and the ability ofthe test compound to bind to a STRIFEl or STRIFE2 receptor is determined.
  • the cell preferably expresses a human STRIFEl or STRIFE2 receptor, e.g., the human receptor encoded by clone AX92_3 contained in ATCC Deposit Number 98101 (described in PCT application number WO 98/01554, published on January 15, 1998) or the human OAF065 receptor (described in PCT application number WO 98/38304, published on September 3, 1998).
  • test compounds can be labeled with 125 I, 35 S, 14 C, or 3 H, either directly or indirectly, and the radioisotope detected by direct counting of radioemmission or by scintillation counting.
  • test compounds can be enzymatically labeled with, for example, horseradish peroxidase, alkaline phosphatase, or luciferase, and the enzymatic label detected by determination of conversion of an appropriate substrate to product.
  • a microphysiometer can be used to detect the interaction of a test compound with a STRIFEl or STRIFE2 receptor without the labeling of either the test compound or the receptor. McConnell, H. M. et al. (1992) Science 257:1906-1912. As used herein, a "microphysiometer" (e.g., CytosensorTM) is - 50 -
  • the assay comprises contacting a cell which expresses a STRIFEl or STRIFE2 receptor on the cell surface with a STRIFEl or STRIFE2 protein or biologically-active portion thereof, to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a STRIFEl or STRIFE2 receptor, wherein determining the ability ofthe test compound to interact with a STRIFEl or STRIFE2 receptor comprises determining the ability ofthe test compound to preferentially bind to the
  • STRIFEl or STRIFE2 receptor as compared to the ability of STRIFEl or STRIFE2, or a biologically active portion thereof, to bind to the receptor.
  • an assay is a cell-based assay comprising contacting a cell which expresses a STRIFEl or STRIFE2 target molecule with a test compound and determining the ability ofthe test compound to modulate the activity ofthe STRIFEl or STRIFE2 target molecule.
  • the activity ofthe target molecule can be determined by detecting induction of a cellular second messenger ofthe target (e.g., intracellular Ca 2+ , diacylglycerol, IP 3 , etc.), detecting catalytic/enzymatic activity ofthe target an appropriate substrate, detecting the induction of a reporter gene (comprising a STRIFEl or STRIFE2 -responsive regulatory element operatively linked to a nucleic acid encoding a detectable marker, e.g., luciferase), or detecting a cellular response, for example, development, differentiation or rate of proliferation.
  • a cellular second messenger ofthe target e.g., intracellular Ca 2+ , diacylglycerol, IP 3 , etc.
  • detecting catalytic/enzymatic activity ofthe target an appropriate substrate detecting the induction of a reporter gene (comprising a STRIFEl or STRIFE2 -responsive regulatory element operatively linked to a nucleic acid en
  • an assay ofthe present invention is a cell-free assay in which a STRIFEl or STRIFE2 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to bind to the STRIFEl or STRIFE2 protein or biologically active portion thereof is determined. Binding ofthe test compound to the STRIFEl or STRIFE2 protein can be determined either directly or indirectly as described above.
  • the assay includes contacting the STRIFEl or STRIFE2 protein or biologically active portion thereof with a known compound which binds STRIFEl or STRIFE2 to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with a STRIFEl or STRIFE2 protein, wherein determining the ability ofthe test compound to interact with a STRIFEl or STRIFE2 protein comprises determining the ability ofthe test compound to preferentially bind to STRIFEl or STRIFE2 or biologically active portion thereof as compared to the known compound.
  • determining the ability ofthe test compound to interact with a STRIFEl or STRIFE2 protein comprises determining the ability ofthe test compound to preferentially bind to STRIFEl or STRIFE2 or biologically active portion thereof as compared to the known compound.
  • the assay is a cell-free assay in which a STRIFEl or STRIFE2 protein or biologically active portion thereof is contacted with a test compound and the ability ofthe test compound to modulate (e.g., stimulate or inhibit) the activity ofthe STRIFEl or STRIFE2 protein or biologically active portion thereof is determined. Determining the ability ofthe test compound to modulate the activity of a STRIFEl or STRIFE2 protein can be accomplished, for example, by determining the ability ofthe STRIFEl or STRIFE2 protein to bind to a STRIFEl or STRIFE2 target molecule by one ofthe methods described above for determining direct binding.
  • STRIFEl or STRIFE2 protein Determining the ability ofthe STRIFEl or STRIFE2 protein to bind to a STRIFEl or STRIFE2 target molecule can also be accomplished using a technology such as real-time Biomolocular Interaction Analysis (BIA).
  • BIOA Biomolocular Interaction Analysis
  • BIOA is a technology for studying biospecific interactions in real time, without labeling any ofthe interactants (e.g., BIAcoreTM). Changes in the optical phenomenon surface plasmon resonance (SPR) can be used as an indication of real-time reactions between biological molecules.
  • determining the ability ofthe test compound to modulate the activity of a STRIFEl or STRIFE2 protein can be accomplished by determining the ability ofthe STRIFEl or STRIFE2 protein to further modulate the activity of a STRIFEl or STRIFE2 target molecule.
  • the catalytic/enzymatic activity ofthe target molecule on an appropriate substrate can be determined as previously described.
  • the cell-free assay involves contacting a STRIFEl or STRIFE2 protein or biologically active portion thereof with a known compound which binds the STRIFEl or STRIFE2 protein to form an assay mixture, contacting the assay mixture with a test compound, and determining the ability ofthe test compound to interact with the STRIFEl or STRIFE2 protein, wherein determining the ability ofthe test compound to interact with the STRIFEl or STRIFE2 protein comprises determining the ability ofthe STRIFEl or STRIFE2 protein to preferentially bind to or modulate the activity of a STRIFEl or STRIFE2 target molecule.
  • the cell-free assays ofthe present invention are amenable to use of both soluble and/or membrane-bound forms of isolated proteins (e.g., STRIFEl or STRIFE2 proteins or biologically active portions thereof or STRIFEl or STRIFE2 target molecules).
  • isolated proteins e.g., STRIFEl or STRIFE2 proteins or biologically active portions thereof or STRIFEl or STRIFE2 target molecules.
  • a membrane-bound form an isolated protein e.g., a STRIFE2 target molecule or receptor
  • solubilizing agent include non-ionic detergents such as n- - 52 -
  • STRIFEl or STRIFE2 it may be desirable to immobilize either STRIFEl or STRIFE2 or its target molecule to facilitate separation of complexed from uncomplexed forms of one or both ofthe proteins, as well as to accommodate automation ofthe assay.
  • Binding of a test compound to a STRIFEl or STRIFE2 protein, or interaction of a STRIFEl or STRIFE2 protein with a target molecule in the presence and absence of a candidate compound can be accomplished in any vessel suitable for containing the reactants. Examples of such vessels include microtitre plates, test tubes, and micro-centrifuge tubes.
  • a fusion protein can be provided which adds a domain that allows one or both ofthe proteins to be bound to a matrix.
  • glutathione-S-transferase/ STRIFEl or STRIFE2 fusion proteins or glutathione-S-transf erase/target fusion proteins can be adsorbed onto glutathione sepharose beads (Sigma Chemical, St. Louis, MO) or glutathione derivatized microtitre plates, which are then combined with the test compound or the test compound and either the non-adsorbed target protein or STRIFEl or STRIFE2 protein, and the mixture incubated under conditions conducive to complex formation (e.g., at physiological conditions for salt and pH).
  • the beads or microtitre plate wells are washed to remove any unbound components, the matrix immobilized in the case of beads, complex determined either directly or indirectly, for example, as described above.
  • the complexes can be dissociated from the matrix, and the level of STRIFEl or STRIFE2 binding or activity determined using standard techniques.
  • STRIFEl or STRIFE2 protein or a STRIFEl or STRIFE2 target molecule can be immobilized utilizing conjugation of biotin and streptavidin.
  • Biotinylated STRIFEl or STRIFE2 protein or target molecules can be prepared from biotin-NHS (N-hydroxy-succinimide) using techniques well known in the art (e.g., biotinylation kit, Pierce Chemicals, Rockford, IL), and immobilized in the wells of streptavidin-coated 96 well plates (Pierce Chemical).
  • antibodies reactive with STRIFEl or STRIFE2 protein or target molecules but which do not interfere with binding ofthe STRIFEl or STRIFE2 protein to its target molecule can be derivatized to the wells ofthe plate, and unbound target or STRIFEl or - 53 -
  • Methods for detecting such complexes include immunodetection of complexes using antibodies reactive with the STRIFEl or STRIFE2 protein or target molecule, as well as enzyme-linked assays which rely on detecting an enzymatic activity associated with the STRIFEl or STRIFE2 protein or target molecule.
  • modulators of STRIFEl or STRIFE2 expression are identified in a method wherein a cell is contacted with a candidate compound and the expression of STRIFEl or STRIFE2 mRNA or protein in the cell is determined. The level of expression of STRIFEl or STRIFE2 mRNA or protein in the presence ofthe candidate compound is compared to the level of expression of STRIFEl or STRIFE2 mRNA or protein in the absence ofthe candidate compound. The candidate compound can then be identified as a modulator of STRIFEl or STRIFE2 expression based on this comparison.
  • STRIFEl or STRIFE2 mRNA or protein when expression of STRIFEl or STRIFE2 mRNA or protein is greater (statistically significantly greater) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as a stimulator of STRIFEl or STRIFE2 mRNA or protein expression.
  • the candidate compound when expression of STRIFEl or STRIFE2 mRNA or protein is less (statistically significantly less) in the presence ofthe candidate compound than in its absence, the candidate compound is identified as an inhibitor of STRIFEl or STRIFE2 mRNA or protein expression.
  • the level of STRIFEl or STRIFE2 mRNA or protein expression in the cells can be determined by methods described herein for detecting STRIFEl or STRIFE2 mRNA or protein.
  • the STRIFEl or STRIFE2 proteins can be used as "bait proteins" in a two-hybrid assay or three-hybrid assay (see, e.g., U.S. Patent No. 5,283,317; Zervos et al. (1993) Cell 72:223-232; Madura et al. (1993) J Biol.
  • STRIFE2-binding proteins are likely to be cell-surface molecules associated with non-STRIFE2-expressing cells, wherein such STRIFE2 -binding proteins are involved in signal transduction.
  • the two-hybrid system is based on the modular nature of most transcription factors, which consist of separable DNA-binding and activation domains. Briefly, the assay utilizes two different DNA constructs. In one construct, the gene that codes for a - 54 -
  • STRIFE1 or STRIFE2 protein is fused to a gene encoding the DNA binding domain of a known transcription factor (e.g., GAL-4).
  • a DNA sequence from a library of DNA sequences, that encodes an unidentified protein ("prey" or “sample") is fused to a gene that codes for the activation domain ofthe known transcription factor. If the "bait" and the "prey” proteins are able to interact, in vivo, forming an STRIFEl or STRIFE2-dependent complex, the DNA-binding and activation domains ofthe transcription factor are brought into close proximity. This proximity allows transcription of a reporter gene (e.g., LacZ) which is operably linked to a transcriptional regulatory site responsive to the transcription factor. Expression ofthe reporter gene can be detected and cell colonies containing the functional transcription factor can be isolated and used to obtain the cloned gene which encodes the protein which interacts with the STRIFEl or STRIFE2 protein.
  • a reporter gene e.g., LacZ
  • This invention further pertains to novel agents identified by the above-described screening assays. Accordingly, it is within the scope of this invention to further use an agent identified as described herein in an appropriate animal model.
  • an agent identified as described herein e.g., a STRIFEl or STRIFE2 modulating agent, an antisense STRIFEl or STRIFE2 nucleic acid molecule, a STRIFEl or STRIFE2-specific antibody, or a STRIFEl or STRIFE2 -binding partner
  • an agent identified as described herein can be used in an animal model to determine the mechanism of action of such an agent.
  • this invention pertains to uses of novel agents identified by the above-described screening assays for treatments as described herein.
  • either the murine STRIFEl or STRIFE2 receptors could be used, or preferably, a human STRIFEl or STRIFE2 receptor, e.g., the human receptor encoded by clone AX92_3 contained in ATCC Deposit Number 98101 (described in PCT application number WO 98/01554, published on January 15, 1998) or the human OAF065 receptor (described in PCT application number WO 98/38304, published on September 3, 1998), may be used.
  • cDNA sequences identified herein can be used in numerous ways as polynucleotide reagents. For example, these sequences can be used to: (i) map their respective genes on a chromosome; and, thus, locate gene regions associated with genetic disease; (ii) identify an individual from a minute biological sample (tissue - 55 -
  • Chromosome Mapping Once the sequence (or a portion ofthe sequence) of a gene has been isolated, this sequence can be used to map the location ofthe gene on a chromosome. This process is called chromosome mapping. Accordingly, portions or fragments ofthe STRIFEl or STRIFE2 nucleotide sequences, described herein, can be used to map the location ofthe STRIFEl or STRIFE2 genes on a chromosome. The mapping ofthe STRIFEl or STRIFE2 sequences to chromosomes is an important first step in correlating these sequences with genes associated with disease.
  • STRIFEl or STRIFE2 genes can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp in length) from the STRIFEl or STRIFE2 nucleotide sequences. Computer analysis ofthe STRIFEl or STRIFE2 sequences can be used to predict primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers can then be used for PCR screening of somatic cell hybrids containing individual human chromosomes. Only those hybrids containing the human gene corresponding to the STRIFEl or STRIFE2 sequences will yield an amplified fragment. Somatic cell hybrids are prepared by fusing somatic cells from different mammals (e.g., human and mouse cells).
  • PCR mapping of somatic cell hybrids is a rapid procedure for assigning a particular sequence to a particular chromosome. Three or more sequences can be assigned per day using a single thermal cycler. Using the STRIFEl or STRIFE2 nucleotide sequences to design oligonucleotide primers, sublocalization can be achieved with panels of fragments from specific chromosomes. Other mapping strategies which can similarly be used to map a STRIFEl or STRIFE2 sequence to its chromosome - 56 -
  • Fluorescence in situ hybridization (FISH) of a DNA sequence to a metaphase chromosomal spread can further be used to provide a precise chromosomal location in one step.
  • Chromosome spreads can be made using cells whose division has been blocked in metaphase by a chemical such as colcemid that disrupts the mitotic spindle.
  • the chromosomes can be treated briefly with trypsin, and then stained with Giemsa. A pattern of light and dark bands develops on each chromosome, so that the chromosomes can be identified individually.
  • the FISH technique can be used with a DNA sequence as short as 500 or 600 bases.
  • clones larger than 1,000 bases have a higher likelihood of binding to a unique chromosomal location with sufficient signal intensity for simple detection.
  • Reagents for chromosome mapping can be used individually to mark a single chromosome or a single site on that chromosome, or panels of reagents can be used for marking multiple sites and/or multiple chromosomes. Reagents corresponding to noncoding regions ofthe genes actually are preferred for mapping pu ⁇ oses. Coding sequences are more likely to be conserved within gene families, thus increasing the chance of cross hybridizations during chromosomal mapping.
  • Comparison of affected and unaffected individuals generally involves first looking for structural alterations in the chromosomes, such as deletions or translocations that are visible from chromosome spreads or detectable using PCR based on that DNA sequence. Ultimately, complete sequencing of genes from several - 57 -
  • the STRIFEl or STRIFE2 sequences ofthe present invention can also be used to identify individuals from minute biological samples.
  • the United States military, for example, is considering the use of restriction fragment length polymo ⁇ hism (RFLP) for identification of its personnel.
  • RFLP restriction fragment length polymo ⁇ hism
  • an individual's genomic DNA is digested with one or more restriction enzymes, and probed on a Southern blot to yield unique bands for identification. This method does not suffer from the current limitations of "Dog Tags" which can be lost, switched, or stolen, making positive identification difficult.
  • the sequences ofthe present invention are useful as additional DNA markers for RFLP (described in U.S. Patent 5,272,057).
  • sequences ofthe present invention can be used to provide an alternative technique which determines the actual base-by-base DNA sequence of selected portions of an individual's genome.
  • the STRIFEl or STRIFE2 nucleotide sequences described herein can be used to prepare two PCR primers from the 5' and 3' ends ofthe sequences. These primers can then be used to amplify an individual's DNA and subsequently sequence it. Panels of corresponding DNA sequences from individuals, prepared in this manner, can provide unique individual identifications, as each individual will have a unique set of such DNA sequences due to allelic differences.
  • the sequences ofthe present invention can be used to obtain such identification sequences from individuals and from tissue.
  • the STRIFEl or STRIFE2 nucleotide sequences ofthe invention uniquely represent portions ofthe human genome. Allelic variation occurs to some degree in the coding regions of these sequences, and to a greater degree in the noncoding regions. It is estimated that allelic variation between individual humans occurs with a frequency of about once per each 500 bases.
  • Each ofthe sequences described herein can, to some degree, be used as a standard against which DNA from an individual can be compared for identification pu ⁇ oses. Because greater numbers of polymo ⁇ hisms occur in the noncoding regions, fewer sequences are necessary to differentiate individuals.
  • the noncoding sequences of SEQ ID NOT or SEQ ID NO:5 can comfortably provide positive individual identification with a panel of perhaps 10 to 1 ,000 primers which each yield a noncoding amplified sequence of 100 bases. If predicted coding sequences, such as those in SEQ ID NO: 3 or SEQ ID NO: 7 are used, a more appropriate number of primers for positive individual identification would be 500-2,000. - 58 -
  • a panel of reagents from STRIFEl or STRIFE2 nucleotide sequences described herein is used to generate a unique identification database for an individual, those same reagents can later be used to identify tissue from that individual.
  • positive identification ofthe individual, living or dead can be made from extremely small tissue samples.
  • DNA-based identification techniques can also be used in forensic biology. Forensic biology is a scientific field employing genetic typing of biological evidence found at a crime scene as a means for positively identifying, for example, a pe ⁇ etrator of a crime.
  • PCR technology can be used to amplify DNA sequences taken from very small biological samples such as tissues, e.g., hair or skin, or body fluids, e.g., blood, saliva, or semen found at a crime scene. The amplified sequence can then be compared to a standard, thereby allowing identification ofthe origin ofthe biological sample.
  • sequences ofthe present invention can be used to provide polynucleotide reagents, e.g., PCR primers, targeted to specific loci in the human genome, which can enhance the reliability of DNA-based forensic identifications by, for example, providing another "identification marker" (i.e. another DNA sequence that is unique to a particular individual).
  • an "identification marker” i.e. another DNA sequence that is unique to a particular individual.
  • actual base sequence information can be used for identification as an accurate alternative to patterns formed by restriction enzyme generated fragments.
  • Sequences targeted to noncoding regions of SEQ ID NOsT or SEQ ID NO: 5 are particularly appropriate for this use as greater numbers of polymo ⁇ hisms occur in the noncoding regions, making it easier to differentiate individuals using this technique.
  • polynucleotide reagents include the
  • STRIFEl or STRIFE2 nucleotide sequences or portions thereof e.g., fragments derived from the noncoding regions of SEQ ID NO: 1 or SEQ ID NO:5, having a length of at least 20 bases, preferably at least 30 bases.
  • the STRIFEl or STRIFE2 nucleotide sequences described herein can further be used to provide polynucleotide reagents, e.g., labeled or labelable probes which can be used in, for example, an in situ hybridization technique, to identify a specific tissue, e.g., brain or lung tissue. This can be very useful in cases where a forensic pathologist is presented with a tissue of unknown origin. Panels of s ⁇ ch STRIFEl or STRIFE2 probes can be used to identify tissue by species and/or by organ type. In a similar fashion, these reagents, e.g., STRIFEl or STRIFE2 primers or probes can be used to screen tissue culture for contamination (i.e. screen for the presence of a mixture of different types of cells in a culture). - 59 -
  • the present invention also pertains to the field of predictive medicine in which diagnostic assays, prognostic assays, and monitoring clinical trails are used for prognostic (predictive) pu ⁇ oses to thereby treat an individual prophylactically.
  • diagnostic assays for determining STRIFEl or STRIFE2 protein and/or nucleic acid expression as well as STRIFEl or STRIFE2 activity, in the context of a biological sample (e.g., blood, serum, cells, tissue) to thereby determine whether an individual is afflicted with a disease or disorder, or is at risk of developing a disorder, associated with aberrant STRIFEl or STRIFE2 expression or activity.
  • a biological sample e.g., blood, serum, cells, tissue
  • the invention also provides for prognostic (or predictive) assays for determining whether an individual is at risk of developing a disorder associated with STRIFEl or STRIFE2 protein, nucleic acid expression or activity. For example, mutations in a STRIFEl or STRIFE2 gene can be assayed in a biological sample. Such assays can be used for prognostic or predictive pu ⁇ ose to thereby phophylactically treat an individual prior to the onset of a disorder characterized by or associated with STRIFEl or STRIFE2 protein, nucleic acid expression or activity.
  • Another aspect ofthe invention pertains to monitoring the influence of agents (e.g., drugs, compounds) on the expression or activity of STRIFEl or STRIFE2 in clinical trials.
  • agents e.g., drugs, compounds
  • An exemplary method for detecting the presence or absence of STRIFEl or STRIFE2 protein or nucleic acid in a biological sample involves obtaining a biological sample from a test subject and contacting the biological sample with a compound or an agent capable of detecting STRIFEl or STRIFE2 protein or nucleic acid (e.g., mRNA, genomic DNA) that encodes STRIFEl or STRIFE2 protein such that the presence of STRIFEl or STRIFE2 protein or nucleic acid is detected in the biological sample.
  • a preferred agent for detecting STRIFEl or STRIFE2 mRNA or genomic DNA is a labeled nucleic acid probe capable of hybridizing to STRIFEl or STRIFE2 mRNA or genomic DNA.
  • the nucleic acid probe can be, for example, a full-length STRIFEl or STRIFE2 nucleic acid, such as the nucleic acid of SEQ ID NO: 1 or SEQ ID NO:5, or a portion thereof, such as an oligonucleotide of at least 15, 30, 50, 100, 250 or 500 nucleotides in length and sufficient to specifically hybridize under stringent conditions to STRIFEl or STRIFE2 mRNA or genomic DNA.
  • Other suitable probes for use in the diagnostic assays ofthe invention are described herein. - 60 -
  • a preferred agent for detecting STRIFEl or STRIFE2 protein is an antibody capable of binding to STRIFEl or STRIFE2 protein, preferably an antibody with a detectable label.
  • Antibodies can be polyclonal, or more preferably, monoclonal. An intact antibody, or a fragment thereof (e.g., Fab or F(ab')2) can be used.
  • the term "labeled", with regard to the probe or antibody, is intended to encompass direct labeling ofthe probe or antibody by coupling (i.e., physically linking) a detectable substance to the probe or antibody, as well as indirect labeling ofthe probe or antibody by reactivity with another reagent that is directly labeled.
  • Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody and end-labeling of a DNA probe with biotin such that it can be detected with fluorescently labeled streptavidin.
  • biological sample is intended to include tissues, cells and biological fluids isolated from a subject, as well as tissues, cells and fluids present within a subject. That is, the detection method ofthe invention can be used to detect STRIFEl or STRIFE2 mRNA, protein, or genomic DNA in a biological sample in vitro as well as in vivo. For example, in vitro techniques for detection of STRIFEl or
  • STRIFE2 mRNA include Northern hybridizations and in situ hybridizations.
  • In vitro techniques for detection of STRIFEl or STRIFE2 protein include enzyme linked immunosorbent assays (ELISAs), Western blots, immunoprecipitations and immunofluorescence.
  • In vitro techniques for detection of STRIFEl or STRIFE2 genomic DNA include Southern hybridizations.
  • in vivo techniques for detection of STRIFEl or STRIFE2 protein include introducing into a subject a labeled anti-STRIFEl or STRIFE2 antibody.
  • the antibody can be labeled with a radioactive marker whose presence and location in a subject can be detected by standard imaging techniques.
  • the biological sample contains protein molecules from the test subject.
  • the biological sample can contain mRNA molecules from the test subject or genomic DNA molecules from the test subject.
  • a preferred biological sample is a serum sample isolated by conventional means from a subject.
  • the methods further involve obtaining a control biological sample from a control subject, contacting the control sample with a compound or agent capable of detecting STRIFEl or STRIFE2 protein, mRNA, or genomic DNA, such that the presence of STRIFEl or STRIFE2 protein, mRNA or genomic DNA is detected in the biological sample, and comparing the presence of STRIFEl or STRIFE2 protein, mRNA or genomic DNA in the control sample with the presence of STRIFEl or STRIFE2 protein, mRNA or genomic DNA in the test sample.
  • kits for detecting the presence of STRIFEl or STRIFE2 in a biological sample can comprise a labeled compound or agent capable of detecting STRIFEl or STRIFE2 protein or mRNA in a biological sample; means for determining the amount of STRIFEl or STRIFE2 in the sample; and means for comparing the amount of STRIFEl or STRIFE2 in the sample with a standard.
  • the compound or agent can be packaged in a suitable container.
  • the kit can further comprise instructions for using the kit to detect STRIFEl or STRIFE2 protein or nucleic acid.
  • the diagnostic methods described herein can furthermore be utilized to identify subjects having or at risk of developing a disease or disorder associated with aberrant STRIFEl or STRIFE2 expression or activity.
  • the assays described herein such as the preceding diagnostic assays or the following assays, can be utilized to identify a subject having or at risk of developing a disorder associated with STRIFEl or STRIFE2 protein, nucleic acid expression or activity such as a TNF-associated disorder, e.g., inflammatory, immune, or neoplastic disorder.
  • the present invention provides a method for identifying a disease or disorder associated with aberrant STRIFEl or STRIFE2 expression or activity in which a test sample is obtained from a subject and STRIFEl or STRIFE2 protein or nucleic acid (e.g, mRNA, genomic DNA) is detected, wherein the presence of STRIFEl or STRIFE2 protein or nucleic acid is diagnostic for a subject having or at risk of developing a disease or disorder associated with aberrant STRIFEl or STRIFE2 expression or activity.
  • a test sample refers to a biological sample obtained from a subject of interest.
  • a test sample can be a biological fluid (e.g., serum), cell sample, or tissue.
  • the prognostic assays described herein can be used to determine whether a subject can be administered an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate) to treat a disease or disorder associated with aberrant STRIFEl or STRIFE2 expression or activity.
  • an agent e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate
  • an agent for a disorder such as a proliferative disorder, a differentiative or developmental disorder, or a hematopoietic disorder.
  • such methods can be used to determine whether a subject can be effectively treated with an agent for a differentiative or proliferative disease (e.g., cancer).
  • the present invention provides methods for determining whether a subject can be effectively treated with an agent for a disorder associated with aberrant STRIFEl or STRIFE2 expression or activity in which a test sample is obtained and STRIFEl or STRIFE2 protein or - 62 -
  • nucleic acid expression or activity is detected (e.g., wherein the abundance of STRIFEl or STRIFE2 protein or nucleic acid expression or activity is diagnostic for a subject that can be administered the agent to treat a disorder associated with aberrant STRIFEl or STRIFE2 expression or activity.)
  • the methods ofthe invention can also be used to detect genetic alterations in an
  • the methods include detecting, in a sample of cells from the subject, the presence or absence of a genetic alteration characterized by at least one of an alteration affecting the integrity of a gene encoding a STRIFEl or STRIFE2-protein, or the mis-expression ofthe STRIFEl or STRIFE2 gene.
  • such genetic alterations can be detected by ascertaining the existence of at least one of 1) a deletion of one or more nucleotides from an STRIFEl or STRIFE2 gene; 2) an addition of one or more nucleotides to a STRIFEl or STRIFE2 gene; 3) a substitution of one or more nucleotides of a STRIFEl or STRIFE2 gene, 4) a chromosomal rearrangement of a STRIFEl or STRIFE2 gene; 5) an alteration in the level of a messenger RNA transcript of a STRIFEl or STRIFE2 gene, 6) aberrant modification of a STRIFEl or STRIFE2 gene, such as ofthe methylation pattern ofthe genomic DNA, 7) the presence of a non- wild type splicing pattern of a messenger RNA transcript of a STRIFEl or STRIFE2 gene, 8) a non- wild type level of a STRIFEl or STRIFE2 -protein, 9) allelic loss
  • detection ofthe alteration involves the use of a probe/primer in a polymerase chain reaction (PCR) (see, e.g., U.S. Patent Nos. 4,683,195 and 4,683,202), such as anchor PCR or RACE PCR, or, alternatively, in a ligation chain reaction (LCR) (see, e.g., Landegran et al. (1988) Science 241 077-1080; and Nakazawa et al. (1994) PNAS 91 : 360-364), the latter of which can be particularly useful for detecting point mutations in the STRIFEl or STRIFE2-gene (see Abravaya et al.
  • PCR polymerase chain reaction
  • LCR ligation chain reaction
  • This method can include the steps of collecting a sample of cells from a patient, isolating nucleic acid (e.g., genomic, mRNA or both) from the cells ofthe sample, contacting the nucleic acid sample with one or more primers which specifically hybridize to a STRIFEl or STRIFE2 gene under conditions such that hybridization and amplification ofthe STRIFEl or STRIFE2-gene - 63 -
  • nucleic acid e.g., genomic, mRNA or both
  • PCR and/or LCR may be desirable to use as a preliminary amplification step in conjunction with any ofthe techniques used for detecting mutations described herein.
  • Alternative amplification methods include: self sustained sequence replication (Guatelli, J.C. et al, 1990, Proc. Natl. Acad. Sci. USA 87:1874-1878), transcriptional amplification system (Kwoh, D.Y. et al, 1989, Proc. Natl. Acad. Sci. USA 86:1173- 1177), Q-Beta Replicase (Lizardi, P.M. et all, 1988, Bio/Technology 6:1197), or any other nucleic acid amplification method, followed by the detection ofthe amplified molecules using techniques well known to those of skill in the art. These detection schemes are especially useful for the detection of nucleic acid molecules if such molecules are present in very low numbers.
  • mutations in a STRIFEl or STRIFE2 gene from a sample cell can be identified by alterations in restriction enzyme cleavage patterns.
  • sample and control DNA is isolated, amplified (optionally), digested with one or more restriction endonucleases, and fragment length sizes are determined by gel electrophoresis and compared. Differences in fragment length sizes between sample and control DNA indicates mutations in the sample DNA.
  • sequence specific ribozymes see, for example, U.S. Patent No. 5,498,531 can be used to score for the presence of specific mutations by development or loss of a ribozyme cleavage site.
  • genetic mutations in STRIFEl or STRIFE2 can be identified by hybridizing a sample and control nucleic acids, e.g., DNA or RNA, to high density arrays containing hundreds or thousands of oligonucleotides probes (Cronin, M.T. et al. (1996) Human Mutation 1: 244-255; Kozal, M.J. et al. (1996) Nature Medicine 1: 753-759).
  • genetic mutations in STRIFEl or STRIFE2 can be identified in two dimensional arrays containing light-generated DNA probes as described in Cronin, M.T. et al. supra.
  • a first hybridization array of probes can be used to scan through long stretches of DNA in a sample and control to identify base changes between the sequences by making linear arrays of sequential ovelapping probes. This step allows the identification of point mutations. This step is followed by a second hybridization array that allows the characterization of specific mutations by using smaller, specialized probe arrays complementary to all variants or mutations detected. Each mutation array is composed of parallel probe sets, one complementary to the wildtype gene and the other complementary to the mutant gene. - 64 -
  • any of a variety of sequencing reactions known in the art can be used to directly sequence the STRIFEl or STRIFE2 gene and detect mutations by comparing the sequence ofthe sample STRIFEl or STRIFE2 with the corresponding wild-type (control) sequence.
  • Examples of sequencing reactions include those based on techniques developed by Maxim and Gilbert ((1977) PNAS 74:560) or Sanger ((1977) PNAS 74:5463).
  • any of a variety of automated sequencing procedures can be utilized when performing the diagnostic assays ((1995) Biotechniques 19:448), including sequencing by mass spectrometry (see, e.g., PCT International Publication No. WO 94/16101 ; Cohen et al. (1996) Adv. Chromatogr. 36:127-162; and Griffin et al. (1993) Appl. Biochem. Biotechnol 38:147-159).
  • RNA/RNA or RNA/DNA heteroduplexes Other methods for detecting mutations in the STRIFEl or STRIFE2 gene include methods in which protection from cleavage agents is used to detect mismatched bases in RNA/RNA or RNA/DNA heteroduplexes (Myers et al. (1985) Science 230:1242).
  • the art technique of "mismatch cleavage" starts by providing heteroduplexes of formed by hybridizing (labeled) RNA or DNA containing the wild-type STRIFEl or STRIFE2 sequence with potentially mutant RNA or DNA obtained from a tissue sample.
  • the double-stranded duplexes are treated with an agent which cleaves single- stranded regions ofthe duplex such as which will exist due to basepair mismatches between the control and sample strands.
  • RNA/DNA duplexes can be treated with RNase and DNA/DNA hybrids treated with S 1 nuclease to enzymatically digesting the mismatched regions.
  • either DNA/DNA or RNA/DNA duplexes can be treated with hydroxylamine or osmium tetroxide and with piperidine in order to digest mismatched regions. After digestion ofthe mismatched regions, the resulting material is then separated by size on denaturing polyacrylamide gels to determine the site of mutation. See, for example, Cotton et al. (1988) Proc. Natl Acad Sci USA 85:4397; Saleeba et ⁇ /. (1992) Methods Enzymol 217:286-295.
  • the control DNA or RNA can be labeled for detection.
  • the mismatch cleavage reaction employs one or more proteins that recognize mismatched base pairs in double-stranded DNA (so called "DNA mismatch repair" enzymes) in defined systems for detecting and mapping point mutations in STRIFEl or STRIFE2 cDNAs obtained from samples of cells.
  • DNA mismatch repair enzymes
  • the mutY enzyme of E. coli cleaves A at G/A mismatches and the thymidine DNA glycosylase from HeLa cells cleaves T at G/T mismatches (Hsu et al. (1994) Carcinogenesis 15:1657-1662).
  • a probe based on an STRIFEl or STRIFE2 sequence e.g., a wild-type STRIFEl or STRIFE2 sequence
  • a cDNA or other DNA product from a test cell(s).
  • the duplex is treated with a DNA mismatch repair enzyme, and the cleavage products, if any, can be - 65 -
  • alterations in electrophoretic mobility will be used to identify mutations in STRIFEl or STRIFE2 genes.
  • single strand conformation polymo ⁇ hism may be used to detect differences in electrophoretic mobility between mutant and wild type nucleic acids (Orita et al. (1989) Proc Natl. Acad. Sci USA: 86:2766, see also Cotton (1993) Mutat Res 285:125-144; and Hayashi (1992) Genet Anal Tech Appl 9:73-79).
  • Single-stranded DNA fragments of sample and control STRIFEl or STRIFE2 nucleic acids will be denatured and allowed to renature.
  • the secondary stracture of single-stranded nucleic acids varies according to sequence, the resulting alteration in electrophoretic mobility enables the detection of even a single base change.
  • the DNA fragments may be labeled or detected with labeled probes.
  • the sensitivity ofthe assay may be enhanced by using RNA (rather than DNA), in which the secondary stracture is more sensitive to a change in sequence.
  • the subject method utilizes heteroduplex analysis to separate double stranded heteroduplex molecules on the basis of changes in electrophoretic mobility (Keen et al. (1991) Trends Genet 7:5).
  • DGGE denaturing gradient gel electrophoresis
  • DGGE DGGE is used as the method of analysis, DNA will be modified to insure that it does not completely denature, for example by adding a GC clamp of approximately 40 bp of high-melting GC-rich DNA by PCR.
  • a temperature gradient is used in place of a denaturing gradient to identify differences in the mobility of control and sample DNA (Rosenbaum and Reissner (1987) Biophys Chem 265:12753).
  • oligonucleotide primers may be prepared in which the known mutation is placed centrally and then hybridized to target DNA under conditions which permit hybridization only if a perfect match is found (Saiki et al. (1986) Nature 324:163); Saiki et al. (1989) Proc. Natl Acad. Sci USA 86:6230).
  • Such allele specific oligonucleotides are hybridized to PCR amplified target DNA or a number of different mutations when the oligonucleotides are attached to the hybridizing membrane and hybridized with labeled target DNA.
  • allele specific amplification technology which depends on selective PCR amplification may be used in conjunction with the instant invention.
  • Oligonucleotides used as primers for specific amplification may carry the mutation of - 66 -
  • amplification depends on differential hybridization
  • mismatch can prevent, or reduce polymerase extension
  • the methods described herein may be performed, for example, by utilizing prepackaged diagnostic kits comprising at least one probe nucleic acid or antibody reagent described herein, which may be conveniently used, e.g., in clinical settings to diagnose patients exhibiting symptoms or family history of a disease or illness involving a STRIFEl or STRIFE2 gene.
  • any cell type or tissue in which STRIFEl or STRIFE2 is expressed may be utilized in the prognostic assays described herein.
  • STRIFEl or STRIFE2 Monitoring the influence of agents (e.g., drags, compounds) on the expression or activity of STRIFEl or STRIFE2 (e.g., modulation of cellular signal transduction, regulation of gene transcription in a cell involved in development or differentiation, regulation of cellular proliferation) can be applied not only in basic drag screening, but also in clinical trials.
  • agents e.g., drags, compounds
  • STRIFE2 e.g., modulation of cellular signal transduction, regulation of gene transcription in a cell involved in development or differentiation, regulation of cellular proliferation
  • the effectiveness of an agent determined by a screening assay as described herein to increase STRIFEl or STRIFE2 gene expression, protein levels, or upregulate STRIFEl or STRIFE2 activity can be monitored in clinical trails of subjects exhibiting decreased STRIFEl or STRIFE2 gene expression, protein levels, or downregulated STRIFEl or STRIFE2 activity.
  • the effectiveness of an agent determined by a screening assay to decrease STRIFEl or STRIFE2 gene expression, protein levels, or downregulate STRIFEl or STRIFE2 activity can be monitored in clinical trails of subjects exhibiting increased STRIFEl or STRIFE2 gene expression, protein levels, or upregulated STRIFEl or STRIFE2 activity.
  • the expression or activity of STRIFEl or STRIFE2 and, preferably, other genes that have been implicated in, for example, a proliferative disorder can be used as a "read out" or markers ofthe phenotype of a particular cell. - 67 -
  • genes including STRIFEl or STRIFE2 that are modulated in cells by treatment with an agent (e.g., compound, drag or small molecule) which modulates STRIFEl or STRIFE2 activity (e.g., identified in a screening assay as described herein) can be identified.
  • an agent e.g., compound, drag or small molecule
  • STRIFEl or STRIFE2 activity e.g., identified in a screening assay as described herein
  • cells can be isolated and RNA prepared and analyzed for the levels of expression of STRIFEl or STRIFE2 and other genes implicated in the proliferative disorder, developmental or differentiative disorder, or hematopoietic disorder, respectively.
  • the levels of gene expression can be quantified by Northern blot analysis or RT-PCR, as described herein, or alternatively by measuring the amount of protein produced, by one ofthe methods as described herein, or by measuring the levels of activity of STRIFEl or STRIFE2 or other genes.
  • the gene expression pattern can serve as a marker, indicative ofthe physiological response ofthe cells to the agent. Accordingly, this response state may be determined before, and at various points during treatment ofthe individual with the agent.
  • the present invention provides a method for monitoring the effectiveness of treatment of a subject with an agent (e.g., an agonist, antagonist, peptidomimetic, protein, peptide, nucleic acid, small molecule, or other drag candidate identified by the screening assays described herein) comprising the steps of (i) obtaining a pre-administration sample from a subject prior to administration ofthe agent; (ii) detecting the level of expression of a STRIFEl or STRIFE2 protein, mRNA, or genomic DNA in the preadministration sample; (iii) obtaining one or more post- administration samples from the subject; (iv) detecting the level of expression or activity of the STRIFE 1 or STRIFE2 protein, mRNA, or genomic DNA in the post- administration samples; (v) comparing the level of expression or activity ofthe STRIFEl or STRIFE2 protein, mRNA, or genomic DNA in the pre-administration sample with the STRIFEl or STRIFE2 protein, mRNA, or genomic DNA in the
  • increased administration ofthe agent may be desirable to increase the expression or activity of STRIFEl or STRIFE2 to higher levels than detected, i.e., to increase the effectiveness ofthe agent.
  • decreased administration ofthe agent may be desirable to decrease expression or activity of STRIFEl or STRIFE2 to lower levels than detected, i.e. to decrease the effectiveness of the agent.
  • STRIFEl or STRIFE2 expression or activity may be used as an indicator ofthe effectiveness of an agent, even in the absence of an observable phenotypic response.
  • the present invention provides for both prophylactic and therapeutic methods of treating a subject at risk of (or susceptible to) a disorder or having a disorder associated with aberrant STRIFEl or STRIFE2 expression or activity.
  • treatments may be specifically tailored or modified, based on knowledge obtained from the field of pharmacogenomics.
  • “Pharmacogenomics”, as used herein refers to the application of genomics technologies such as gene sequencing, statistical genetics, and gene expression analysis to drags in clinical development and on the market. More specifically, the term refers to the study of how a patient's genes determine his or her response to a drag (e.g., a patient's "drag response phenotype", or “drug response genotype”).
  • another aspect ofthe invention provides methods for tailoring an individual's prophylactic or therapeutic treatment with either the STRIFEl or STRIFE2 molecules ofthe present invention or STRIFEl or STRIFE2 modulators according to that individual's drag response genotype.
  • Pharmacogenomics allows a clinician or physician to target prophylactic or therapeutic treatments to patients who will most benefit from the treatment and to avoid treatment of patients who will experience toxic drug-related side effects.
  • the invention provides a method for preventing in a subject, a disease or condition associated with an aberrant STRIFEl or STRIFE2 expression or activity, by administering to the subject an agent which modulates STRIFEl or STRIFE2 expression or at least one STRIFEl or STRIFE2 activity.
  • Subjects at risk for a disease which is caused or contributed to by aberrant STRIFEl or STRIFE2 expression or activity can be identified by, for example, any or a combination of diagnostic or prognostic assays as described herein.
  • Administration of a prophylactic agent can occur prior to the manifestation of symptoms characteristic ofthe STRIFEl or STRIFE2 aberrancy, such that a disease or disorder is prevented or, alternatively, delayed in its progression.
  • an STRIFEl or STRIFE2 agonist or STRIFEl or STRIFE2 antagonist agent can be used for treating the subject.
  • the appropriate agent can be determined based on screening assays described herein. The prophylactic methods ofthe present invention are further discussed in the following subsections. - 69 -
  • the modulatory method ofthe invention involves contacting a cell with an agent that modulates one or more ofthe activities of STRIFEl or STRIFE2 protein activity associated with the cell.
  • An agent that modulates STRIFEl or STRIFE2 protein activity can be an agent as described herein, such as a nucleic acid or a protein, a naturally-occurring target molecule of a STRIFEl or STRIFE2 protein, a peptide, a STRIFEl or STRIFE2 peptidomimetic, or other small molecule.
  • the agent stimulates one or more STRIFEl or STRIFE2 protein activity.
  • stimulatory agents include active STRIFEl or STRIFE2 protein and a nucleic acid molecule encoding STRIFEl or STRIFE2 that has been introduced into the cell.
  • the agent inhibits one or more STRIFEl or STRIFE2 protein activity.
  • inhibitory agents include antisense STRIFEl or STRIFE2 nucleic acid molecules and anti-STRIFEl or STRIFE2 antibodies.
  • the present invention provides methods of treating an individual afflicted with a disease or disorder characterized by aberrant expression or activity of a STRIFEl or STRIFE2 protein or nucleic acid molecule.
  • the method involves administering an agent (e.g., an agent identified by a screening assay described herein), or combination of agents that modulates (e.g., upregulates or downregulates) STRIFEl or STRIFE2 expression or activity.
  • the method involves administering a STRIFEl or STRIFE2 protein or nucleic acid molecule as therapy to compensate for reduced or aberrant STRIFEl or STRIFE2 expression or activity.
  • Stimulation of STRIFEl or STRIFE2 activity is desirable in situations in which STRIFEl or STRIFE2 is abnormally downregulated and/or in which increased STRIFEl or STRIFE2 activity is likely to have a beneficial effect.
  • inhibition of STRIFEl or STRIFE2 activity is desirable in situations in which STRIFEl or STRIFE2 is abnormally upregulated and/or in which decreased STRIFEl or STRIFE2 activity is likely to have a beneficial effect.
  • TNF-associated disorder e.g., an inflammatory, immune, or neoplastic disorder.
  • STRIFEl or STRIFE2 molecules ofthe present invention as well as agents, or modulators which have a stimulatory or inhibitory effect on STRIFEl or STRIFE2 activity (e.g., STRIFEl or STRIFE2 gene expression) as identified by a screening assay - 70 -
  • pharmacogenomics i.e., the study ofthe relationship between an individual's genotype and that individual's response to a foreign compound or drag
  • Differences in metabolism of therapeutics can lead to severe toxicity or therapeutic failure by altering the relation between dose and blood concentration ofthe pharmacologically active drag.
  • a physician or clinician may consider applying knowledge obtained in relevant pharmacogenomics studies in determining whether to administer an STRIFEl or STRIFE2 molecule or STRIFEl or STRIFE2 modulator as well as tailoring the dosage and/or therapeutic regimen of treatment with an STRIFEl or STRIFE2 molecule or STRIFEl or STRIFE2 modulator.
  • Pharmacogenomics deals with clinically significant hereditary variations in the response to drugs due to altered drug disposition and abnormal action in affected persons. See e.g., Eichelbaum, M., Clin Exp Pharmacol Physiol, 1996, 23(10-11) :983- 985 and Linder, M.W., Clin Chem, 1997, 43(2):254-266.
  • two types of pharmacogenetic conditions can be differentiated. Genetic conditions transmitted as a single factor altering the way drugs act on the body (altered drag action) or genetic conditions transmitted as single factors altering the way the body acts on drugs (altered drug metabolism). These pharmacogenetic conditions can occur either as rare genetic defects or as naturally-occurring polymo ⁇ hisms.
  • G6PD glucose-6-phosphate dehydrogenase deficiency
  • oxidant drags anti-malarials, sulfonamides, analgesics, nitrofurans
  • a genome-wide association relies primarily on a high-resolution map ofthe human genome consisting of already known gene-related markers (e.g., a "bi- allelic" gene marker map which consists of 60,000-100,000 polymo ⁇ hic or variable sites on the human genome, each of which has two variants).
  • Such a high-resolution genetic map can be compared to a map ofthe genome of each of a statistically significant number of patients taking part in a Phase II/III drug trial to identify markers associated with a particular observed drug response or side effect.
  • a high resolution map can be generated from a combination of some ten-million known single nucleotide polymo ⁇ hisms (SNPs) in the human genome.
  • SNPs single nucleotide polymo ⁇ hisms
  • a SNP may occur once per every 1000 bases of DNA.
  • a SNP may be involved in a disease process, however, the vast majority may not be disease- - 71 -
  • a method termed the "candidate gene approach” can be utilized to identify genes that predict drag response.
  • a gene that encodes a drag target e.g., a STRIFEl or STRIFE2 protein or a STRIFEl receptor ofthe present invention
  • all common variants of that gene can be identified in the population and a particular drag response can be associated with one or more genes.
  • the activity of drug metabolizing enzymes is a major determinant of both the intensity and duration of drag action.
  • the gene coding for CYP2D6 is highly polymo ⁇ hic and several mutations have been identified in PM, which all lead to the absence of functional CYP2D6. Poor metabolizers of CYP2D6 and CYP2C19 quite frequently experience exaggerated drug response and side effects when they receive standard doses. If a metabolite is the active therapeutic moiety, PM show no therapeutic response, as demonstrated for the analgesic effect of codeine mediated by its C YP2D6-formed metabolite mo ⁇ hine. The other extreme are the so called ultra-rapid metabolizers who do not respond to standard doses. Recently, the molecular basis of ultra-rapid metabolism has been identified to be due to CYP2D6 gene amplification.
  • a method termed the "gene expression profiling" can be utilized to identify genes that predict drag response.
  • a drug e.g., a STRIFEl or STRIFE2 molecule or STRIFEl or STRIFE2 modulator ofthe present invention
  • a drug e.g., a STRIFEl or STRIFE2 molecule or STRIFEl or STRIFE2 modulator ofthe present invention
  • Information generated from more than one ofthe above pharmacogenomics approaches can be used to determine appropriate dosage and treatment regimens for prophylactic or therapeutic treatment an individual. This knowledge, when applied to dosing or drag selection, can avoid adverse reactions or therapeutic failure and thus enhance therapeutic or prophylactic efficiency when treating a subject with a STRIFEl - 72 -
  • STRIFE2 molecule or STRIFEl or STRIFE2 modulator such as a modulator identified by one ofthe exemplary screening assays described herein.
  • STRIFE is a mouse gene which encodes a protein belonging to the TNFR family. Two splice forms have been identified, one that is predicted to be membrane bound (STRIFEl) and one that is secreted (STRIFE2).
  • STRIFE was identified as a TNFR homologue by a computer-based search ofthe public EST databases. More specifically, the murine STRIFEl and STRIFE2 cDNA were identified by searching against a copy ofthe GenBank nucleotide database using the BLASTNTM program (BLASTN 1.3MP: Altschul et al., J Mol Bio. 215:403, 1990). Numerous clones that consisted mostly of 3' reads and some that were 5' reads within the 3' untranslated region were found by this search. The sequences were analyzed against a non-redundant protein database with the BLASTXTM program, which translates a nucleic acid sequence in all six frames and compares it against available protein databases
  • BLASTX 1.3MP:Altschul et al., supra This protein database is a combination ofthe Swiss-Prot, PIR, and NCBI GenPept protein databases. Two clones (Accession Numbers AA036247 and AA003356) were obtained from the IMAGE consortium, and fully sequenced. The additional sequencing of AA036247 (T 127a; STRIFEl) extended the original EST by 623 nucleotides (see SEQ ID NOT) and the further sequencing of AA003356 (T127b; STRIFE2) extended the original EST by 254 nucleotides (see SEQ ID NO:5).
  • Human I and mouse multiple tissue northern (MTN) blots (Clontech, Palo Alto, CA) containing 2 ⁇ g of poly A+ RNA per lane were probed with a 750bp EcoRRNot/ fragment ofthe mouse STRIFEl cDNA.
  • the filters were prehybridized in 10 ml of Express Hyb hybridization solution (Clontech, Palo Alto, CA) at 68°C for 1 hour, after which 100 ng of 3 P labeled probe was added.
  • the probe was generated using the Stratagene Prime-It kit, Catalog Number 300392 (Clontech, Palo Alto, CA). Hybridization was allowed to proceed at 68°C for approximately 2 hours.
  • the filters were washed in a 0.05% SDS/2X SSC solution for 15 minutes at room temperature and then twice with a 0.1% SDS/0.1X SSC solution for 20 minutes at 50°C and then exposed to autoradiography film overnight at -80°C with one screen.
  • the mouse tissues tested included: heart, brain, spleen, lung, liver, skeletal muscle, kidney, and testis.
  • the human tissues tested included: heart, placenta, lung, liver, skeletal muscle, kidney, and pancreas.
  • STRIFEl or STRIFE2 is expressed as a recombinant glutathione-S-transferase (GST) fusion polypeptide in E. coli and the fusion polypeptide is isolated and characterized.
  • GST glutathione-S-transferase
  • STRIFEl or STRIFE2 is fused to GST and this fusion polypeptide is expressed in E. coli, e.g., strain PEB199.
  • the murine STRIFEl and STRIFE2 proteins are predicted to be approximately 23.55 kDa and 16.72 kDa, respectively, and GST is predicted to be 26 kDa
  • the fusion polypeptides are predicted to be approximately 49.55 kDa and 42.72 kDa, respectively, in molecular weight.
  • Expression ofthe GST-STRIFE1 or STRIFE2 fusion protein in PEB199 is induced with IPTG.
  • the recombinant fusion polypeptide is purified from crude bacterial -
  • the pcDNA/Amp vector by Invitrogen Co ⁇ oration (San Diego, CA) is used.
  • This vector contains an SV40 origin of replication, an ampicillin resistance gene, an E. coli replication origin, a CMV promoter followed by a polylinker region, and an SV40 intron and polyadenylation site.
  • a DNA fragment encoding the entire STRIFEl or STRIFE2 protein and a HA tag (Wilson et al.
  • the STRIFEl or STRIFE2 DNA sequence is amplified by PCR using two primers.
  • the 5' primer contains the restriction site of interest followed by approximately twenty nucleotides ofthe STRIFEl or STRIFE2 coding sequence starting from the initiation codon; the 3' end sequence contains complementary sequences to the other restriction site of interest, a translation stop codon, the HA tag and the last 20 nucleotides ofthe STRIFEl or STRIFE2 coding sequence.
  • the PCR amplified fragment and the pCDNA/Amp vector are digested with the appropriate restriction enzymes and the vector is dephosphorylated using the CIAP enzyme (New England Biolabs, Beverly, MA).
  • the two restriction sites chosen are different so that the STRIFEl or STRIFE2 gene is inserted in the correct orientation.
  • the ligation mixture is transformed into E. coli cells (strains HBlOl , DH5a, SURE, available from Stratagene Cloning Systems, La Jolla, CA, can be used), the transformed culture is plated on ampicillin media plates, and resistant colonies are selected. Plasmid DNA is isolated from transformants and examined by restriction analysis for the presence ofthe correct fragment.
  • COS cells are subsequently transfected with the STRIFEl or STRIFE2- pcDNA/Amp plasmid DNA using the calcium phosphate or calcium chloride co- precipitation methods, DEAE-dextran-mediated transfection, lipofection, or electroporation.
  • Other suitable methods for transfecting host cells can be found in Sambrook, J., Fritsh, E. F., and Maniatis, T. Molecular Cloning: A Laboratory Manual. 2nd, ed., Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989.
  • the expression ofthe STRIFEl and STRIFE2 polypeptide is detected by radiolabelling ( 35 S-methionine or 35 S-cysteine available from NEN, Boston, - 75 -
  • the cells are labelled for 8 hours with 35 S-methionine (or 35 S-cysteine).
  • the culture media are then collected and the cells are lysed using detergents (RIPA buffer, 150 mM NaCI, 1 % NP-40, 0.1 % SDS, 0.5% DOC, 50 mM Tris, pH 7.5). Both the cell lysate and the culture media are precipitated with an HA specific monoclonal antibody. Precipitated polypeptides are then analyzed by SDS-PAGE.
  • DNA containing the STRIFEl or STRIFE2 coding sequence is cloned directly into the polylinker ofthe pCDNA Amp vector using the appropriate restriction sites.
  • the resulting plasmid is transfected into COS cells in the manner described above, and the expression ofthe STRIFEl or STRIFE2 polypeptide is detected by radiolabelling and immunoprecipitation using an STRIFEl or STRIFE2 specific monoclonal antibody.
  • STRIFEl is approximately 981 nucleotides in length and has an open reading frame of 645 nucleotides that is predicted to encode a protein of 214 amino acids.
  • STRIFE2 is approximately 655 nucleotides long with an open reading frame of 453 nucleotides predicted to encode a protein of 150 amino acids. Both clones have been subcloned into a variety of expression vectors including those for retroviral delivery and for expression in bacterial, yeast and mammalian cells.
  • STRIFE2 are approximately 40% identical to OX40. Importantly, a number of cysteine residues within the extracellular domains of STRIFEl and STRIFE2 match the cysteine- rich domain signature ofthe TNFR/NGFR family (Prosite Accession PDOC00561). The program SignalP (Nielsen et al, 1997) predicts a 30 amino acid signal peptide at the very N-terminus of both STRIFEl and STRIFE2 (i.e., aa 1 -29 of SEQ ID NOs: 1 and 5).
  • the predicted molecular weight for STRIFEl is approximately 23.55 kDa with the signal peptide and 20.34 kDa without the signal peptide which is presumed to be cleaved in the mature protein. There are no obvious motifs in the small intracellular domain of STRIFEl . STRIFE2 is predicted to be 16.72 kDa with the signal peptide and 13.51 kDa without the signal peptide.
  • STRIFEl is 70.6% identical to the nucleic acid molecule encoding the human OAF065 receptor (Accession number V33362; described in PCT application number WO 98/38304, published on September 3, 1998) over nucleotide residues 65- 981. The results from this search are shown in Figure 5.
  • STRIFEl or STRIFE2 The entire open reading frame of STRIFEl or STRIFE2 is subcloned into the retroviral vector MSCVneo, described in Hawley et al.(1994) Gene Therapy 1:136-138.
  • Cells (293Ebna, Invitrogen) are then transiently transfected with the STRIFEl or STRIFE2 construct and with constructs containing viral regulatory elements, to produce high titre retrovirus containing the STRIFEl or STRIFE2 gene. This virus is then used to transfect mice. These mice are then tested for any gross pathology and for changes in their immune response using standard assays.

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WO1999011791A2 (en) * 1997-09-05 1999-03-11 University Of Washington Tumor necrosis factor family receptors and ligands, encoding nucleic acids and related binding agents
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