WO1999011791A2

WO1999011791A2 - Tumor necrosis factor family receptors and ligands, encoding nucleic acids and related binding agents

Info

Publication number: WO1999011791A2
Application number: PCT/US1998/018393
Authority: WO
Inventors: Preet M. Chaudhary
Original assignee: University Of Washington
Priority date: 1997-09-05
Filing date: 1998-09-04
Publication date: 1999-03-11
Also published as: WO1999011791A3; AU9376498A

Abstract

The invention provides novel receptors of the tumor necrosis factor receptor family as well as ligands of the tumor necrosis factor family. In addition to the isolated receptors and ligands of the invention, there are provided encoding nucleic acids and related selective binding agents.

Description

TUMOR NECROSIS FACTOR FAMILY RECEPTORS AND LIGANDS. ENCODING NUCLEIC ACIDS AND RELATED BINDING AGENTS

BACKGROUND OF THE INVENTION

This invention relates to molecules involved in apoptosis and more specifically to tumor necrosis family receptors and ligands.

The tumor necrosis factor (TNF) family of ligands are a superfamily of polypeptides involved in cell-cell signaling. The prototypic TNF molecule was first identified by its ability to kill a variety of transformed cell lines. In addition to their role in cell survival and death, TNF-like molecules and their cognate receptors are now known to be involved in the regulation of a variety of cellular functions including cellular proliferation, differentiation and cytokine secretion.

Ligands of the TNF superfamily are type II membrane glycoproteins with a large C-terminal extracellular domain, a single transmembrane spanning region and a variable cytoplasmic domain. Like the prototypic TNF- molecule, most are acidic molecules with approximately 20% sequence homology in the extracellular receptor-binding domain. The conservation of the more variable cytoplasmic domains among species homologs indicates that the cytoplasmic domains also can serve an important function in signal transduction. The TNFR ligand family includes TNF-α, TNF-β, LTβ, FAS ligand, CD27 ligand, CD30 ligand, CD40 ligand, OX40 ligand and 4-1BB ligand. Although ligands of the TNF family are typically expressed on the cell surface, soluble forms of TNF-α, TNF-β and FAS ligand also have been identified. TNF receptor family members generally are type I membrane glycoproteins with sequence homology typically confined to the extracellular domain. This amino-terminal extracellular domain includes a variable number of cysteine-rich pseudo repeats, which are motifs of approximately 40 amino acids containing about six cysteine residues. Numerous cellular and viral members of the TNF receptor superfamily have been described, including TNFR-1 and TNFR-2, TNFR-RP/TNFR-3, nerve growth factor receptor (NGFR) , CD27, CD30, CD40, 4-1BB, OX-40, FAS/APO-1 (CD95), DR3 ( sl-l/APO-3/TRAMP) and DR . In addition, related viral proteins PV-T2 and PV-A53R encode soluble secreted members of the TNF receptor family.

Several of the tumor necrosis factor receptors function in the process of regulated cell death, termed programmed cell death, or "apoptosis." TNFR-1, FAS/APO-1 and DR3 are TNF receptors that transmit a suicidal signal through a conserved 80 amino acid cytoplasmic region known as a "death domain." Cytoplasmic death-domain containing proteins including FADD/MORTl (Fas-associated protein with death domain) and TRADD (TNFR-1-associated death domain protein) act as intracellular adaptor molecules in transmitting the ligand-dependent apoptotic signal initiated by a "death receptor." The FAS/APO-1 receptor appears to directly interact with FADD/MORTl, while other death receptors such as TNFR-1 and DR3 associate with TRADD, which acts as an adaptor molecule and recruits FADD/MORT . Despite its sequence homology to the similar domains present in the death receptors, the death domain of FADD does not induce apoptosis when over-expressed in mammalian cells and blocks apoptosis mediated by full-length FADD in a dominant negative fashion. However, FADD possesses another conserved domain denoted a "death effector domain" (DED) , which mediates apoptosis when over-expressed in mammalian cells. Through its DED, FADD binds to homologous domains present in the cysteine protease (caspase) FLICE/MACHl (caspase 8). FLICE/MACHl is the most proximal caspase in a cascade of cysteine proteases that are triggered by ligand-dependent activation of the death receptors, ultimating resulting in programmed cell death.

In addition to mediating apoptosis through FADD, the TRADD adaptor molecule can recruit other molecules to the aggregated receptor complex of TNFR-1 or DR3, including the death domain-containing protein RIP (receptor-interacting protein) and TRAF2, which lacks a death domain. While recruitment of FADD leads to activation of caspases and eventual cell death, recruitment of RIP and TRAF2 leads to activation of the NF-κB (nuclear factor kappa-B) pathway, which may protect cells from TNF-induced apoptosis.

Programmed cell death plays a major role throughout development, as well as in the homeostatic control of cell numbers and in defense against intracellular pathogens. Furthermore, abnormalities in programmed cell death contribute to the pathogenesis of a number of diseases. For example, the failure of cells to undergo apoptotic cell death can be involved in cancers such as breast, prostate and ovarian cancer, in autoimmune diseases and in viral infections. Furthermore, a number of diseases are characterized by excessive apoptosis including acquired immunodeficiency syndrome (AIDS) ; neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease and retinitis pigmentosa; osteoporosis; ischemic injury; vasculitis; hepatic necrosis; and cerebral or myocardial infarction. Excessive cell death also can contribute to organ-specific autoimmune diseases such as Hashimoto thyroiditis. Therapies which are specifically designed to modulate apoptotic pathways can change the natural progression of these and other pathologies characterized by inadequate or excessive apoptosis.

Unfortunately, the ability to provide effective therapeutic intervention is limited by the fact that critical polypeptide receptors and ligands involved in the process of apoptosis remain to be isolated. For example, novel TNF superfamily receptors, which can play a critical role in regulating apoptosis in particular cell types or under certain conditions, remain to be isolated.

Thus, there exists a need to identify and isolate novel TNF superfamily receptors and ligands and their encoding nucleic acids . The present invention satisfies this need and provides related advantages as well .

SUMMARY OF THE INVENTION

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1. Alignment of the extracellular domains of several representative tumor necrosis family receptors. Alignment was done by pileup program of GCG software (Genetic Computer Group, Madison, WI). Dominant residues are shown in upper case, or determined by the pretty program of the GCG software.

Figure 2. hAP08 nucleotide sequence SEQ ID N0:1 and amino acid sequence SEQ ID NO: 2.

Figure 3. AP08-related polypeptide (AP08RP) nucleotide sequence SEQ ID NO: 3 and amino acid sequence SEQ ID NO: 4.

Figure 4. (A) Alignment of the predicted amino acid sequence of AP08 with AP08RP. Identical amino acids are shaded black and homologous residues are shaded gray. Predicted signal peptides, transmembrane regions (TM regions) and death domains are indicated. (B) Sequence alignment of the death domains of AP08 , DR4 , DR3, TNFR-1 and FAS/APO-1. Residues identical in more than 30% of sequences are shaded black and those homologous in greater than 30% of sequences are shaded gray. Residues corresponding to the site of the lpr mutation in the murine FAS receptor are indicated by a Δ symbol.

Figure 5. (A) Induction of apoptosis in MCF-7 cells by overexpression of either the full-length AP08 construct or AP08 mutant constructs. (B) Inhibition of AP08, AP08RP and DR3 induced apoptosis by progressive increasing amounts of dominant negative FADD (DN-FD) in 293T cells. The DNA ratio of receptor to DN-FADD is shown in parenthesis. © Inhibition of AP08-induced apoptosis in MCF-7 cells by dominant negative FADD (DN-FD), dominant negative FLICE (FL-C360S) and the cysteine protease inhibitors CrmA and z-VAD-fmk.

(D) AP08 and AP08RP activate the NF-κB pathway. Shown is the relative luciferase activity from an NF-κB luciferase reporter in 293T cells cotransfected with the indicated reporter construct.

Figure 6. hAP09 nucleotide sequence SEQ ID NO: 5 and amino acid sequence SEQ ID NO: 6.

Figure 7. (A) mAP04-α long nucleotide sequence SEQ ID NO: 7 and amino acid sequence SEQ ID NO: 8. (B) mAP04-α short nucleotide sequence SEQ ID NO: 9 and amino acid sequence SEQ ID NO: 10. © hAP04-α nucleotide sequence SEQ ID NO: 11 and amino acid sequence SEQ ID

NO: 12. (D) rAP04-α nucleotide sequence SEQ ID NO: 13 and amino acid sequence SEQ ID NO: 14. (E) mAP04-γ nucleotide sequence SEQ ID NO: 31 and amino acid sequence SEQ ID NO:32.

Figure 8. mAP04-β nucleotide sequence SEQ ID

NO: 15 and amino acid sequence SEQ ID NO: 16.

Figure 9. hAP06 nucleotide sequence SEQ ID NO: 17 and amino acid sequence SEQ ID NO: 18.

Figure 10. Alignment of representative ligands of the tumor necrosis factor family. Residues identical in more than 30% of sequences are shaded black and those homologous in greater than 30% of sequences are shaded gray.

Figure 11. (A) hTNRLl-α nucleotide sequence SEQ ID NO: 17 and amino acid sequence SEQ ID NO: 20. (B) mTNRLl-α nucleotide sequence SEQ ID NO: 21 and amino acid sequence SEQ ID NO: 22. Figure 12. (A) hTNRLl-β nucleotide sequence SEQ ID NO:23 and amino acid sequence SEQ ID NO:24. (B) mTNRLl-β nucleotide sequence SEQ ID NO: 25 and amino acid sequence SEQ ID NO: 26.

Figure 13. (A) hTNRL3 nucleotide sequence SEQ

ID NO: 27 and amino acid sequence SEQ ID NO: 28. (B) mTNRL3 nucleotide sequence SEQ ID NO: 29 and amino acid sequence SEQ ID NO: 30.

Figure 14. (A) Induction of apoptosis in 293T cells by expression of full-length human AP04 (hAP04). Inhibition of AP04-induced apoptosis by various inhibitors of the caspase or JNK pathway are also shown. (B) hAP04 activates the JNK pathway. Shown is the relative luciferase activity from a c-Jun transactivating reporter in 293T cells cotransfected with hAP04 in the presence or absence of the indicated inhibitor plasmid (JBD of JIP-1, MEKK1-D1369A, or DN-TRAF2) . (C) hAP04 activates the NF-κB pathway. Shown is the relative luciferase activity from an NF-κB reporter construct in 293T cells cotransfected with hAP04 and empty vector, dominant negative TRAF2 (DN-TRAF2) or IκBα-S32/36A.

DETAILED DESCRIPTION OF THE INVENTION

Members of the TNFR family are type I transmembrane receptors characterized by the presence of cysteine-rich pseudo-repeats in the extracellular domain. These cysteine rich regions, which are motifs of approximately 40 amino acids with about 6 cysteines, are involved in ligand binding. The average homology in the cysteine-rich extracellular region is in the range of 25% to 30%. In addition to a signal peptide, a large extracellular domain and hydrophobic transmembrane region, receptors of the TNF family have cytoplasmic domains, which function in transmitting a ligand-dependent signal.

The TNF receptor superfamily contains at least 10 different cellular membrane proteins and several viral proteins encoding TNF receptor related molecules. The first cloned receptor of this family was the nerve growth factor receptor (NGFR) , and the TNF receptor superfamily of type I transmembrane receptors now includes TNFR-1 and TNFR-2, TNFR-RP/TNFR-3, nerve growth factor receptor

(NGFR), CD27, CD30, CD40, 4-1BB, OX-40, FAS/APO-1 (CD95), DR3 (Wsl-l/APO-3/TRAMP) and DR . In addition, related viral proteins PV-T2 and PV-A53R encode soluble secreted members of the TNF receptor family. Several members such as TNFR-1, TNFR-2 and NGFR have a broad tissue distribution, while CD27, CD30, CD40, 4-1BB and OX40 are chiefly restricted to cells of the lymphoid or hematopoietic systems.

The present invention is directed to the discovery of several new receptors belonging to the TNF family, designated an AP08 polypeptide, an AP08 related polypeptide (AP08RP) , an AP09 polypeptide, an AP04 polypeptide, and an AP06 polypeptide. In general, the receptors of the invention are transmembrane proteins with large extracellular ligand-binding domains sharing 16-60% sequence identity in this region with other members of the TNF receptor family (Figure 1 and Table 1 above) . A preferred method for determining amino acid identity is by pairwise alignment using the gap program of GCG software (Genetic Computer Group, Madison, WI) using the default parameters. The present invention provides an isolated AP08 polypeptide, which is a novel TNF receptor having substantially the same amino acid sequence as AP08, or an active fragment thereof. An isolated AP08 polypeptide of the invention can have substantially the same amino acid sequence as the AP08 sequence SEQ ID NO: 2 shown in Figure 2.

An exemplary human AP08 polypeptide (hAP08) is provided herein. hAP08 is a widely expressed polypeptide containing structural features reminiscent of known "death receptors," including an intracellular death domain. As disclosed herein, overexpression of hAP08 results in apoptosis, which is caspase-dependent and also dependent upon the FADD/MORT adaptor molecule (Example II). hAP08 also activates NF-κB expression through the TRADD adaptor molecule and, thus, can have anti-apoptotic activity. As further disclosed herein, hAP08 interacts directly with TRADD, thereby recruiting FADD/MORT. The hAP08 polypeptide of the invention can be useful in identifying selective enhancers or inhibitors of AP08 activity, which can be used as novel therapeutics for the management of AP08-mediated diseases or diseases involving the dysfunction of programmed cell death. In addition, nucleic acid molecules encoding hAP08 or apoptotic fragments of hAP08 can themselves be used as inducers or inhibitors of programmed cell death.

The term "isolated," as used herein in reference to a polypeptide of the invention, means a polypeptide that is in a form that is relatively free from contaminating lipids, unrelated polypeptides, nucleic acids and other cellular material normally associated with the polypeptide in a cell. As used herein, the term "AP08" means an AP08 polypeptide and includes polypeptides having substantially the same amino acid sequence as the hAP08 polypeptide having amino acid sequence SEQ ID NO: 2. hAP08 is a transmembrane protein of 411 amino acids having the amino acid sequence shown in Figure 2. hAP08 contains an N-terminal signal peptide of 51 amino acids, a large extracellular domain with characteristic TNF receptor family cysteine-rich regions, a hydrophobic transmembrane domain of 27 amino acids (residues 185 to 212) and a C-terminal cytoplasmic domain (residues 213 to 411) . The AP08 cytoplasmic domain includes a "death domain, " which is a conserved domain of about 80 amino acids present in pro-apoptotic TNF receptors such as TNFR-1, FAS/APO-1 and DR3 and that is required for apoptosis. Death domains also are present in several cytoplasmic proteins, and a homotypic association between death domains appears responsible for the interaction of these cytoplasmic proteins with TNFR-1, FAS/APO-1 and DR3.

As disclosed in Example I, hAPOδ RNA is expressed in a variety of tissues, with a particularly high level of expression observed in peripheral blood lymphocytes (PBL) . The results disclosed in Example II demonstrate that the hAP08 polypeptide of the invention exhibits caspase-dependent apoptotic activity and activates NF-κB gene expression. Thus, hAP08 is a TNF family receptor, which, like TNFR-1 and DR3, activates NF- B expression and promotes apoptosis through a cascade of cysteine proteases.

The term AP08 encompasses a polypeptide having the sequence of the naturally occurring human AP08 polypeptide (SEQ ID NO: 2) and is intended to include related polypeptides having substantial amino acid sequence similarity to hAP08 (SEQ ID N0:2) . Such related polypeptides exhibit greater sequence similarity to hAP08 than to other polypeptides containing cysteine-rich pseudo-repeats or other death domain containing polypeptides and include alternatively spliced forms of hAP08, species homologues, and isotype variants of the amino acid sequence shown in Figure 2. The hAP08 polypeptide disclosed herein has about 5δ% amino acid identity and about 71% amino acid similarity with the Death Receptor (DR4). As used herein, the term APOδ describes polypeptides generally having an amino acid sequence with greater than about 65% amino acid sequence identity with hAPOδ (SEQ ID NO: 2), preferably greater than about 75% amino acid identity with hAP08 (SEQ ID

N0:2), more preferably greater than about 85% amino acid identity with hAP08 (SEQ ID N0:2), and includes polypeptides having greater than about 90%, 95% or 97% amino acid identity with hAP08 (SEQ ID NO:2).

The present invention also provides active fragments of the AP08 polypeptide of the invention. In general, the term "active fragment" means a polypeptide fragment having substantially the same amino acid sequence as a portion of the indicated polypeptide of the invention, provided that the fragment retains at least one biological activity of the polypeptide. A portion of a polypeptide generally has an amino acid sequence of about 15 to about 400 contiguous residues and can have, for example, an amino acid sequence of at least about 18, 20, 25, 30, 35, 40, 50, 100, 150 or 200 contiguous residues. As described further below, the invention also provides active segments of the AP08 polypeptide of the invention. In general, an active segment is characterized, in part, by having substantially the same amino acid sequence as a portion of the indicated polypeptide. Such an active segment generally has an amino acid sequence of about 15 to about 400 contiguous residues and can have, for example, an amino acid sequence of at least about 18, 20, 25, 30, 35, 40, 50 100, 150 or 200 contiguous residues.

As used herein, an "active fragment of an AP08 polypeptide" is synonymous with "active fragment of AP08" or "active APOδ fragment" and means a polypeptide fragment having substantially the same amino acid sequence as a portion of an AP08 polypeptide, provided that the fragment retains at least one biological activity of an AP08 polypeptide. An active fragment of an AP08 polypeptide can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hAPOδ (SEQ ID NO: 2), provided that the fragment retains at least one biological activity of an AP08 polypeptide. A biological activity of AP08 can be, for example, the ability to bind ligand, bind TRADD or RIP, induce or suppress apoptosis, activate NF- B expression, or induce or suppress cell proliferation, differentiation or cytokine secretion. Particularly useful active fragment of APOδ are polypeptide fragments having pro-apoptotic or anti-apoptotic activity. A biological activity of an APOδ polypeptide or fragment can be routinely assayed; for example, apoptotic activity can be analyzed by transfecting an AP08 encoding nucleic acid and measuring the number of cells with apoptotic morphology, as set forth in Example II.

An "active fragment of an AP08 polypeptide" also can be an active AP08 segment, which is a polypeptide portion having substantially the same amino acid sequence as a portion of an APOδ polypeptide, provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA223122 or AA232440 and provided that the segment retains at least one biological activity of an APOδ polypeptide. An active APOδ segment can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hAP08 (SEQ ID N0:2), provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA223122 or AA232440 and provided that the segment retains at least one biological activity of an AP08 polypeptide.

In one embodiment, the invention provides active fragments that are soluble, extracellularly expressed forms of the receptors of the invention. Such a soluble active fragment includes the receptor ligand binding domain. Such a soluble active fragment can be an inhibitory polypeptide that binds ligand and opposes the biological function of full-length or membrane-bound receptor. For example, as disclosed herein, AP08 induces caspase-dependent apoptosis when expressed in BHK cells; a soluble form of the APOδ receptor can compete for binding to an APOδ ligand and reduce or inhibit the pro-apoptotic activity of AP08. Another use of such soluble forms of the receptors of the invention can be to transduce a signal across the surface of a cell which expresses the cognate ligand of the receptor. One skilled in the art understands that a soluble receptor can act in a systemic fashion, for example, to block the activity of full-length or membrane-bound receptors wherever the full-length receptor is expressed. As used herein, the term "soluble" refers to a polypeptide that is not membrane bound. A particularly useful soluble polypeptide of the invention is secreted and, thus, expressed extracellularly .

Provided herein is a soluble APOδ active fragment that includes an APOδ ligand binding domain. A soluble AP08 active fragment of the invention can be, for example, a truncated polypeptide encoding the extracellular domain of APOδ. Such a soluble AP08 active fragment can have, for example, substantially the same amino acid sequence as the signal peptide and ligand-binding domain of hAP08 (amino acids 1 to 184 of hAPOδ shown in Figure 1) . One skilled in the art understands that a soluble APOδ active fragment is distinguished from a membrane-bound fragment by the deletion or inactivation of the transmembrane domain, which is shown as amino acids 185 to 212 in Figure 2. A soluble AP08 active fragment also can be, for example, an active APOδ segment. Active APOδ segments are described hereinabove.

The present invention also provides an isolated AP08-related polypeptide, which is a novel TNF superfamily receptor having substantially the same amino acid sequence as APOδ or an APOδ-related polypeptide. An isolated APOδ related polypeptide of the invention can have substantially the same amino acid sequence as the AP08 sequence SEQ ID NO: 2 shown in Figure 2 or substantially the same amino acid sequence as the AP08RP sequence SEQ ID NO: 4 shown in Figure 3. An APOβ-related polypeptide is characterized, in part, by the ability to induce FADD-dependent apoptosis or the ability to activate NF- B. As disclosed herein, AP08 and AP08RP activate NF-κB by a TRADD dependent pathway. As further disclosed herein, AP08 and AP08RP interact directly with TRADD in co-immunoprecipitation experiments, and FADD is indirectly recruited to AP08 or AP08RP through TRADD. An AP08-related polypeptide of the invention can be useful in diagnosing an AP08-related polypeptide-mediated disease or in treating or reducing the severity of such a disease, as described further below.

hAPOδ related polypeptides include hAP08RP, a polypeptide of 410 amino acids, and hAPOδ, a polypeptide of 411 amino acids. hAPOδRP (SEQ ID NO: 4) and hAPOδ (SEQ ID NO: 2) share an amino-terminal signal peptide of 51 amino acids with about 53% amino acid identity (see Figure 4A) . An AP08 related polypeptide of the invention is characterized, in part, in that the amino-terminal 50 residues of the mature polypeptide (residues 52 to 101) have at least about 30% amino acid identity with SEQ ID NO: 2. Although the DR4 sequence described in Pan et al., Science 276:111-113 (1997) shares some similarity with hAPOδ and hAPOδRP, the DR4 sequence has only about 26% amino acid identity in the amino-terminal region of the mature polypeptide (shown as SEQ ID NO:2). Thus, DR4 is not an APOδ-related polypeptide, as defined herein.

The term APOδ-related polypeptide encompasses a polypeptide having the sequence of the naturally occurring human APOδ polypeptide (SEQ ID NO: 2) or the naturally occurring AP08-related polypeptide (AP08RP; SEQ ID NO: 4) and is intended to include related polypeptides having substantial amino acid sequence similarity to hAP08 (SEQ ID NO : 2 ) or hAP08RP (SEQ ID NO:4). Such related polypeptides exhibit greater sequence similarity to hAPOδ or hAPOδRP than to other polypeptide containing cysteine-rich pseudo-repeats such as TNFR-1 or DR4 and include alternatively spliced forms of hAPOδ and hAP08RP, species homologues, and isotype variants of the amino acid sequences shown in Figures 2 and 3A. As used herein, the term AP08-related polypeptide describes a polypeptide generally having an amino acid sequence with greater than about 50% amino acid sequence identity with hAPOδ (SEQ ID NO: 2) and, in particular, having a sequence in which the amino terminal 50 residues of the mature polypeptide have at least about 30% identity with amino acids 52 to 101 of SEQ ID NO: 2. An APOδ-related polypeptide preferably has an amino acid sequence in which the amino terminal 50 residues of the mature polypeptide have at least about 50% amino acid identity with amino acids 52 to 101 of SEQ ID NO: 2, more preferably greater than about 75% amino acid identity with amino acids 52 to 101 of SEQ ID NO: 2, and can be a polypeptide in which the amino terminal 50 residues of the mature polypeptide have greater than about 85%, 90%, 95% or 97% amino acid identity with amino acids 52 to 101 of SEQ ID NO: 2. One skilled in the art understands that the term APOδ-related polypeptide encompasses fusion proteins, in which a heterologous polypeptide sequence is fused N- or C- terminally, or is fused internally, for example, near the cleavage site of the signal peptide.

The present invention also provides an isolated AP09 polypeptide having substantially the same amino acid sequence as AP09, or an active fragment thereof. An isolated AP09 polypeptide of the invention can have substantially the same amino acid sequence as the human AP09 sequence SEQ ID NO: 6 shown in Figure 6.

As used herein, the term "AP09" means an AP09 polypeptide and includes polypeptides having substantially the same amino acid sequence as the hAP09 polypeptide having amino acid sequence SEQ ID NO: 6. A short, alternatively spliced form of hAP09 having 259 amino acids is shown in Figure 6. This hAP09 sequence contains an amino-terminal signal peptide (residues 1 to 22) , a large extracellular ligand binding domain with characteristic TNF receptor family cysteine-rich regions (residues 23 to 241) and a hydrophobic membrane-anchoring domain (residues 242 to 259) at the 3' end. The hAP09 extracellular domain has significant homology to other TNF receptor polypeptides as shown in Figure 1. Human AP09 is most closely related to the DR4 death receptor, with 57% amino acid sequence identity and 71% amino acid sequence similarity in the extracellular domain. A stop codon is present immediately after the membrane-anchoring domain such that the short form of hAP09 shown in Figure 6 lacks a cytoplasmic domain. Alternatively spliced, full-length forms of AP09 having a cytoplasmic signaling domain can mediate apoptosis; their function can be opposed by the short AP09 form shown in Figure 6.

The term AP09 encompasses a polypeptide having the sequence of the naturally occurring human AP09 polypeptide (SEQ ID NO: 6) and is intended to include related polypeptides having substantial amino acid sequence similarity to hAP09 (SEQ ID NO: 6). Such related polypeptides exhibit greater sequence similarity to hAP09 than to members of the TNF receptor superfamily and include alternatively spliced forms of hAP09, full-length forms having a cytoplasmic signaling domain, species homologues, and isotype variants of the amino acid sequence shown in Figure 6. Such full-length forms or AP09 species homologues can be readily obtained by the skilled artisan using routine molecular techniques, for example, by screening an appropriate cDNA library with a portion of SEQ ID NO: 5 as a probe. As used herein, the term AP09 describes polypeptides generally having an amino acid sequence with greater than about 65% amino acid sequence identity in the extracellular domain and transmembrane domain with hAP09 (SEQ ID NO: 6), preferably greater than about 75% amino acid identity in the extracellular domain and transmembrane domain with hAP09 (SEQ ID NO: 6), more preferably greater than about δ5% amino acid identity in the extracellular domain and transmembrane domain with hAP09 (SEQ ID NO: 6), and includes polypeptides having greater than about 90%, 95% or 97% amino acid identity in the extracellular domain and transmembrane domain with hAP09 (SEQ ID NO: 6).

The present invention also provides active fragments of the AP09 polypeptide of the invention. As used herein, the term "active fragment of an AP09 polypeptide" is synonymous with "active fragment of AP09" or "active AP09 fragment" and means a polypeptide fragment having substantially the same amino acid sequence as a portion of an AP09 polypeptide, provided that the fragment retains at least one biological activity of an AP09 polypeptide. An active fragment of an AP09 polypeptide can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hAP09 (SEQ ID NO: 6), provided that the fragment retains at least one biological activity of an AP09 polypeptide. A biological activity of AP09 can be, for example, the ability to bind ligand, the ability to induce or suppress apoptosis or the ability to induce or suppress cell proliferation, differentiation or cytokine secretion. A biological activity of an AP09 polypeptide or fragment can be routinely assayed; for example, apoptotic activity can be analyzed by transfecting an AP09 encoding nucleic acid and measuring the number of cells with apoptotic morphology, as set forth in Example II. An "active fragment of an AP09 polypeptide" can be an active AP09 segment, which is a polypeptide portion having substantially the same amino acid sequence as a portion of an AP09 polypeptide, provided that the segment does not consist of the identical amino acid sequence by an expressed sequence tag having GenBank accession number AA031883, AA150849, T71406 or R10995 and provided that the segment retains at least one biological activity of an AP09 polypeptide. An active AP09 segment can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hAP09 (SEQ ID NO: 6), provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA031863, AA150849, T71406 or

R10995 and provided that the segment retains at least one biological activity of an AP09 polypeptide.

In one embodiment, the invention provides a soluble AP09 active fragment that includes an AP09 ligand binding domain. A soluble AP09 active fragment of the invention can be, for example, a truncated polypeptide encoding the extracellular domain of AP09. An exemplary soluble AP09 active fragment is provided in Figure 1 as amino acids 1 to 241 of hAP09 and includes the signal peptide and ligand binding domain. One skilled in the art understands that a soluble AP09 active fragment is distinguished from a membrane-bound fragment of AP09 by the deletion or inactivation of the hydrophobic membrane-anchoring domain, shown as amino acids 242 to 259 in Figure 6. A soluble AP09 active fragment also can be, for example, an active AP09 segment. Active AP09 segments are described hereinabove. The present invention also provides an isolated AP04 polypeptide having substantially the same amino acid sequence as an AP04 polypeptide, or an active fragment thereof. An isolated AP04 polypeptide of the invention can have substantially the same amino acid sequence as murine AP04-αL (SEQ ID NO: 8) shown in Figure 7A, murine AP04-αS (SEQ ID NO: 10) shown in Figure 7B, human AP04-α (SEQ ID NO: 12) shown in Figure 7C, or rat AP04-α (SEQ ID NO: 14) shown in Figure 7D. An isolated AP04 polypeptide of the invention also can have substantially the same amino acid sequence as murine AP04-β (SEQ ID NO: 16) as shown in Figure 8.

As used herein, the term "AP04 polypeptide" means an AP04-α or AP04-β polypeptide and includes polypeptides having substantially the same amino acid sequence as the mAP04-αL polypeptide (SEQ ID NO: 8), the mAP04-αS polypeptide (SEQ ID NO: 10), the hAP04-α polypeptide (SEQ ID N0:12), the rAP04-α polypeptide (SEQ ID NO:14) or the mAP04-β polypeptide (SEQ ID NO:16). Murine AP04-α is an alternatively spliced protein including an amino-terminal signal peptide, cysteine-rich pseudo-repeats characteristic of the TNF receptor family and a highly hydrophobic stretch of amino acids representing a transmembrane domain. The short mAP04-α isoform (mAP04-αS) is encoded by an open reading frame of 214 amino acids and includes a short cytoplasmic tail, while the long mAP04-αL isoform (mAP04-αL) is a polypeptide of 416 amino acids with a unique cytoplasmic tail having no significant homology to other members of the TNF receptor family. The truncated mAP04-αS polypeptide can compete for binding to an AP04-α ligand, thereby opposing the function of a long form of AP04-α such as mAP04-αL. Human AP04-α (hAP04-α) is another AP04 polypeptide of the invention. The nucleotide sequence encoding hAP04-α is shown in Figure 7C. This sequence encodes an hAP04-α polypeptide of 423 amino acids with 70.7% amino acid identity and 81.1% amino acid similarity to mAP04-α. As shown in Figure 7C, the hAP04-α polypeptide possesses a signal peptide (residues 1 to 23), a putative transmembrane domain (residues 178 to 191) and the characteristic cytoplasmic domain of AP04 polypeptides. Rat AP04-α (rAP04-α) is another AP04 polypeptide of the invention. The available sequence of the rAP04-α cDNA is shown in Figure 7D. The cytoplasmic tail of rAP04-α contains the unique AP04 cytoplasmic domain and is 80% identical with mAP04-α at the amino acid level. Murine AP04-β (mAP04-β) is another AP04 polypeptide of the invention; the available sequence of mAP04-β is shown in Figure 8. Finally, murine AP04-γ (mAP04-γ) is another AP04 peptide of the invention whose sequence is shown in Figure 7E. mAP04-γ lacks a transmembrane domain and therefore represents a soluble receptor which can compete for binding to the AP04 ligand, thereby opposing the function of the long forms of mAP04 such as mAP04-αL.

The term AP04 polypeptide encompasses a polypeptide having the sequence of naturally occurring murine AP04-αL (SEQ ID N0:8) or AP04-αS (SEQ ID NO:10), naturally occurring human AP04-α (SEQ ID NO: 12) or rat AP04-α (SEQ ID NO: 14) or naturally occurring murine AP04-β (SEQ ID NO: 16) or naturally occurring murine AP04- Y (SEQ ID NO: 32) and is intended to include related polypeptides having substantial amino acid sequence similarity to SEQ ID NOS : 8 , 10, 12, 14, 16 or 32. Such related polypeptides exhibit greater sequence similarity to mAP04-αL, mAP04-αS, hAP04-α, rAP04-α, mAP04-β or mAP04-γ than to other TNF family receptors and include alternatively spliced forms of human, murine or rat AP04-α, AP04-β or AP04-γ full-length forms of AP04, species homologues, and isotype variants of the amino acid sequences shown in Figures 7 and 8. As used herein, the term AP04 polypeptide describes polypeptides generally including an amino acid region with greater than about 35% amino acid sequence identity with mAP04-αL (SEQ ID N0:8), mAP04-αS (SEQ ID NO: 10), hAP04-α (SEQ ID NO:12), rAP04-α (SEQ ID NO:14) or mAP04-β (SEQ ID NO:16). In particular, an AP04 polypeptide can have greater than about 55% amino acid identity, preferably greater than about 65% amino acid identity, more preferably greater than about 75% amino acid identity, still more preferably greater than about 85% amino acid identity and most preferably greater than about 90%, 95% or 97% amino acid identity with mAP04-αL (SEQ ID NO:8), mAP04-αS (SEQ ID NO:10), hAP04-α (SEQ ID NO:12), rAP04-α (SEQ ID N0:14), mAP04-β (SEQ ID NO:16) or mAP04-γ (SEQ ID NO:32).

The present invention also provides active fragments of the AP04 polypeptides of the invention. As used herein, the term "active fragment of an AP04 polypeptide" is synonymous with "active fragment of AP04" or "active AP04 fragment" and means a polypeptide fragment having substantially the same amino acid sequence as a portion of an AP04-α, AP04-β or AP04-γ polypeptide, provided that the fragment retains at least one biological activity of an AP04 polypeptide. An active fragment of an AP04 polypeptide can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of mAP04-αL (SEQ ID NO:8), mAP04-αS (SEQ ID NO: 10), hAP04-α (SEQ ID NO: 12), rAP04-α (SEQ ID NO: 14), mAP04-β (SEQ ID NO:16), or AP04-γ (SEQ ID NO:32), provided that the fragment retains at least one biological activity of an AP04 polypeptide. A biological activity of an AP04 polypeptide can be, for example, the ability to bind an AP04 ligand, the ability to induce or suppress apoptosis, the ability to activate the JNK pathway, or the ability to induce or suppress cell proliferation, differentiation or cytokine secretion. A biological activity of an AP04 polypeptide or fragment can be routinely assayed; for example, apoptotic activity can be analyzed by transfecting an AP04 encoding nucleic acid and measuring the number of cells with apoptotic morphology, as set forth in Example II.

An active fragment of AP04 also be, for example, a cytoplasmic fragment of AP04. An active fragment of AP04 can be an N-terminal fragment having the amino terminal 355 amino acids of AP04, which was able to activate the JNK pathway in 293 EBNA cells.

An "active fragment of an AP04 polypeptide" also can be an active segment of an AP04 polypeptide. Such an active segment is a polypeptide portion having substantially the same amino acid sequence as a portion of an AP04 polypeptide, provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA036247, AA003356, W55289, AA445δ05 or W56629 and provided that the segment retains at least one biological activity of an AP04 polypeptide. An active AP04 polypeptide segment can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of mAP04-αL (SEQ ID NO: 8), mAP04-αS (SEQ ID NO: 10), hAP04-α (SEQ ID NO: 12), rAP04-α (SEQ ID N0:14), mAP04-β (SEQ ID NO:16) or AP04-γ (SEQ ID NO:32), provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA036247, AA003356, W55289, AA445805 or W56629 and provided that the segment retains at least one biological activity of an AP04 polypeptide.

In one embodiment, the invention provides a soluble active fragment of an AP04 polypeptide. Such a soluble active fragment includes the ligand binding domain of an AP04 polypeptide and can be, for example, a truncated polypeptide encoding the extracellular domain of an AP04 polypeptide. A soluble active fragment of an AP04 polypeptide can have, for example, substantially the same amino acid sequence as the signal peptide and ligand-binding domain of mAP04-α (amino acids 1 to 177 of mAP04-α shown in Figure 7A) or substantially the same sequence as mAP04-γ (Figure 7E) or substantially the same sequence as the signal peptide and ligand-binding domain of hAP04-α (amino acids 1 to 177 of hAP04-α shown in Figure 7C) . A specific example of such a soluble fragment is mAP04-γ. One skilled in the art understands that a soluble active fragment of an AP04 polypeptide is distinguished from a membrane-bound form by deletion or inactivation of the transmembrane domain. The transmembrane domain of murine AP04-α is shown in Figure 7A as amino acids 173 to 191, and the transmembrane domain of human AP04-α is shown in Figure 7C as amino acids 178 to 191. A soluble active fragment of an AP04 polypeptide also can be, for example, an active segment of an AP04 polypeptide. Such active segments of AP04 polypeptides are described hereinabove.

In another embodiment, the invention provides an AP04-related polypeptide, which has a sequence with substantial similarity to the unique cytoplasmic domain of an AP04 polypeptide. The AP04 cytoplasmic domain is a conserved region that can function to promote self-association or association with a different protein. The AP04 cytoplasmic domain also can promote ligand-dependent cell survival, proliferation, differentiation or death. An AP04-related polypeptide can be, for example, a transmembrane receptor including a cytoplasmic domain having substantial similarity to the cytoplasmic domain of mAP04-αL or hAP04-α. The extracellular domain of an AP04-related polypeptide receptor can be substantially the same as, or can be entirely different than the extracellular domain of an AP04 polypeptide. AP04-related polypeptides also include intracellularly expressed polypeptides that contain one or more AP04 cytoplasmic domains . An AP04 cytoplasmic domain can have, for example, the amino acid sequence shown as amino acids 192 to 416 in Figure 7A, the amino acid sequence shown as amino acids 192 to 423 in Figure 7C, or an amino acid sequence with substantial similarity to one of these sequences.

The invention further provides an isolated AP06 polypeptide having substantially the same amino acid sequence as AP06, or an active fragment thereof. An isolated AP06 polypeptide of the invention can have, for example, substantially the same amino acid sequence as the human AP06 sequence SEQ ID NO: 18 shown in Figure 9.

As used herein, the term "AP06" means an AP06 polypeptide and includes polypeptides having substantially the same amino acid sequence as the hAP06 polypeptide having amino acid sequence SEQ ID NO: 18. A partial nucleotide sequence encoding hAPOδ, which lacks 5' and 3' sequence, is shown in Figure 9. The available hAPOδ sequence shows about 33% homology to the extracellular domain of TNFR-2. The partial APOδ sequence shown in Figure 9 can be part of a full-length APOδ transmembrane receptor.

The term APOδ encompasses a polypeptide having the sequence of the naturally occurring human APOδ polypeptide (SEQ ID NO: 18) and is intended to include related polypeptides having substantial amino acid sequence similarity to hAPOδ (SEQ ID N0:18). Such related polypeptides exhibit greater sequence similarity to hAPOδ than to other members of the TNF receptor superfamily and include alternatively spliced forms of hAPOδ, full-length forms of AP06 having a cytoplasmic signaling domain, species homologues, and isotype variants of the amino acid sequence shown in Figure 9. Such full-length forms or AP06 species homologues can be readily obtained by the skilled artisan using routine molecular techniques, for example, by screening an appropriate cDNA library with a portion of SEQ ID NO: 17 as a probe. As used herein, the term APOδ describes polypeptides generally having an amino acid sequence with greater than about 40% amino acid identity with the extracellular domain of hAP06 (SEQ ID NO:18), preferably greater than about 65% amino acid identity with the extracellular domain of hAPOδ (SEQ ID NO:18), more preferably greater than about 75% amino acid identity with the extracellular domain of hAPOδ (SEQ ID NO: 16), still more preferably greater than about 75% amino acid identity with the extracellular domain of hAPOδ (SEQ ID NO:18), and most preferably about 90%, 95% or 97% amino acid identity with the extracellular domain of hAPOδ (SEQ ID NO:18) .

The present invention also provides active fragments of the APOδ polypeptide of the invention. As used herein, the term "active fragment of an APOδ polypeptide" is synonymous with "active fragment of AP06" or "active APOδ fragment" and means a polypeptide fragment having substantially the same amino acid sequence as a portion of an APOδ polypeptide, provided that the fragment retains at least one biological activity of an AP06 polypeptide. An active fragment of an AP06 polypeptide can have, for example, an amino acid sequence that is identical or substantially the same as a portion of an amino acid sequence of hAP06 (SEQ ID

NO: 18), provided that the fragment retains at least one biological activity of an APOδ polypeptide. Biological activities of APOδ include the ability to bind an APOδ ligand, the ability to induce or suppress apoptosis and the ability to induce or suppress cell proliferation, differentiation or cytokine secretion. A biological activity of an AP06 polypeptide or fragment can be routinely assayed; for example, apoptotic activity can be analyzed by transfecting an AP06 encoding nucleic acid and measuring the number of cells with apoptotic morphology, as set forth in Example II.

An active fragment of an AP06 polypeptide also can be an active APOδ segment, which is a polypeptide segment having substantially the same amino acid sequence as a portion of an AP06 polypeptide, provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA025673, AA025672, AA155701, AA155646, W67560 or AA158406 and provided that the segment retains at least one biological activity of an AP06 polypeptide. An active AP06 segment can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hAP06 (SEQ ID N0:18), provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA025673, AA025672, AA155701, AA155646, W67560 or AA158406 and provided that the segment retains at least one biological activity of an AP06 polypeptide.

In one embodiment, the invention provides a soluble AP06 active fragment that includes an APOδ ligand binding domain. A soluble APOδ active fragment can be, for example, a truncated polypeptide encoding the extracellular domain of an AP06 polypeptide. A soluble AP06 active fragment can have, for example, substantially the same amino acid sequence as amino acids 1 to 168 of the hAP06 sequence shown in Figure 1. Soluble AP06 active fragments are distinguished from membrane-bound forms by the absence of a functional transmembrane domain. A soluble APOδ active fragment also can be, for example, an active APOδ segment. Active AP06 segments are described hereinabove.

Ligands of the TNF ligand superfamily are acidic, TNF-like molecules with approximately 20% sequence homology in the extracellular receptor-binding domain. Most exist principally as type II membrane glycoproteins, with the biologically active form a trimeric or multimeric complex. Like the prototypic TNF-α molecule, most have a large C-terminal extracellular domain, a single transmembrane spanning region and a variable amino-terminal cytoplasmic domain. The TNFR ligand family includes TNF-α, TNF-β, LTβ, FAS ligand, CD27 ligand, CD30 ligand, CD40 ligand, 4-1BB ligand, OX40 ligand and FAS/APO-1 ligand. Although members of the TNF ligand superfamily typically are cell surface-expressed molecules, soluble forms of TNF-α, TNF-β and FAS ligand also have been identified.

Disclosed herein is subfamily of polypeptide ligands designated the tumor necrosis factor related ligand 1 (TNRL1) subfamily. Thus, the present invention provides an isolated TNRL1 polypeptide having substantially the same amino acid sequence as TNRL1, or an active fragment thereof. An isolated TNRL1 polypeptide of the invention can have, for example, substantially the same amino acid sequence as the sequence of human TNRLl-α (SEQ ID NO:20); murine TNRLl-α (SEQ ID NO:22); human TNRLl-β (SEQ ID NO:24) or murine TNRLl-β (SEQ ID NO:26).

As used herein, the term "TNRL1" means a TNRL1 polypeptide and includes polypeptides having substantially the same amino acid sequence as the hTNRLl-α polypeptide having amino acid sequence SEQ ID NO: 20; the mTNRLl-α polypeptide having amino acid sequence SEQ ID NO: 22; the hTNRLl-β polypeptide having amino acid sequence SEQ ID NO: 24; and the mTNRLl-β polypeptide having amino acid sequence SEQ ID NO: 26. Human TNRLl-α (hTNRLl-α) is a polypeptide of at least 230 (?) amino acids; the available hTNRLl-α nucleotide (SEQ ID NO: 19) and amino acid sequence (SEQ ID NO: 20) are shown in Figure 11A. Human TNRLl-α (SEQ ID NO: 20) exhibits about 25% amino acid identity and about 49% amino acid similarity to TNF-α.

Murine TNRLl-α (mTNRLl-α) is the homolog of human TNRLl-α and also is an TNRL1 polypeptide of the invention. mTNRLl-α is a polypeptide of at least 290 amino acids; the available mTNRLl-α nucleotide (SEQ ID NO: 21) and amino acid sequence (SEQ ID NO: 22) are shown in Figure 11B. Murine TNRLl-α (SEQ ID NO: 22) is about 68% identical and 81% similar to human TNRLl-α at the amino acid level.

Human TNRLl-β (hTNRLl-β) also is a TNRL1 polypeptide of the invention. Human TNRLl-β is a polypeptide of 250 amino acids; the hTNRLl-β nucleotide sequence (SEQ ID NO: 23) and amino acid sequence (SEQ ID NO: 24) are shown in Figure 12A. The hTNRLl-β polypeptide (SEQ ID NO:24) is about 31% identical and 55% similar to hTNRLl-α at the amino acid level and is more distantly related to TNF-α (see Figure 10) .

Murine TNRLl-β (mTNRLl-β) , the homolog of human TNRLl-β, also is a TNRL1 polypeptide of the invention. The nucleotide (SEQ ID NO: 25) and corresponding amino acid sequence (SEQ ID NO: 26) of full-length mTNRLl-β are shown in Figure 12B.

The term TNRL1 encompasses a polypeptide having the sequence of the naturally occurring human TNRLl-α polypeptide SEQ ID NO: 20, the murine TNRLl-α polypeptide SEQ ID NO: 22, the human TNRLl-β polypeptide SEQ ID NO: 24, or the murine TNRLl-β polypeptide SEQ ID NO: 26 and is intended to include related polypeptides having substantial amino acid sequence similarity to hTNRLl-α, mTNRLl-α, hTNRLl-β or mTNRLl-β. Such related polypeptides exhibit greater sequence similarity to hTNRLl-α, mTNRLl-α, hTNRLl-β or mTNRLl-β than to other members of the TNF ligand superfamily, such as TNF-α, and include alternatively spliced forms of hTNRLl-α, full- length forms of hTNRLl-α, mTNRLl-α, hTNRLl-β or mTNRLl-β, species homologues, and isotype variants of the amino acid sequences shown in Figures 11 and 12. As used herein, the term TNRL1 describes polypeptides generally having an amino acid sequence with greater than about 30% amino acid identity with hTNRLl-α (SEQ ID NO: 20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO:26), preferably greater than about 35% amino acid identity with hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO:26), more preferably greater than about 45% amino acid identity with hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO:26), and most preferably about

65%, 75%, 85%, 90%, 95% or 97% amino acid identity with hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO:26).

The present invention also provides an isolated TNRLl polypeptide having substantially the same amino acid sequence as TNRLl, or an active fragment of a TNRLl polypeptide of the invention. An isolated TNRLl polypeptide of the invention can have, for example, substantially the same amino acid sequence as the human TNRLl (SEQ ID NO: 20) shown in Figure 11.

The present invention also provides active fragments of a TNRLl polypeptide of the invention. As used herein, the term "active fragment of an TNRLl polypeptide" is synonymous with "active fragment of TNRLl" or "active TNRLl fragment" and means a polypeptide fragment having substantially the same amino acid sequence as a portion of a TNRLl polypeptide, provided that the fragment retains at least one biological activity of a TNRLl polypeptide. An active fragment of a TNRLl polypeptide can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO:26), provided that the fragment retains at least one biological activity of a TNRLl polypeptide. A biological activity of a TNRLl polypeptide can be, for example, the ability to bind a TNRLl receptor, the ability to induce or suppress apoptosis or the ability to induce or suppress cell proliferation, differentiation or cytokine secretion. A biological activity of a TNRLl polypeptide or fragment can be routinely assayed; for example, apoptotic activity can be analyzed by treating BJAB cells with polypeptide and assaying cell survival as set forth in Example VI.

An "active fragment of a TNRLl polypeptide" also can be an active TNRLl segment, which is a polypeptide segment having substantially the same amino acid sequence as a portion of a TNRLl polypeptide, provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA166695, T87299, R168δ2, AA254047, AA056924, AA057069, N91002, N7901δ or AA497494 or TIGR sequence number M76231 and provided that the segment retains at least one biological activity of a TNRLl polypeptide. An active TNRLl segment can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO:26), provided that the segment does not consist of the identical amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA166695, T67299, R16δδ2, AA254047, AA056924, AA057069, N91002, N7901δ or AA497494 or TIGR sequence number M78231 and provided that the segment retains at least one biological activity of a TNRLl polypeptide. In one embodiment, the invention provides a soluble active fragment of a TNRLl ligand of the invention, where the active fragment includes the TNRLl receptor binding domain. A TNRLl ligand of the invention can be, for example, a membrane-bound ligand that transduces a signal when the cell on which it is expressed contacts a cell bearing its cognate receptor or, alternatively, a soluble form of TNRLl can be expressed extracellularly and bind a distant receptor-bearing cell. Such a soluble TNRLl active fragment can have the activity of a full-length TNRLl of the invention or can be an inhibitory polypeptide that opposes the biological function of full-length ligand.

Thus, the invention provides a soluble TNRLl active fragment that includes a TNRLl receptor binding domain. A soluble TNRLl active fragment can be, for example, a truncated polypeptide encoding the extracellular domain of a TNRLl polypeptide and can have, for example, an amino acid sequence that is identical or substantially the same as amino acids 22 to 225 of hTNRLl-α, amino acids 32 to 243 of mTNRLl-α, amino acids 50 to 250 of hTNRLl-β or amino acids 42 to 241 of mRNRL-β, shown in Figure 10. One skilled in the art understands that soluble TNRLl active fragments lack a functional TNRLl transmembrane domain. A soluble TNRLl active fragment also can be an active TNRLl segment. Segments having an amino acid sequence identical to the amino acid sequence encoded by an expressed sequence tag having GenBank accession number AA166695, Tδ7299, R168δ2, AA254047, AA056924, AA057069, N91002, N79016 or AA497494 or TIGR sequence number M76231 are excluded from the definition of an active TNRLl segment, as defined herein. The present invention further provides an isolated TNRL3 polypeptide having substantially the same amino acid sequence as TNRL3, or an active fragment thereof. An isolated TNRL3 polypeptide of the invention can have, for example, substantially the same amino acid sequence as the human TNRL3 (SEQ ID NO: 28) or murine TNRL3 (SEQ ID NO: 30) sequence shown in Figure 13.

As used herein, the term "TNRL3" means a TNRL3 polypeptide and includes polypeptides having substantially the same amino acid sequence as the hTNRL3 polypeptide having amino acid sequence SEQ ID NO: 28 or the mTNRL3 polypeptide having amino acid sequence SEQ ID NO: 30. A partial nucleotide sequence encoding the 3' portion of hTNRL3 is shown in Figure 13A. The available sequence shows homology to TRAIL ligand and to other members of the TNF ligand superfamily (see Figure 10). The murine TNRL3 homolog, mTNRL3, also is a TNRL3 polypeptide of the invention. The partial nucleotide and amino acid sequence of murine TNRL3 lacks some 5 ' sequence; the available sequence is shown in Figure 13B.

The term TNRL3 encompasses a polypeptide having the sequence of naturally occurring human TNRL3 (SEQ ID NO: 28) or murine TNRL3 (SEQ ID NO: 30) and is intended to include related polypeptides having substantial amino acid sequence similarity to the human or murine TNRL3 polypeptides SEQ ID NOS: 28 or 30. Such related polypeptides exhibit greater sequence similarity to hTNRL3 or mTNRL3 than to other members of the TNF ligand superfamily such as TRAIL and include alternatively spliced forms of hTNRL3 or mTNRL3, species homologues, full-length forms and isotype variants of the amino acid sequences shown in Figure 13. As used herein, the term TNRL3 describes polypeptides generally having an amino acid sequence with greater than about 30% amino acid sequence identity with hTNRL3 or mTNRL3 (SEQ ID NO: 28 or SEQ ID NO: 30), preferably greater than about 35% amino acid identity with hTNRL3 or mTNRL3, more preferably greater than about 45% amino acid identity with hTNRL3 or mTNRL3 (SEQ ID NO:28 or SEQ ID NO:30), still more preferably greater than about 65% amino acid identity with hTNRL3 or mTNRL3 (SEQ ID NO:28 or SEQ ID NO:30), and most preferably about 75%, 85%, 90%, 95% or 97% amino acid identity with hTNRL3 or mTNRL3 (SEQ ID NO: 28 or SEQ ID NO:30) .

Further provided herein is an active fragment of a TNRL3 polypeptide of the invention. As used herein, the term "active fragment of an TNRL3 polypeptide" is synonymous with "active fragment of TNRL3" or "active TNRL3 fragment" and means a polypeptide fragment having substantially the same amino acid sequence as a portion of a TNRL3 polypeptide, provided that the fragment retains at least one biological activity of a TNRL3 polypeptide. An active fragment of a TNRL3 polypeptide can have, for example, substantially the same amino acid sequence as a portion of hTNRL3 (SEQ ID NO: 28) or mTNRL3 (SEQ ID NO: 30), provided that the fragment retains at least one biological activity of a TNRL3 polypeptide. A biological activity of a TNRL3 polypeptide can be, for example, the ability to bind a TNRL3 receptor, the ability to induce or suppress apoptosis or the ability to induce or suppress cell proliferation, differentiation or cytokine secretion. A biological activity of a TNRL3 polypeptide or fragment can be routinely assayed; for example, apoptotic activity can be analyzed by treating BJAB cells with polypeptide and assaying cell survival as set forth in Example VI. An "active fragment of a TNRL3 polypeptide" also can be an active TNRL3 segment, which is a polypeptide segment having substantially the same amino acid sequence as a portion of a TNRL3 polypeptide, provided that the segment does not consist of the amino acid sequence, or a segment thereof, encoded by an expressed sequence tag having GenBank accession number R55285, N35070, C00994 or AA221610 and provided that the segment retains at least one biological activity of a TNRL3 polypeptide. An active TNRL3 segment can have, for example, an amino acid sequence that is identical or substantially the same as a portion of the amino acid sequence of hTNRL3 (SEQ ID NO: 28) or mTNRL3 (SEQ ID NO: 30), provided that the segment does not consist of the amino acid sequence, or a segment thereof, encoded by an expressed sequence tag having GenBank accession number R55285, N35070, C00994 or AA221610 and provided that the segment retains at least one biological activity of a TNRL3 polypeptide.

In one embodiment, the invention provides a soluble TNRL3 active fragment that includes a TNRL3 receptor binding domain. Such a soluble TNRL3 active fragment can be, for example, a truncated polypeptide encoding the extracellular domain of a TNRL3 polypeptide. Soluble TNRL3 active fragments can have, for example, an amino acid sequence that is identical or substantially the same as amino acids 4 to 20δ of hTNRL3 or amino acids 7 to 211 of mTNRL3, as shown in Figure 10. Such a soluble TNRL3 active fragment lacks a functional transmembrane domain. A soluble TNRL3 active fragment also can be, for example, an active TNRL3 segment. Such active TNRL3 segments are described hereinabove. As used herein, the term "substantially the same amino acid sequence," when used in reference to a polypeptide, fragment or segment of the invention, is intended to mean a polypeptide, fragment or segment having an identical amino acid sequence, or a polypeptide, fragment or segment having a similar, non-identical sequence that is considered by those skilled in the art to be a functionally equivalent amino acid sequence. For example, polypeptide including substantially the same amino acid sequence as APOδ can have an amino acid sequence identical to the sequence of hAP08 (SEQ ID NO: 2) shown in Figure 2, or a similar, non-identical sequence that is functionally equivalent. An amino acid sequence that is "substantially the same" can have one or more modifications such as amino acid additions or substitutions relative to the amino acid sequence shown, provided that the modified polypeptide retains substantially at least one biological activity of the polypeptide. Biological activities of the polypeptides of the invention are described herein; for example, biological activities of APOδ include the ability to bind an AP08 ligand, bind the TRADD or RIP adaptor molecules, induce or suppress apoptosis, activate NF-κB expression, or induce or suppress cell proliferation, differentiation or cytokine secretion.

Comparison of sequences for substantial similarity can be performed between two sequences of any length and usually is performed with nucleotide sequences of between 5 and 3500 nucleotides, preferably between about 10 and 300 nucleotides and more preferably between about 15 and 50 nucleotides. Comparison for substantial similarity between amino acid sequences is usually performed with sequences between about 6 and 1200 residues, preferably between about 10 and 100 residues and more preferably between about 25 and 35 residues. Such comparisons for substantial similarity are performed using methodology routine in the art.

Therefore, it is understood that limited modifications can be made without destroying the biological function of a polypeptide or ligand of the invention. For example, minor modifications of hAPOδ (SEQ ID NO: 2) that do not destroy polypeptide activity also fall within the definition of APOδ and within the definition of the polypeptide claimed as such. Similarly, minor modifications of hAP09 that do not destroy polypeptide activity fall within the definition of AP09; minor modifications of mAP04-αL, mAP04-αS, hAP04-α, rAP04-α or mAP04-β that do not destroy polypeptide activity fall within the definition of an AP04 polypeptide; and minor modifications of hAPOδ that do not destroy polypeptide activity fall within the definition of APOδ. Similarly, minor modifications of hTNRLl-α, mTNRLl-α, hTNRLl-β or mTNRLl-β that do not destroy polypeptide activity fall within the definition of TNRLl, and minor modifications of hTNRL3 and mTNRL3 that do not destroy polypeptide activity fall within the definition of TNRL3. Also, for example, genetically engineered fusion proteins that retain at least one measurable biological activity of a polypeptide of the invention fall within the definition of the polypeptides claimed as such.

It is understood that minor modifications of primary amino acid sequence can result in polypeptides which have substantially equivalent or enhanced function as compared to the APOδ polypeptide sequence set forth in Figure 2, substantially equivalent or enhanced function as compared to the hAP09 sequence set forth in Figure 6, substantially equivalent or enhanced function as compared to the AP04 polypeptide sequences set forth in Figures 6, 7 and δ, or substantially equivalent or enhanced function as compared to the hAPOδ sequence set forth in Figure 9. Also, minor modifications of primary amino acid sequence can result in polypeptides having substantially equivalent or enhanced function as compared to the TNRLl polypeptide sequences shown in Figures 11 and 12, or in polypeptides having substantially equivalent or enhanced function as compared to the TNRL3 polypeptide sequences shown in Figure 13. These modifications can be deliberate, as through site-directed mutagenesis, or can be accidental such as through mutation in hosts harboring an encoding nucleic acid. All such modified polypeptides are included in the definition of an APOδ polypeptide as long as at least one biological function of AP08 is retained. Similarly, all such modified polypeptides are included in the definition of an AP09, AP04, AP06, TNRLl or TNRL3 polypeptide as long as at least one biological function of an AP09, AP04, APOδ, TNRLl or TNRL3 polypeptide, respectively, is retained. Further, various molecules can be attached to an AP08, AP09, AP04, APOδ, TNRLl or TNRL3 polypeptide, for example, other polypeptides, carbohydrates, lipids, or chemical moieties. Such modifications are included within the definition of each of the polypeptides of the invention.

Polypeptide fragments or segments to be screened for activity can be produced, for example, by recombinant methods or by chemical or proteolytic cleavage of the isolated polypeptide. Methods for chemical and proteolytic cleavage and for purification of the resultant polypeptide fragments are well known in the art as described above. (See, for example, Deutscher, Methods in Enzymology_r Vol. 162, "Guide to Protein Purification," San Diego: Academic Press, Inc. (1990), which is incorporated herein by reference) .

A polypeptide fragment or segment of the invention can be screened, for example, for pro-apoptotic or anti-apoptotic activity. Pro-apoptotic activity is the ability either alone, or in combination with another molecule, to produce cell death accompanied by at least one of the morphological or biochemical alterations characteristic of apoptosis. Morphological alterations characteristic of apoptosis are well known in the art and include, for example, condensed and rounded cellular morphology; membrane blebbing; the formation of apoptotic bodies, which are membrane-bound bodies containing cytoplasmic and nuclear components; and condensation of the nucleus, with cytoplasmic organelles being relatively well maintained (Studzinski (Ed.), Cell Growth and Apoptosis , Oxford: Oxford University Press (1995), which is incorporated herein by reference) . Biochemical alterations characteristic of apoptosis also are well known in the art. The classical biochemical alteration characteristic of apoptosis is the appearance of oligonucleosome-sized fragments of DNA, which produce a "ladder" upon agarose gel electrophoresis . This extensive fragmentation can be preceded by an earlier endonucleolytic cleavage of chromatin, producing DNA fragments of about 50 kb to 300 kb in size.

A variety of assays for determining whether a polypeptide fragment or segment has pro-apoptotic or anti-apoptotic activity are well known in the art. Such methods include light microscopy for determining the presence of one or more morphological characteristics of apoptosis, such as condensed or rounded morphology, shrinking and blebbing of the cytoplasm, preservation of structure of cellular organelles including mitochondria, and condensation and margination of chromatin. As described in Example II, cells expressing fragments of hAPOδ were determined to be apoptotic by scoring for condensed and rounded morphology.

A polypeptide fragment or segment of the invention also can be assayed for pro-apoptotic or anti-apoptotic activity using terminal deoxytransferase-mediated (TdT) dUTP biotin nick end-labeling (TUNEL) (Gavriel et al., J. Cell Biol. 119:493 (1992), which is incorporated herein by reference; Gorczyca et al . , Int. J. Oncol. 1:639 (1992); Studzinski, supra , 1995). ApopTag™ (ONCOR, Inc., Gaithersburg, MD) is a commercially available kit for identification of apoptotic cells using digoxygenin labeling. In addition, a polypeptide fragment or segment of the invention can be assayed for pro-apoptotic or anti-apoptotic activity by detecting nucleosomal DNA fragments using agarose gel electrophoresis (Studzinski, supra , 1995; Gong et al., Anal. Biochem. 21δ:314 (1994)).

DNA filter elution methodology also can be used to detect apoptosis-associated DNA fragmentation and to determine pro-apoptotic or anti-apoptotic activity (Studzinski, supra , 1995; Bertrand et al . , Drug Devel. 34:136 (1995), which is incorporated herein by reference) . Pro-apoptotic or anti-apoptotic activity also can be detected and quantitated by determining an altered mitochondrial to nuclear DNA ratio as described in Tepper et al., Anal. Biochem. 203:127 (1992) and Tepper and Studzinski, J. Cell Biochem. 52:352 (1993), each of which is incorporated herein by reference. One skilled in the art understands that these, or other assays for pro-apoptotic or anti-apoptotic activity, can be performed using methodology routine in the art.

A nucleic acid to be assayed can encode a polypeptide fragment or segment corresponding to a portion of a native polypeptide of the invention or can be modified to encode one or more amino acid substitutions, deletions or insertions. One or more point mutations can be introduced into the nucleic acid encoding the modified polypeptide, fragment or segment to be assayed using, for example, site-directed mutagenesis (see Wu (Ed.), Meth. In Enzymol. Vol. 217, San Diego: Academic Press (1993); Chapter 22 of Innis et al. (Ed.), PCR Protocols, San Diego: Academic Press, Inc. (1990), each of which is incorporated herein by reference) . Such mutagenesis can be used to introduce a specific, desired amino acid substitution, deletion or insertion; alternatively, a nucleic acid sequence can be synthesized having random nucleotides at one or more predetermined positions to generate random amino acid substitutions. Scanning mutagenesis also can be useful in generating nucleic acid molecules encoding, for example, an APOδ polypeptide, fragment or segment that is modified throughout the entire sequence. Such a modified fragment or segment can be screened, for example, for the ability to bind an APOδ ligand; the ability to bind TRADD or RIP; the ability to induce or suppress apoptosis or the ability to activate NF-κB.

A polypeptide of the invention can be obtained by a variety of methods known within the art, including biochemical, recombinant and chemical synthesis methods. Biochemical methods for isolating a polypeptide of the invention, or active fragment thereof, include preparative gel electrophoresis, gel filtration, affinity chromatography, ion exchange and reversed phase chromatography, chromatofocusing, isoelectric focusing and sucrose or glycerol density gradients (see, for example, Chapter 3δ of Deutscher, Methods in Enzymology: Guide to Protein Purification, Vol. 182, Academic Press, Inc., San Diego (1990) and Chapter δ of Balch et al., Methods in Enzymology, Vol. 257, Academic Press, Inc., San Diego (1995), each of which is incorporated herein by reference in its entirety) . For example, as disclosed herein in Example I, human APOδ RNA is expressed in a variety of human tissues including spleen, thy us, prostate, testis, ovary, small intestine, colonic mucosa and peripheral blood lymphocyte. From these results, one skilled in the art knows that one of these tissues can be used as a source of material for isolating an hAP08 polypeptide of the invention.

Preparative gel electrophoresis can be useful in preparing an isolated polypeptide, active fragment or active segment of the invention. For example, an AP08 polypeptide, or an active fragment or segment thereof, can be isolated by preparative polyacrylamide gel electrophoresis and elution by diffusion or electroelution (see, for example, Chapter 33 of Deutscher, supra , 1990). Continuous elution gel electrophoresis using a system such as the Model 491 Prep Cell (BioRad, Hercules, CA) can be used to purify a polypeptide or an active fragment or segment of the invention. If desired, continuous elution gel electrophoresis can be combined with further purification steps such as liquid phase preparative isoelectric focusing using, for example, the Rotofor system (BioRad) .

Affinity chromatography is particularly useful in preparing an isolated polypeptide or an active fragment or segment of the invention. For example, a polypeptide that interacts with an APOδ polypeptide can be useful as an affinity matrix for isolating an AP08 polypeptide or an active AP08 fragment or segment of the invention. As disclosed herein, AP08 interacts physically with TRADD and RIP (Example II), indicating that TRADD or RIP can be used as an affinity matrix for isolating an AP08 polypeptide. Death domain containing polypeptides or fragments also can be useful as an affinity matrix for isolating an AP08 polypeptide or an active APOδ fragment or segment of the invention.

Immunoaffinity chromatography can be particularly useful in isolating a polypeptide or an active fragment or segment of the invention. For example, immunoprecipitation or column chromatography with an antibody that selectively binds APOδ can be used to isolate an APOδ polypeptide or active fragment thereof. An anti-AP08 monoclonal or polyclonal antibody that selectively binds APOδ can be prepared using an immunogen such as the sequence shown as SEQ ID NO: 2, or a synthetic peptide fragment thereof, as described further below. Methods of affinity chromatography are well known in the art and are described, for example, in Chapters 29, 30 and 3δ of Deutscher, supra , 1990, which has been incorporated herein by reference.

Recombinant methods for producing a polypeptide through expression of a nucleic acid sequence in a suitable host cell also are well known in the art and are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd Ed, Vols 1 to 3, Cold Spring Harbor Laboratory Press, New York (1989), which is incorporated herein by reference. A nucleic acid for expression of an APOδ polypeptide is provided herein as SEQ ID NO:l and a nucleic acid for expression of an AP08RP polypeptide is provided herein as SEQ ID NO: 3. A nucleic acid for expression of an AP09 polypeptide is provided herein as SEQ ID NO: 5; nucleic acids for expression of AP04 polypeptides are provided herein as SEQ ID NOS: 7, 9, 11, 13 and 15; and a nucleic acid for expression of an AP06 polypeptide is provided herein as SEQ ID NO: 17. Nucleic acids for expression of a TNRLl polypeptide also are provided as SEQ ID NOS: 19, 21, 23 and 25; and nucleic acids for expression of a TNRL3 polypeptide are provided herein as SEQ ID NOS: 27 and 29.

A recombinant polypeptide of the invention, or active fragment or segment thereof, also can be expressed as a fusion protein with a heterologous "tag" for convenient isolation from bacterial or mammalian host proteins. For example, histidine-tagged recombinant APOδ can be isolated by nickel-chelate chromatography. Similarly, a glutathione-S-transferase tag or an antigenic tag such as "FLAG, " "AU" or a myc epitope tag also can be included in a recombinant polypeptide or active fragment of the invention (Sambrook et al., supra , 1969) . The PinPoint™ expression system is a commercially available system for expression of a polypeptide or active fragment as a fusion protein with a heterologous biotinylated peptide.

A polypeptide or an active fragment or segment of the invention also can be produced by chemical synthesis, for example, by the solid phase peptide synthesis method of Merrifield et al., J. Am. Chem. Soc. 85:2149 (1964), which is incorporated herein by reference. Standard solution methods well known in the art also can be used to synthesize a polypeptide or an active fragment or segment useful in the invention (see, for example, Bodanszky, Principles of Peptide Synthesis, Springer-Verlag, Berlin (1984) and Bodanszky, Peptide Chemistry, Springer-Verlag, Berlin (1993), each of which is incorporated herein by reference) . A newly synthesized polypeptide, fragment or segment can be purified, for example, by high performance liquid chromatography (HPLC) and can be characterized using mass spectrometry or amino acid sequence analysis.

Also provided herein are nucleic acid molecules encoding the tumor necrosis family receptors and ligands of the invention. These nucleic acid molecules are useful, for example, in producing recombinant polypeptides and as probes for diagnosing diseases mediated by the polypeptides of the invention, as described further below. A nucleic acid molecule of the invention can have a nucleotide sequence of, for example, about 15 to about 1500 nucleotides. In particular, a nucleic acid molecule of the invention can have a sequence of about 15, 18, 20, 25, 30, 35, 50, 100, 200, 500 or more nucleotides.

The term "isolated, " as used herein in reference to a nucleic acid molecule of the invention, means a nucleic acid molecule that is in a form that is relatively free from contaminating lipids, polypeptides, unrelated nucleic acids and other cellular material normally associated with a nucleic acid molecule in a cell.

Provided herein is an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as AP08, or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA223122 or AA232440. An isolated nucleic acid molecule of the invention can have, for example, a nucleotide sequence encoding substantially the same amino acid sequence as hAPOδ (SEQ ID NO: 2), or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA223122 or AA232440. An exemplary nucleic acid molecule of the invention encodes human APOδ and is provided herein as SEQ ID NO:l (see Figure 2). In one embodiment, the invention provides an isolated nucleic acid molecule containing a nucleotide sequence encoding a soluble APOδ active segment having an AP08 ligand binding domain.

The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as AP09, or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA031863, AA150849, T71406 or R10995. For example, the invention provides an isolated nucleic acid molecule of the invention having a nucleotide sequence encoding substantially the same amino acid sequence as hAP09 (SEQ ID NO: 6), or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA031883, AA150849, T71406 or R10995. An exemplary nucleic acid molecule of the invention encodes human AP09 and is provided herein as SEQ ID NO: 5 (see Figure 6) . In one embodiment, the invention provides an isolated nucleic acid molecule containing a nucleotide sequence encoding a soluble AP09 active segment having an AP09 ligand binding domain.

In addition, the invention provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as an AP04 polypeptide, or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA036247, AA003356, W55289, AA445805 or W56629. Such a nucleic acid molecule can have a nucleotide sequence encoding substantially the same amino acid sequence as mAP04-αL (SEQ ID NO: 8), mAP04-αS (SEQ ID NO:10), hAP04-α (SEQ ID NO:12), rAP04-α (SEQ ID NO: 14), mAP04-β (SEQ ID NO: 16), or mAP04-γ (SEQ ID NO:32), or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA036247, AA003356, W55289, AA445805 or W56629. Exemplary nucleic acid molecules encoding the AP04 polypeptides of the invention are provided herein. As shown in Figure 7A-E, SEQ ID NO: 7 is a nucleotide sequence encoding murine AP04-αL; SEQ ID NO: 9 is a nucleotide sequence encoding murine AP04-αS; SEQ ID NO: 11 is a nucleotide sequence encoding human AP04-α; and SEQ ID NO: 13 is a nucleotide sequence encoding rat AP04-α and SEQ ID NO: 31 is a nucleotide sequence encoding murine AP04-γ. Figure δ shows SEQ ID NO: 15, which is a nucleotide sequence encoding murine AP04-β. The invention also provides an isolated nucleic acid molecule containing a nucleotide sequence encoding a soluble active segment of an AP04 polypeptide having an AP04 ligand binding domain. Also provided by the invention is an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as AP06, or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA025673, AA025672, AA155701, AA155646, W67560 or AA156406. For example, the invention provides an isolated nucleic acid molecule of the invention having a nucleotide sequence encoding substantially the same amino acid sequence as hAPOδ (SEQ ID NO:18), or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA025673, AA025672, AA155701, AA155646, W67560 or AA158406. Provided in Figure 9 is an exemplary nucleic acid molecule of the invention, SEQ ID NO: 17, which encodes human APOδ. In one embodiment, the invention provides an isolated nucleic acid molecule containing a nucleotide sequence encoding a soluble AP06 active segment having an APOδ ligand binding domain.

The invention also provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as a TNRLl polypeptide, or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number AA166695, Tδ7299, R166δ2, AA254047, AA056924, AA057069, N91002, N79018 or AA497494 or TIGR sequence number M78231. The invention provides, for example, an isolated nucleic acid molecule having a nucleotide sequence encoding substantially the same amino acid sequence as hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO: 26) or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence, or a segment thereof, of an expressed sequence tag having GenBank accession number AA166695, T87299, R16862, AA254047, AA056924, AA057069, N91002, N79018 or AA497494 or TIGR sequence number M78231. Provided in Figures 11 and 12 are exemplary nucleic acid molecules of the invention: SEQ ID NO: 19 encodes human TNRLl-α; SEQ ID NO: 21 encodes murine TNRLl-α; SEQ ID NO: 23 encodes human TNRLl-β; and SEQ ID NO: 25 encodes murine TNRLl-β. The invention also provides an isolated nucleic acid molecule containing a nucleotide sequence encoding a soluble TNRLl active segment having a TNRLl ligand binding domain.

The invention further provides an isolated nucleic acid molecule containing a nucleotide sequence encoding substantially the same amino acid sequence as TNRL3, or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number R55285, N35070, C00994 or AA221610. For example, the invention provides an isolated nucleic acid molecule of the invention having a nucleotide sequence encoding substantially the same amino acid sequence as hTNRL3 (SEQ ID NO: 28) or mTNRL3 (SEQ ID NO: 30), or an active segment thereof, provided that the nucleic acid molecule does not consist of the identical nucleotide sequence of an expressed sequence tag having GenBank accession number R55285, N35070, C00994 or

AA221610. Provided in Figure 13 are exemplary nucleic acid molecules of the invention. SEQ ID NO: 27 encodes human TNRL3, and SEQ ID NO: 29 encodes murine TNRL3. The invention also provides an isolated nucleic acid molecule containing a nucleotide sequence encoding a soluble TNRL3 active segment having a TNRL3 ligand binding domain.

The invention also provides selective binding agents, which are agents that exhibit selective binding for a receptor or ligand of the invention.

Provided herein are APOδ selective binding agents, which are agents that selectively bind an AP08 polypeptide or an active fragment thereof. Such an AP08 selective binding agent exhibits selective binding affinity for an AP08 polypeptide, such as a polypeptide having substantially the same amino acid sequence as hAPOδ (SEQ ID NO: 2) or active fragment thereof. In addition to exhibiting selective binding affinity for AP08, such AP08 selective binding agents can also function to selectively enhance or inhibit the function of APOδ . For example, an APOδ selective binding agent can function to enhance or inhibit binding of an APOδ polypeptide to its natural APOδ ligand, enhance or inhibit binding to TRADD or RIP, enhance or inhibit apoptotic activity, enhance or inhibit the capacity of

APOδ to activate NF-κB, or enhance or inhibit the ability of an APOδ polypeptide to effect cell proliferation, differentiation or cytokine secretion.

The invention also provides AP09 selective binding agents, which are agents that selectively bind an AP09 polypeptide or active fragment thereof, such as a polypeptide having, for example, substantially the same amino acid sequence as hAP09 (SEQ ID NO: 6). Such AP09 selective binding agents exhibit selective binding affinity for AP09 and, in addition, can function to selectively enhance or inhibit the function of AP09. An AP09 selective binding agent can, for example, enhance or inhibit AP09 binding to a ligand, enhance or inhibit the pro-apoptotic or anti-apoptotic activity of an AP09 polypeptide, or enhance or inhibit the ability of an AP09 polypeptide to effect cell proliferation, differentiation or cytokine secretion.

Further provided herein are AP04 selective binding agents, which are agents that selectively bind an AP04 polypeptide or active fragment thereof. An AP04 selective binding agent can exhibit selective binding affinity for an AP04 polypeptide having, for example, substantially the same amino acid sequence as mAP04-αL (SEQ ID NO:8), mAP04-αS (SEQ ID NO:10), hAP04-α (SEQ ID N0:12), rAP04-α (SEQ ID N0:14), mAP04-β (SEQ ID N0:16) or mAP04-γ (SEQ ID NO:32), or an active fragment thereof. Such AP04 selective binding agents exhibit selective binding affinity for an AP04 polypeptide and, in addition, can selectively enhance or inhibit the function of an AP04 polypeptide. An AP04 selective binding agent can, for example, enhance or inhibit binding of an AP04 polypeptide to a ligand, enhance or inhibit pro-apoptotic or anti-apoptotic activity of an AP04 polypeptide, or enhance or inhibit the ability of an AP04 polypeptide to effect cell proliferation, differentiation or cytokine secretion.

APOδ selective binding agents also are provided by the present invention. Such APOδ selective binding agents are agents that selectively bind an AP06 polypeptide of the invention or active fragment thereof. An AP06 selective binding agent can exhibit selective binding affinity for an AP06 polypeptide having, for example, substantially the same amino acid sequence as hAP06 (SEQ ID NO: 18), or active fragment thereof. Such APOδ selective binding agents exhibit selective binding affinity for an APOδ polypeptide and, in addition, can selectively enhance or inhibit the function of an APOδ polypeptide. For example, an APOδ selective binding agent can enhance or inhibit the binding of an APOδ polypeptide to a ligand, enhance or inhibit the pro-apoptotic or anti-apoptotic activity of an AP06 polypeptide, or enhance or inhibit the ability of an APOδ polypeptide to effect cell proliferation, differentiation or cytokine secretion.

The invention additionally provides binding agents that selectively bind a ligand of the invention. Provided herein are TNRLl selective binding agents, which are agents that selectively bind a TNRLl polypeptide or active fragment thereof. A TNRLl selective binding agent can exhibit selective binding affinity for a TNRLl polypeptide having, for example, substantially the same amino acid sequence as hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO:26), or active fragments thereof. Such TNRLl selective binding agents exhibit selective binding affinity for a TNRLl polypeptide and, in addition, can selectively enhance or inhibit the function of a TNRLl polypeptide. For example, a TNRLl selective binding agent can enhance or inhibit the binding of a TNRLl polypeptide to its cognate receptor.

The invention further provides a TNRL3 selective binding agent, which is an agent that selectively binds a TNRL3 polypeptide or active fragment thereof. A TNRL3 selective binding agent can exhibit selective binding affinity for a TNRL3 polypeptide having, for example, substantially the same amino acid sequence as hTNRL3 (SEQ ID NO: 28) or mTNRL3 (SEQ ID NO:30), or active fragments thereof. Such TNRL3 selective binding agents exhibit selective binding affinity for a TNRL3 polypeptide and, additionally, can selectively enhance or inhibit the function of a TNRL3 polypeptide. For example, a TNRL3 selective binding agent can enhance or inhibit the binding of a TNRL3 polypeptide to a cognate receptor.

The term "selective binding agent, " as used herein, means an agent that exhibits selective binding to the indicated polypeptide or polypeptide family. As used herein, the term "agent" encompasses simple or complex organic molecules, peptides, peptidomimetics, polypeptides and antibodies as well as nucleic acids, carbohydrates or lipids. A selective binding agent is any such agent that binds with substantially higher affinity to the indicated polypeptide or nucleic acid than to an unrelated polypeptide or nucleic acid.

Selective binding agents include natural ligands as well as other agents that exhibit selective binding for the indicated polypeptide of the invention. Such agents include polyclonal or monoclonal antibodies as well as selective binding agents isolated, for example, from random bacteriophage libraries or combinatorial libraries. Particularly useful selective binding agents include activating antibodies that selectively bind an AP08, AP09, AP04, APOδ, TNRLl or TNRL3 polypeptide of the invention. Such activating antibodies can, for example, function to cross-link receptors and trigger receptor signaling in the absence of ligand. Particularly useful selective binding agents also include neutralizing antibodies, which can bind and dampen or inactivate the function of an AP08, AP09, AP04, APOδ, TNRLl or TNRL3 polypeptide. Particularly useful selective binding agents that selectively bind a TNRLl or TNRL3 polypeptide of the invention also include soluble or full-length cognate receptors, which bind their respective TNRLl or TNRL3 polypeptide ligand.

The term "selective binding," as used in reference to a selective binding agent and an indicated polypeptide, means that the agent binds with substantially higher affinity to the indicated polypeptide, or fragment thereof, than to another polypeptide. For example, an AP08 selective binding agent, which selectively binds an AP08 polypeptide, binds with substantially higher affinity to an AP08 polypeptide, or fragment thereof, than to another member of the TNFR superfamily such as TNFR-1 or another death domain containing polypeptide such as FADD. Thus, for example, an agent that binds the death domains of TNFR-1, FAS/APO-1 and AP08 with similar affinity is not a selective binding agent as defined herein. However, an agent that has substantially higher affinity for an AP08 death domain than for the TNFR-1 and FAS/APO-1 death domains exhibits selective binding for APOδ and, therefore, is encompassed within the definition of an AP08 selective binding agent. Similarly, a TNRLl selective binding agent, which as defined herein is an agent that selectively binds a TNRLl polypeptide, binds with substantially higher affinity to a TNRLl polypeptide than to another member of the TNF family such as TNF-α.

The invention also provides a method of identifying an AP08 selective binding agent by contacting an AP08 polypeptide, or fragment thereof, with an agent and determining selective binding of the agent to the AP08 polypeptide, where said the selective binding indicates that the agent is an APOδ selective binding agent . A variety of methods for determining the selective binding of an agent to a polypeptide or fragment of the invention are well known in the art. Such methods include, for example, ELISA, RIA, receptor/ligand binding assays and other affinity assays such as chromatographic methods and panning. These screening methods are well known in the art and are described, for example in Harlow et al., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York (1992); Ansubel et al., Current Protocols in

Molecular Biology, John Wiley and Sons, Baltimore, MD (1989) and in Devlin, T.M., Textbook of Biochemistry with Clinical Correlations, Wiley-Liss, New York, NY (1992) .

Screening assays for detecting selective binding also include two-hybrid systems such as the yeast two hybrid system, which can be used to screen a panel of agents to detect selective binding to a polypeptide of the invention or fragment thereof (see Fields and Song, Nature 340:245-246 (1989), which is incorporated herein by reference) or a two-hybrid system adapted for use in mammalian cells (see Fearon et al., Proc. Natl. Acad. Sci., USA 89:7958-7962 (1992), which is incorporated herein by reference) .

Using one of the assays described above, or another assay well known in the art, a large collection, or library, of random agents or agents or interest can be screened for selective binding activity. Polypeptide libraries and tagged chemical libraries including those comprising polypeptides, peptides and peptidomimetic molecules can be screened. Polypeptide libraries also include those generated by phage display technology. Phage display technology includes the expression of polypeptide molecules on the surface of phage as well as other methodologies by which a protein ligand is or can be associated with the nucleic acid which encodes it. Methods for production of phage display libraries, including vectors and methods of diversifying the population of peptides which are expressed, are well known in the art (see, for example, Smith and Scott, Methods Enzymol. 217:228-257 (1993); Scott and Smith, Science 249:386-390 (1990); and Huse, WO 91/07141 and WO 91/07149, each of which is incorporated herein by reference) . These or other well known methods can be used to produce a phage display library which can be screened, for example, to identify a selective binding agent of the invention.

Each of the isolated receptor polypeptides provided herein are useful as immunogens for preparing an antibodies. Thus, the invention provides an antibody that selectively binds an AP08 polypeptide, for example, hAPOδ (SEQ ID NO:2). Such an antibody is useful, for example, in purifying an APOδ polypeptide by immunoaffinity chromatography.

The invention further provides an antibody that selectively binds an AP09 polypeptide of the invention, for example, hAP09 (SEQ ID NO: 6). Similarly, the invention provides an antibody that selectively binds an AP04 polypeptide; such an antibody can selectively bind, for example, mAP04-αL (SEQ ID NO:δ), mAP04-αS (SEQ ID NO: 10), hAP04-α(SEQ ID NO: 12), rAP04-α (SEQ ID NO: 14), mAP04-β (SEQ ID NO:16) or mAP04-γ (SEQ ID NO:32). Further provided by the invention is an antibody that selectively binds an APOδ polypeptide, for example, hAPOδ (SEQ ID NO:18). Each of these antibodies are useful in purifying the polypeptide to which it selectively binds using immunoaffinity chromatography. The isolated ligand polypeptides provided herein also are useful as immunogens for preparing antibodies. Thus, the invention provides antibodies that selectively bind a ligand of the invention. Provided herein is an antibody that selectively binds a TNRLl polypeptide, for example, hTNRLl-α (SEQ ID NO:20), mTNRLl-α (SEQ ID NO:22), hTNRLl-β (SEQ ID NO:24) or mTNRLl-β (SEQ ID NO: 26) or an active fragment thereof. Also provided herein is an antibody that selectively binds a TNRL3 polypeptide of the invention. Such an antibody can selectively bind, for example, hTNRL3 (SEQ ID NO:28) or mTNRL3 (SEQ ID NO:30).

As used herein, the term "antibody" is used in its broadest sense to include polyclonal and monoclonal antibodies, as well as polypeptide fragments of antibodies that retain a selective binding activity for a polypeptide of the invention of at least about 1 x 10⁵ M One skilled in the art would know that antibody fragments such as Fab, F(ab')₂ and Fv fragments can retain selective binding activity for a polypeptide of the invention and, thus, are included within the definition of an antibody. In addition, the term antibody as used herein includes naturally occurring antibodies, as well as non-naturally occurring antibodies and fragments such as chimeric antibodies and humanized antibodies that have selective binding activity. Such non-naturally occurring antibodies can be constructed using solid phase peptide synthesis, produced recombinantly or obtained, for example, by screening combinatorial libraries consisting of variable heavy chains and variable light chains as described by Borrebaeck (Ed.), Antibody Engineering (Second edition) New York: Oxford University Press (1995), which is incorporated herein by reference. Methods for producing antibodies are routine in the art. A purified polypeptide of the invention, which can be prepared from natural sources or produced recombinantly as described above, or a fragment thereof, such as a synthetic peptide, can be used as an immunogen. Non-immunogenic fragments or synthetic peptides can be made immunogenic by coupling the hapten to a carrier molecule such as bovine serum albumin (BSA) or keyhole limpet hemocyanin (KLH) . In addition, various other carrier molecules and methods for coupling a hapten to a carrier molecule are well known in the art as described, for example, by Harlow and Lane, Antibodies: A Laboratory Manual (Cold Spring Harbor Laboratory Press, 1988), which is incorporated herein by reference.

An antibody "having selective binding for" a polypeptide, or that "selectively binds" a polypeptide, binds with substantially higher affinity to that polypeptide than to another polypeptide.

The AP08 encoding nucleic acids and polypeptides of the invention can be used to diagnose, treat or reduce the severity of an AP08-mediated disease. For example, the AP08 encoding nucleic acids, polypeptides and active fragments and segments thereof can be used for diagnosis of an AP08-mediated disease or can be used to generate reagents useful for such diagnosis. Diagnosis can be performed, for example, by nucleic acid probe hybridization, or by amplification of AP08 encoding nucleotide sequences and subsequent electrophoretic analysis such as DNA sequencing. Diagnosis also can be performed, for example, using antibody or ligand based detection with an AP08 selective binding agent. Detection can be performed ex vivo, for example, by removing a cell or tissue sample from an individual at risk of, suspected of having or exhibiting one or more symptoms of an AP08-medιated disease. Altered AP08 expression or activity is indicative of an APOδ-mediated disease. As used herein, the term "AP08-medιated disease" means a disease, pathology, or other condition resulting from an abnormality the expression or activity of an AP08 polypeptide. Included within the meaning of the term "AP08-medιated disease" are disorders of cell loss, disorders of cell accumulation and disorders of differentiation, as described further below.

The present invention provides a method of treating or reducing the severity of an AP08-medιated disease in a subject by administering an AP08 polypeptide or active fragment thereof or by administering a nucleic acid molecule encoding the APOδ polypeptide or fragment. An APOδ polypeptide or nucleic acid molecule useful m such a method can be or encode, for example, an ammo acid sequence that is identical or substantially the same as hAPOδ (SEQ ID NO:2), or an active fragment thereof. An APOδ antisense nucleic acid molecule, which has a nucleotide sequence that is identically or substantially complementary to an APOδ encoding nucleic acid or active fragment thereof, also can be used to treat or reduce the severity of an APOδ-mediated disease according to a method of the invention.

Also provided herein is a method of treating or reducing the severity of an APOδ-mediated disease in a subject by administering an APOδ regulatory agent to the subject. As used herein, an "APOδ regulatory agent" is an agent that inhibits or enhances a biological activity of an APOδ polypeptide. Such an APOδ regulatory agent can be, for example, an AP06 inhibitory agent such as a dominant negative form of AP08, an AP08 selective binding agent that inhibits a biological activity of an AP08 polypeptide, or a cysteine protease inhibitor. Dominant negative forms of AP08 include, for example, soluble forms of AP08 having an AP08 ligand binding domain, as well as membrane bound forms of AP08 having an AP08 ligand binding domain but lacking a functional cytoplasmic tail, known as "decoy receptors." Examples of such "decoy receptor" AP08 inhibitory agents are provided herein as APOδΔCP and AP08ΔDD (see Example II) .

An AP08 regulatory agent also can be an agent that inhibits or enhances the activity or expression of FADD, FLICE, TRADD or RIP or that inhibits or enhances the activity or expression of NF-κB. One skilled in the art understands that such an AP08 regulatory agent can be an agent that selectively regulates a biological activity of an APOδ polypeptide or, alternatively, can be a non-selective agent that, in addition to regulating a biological activity of an AP08 polypeptide, also regulates the activity of, for example, other death domain containing receptors. As shown in Example II, the FADD dominant negative construct DN-FADD, the FLICE dominant negative construct (FL-C360S) and the cysteine protease inhibitors CrmA and z-VAD-fmk each inhibit AP08 induced apoptosis. In addition, the TRADD dominant negative construct ND-TRADD inhibits APOδ-mediated NF-κB activation, which can protect cells from apoptosis (Example II). Thus, each of these molecules are examples of APOδ regulatory agents as defined herein.

As described above, an agent can be a simple or complex organic molecule, peptide, peptidomimetic, polypeptide, antibody, nucleic acid, carbohydrate or lipid molecule. The present invention also provides a method of treating or reducing the severity of an APOδRP-mediated disease in a subject by administering to the subject a FADD regulatory agent. Such a FADD regulatory agent is an agent, such as a simple or complex organic molecule, peptide, peptidomimetic, polypeptide, antibody, nucleic acid, carbohydrate or lipid molecule, that inhibits or enhances FADD activity or expression. As disclosed herein, hAPOδ (SEQ ID N0:2) and hAPOδRP (SEQ ID NO: 4) induce apoptosis through the FADD adaptor molecule. The data shown in Figure 5B demonstrate that a FADD dominant negative construct (DN-FD) reduces APOδRP-mediated apoptosis. These results indicate that a FADD regulatory agent, such as a FADD dominant negative construct, can be used to reduce APOδRP-induced apoptosis and, thus, treat or reduce the severity of an APOδRP-mediated disease. An "AP08 related polypeptide-mediated disease, " as used herein, is synonymous with "APOδRP-mediated disease" and means a disease, pathology, or other condition resulting from an abnormality in the expression or activity of an AP08RP polypeptide and can be, for example, a disorder of cell loss, disorder of cell accumulation or disorder of cell differentiation. An AP08RP-mediated disease also can be, for example, an autoimmune disorder.

Further provided by the present invention is a method of treating or reducing the severity of an APOδRP-mediated disease in a subject by administering to the subject a TRADD regulatory agent or a RIP regulatory agent. Such an agent enhances or inhibits the activity or expression of TRADD or RIP. As disclosed herein in Example II, both AP08 and AP08RP directly interact with the TRADD and RIP adaptor molecules. Example II further demonstrates that a TRADD dominant negative construct blocks NF-KB activation by AP08 and AP08RP. Thus, a TRADD regulatory agent or a RIP regulatory agent, such as a TRADD dominant negative construct, can be used to treat or reduce the severity of an AP08RP-mediated disease.

The present invention also provides a method of treating or reducing the severity of an AP08RP-mediated disease in a subject by administering to the subject an NF-κB regulatory agent, which is an agent that enhances or inhibits the activity or expression of NF-κB. Activation of the NF- B pathway is known to protect cells from apoptosis, and, as disclosed herein, expression of AP08 or APOδRP results in NF-κB activation (see Example II) . Thus, an NF-κB regulatory agent can block, for example, apoptosis and, therefore, can be useful in treating or reducing the severity of an APOδRP-mediated disease that results from excessive cell death.

The AP09 encoding nucleic acids and polypeptides of the invention can be used to diagnose, treat or reduce the severity of an AP09-mediated disease. For example, the AP09 encoding nucleic acids, polypeptides and active fragments thereof can be used for diagnosis of an AP09-mediated disease or can be used to generate reagents useful for such diagnosis. Diagnosis can be performed, for example, by nucleic acid probe hybridization, or by amplification of AP09 encoding nucleotide sequences and subsequent electrophoretic analysis such as DNA sequencing. Diagnosis also can be performed using, for example, antibody or ligand based detection with an AP09 selective binding agent. Detection can be performed ex vivo, for example, by removing a cell or tissue sample from an individual at risk of, suspected of having or exhibiting one or more symptoms of an APOδ-mediated disease. Altered AP09 expression or activity is indicative of an AP09-mediated disease. As used herein, the term "AP09-mediated disease" means a disease, pathology, or other condition resulting from an abnormality in the expression or activity of an AP09 polypeptide. Included within the meaning of the term "AP09-mediated disease" are disorders of cell loss, disorders of cell accumulation and disorders of cell differentiation.

The present invention provides a method of treating or reducing the severity of an AP09-mediated disease in a subject by administering an AP09 polypeptide or active fragment thereof or by administering a nucleic acid molecule encoding the AP09 polypeptide or fragment. An AP09 polypeptide or nucleic acid molecule useful in such a method can be or encode, for example, an amino acid sequence identical or substantially the same as hAP09 (SEQ ID NO: 6), or an active fragment thereof. An AP09 antisense nucleic acid molecule, which has a nucleotide sequence that is identically or substantially complementary to an AP09 encoding nucleic acid or active fragment thereof, also can be used to treat or reduce the severity of an AP09-mediated disease according to a method of the invention.

Further provided herein is a method of treating or reducing the severity of an AP09-mediated disease in a subject by administering an AP09 regulatory agent to the subject. As used herein, an "AP09 regulatory agent" is an agent that inhibits or enhances a biological activity of an AP09 polypeptide. Such an AP09 regulatory agent can be, for example, an AP09 inhibitory agent such as a dominant negative form of AP09, an alternative spliced full-length form of AP09, or an AP09 selective binding agent that inhibits a biological activity of an AP09 polypeptide. Dominant negative forms of AP09 include, for example, soluble forms of AP09 having an AP09 ligand binding domain, as well as membrane bound forms of AP09 having an AP09 ligand binding domain but lacking a functional cytoplasmic tail, known as "decoy receptors." An AP09 regulatory agent can also be an agent that selectively binds an alternative spliced full-length form of AP09, thereby blocking or transmitting a signal through full-length AP09. One skilled in the art understands that such an AP09 regulatory agent can be an agent that selectively regulates a biological activity of an AP09 polypeptide or, alternatively, can be a non-selective agent that, in addition to regulating a biological activity of an AP09 polypeptide, also regulates the activity of, for example, other TNF family receptors .

The AP04 encoding nucleic acids and polypeptides of the invention can be used to diagnose, treat or reduce the severity of an AP04-mediated disease. The AP04 encoding nucleic acids, polypeptides and active fragments thereof can be used for diagnosis of an

AP04-mediated disease or can be used to generate reagents useful for such diagnosis. Diagnosis can be performed, for example, by nucleic acid probe hybridization, or by amplification of AP04 encoding nucleotide sequences and subsequent electrophoretic analysis such as DNA sequencing. Diagnosis also can be performed, for example, using antibody or ligand based detection with an AP04 selective binding agent. Detection can be performed ex vivo, for example, by removing a cell or tissue sample from an individual at risk of, suspected of having or exhibiting one or more symptoms of an AP04-mediated disease. Altered AP04 polypeptide expression or activity can be indicative of an AP04-mediated disease. As used herein, the term "AP04-mediated disease" means a disease, pathology, or other condition resulting from an abnormality in the expression or activity of an AP04 polypeptide. Included within the meaning of the term "AP04-mediated disease" are disorders of cell loss, disorders of cell accumulation and disorders of cell differentiation.

As disclosed herein, AP04 was expressed highly in embryonic tissue at day 11, 15 and 17, indicating that AP04 can play a role in early development (see Example IV) . AP04 expression in embryonic tissues also indicates that AP04 can play a role in developmental abnormalities or in gestation. Thus, AP04 and can be used in the diagnosis or treatment of developmental or gestational abnormalities.

The present invention provides, for example, a method a diagnosing a developmental abnormality caused by the aberrant expression of AP04 during development by determining the level or pattern of expression of AP04 mRNA or protein in embryonic cells or tissue. The invention further provides a method of diagnosing a developmental abnormality casued by the aberrant expression of AP04 during development by detecting a mutation in an AP04 gene in a cell or tissue derived from embryos .

In adult tissues, AP04 expression was highest in adult prostate, with lower levels of expression seen in adult spleen, thymus, testis, uterus, small intestine, colon and peripheral blood leukocytes. AP04 also was highly expressed in a prostate carcinoma epithelial cell line, LNCaP (see Example IV) . Since the glandular epithelial cells of the prostate represent the site of origin of prostate cancer and the cell type that increases during prostate cancer growth, increased AP04 levels can be used as a marker diagnostic of the presence of prostate cancer. For example, as the levels of AP04 secreted by prostatic tissue increase with the increasing mass of the prostate epithelial cells, elevated levels of AP04 either in the serum or from a tissue biopsy of an individual suspected of having prostate cancer can be used to diagnose the existence of prostate cancer. In addition, altered AP04 levels can be assayed in an individual with prostate cancer as a method of staging the degree or severity of the cancer, thereby providing critical information for determining the most advantageous therapeutic strategy. Altered AP04 levels also can be useful for the diagnosis of local and distant metastatic spread of prostate cancer and for assessment of disease volume. Such a diagnostic indicator is particularly useful in planning radiation therapy for prostate cancer by defining the limits of the spread of disease .

Thus, the present invention provides a method of diagnosing prostate cancer in an individual by determining the level of AP04 in the individual. A method of the invention can be particularly useful in diagnosing prostate cancer in an individual at risk for this cancer, such as in men over the age of fifty or with a family history of prostate cancer.

Soluble AP04 can be a naturally occurring form secreted into the bloodstream. For example, murine AP04 form occurs naturally in soluble form and is secreted into the bloodstream. In one embodiment, the invention provides a method of diagnosing prostate cancer in an individual by determining the level of soluble AP04 in the individual. Such methods can be particularly useful since a non-invasive blood test can be used in order to determine the level of soluble AP04.

A variety of methods can be used to assay for the level of AP04 according to a method of the invention for diagnosing prostate cancer. Such methods include assays for AP04 RNA levels, for example, reverse transcriptase polymerase chain reaction (RT-PCR) analysis. Such methods further include immunoassays for determining the level of AP04 polypeptide, for example, radioimmunoassays and enzyme-linked immunoassays. The level of AP04 in an individual suspected of having prostate cancer can be determined, for example, in a sample such as a serum sample from the individual. If desired, the level of AP04 also can be assayed in situ, for example, by administering an anti-AP04 antibody linked to a detectable moiety and subsequent detection of the moiety.

The invention also provides a method of identifying an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity. The method includes the steps of contacting an AP04 polypeptide or active fragment thereof with an agent; and determining selective binding of the agent to the AP04 polypeptide or active fragment thereof, where the selective binding indicates that the agent is an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity. An AP04 polypeptide or active fragment thereof useful in the invention can be, for example, an AP04 extracellular ligand binding domain. In one embodiment, the AP04 polypeptide or active fragment thereof is expressed in a cell or on the surface of a cell. In another embodiment, the AP04 polypeptide or active fragment thereof is contacted with an agent in vitro .

Methods for determining selective binding are known in the art and are described hereinabove. For example, assays to determine selective binding include, two-hybrid systems, ELISA, RIA, receptor/ligand binding assays and other affinity assays such as chromatographic methods and panning.

The present invention also provides a method of screening for an AP04 agonist useful in treating prostate cancer. The method includes the steps of contacting a cell expressing an AP04 polypeptide or active fragment thereof with an agent; and assaying for increased AP04 activity, where increased AP04 activity indicates that the agent is an AP04 agonist useful in treating prostate cancer.

An AP04 activity can be, for example, AP04 binding to a ligand, activation of the JNK pathway or activation of NF-κB activity. AP04 activity also includes apoptotic activity in a cell in response to increased AP04 expression or contact with AP04. The stimulation or induction of apoptosis in the cell includes any increased level of apoptosis compared to the level observed prior to contact with the AP04 agonist.

Further provided herein is a method of screening for an AP04 antagonist. The method includes the steps of contacting a cell expressing an AP04 polypeptide or active fragment thereof with an agent; and assaying for decreased AP04 activity, where decreased AP04 activity indicates that the agent is an AP04 antagonist . The invention also provides a method of identifying an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity by forming a mixture including an AP04 polypeptide or active fragment thereof, an AP04 signal transducer molecule that specifically interacts with a cytoplasmic domain of the AP04 polypeptide or active fragment thereof, and an agent; and detecting the level of AP04 activity in the presence of the agent, where an alteration in the AP04 activity relative to control activity indicates that the agent is an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity. An alteration in AP04 activity can be an increased or decreased activity. Examples of AP04 activity include, for example, activation of the JNK pathway, activation of NF-κB, apoptosis, cell proliferation or cell differentiation. In such a method of the invention, the AP04 activity can be measured, for example, in vivo or in vi tro .

As discussed above, AP04 can be highly expressed in prostate cancer epithelial cells. Based on this expression, the present invention also provides a method of treating prostate cancer by administering to an individual having prostate cancer a conjugate including an AP04 selective binding agent linked to a therapeutic moiety. Administration of a conjugate including an AP04 selective binding agent, for example, an anti-AP04 monoclonal antibody, facilitates targeted delivery of a linked therapeutic moiety such as doxorubicin to prostate cancer epithelial cells.

As used herein, the term "AP04 selective binding agent" means a simple or complex organic molecule, peptide, peptidomimetic, polypeptide, antibody, nucleic acid, carbohydrate or lipid that binds with substantially higher affinity to an AP04 polypeptide than to an unrelated polypeptide. Methods for identifying a selective binding agent are described hereinabove.

As disclosed herein, a therapeutic moiety can be, for example, a cancer chemotherapeutic agent linked to an AP04 selective binding agent to produce a conjugate containing an AP04 selective binding agent linked to a therapeutic moiety. Cytotoxic chemotherapy is the basis of the systemic treatment of disseminated malignant tumors. However, a major limitation of the currently used chemotherapeutic agents is that these drugs have the narrowest therapeutic index in all of medicine. As such, the dose of cancer chemotherapeutic agents generally is limited by undesirable toxicity to the patient being treated. Thus, the ability of an AP04 selective binding agent to target a drug to cancerous prostate tissue allows for a higher amount of chemotherapeutic agent to be delivered directly to the cancer with reduced side effects .

As used herein, the term "therapeutic moiety" means a physical, chemical, or biological material that is linked to an AP04 selective binding agent for the purpose of being targeted in vivo to an AP04 polypeptide. A therapeutic moiety can be a cancer chemotherapeutic agent such as doxorubicin, which, when linked to an AP04 selective binding agent, provides a conjugate useful for treating a cancer in a subject. In addition, a therapeutic moiety can be a drug delivery vehicle such as a chambered microdevice, a cell, a liposome or a virus, which can contain an agent such as a drug or a nucleic acid. An AP04 selective binding agent also can be linked to a therapeutic moiety expressed by a virus, for example, the adenovirus penton base coat protein, thus providing a means to target a virus to cancerous prostate cells (Wickman et al., Gene Ther. 2:750-756 (1995); Weitzman et al . , In: "Gene Therapy and Vector Systems" 2:17-25 (1997), each of which is incorporated herein by reference) .

A therapeutic moiety also can be, for example, a radioactive moiety or can be a cytotoxic agent, including a toxin such as ricin or a drug such as a chemotherapeutic agent or can be a physical, chemical or biological material such as a liposome, microcapsule, micropump or other chambered microdevice, which can be used, for example, as a drug delivery system. Generally, such microdevices, should be nontoxic and, if desired, biodegradable. Various moieties including microcapsules, which can contain an agent, are well known in the art and commercially available (see, for example, "Remington's Pharmaceutical Sciences" 18th ed. (Mack Publishing Co. 1990), chapters 89-91; Harlow and Lane, Antibodies: A laboratory manual (Cold Spring Harbor Laboratory Press 1988), each of which is incorporated herein by reference) .

In one embodiment, an AP04 selective binding agent for treating prostate cancer is a monoclonal or polyclonal antibody. Use of antibody-conjugated immunotoxins and radioactive moieties for the treatment of cancer are well known in the art, as described, for example, by Geissler et al. Cancer Res. , 52:2907-2915 (1992); Thrush et al., Ann Rev. Immunol.. 14:49-71 (1996); and Trail et al . , Science. 261:212-215 (1993). For example, the anthracyclin, doxorubicin, has been linked to antibodies and the antibody/doxorubicin conjugated have been therapeutically effective in treating tumors (Sivan et al., Cancer Res. 55:2352-2356 (1995); Lau et al . , Bioorα. Med. Chem. 3:1299-1304 (1995); Shih et al., Cancer Immunol. Immunother. 38:92-98 (1994), which are incorporated herein by reference).

Similarly, other anthracyclins, including idarubicin and daunorubicin, have been chemically conjugated to antibodies, which have delivered effective doses of the therapeutic moiety to tumors (Rowland et al . , Cancer Immunol. Immunother. 37:195-202 (1993); Aboud-Pirak et al., Biochem. Pharmacol. 38:641-648 (1989)). Thus, an anthracyclin, such as doxorubicin, idarubicin or daunorubicin, is a therapeutic moiety particularly useful in the methods of the invention.

A therapeutic moiety can be conjugated to a selective binding agent using a variety of methods. A covalent bond can be formed, for example, using glutaraldehyde, a heterobifunctional cross-linker or a homobifunctional cross-linker. Additional conjugation methods include carbodiimide conjugation methods

(Bauminger and Wilchek, Meth. Enzymol. 70:151-159 (1980), which is incorporated herein by reference) . Carbodiimides comprise a group of compounds that have the general formula R-N=C=N-R' , where R and R' can be aliphatic or aromatic, and are used for synthesis of peptide bonds. The preparative procedure is simple, relatively fast, and is carried out under mild conditions. Carbodiimide compounds attack carboxylic groups to change them into reactive sites for free amino groups. Carbodiimide conjugation has been used to conjugate a variety of compounds to carriers for the production of antibodies. In addition to using carbodiimides for conjugation, the water soluble carbodiimide, l-ethyl-3- (3-dimethylaminopropyl) carbodiimide (EDC) also can be used for conjugating a therapeutic moiety to an AP04 selective binding agent. EDC can also be used to prepare active esters such as N-hydroxysuccinimide (NHS) ester. The NHS ester, which binds only to amino groups, then can be used to induce the formation of an amide bond with the single amino group of the doxorubicin. The use of EDC and NHS in combination is commonly used for conjugation in order to increase the yield of conjugate formation (Bauminger and Wichek, supra , 1980) .

Other methods for conjugating a therapeutic moiety to an AP04 selective binding agent also can be used. For example, sodium periodate oxidation followed by reductive alkylation of appropriate reactants can be used, as can glutaraldehyde cross-linking. The cross-linking of proteins can additionally be accomplished by using reactive groups within the individual protein such as carbohydrate, disulfide, carboxyl or amino groups. Coupling can also be accomplished by oxidation or reduction of the native protein, or treatment with an enzyme, for example. However, it is recognized that, regardless of which method of producing a conjugate is selected, a determination must be made that the AP04 selective binding agent maintains its targeting ability and that the therapeutic moiety maintains its relevant function.

The yield of therapeutic moiety/selective binding agent conjugate formed is determined using routine methods. For example, HPLC or capillary electrophoresis or other qualitative or quantitative method can be used (see, for example, Liu et al . , J_^. Chromatoσ . 735:357-366 (1996); Rose et al . , J. Chromatocrr. 425:419-412 (1988), each of which is incorporated herein by reference) . In particular, the skilled artisan will recognize that the choice of a method for determining yield of a conjugation reaction depends, in part, on the physical and chemical characteristics of the specific therapeutic moiety and selective binding agent. Following conjugation, the reaction products are desalted to remove any free peptide and free drug.

A conjugate made up of an AP04 selective binding agent linked to a therapeutic moiety can be administered to an individual having prostate cancer, or, if desired, to an individual suspected of having prostate cancer or an individual at risk of developing prostate cancer. Administration can be accomplished, for example, by intravenous, intraperitoneal or subcutaneous injection. A conjugate made up of an AP04 selective binding agent linked to a therapeutic moiety can be administered by conventional methods using a dosage sufficient to reduce the growth or severity of the prostate cancer. Such dosages can be readily determined by those skilled in the art and include a variety of different regimes, including single high dose administration or repeated small dose administration or a combination of both. The dosing will depend on the progression of the disease and overall health of the individual and can be determined by those skilled in the ar .

The present invention also provides a method of treating or reducing the severity of an AP04-mediated disease in a subject by administering an AP04 polypeptide or active fragment thereof or by administering a nucleic acid molecule encoding the AP04 polypeptide or fragment. An AP04 polypeptide or nucleic acid molecule useful in such a method can be or encode, for example, an amino acid sequence identical or substantially the same as mAP04-αL (SEQ ID NO: 8) or an active fragment thereof, mAP04-αS (SEQ ID NO: 10) or an active fragment thereof, hAP04-α (SEQ ID NO: 12) or an active fragment thereof, rAP04-α (SEQ ID NO: 14) or an active fragment thereof or mAP04-β (SEQ ID NO: 16) or an active fragment thereof, or mAP04-γ (SEQ ID NO: 32) or an active fragment thereof. An AP04 antisense nucleic acid molecule, which has a nucleotide sequence that is identically or substantially complementary to an AP04 encoding nucleic acid or active fragment thereof, also can be used to treat or reduce the severity of an AP04-mediated disease according to a method of the invention.

The present invention also provides a method of treating or reducing the severity of an AP04-mediated disease in a subject such as, for example, prostate cancer, by administering an AP04 regulatory agent to the subject. As used herein, an "AP04 regulatory agent" is an agent that inhibits or enhances a biological activity of an AP04 polypeptide. Such an AP04 regulatory agent can be, for example, an inhibitory agent capable of inhibiting the activity of AP04 or an AP04-mediated activity. For example, in the case of prostate cancer, individuals with this disease can be treated with inhibitors of a JNK pathway to reduce the severity of the disease .

An AP04 regulatory agent can also be an AP04 inhibitory agent such as a dominant negative form of

AP04, or an AP04 selective binding agent that inhibits a biological activity of an AP04 polypeptide. Dominant negative forms of AP04 include, for example, soluble forms of AP04 having an AP04 ligand binding domain such as mAP04-β, as well as membrane bound forms of AP04 having an AP04 ligand binding domain but lacking a functional cytoplasmic tail such as mAP04-αS, known as "decoy receptors." One skilled in the art understands that such an AP04 regulatory agent can be an agent that selectively regulates a biological activity of an AP04 polypeptide or, alternatively, can be a non-selective agent that, in addition to regulating a biological activity of an AP04 polypeptide, also regulates the activity of, for example, other TNF family receptors.

As disclosed herein, amino acids 194 to 355 are important for activation of the JNK pathway and apoptosis. Since the mAP04α-short and mAP04-γ forms lack the above domains, these and similar isoforms of AP04 can lack the ability to induce the JNK pathway or cell death. However, since these isoforms of AP04 possess a complete or almost complete ligand-binding extracellular domain, they can still compete with the full-length form of AP04 for binding to the ligand, thereby blocking signal transduction mediated by the full-length receptor. As such, mAP04α-short, mAP04-γ, their species homologs and isoforms, and natural or synthetic compounds mimicking their ligand-binding ability but lacking the complete cytoplasmic domain or the domain encoding the amino acids 194 to 355 (or homologous domains in other species) can be useful therapeutic tools for the treatment of diseases resulting from the dysregulation of AP04 signal transduction pathway. Such agents also can serve as lead compounds for the development of therapeutic compounds useful in the diagnosis and treatment of diseases resulting from the dysregulation of AP04 function. The AP06 encoding nucleic acids and polypeptides of the invention can be used to diagnose, treat or reduce the severity of an AP06-mediated disease. For example, the APOδ encoding nucleic acids, polypeptides and active fragments thereof can be used for diagnosis of an AP06-mediated disease or can be used to generate reagents useful for such diagnosis. Diagnosis can be performed, for example, by nucleic acid probe hybridization, or by amplification of APOδ encoding nucleotide sequences and subsequent electrophoretic analysis such as DNA sequencing. Diagnosis also can be performed, for example using antibody or ligand based detection with an AP06 selective binding agent. Detection can be performed ex vivo, for example, by removing a cell or tissue sample from an individual at risk of, suspected of having or exhibiting one or more symptoms of an APOδ-mediated disease. Altered AP06 expression or activity is indicative of an AP06-mediated disease. As used herein, the term "AP06-mediated disease" means a disease, pathology, or other condition resulting from an abnormality in the expression or activity of an APOδ polypeptide. Included within the meaning of the term "APOδ-mediated disease" are disorders of cell loss, disorders of cell accumulation and disorders of cell differentiation.

The invention provides a method of treating or reducing the severity of an AP06-mediated disease in a subject by administering an APOδ polypeptide or active fragment thereof or by administering a nucleic acid molecule encoding the APOδ polypeptide or fragment. An APOδ polypeptide or nucleic acid molecule useful in such a method can be or encode, for example, an amino acid sequence identical or substantially the same as hAPOδ (SEQ ID NO: 18), or an active fragment thereof. An AP06 antisense nucleic acid molecule, which has a nucleotide sequence that is identically or substantially complementary to an AP06 encoding nucleic acid or active fragment thereof, also can be used to treat or reduce the severity of an APOδ-mediated disease according to a method of the invention.

In addition, the invention provides a method of treating or reducing the severity of an APOδ-mediated disease in a subject by administering an APOδ regulatory agent to the subject. An "APOδ regulatory agent," as used herein, is an agent that inhibits or enhances a biological activity of an AP06 polypeptide. Such an APOδ regulatory agent can be, for example, an AP06 inhibitory agent such as a dominant negative form of AP06, an alternatively spliced full-length form of AP06 polypeptide or an AP06 selective binding agent that inhibits a biological activity of an APOδ polypeptide. An APOδ regulatory agent also can be an agent that selectively binds an alternatively spliced full-length form of APOδ, thereby blocking or transmitting a signal through full-length APOδ. One skilled in the art understands that an AP06 regulatory agent can be an agent that selectively regulates a biological activity of an APOδ polypeptide or, alternatively, can be a non-selective agent that, in addition to regulating a biological activity of an AP06 polypeptide, also regulates the activity of, for example, other TNF family receptors .

The TNRLl encoding nucleic acids and polypeptides of the invention can be used to diagnose, treat or reduce the severity of a TNRLl-mediated disease. For example, the TNRLl encoding nucleic acids, polypeptides and active fragments thereof can be used for diagnosis of a TNRLl-mediated disease or can be used to generate reagents useful for such diagnosis. Diagnosis can be performed, for example, by nucleic acid probe hybridization, or by amplification of TNRLl encoding nucleotide sequences and subsequent electrophoretic analysis such as DNA sequencing. Diagnosis also can be performed using, for example, antibody or ligand based detection with a TNRLl selective binding agent. Detection can be performed ex vivo, for example, by removing a cell or tissue sample from an individual at risk of, suspected of having or exhibiting one or more symptoms of a TNRLl-mediated disease. Altered TNRLl expression or activity is indicative of a TNRLl-mediated disease. As used herein, the term "TNRLl-mediated disease" means a disease, pathology, or other condition resulting from an abnormality in the expression or activity of a TNRLl polypeptide. Included within the meaning of the term "TNRLl-mediated disease" are disorders of cell loss, disorders of cell accumulation and disorders of cell differentiation.

The invention also provides a method of treating or reducing the severity of a TNRLl-mediated disease in a subject by administering a TNRLl polypeptide or active fragment thereof or by administering a nucleic acid molecule encoding the TNRLl polypeptide or fragment. A TNRLl polypeptide or nucleic acid molecule useful in the method can be or encode, for example, an amino acid sequence identical or substantially the same as hTNRLl-α (SEQ ID NO: 20) or an active fragment thereof, mTNRLl-α (SEQ ID NO:22) or an active fragment thereof, hTNRLl-β

(SEQ ID NO: 24) or an active fragment thereof, or mTNRLl-β (SEQ ID NO:26) or an active fragment thereof. A TNRLl antisense nucleic acid molecule, which has a nucleotide sequence that is identically or substantially complementary to a TNRLl encoding nucleic acid or active fragment thereof, also can be used to treat or reduce the severity of a TNRLl-mediated disease.

In addition, the invention provides a method of treating or reducing the severity of a TNRLl-mediated disease in a subject by administering a TNRLl regulatory agent to the subject. A "TNRLl regulatory agent," as used herein, is an agent that inhibits or enhances a biological activity of a TNRLl polypeptide. A TNRLl regulatory agent can be, for example, a TNRLl stimulatory agent such as a soluble form of TNRLl having a TNRLl receptor binding domain. A TNRLl regulatory agent also can be, for example, a TNRLl selective binding agent that inhibits a biological activity of a TNRLl polypeptide. Such a TNRLl selective binding agent can be, for example, a small peptide, organic molecule or other agent which competes with TNRLl for binding to a TNRLl receptor but which, when bound, does not lead to receptor aggregation or signal transduction. One skilled in the art understands that a TNRLl regulatory agent can be an agent that selectively regulates a biological activity of a TNRLl polypeptide or, alternatively, can be a non-selective agent that, in addition to regulating a biological activity of a TNRLl polypeptide, also regulates the activity of, for example, other TNF family ligands .

The TNRL3 encoding nucleic acids and polypeptides of the invention also can be used to diagnose, treat or reduce the severity of a TNRL3-mediated disease. For example, the TNRL3 encoding nucleic acids, polypeptides and active fragments thereof can be used for diagnosis of a TNRL3-mediated disease or can be used to generate reagents useful for such diagnosis. Diagnosis can be performed, for example, by nucleic acid probe hybridization, or by amplification of TNRL3 encoding nucleotide sequences and subsequent electrophoretic analysis such as DNA sequencing. Diagnosis also can be performed, for example, using antibody or ligand based detection with a TNRL3 selective binding agent. Detection can be performed ex vivo, for example, by removing a cell or tissue sample from an individual at risk of, suspected of having or exhibiting one or more symptoms of a TNRL3-mediated disease.

Altered TNRL3 expression or activity is indicative of a TNRL3-mediated disease. As used herein, the term "TNRL3-mediated disease" means a disease, pathology, or other condition resulting from an abnormality in the expression or activity of a TNRL3 polypeptide. Included within the meaning of the term "TNRL3-mediated disease" are disorders of cell loss, disorders of cell accumulation and disorders of cell differentiation.

The invention also provides a method of treating or reducing the severity of a TNRL3-mediated disease in a subject by administering a TNRL3 polypeptide or active fragment thereof or by administering a nucleic acid molecule encoding the TNRL3 polypeptide or fragment. A TNRL3 polypeptide or nucleic acid molecule useful in the method can be or encode, for example, an amino acid sequence identical or substantially the same as hTNRL3 (SEQ ID NO: 28) or an active fragment thereof, or mTNRL3 (SEQ ID NO: 30) or an active fragment thereof. A TNRL3 antisense nucleic acid molecule, which has a nucleotide sequence that is identically or substantially complementary to a TNRL3 encoding nucleic acid or active fragment thereof, also can be used to treat or reduce the severity of a TNRL3-mediated disease. In addition, the invention provides a method of treating or reducing the severity of a TNRL3-mediated disease in a subject by administering a TNRL3 regulatory agent to the subject. A "TNRL3 regulatory agent," as used herein, is an agent that inhibits or enhances a biological activity of a TNRL3 polypeptide. A TNRL3 regulatory agent can be, for example, a TNRL3 stimulatory agent such as a soluble form of TNRL3 having a TNRL3 receptor binding domain. A TNRL3 regulatory agent also can be, for example, a TNRL3 selective binding agent that inhibits a biological activity of a TNRL3 polypeptide. Such a TNRL3 selective binding agent can be, for example, a small peptide, organic molecule or other agent which competes with TNRL3 for binding to a TNRL3 receptor but which, when bound, does not lead to receptor aggregation or signal transduction. One skilled in the art understands that such a TNRL3 regulatory agent can be an agent that selectively regulates a biological activity of a TNRL3 polypeptide or, alternatively, can be a non-selective agent that, in addition to regulating a biological activity of a TNRL3 polypeptide, also regulates the activity of, for example, other TNF family ligands .

The present invention also provides a method of treating or reducing the severity of a disorder of cell loss. Examples of such pathologies, which can result from excessive programmed cell death, include AIDS and degenerative disorders such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, retinitis pigmentosa, and cerebellar degeneration.

Disorders of cell loss caused by excessive programmed cell death also include myelodysplastic syndromes such as aplastic anemia and ischemic injuries such as myocardial infarction, stroke and reperfusion injury. Any such pathology is encompassed by the term "disorder of cell loss," which, as used herein, means a disease, pathology or other condition characterized by excessive cell death or differentiation or inadequate cellular proliferation resulting in an abnormally reduced number of cells.

Thus, the invention provides a method of treating or reducing the severity of a disorder of cell loss in a subject by administering an agent that selectively decreases AP08 apoptotic activity, thereby inhibiting apoptosis in the subject. The invention also provides a method of treating or reducing the severity of a disorder of cell loss in a subject by administering an agent that selectively decreases apoptotic activity, where the apoptotic activity is selected from the group selected from AP09 apoptotic activity, AP04 apoptotic activity or AP06 apoptotic activity. Such methods can be used to treat, for example, acquired immunodeficiency syndrome, which is characterized by an increased level of apoptosis in an infected T cell as compared to a normal T cell. The methods of the invention for treating or reducing the severity of a disorder of cell loss in a subject also can be used to treat neurodegenerative disorders such as Alzheimer' s disease or Parkinson' s disease, the disorders of cell loss described above or another disorder of cell loss known in the art.

As used herein, an agent that "selectively decreases AP08 apoptotic activity" produces a substantially greater reduction in the apoptotic activity of an AP06 polypeptide relative to any reduction in the apoptotic activity of another polypeptide, for example, another pro-apoptotic TNF receptor such as TNFR-1. Similarly, an agent that selectively decreases AP09, AP04 or APOδ apoptotic activity produces a substantially greater reduction in the apoptotic activity of an AP09 polypeptide, an AP04 polypeptide, or an AP06 polypeptide, respectively, relative to any reduction in the apoptotic activity of an unrelated polypeptide.

The present invention also provides a method of treating or reducing the severity of a disorder of increased cell accumulation. Disorders of increased cell accumulation include cancers such as lymphomas, carcinomas, and hormone dependent tumors such as breast, prostrate and ovarian tumors. Additionally, autoimmune diseases, such as myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus and immune-mediated glomerulonephritis, and viral infections such as herpesvirus, poxvirus and adenovirus can be a disorder of increased cell accumulation. Pathological conditions involving smooth or cardiac muscle cells, such as hepatic necrosis vasculitis, angiogenesis, atherosclerosis and myocarditis, also can result from increased cell accumulation. Any such pathology is encompassed within the term "disorder of increased cell accumulation, " which, as used herein, means a disease, pathology or other condition characterized by diminished cell death or differentiation or excess cell proliferation resulting in an abnormally high number of cells.

Provided herein is a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering an AP08 polypeptide or active fragment thereof, or a nucleic acid molecule encoding the AP08 polypeptide or active fragment, to the subject. The present invention also provides a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering an AP09 polypeptide or active fragment thereof, or a nucleic acid molecule encoding the AP09 polypeptide or fragment, to the subject. Further provided herein is a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering an AP04 polypeptide or active fragment thereof, or a nucleic acid molecule encoding the AP04 polypeptide or fragment, to the subject. The invention also provides a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering an AP06 polypeptide or active fragment thereof, or a nucleic acid molecule encoding the APOδ polypeptide or fragment, to the subject.

The TNRLl and TNRL3 polypeptides of the invention also can be useful in treating or reducing the severity of the disorder of cell accumulation. The present invention provides a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering a TNRLl polypeptide or active fragment thereof, or a nucleic acid molecule encoding the TNRLl polypeptide or fragment, to the subject. Also provided herein is a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering a TNRL3 polypeptide or active fragment thereof, or a nucleic acid molecule encoding the TNRL3 polypeptide or fragment, to the subject .

Further provided herein is a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering an agent that selectively increases APOδ apoptotic activity, thereby enhancing apoptosis in the subject. The invention also provides a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering an agent that selectively increases apoptotic activity, where the apoptotic activity is selected from the group consisting of AP09 apoptotic activity, AP04 apoptotic activity or AP06 apoptotic activity. The present invention further provides a method of treating or reducing the severity of a disorder of cell accumulation in a subject by administering an agent that selectively increases apoptotic activity, where the apoptotic activity is selected from the group consisting of TNRLl apoptotic activity and TNRL3 apoptotic activity. The methods of the invention for treating or reducing the severity of a disorder of cell accumulation can be used to treat, for example, cancers such as breast, prostrate and ovarian tumors, lymphomas and carcinomas as well as the disorders of cell accumulation described above or another disorder of cell accumulation known in the art.

As used herein, an agent that "selectively increases APOδ apoptotic activity" means an agent that produces a substantially greater enhancement of the apoptotic activity of an APOδ polypeptide relative to any enhancement of the apoptotic activity of another polypeptide, for example, another pro-apoptotic TNF receptor such as CD27. Similarly, an agent that selectively increases AP09, AP04, or APOδ apoptotic activity produces a substantially greater enhancement of the apoptotic activity of an AP09 polypeptide, an AP04 polypeptide, or an AP06 polypeptide, respectively, relative to any enhancement of the apoptotic activity of an unrelated polypeptide. In addition, an agent that selectively increases TNRLl or TNRL3 apoptotic activity produces a substantially greater enhancement of the apoptotic activity of a TNRLl polypeptide or TNRL3 polypeptide, respectively, relative to any enhancement of an unrelated polypeptide such as TNF-α.

The following examples are intended to illustrate but not limit the present invention.

EXAMPLE I

Identification and Characterization of human APQ8 and AP08 related polypeptide

This example describes the identification and characterization of nucleic acid sequences encoding full-length human AP08 and the AP08 related polypeptide AP08RP.

Isolation and characterization of the hAPOβ cDNA

APOδ is a human cDNA related to IMAGE consortium clones 650744 and 664665, which were derived from a Stratagene NT neuronal library. These clones were identified by searching the EST database (dbEST) for sequences with homology to the extracellular domain of human TNFR-1 protein using the TBLASTN algorithm 5' and 3' RACE was performed on human fetal brain Marathon-ready mRNA (Clontech, Palo Alto, CA) according to the manufacturer's instructions. 3' RACE was performed with primer SEQ ID NO: 34

(5 ' -CTGTTGGATCCAGCTGAGTCTGCTCTGATCACCCAAC-3 ' ) and API primer SEQ ID NO: 38 (5 ' -CCATCCTAATACGACTCATATAGGGC-3 ' ) . The subsequent round of nested PCR was performed with primer SEQ ID NO: 35 (5 ' -GGACACCATATCTCAGAAGACGGTAGAG-3 ' ) AP2 primer SEQ ID NO: 36 (5 ' -ACTCATATAGGGCTCGAGCGGC-3 ' ) . 5' RACE was performed with primer SEQ ID NO: 37 (5'-TATAGTCCTGTCCATATTTGCAGGAGATG-3' ) and the API primer SEQ ID NO: 38. The subsequent round of nested PCR was performed with primer SEQ ID NO: 38

(5'-CTCTACCGTCTTCTGAGATATGGTGTCC-3' ) and the AP2 primer SEQ ID NO: 36. RACE fragments were cloned into the PCR2.1 vector using the Original TA Cloning kit (Invitrogen, Carlsbad, CA) . Inserts from eight or more individual clones were PCR amplified using flanking vector primers Vn26 (SEQ ID NO: 39; 5 ' -TTTCCCAGTCACGACGTTGTA-3 ' ) and Vn27 (SEQ ID NO: 40; 5 ' -GTGAGCGGATAACAATTTCAC-3 ' ) and purified using the Wizard PCR kit (Promega, Madison, WI ) . The inserts were sequenced on an ABI 373 automated fluorescent sequencer with dye terminators using purified PCR product or plasmid DNA as template and primers SEQ ID NOS: 39 and 40 as the sequencing primers. Percentage identity and similarity scores were obtained using the Gap program (Genetic Computer Group, Madison, WI) .

The isolated full-length APOδ cDNA nucleotide sequence, provided as SEQ ID NO:l, encodes a polypeptide of 411 amino acids with characteristics of a cell surface receptor, including an N-terminal signal peptide of 51 amino acids and a transmembrane region of 27 amino acids (residues lδ5 to 212). The context of the initiating methionine (CCGCCATGG) conforms to the Kozak consensus sequence, and an in-frame stop codon ten amino acids upstream of this methionine confirmed that it is the true start site. The extracellular domain (residues 52 to 184) of hAP08 contains the cysteine-rich pseudo-repeats that are a hallmark of the TNF receptor family and exhibits a high degree of amino acid sequence homology to the TRAIL receptor (DR4) (5δ% identity and 70% similarity; Pan et al., supra , 1997, which is incorporated herein by reference) . To a lesser extent, the extracellular domain of hAP08 is homologous to TNFR-1 (27% amino acid identity and 46% similarity), FAS/APO-1 (25% amino acid identity and 45% similarity), DR3 (21% amino acid identity and 40% similarity) and other members of the TNFR family (see Table 1) . Based on this homology, the AP08 polypeptide is classified as a new member of the TNFR family.

hAPOδ has a cytoplasmic tail of 212 amino acids

(residues 213 to 411) with a death domain near the C-terminus . As shown in Figure 4B, the APOδ death domain exhibits significant sequence homology to the death domains of DR4, TNFR-1, FAS/APO-1 and DR3. Furthermore, several amino acids that are essential for transmission of the apoptotic signal by TNFR-1 and FAS/APO-1 also are conserved in the death domain of AP08. This includes leucine residue 334, which corresponds to the site of the lpr mutation in the murine FAS receptor (see Figure 4B) .

Expression of hAP08 was studied by Northern analysis. A major transcript of about 4.4 kb was seen in all tissues tested, including spleen, thymus, prostate, testis, ovary, small intestine, colonic mucosa and peripheral blood lymphocyte (PBL), with the highest level of expression in PBL. A more slowly migrating minor transcript, which may represent the product of alternative splicing, was present in all tissues assayed. These results demonstrate that RNA encoding the human AP08 polypeptide is widely expressed.

Northern analysis was performed using a multiple human tissue RNA blot (#7759-1) from Clontech. The blot was hybridized with a ³²P-labeled AP08 cDNA according to the manufacturer's instructions. Characterization of the AP08 related polypeptide (APOβRP) cDNA

The AP08RP nucleotide sequence, shown in Figure 3A, encodes a polypeptide of 410 amino acids. Alignment of the amino acid sequences of human AP08 (SEQ ID NO: 2) and human AP08RP (SEQ ID NO: 4) revealed that hAPOδRP exhibits substantial homology to hAPOδ throughout the entire polypeptide sequence. The hAPOδ and hAPOδRP predicted signal peptides, transmembrane regions and death domains are shown in Figure 4A. In particular, the hAPOδRP signal peptide sequence (residues 1 to 51) is homologous to the hAP08 signal peptide (residues 1 to 51), indicating that the N-terminal portion of the hAPOδRP signal is Met-Gly-Gln-His . Hydrophobicity plots of the hAPOδ and hAPOδRP sequences support the conclusion that the hAP08 signal peptide corresponds to residues 1 to 51 shown in Figure 4A and that the AP08RP signal peptide corresponds to residues 1 to 51 shown in Figure 4A. Furthermore, two artificial neural networks (Nielsen et al., Protein Enα. 10:1-6 (1997), which is incorporated by reference herein) , trained at recognizing eukaryotic signal peptides and their cleavage sites, identified cleavable signal peptides between amino acids 1 to 51 of AP08 and between amino acids 1 to 51 of hAPOδRP, respectively. The results with hAPOδRP contrast with those of Pan et al . , supra , 1997, which describe a related protein containing an additional 5δ N-terminal residues .

Hydrophobicity plots were generated by using the Pepplot program (Genetic Computer Group) . Artificial neural networks analysis for recognizing the signal peptides and their cleavage sites was performed using the Signal VI .1 World Wide Web Server (http://www.cbs.dtu.dk/service

EXAMPLE II

APQ8 and DR4 regulate NF-κB expression and induce

FADD-dependent apoptosis

This example demonstrates that APOδ and AP06RP induce cell death through the cytoplasmic protein FADD. In addition, the APOδ and AP08RP polypeptides activate NF-κB expression.

AP08 induces apoptosis

The ability of hAPOδ to induce apoptosis was assayed in the human breast carcinoma cell line MCF-7, the human embryonic kidney cell 293T subclone and the baby hamster kidney BHK cell line. Transient transfection of full-length hAPOδ induced rapid apoptosis in all three cell lines tested. In contrast, expression of an APOδ deletion mutant lacking the C-terminal 192 amino acids ("AP08ΔCP") or an APOδ deletion mutant lacking the C-terminal 134 amino acids, which includes the death domain, ("AP08ΔDD") failed to induce apoptosis. Exemplary results in MCF-7 cells are shown in Figure 5A. These data demonstrate that the C-terminal 134 amino acids of hAP08 including its death domain are essential for transmitting the apoptotic signal. Furthermore, a mutant construct containing a leucine to asparagine substitution at residue 334 ("AP08-L334N" ) also failed to induce apoptosis (Figure 4A) . Leucine 334 corresponds to the site of the lpr mutation and is essential for apoptotic signaling in TNFR-1, FAS/APO-1 and DR3. These results indicate that, like TNFR-1, FAS/APO-1 and DR3, leucine residue 334 is required for apoptotic activity of hAPOδ . AP08 and AP08RP induced apoptosis is mediated by FADD/MORTl

The adaptor molecule FADD/MORTl mediates apoptosis by the death domain containing receptors TNFR-1, FAS/APO-1 and DR3 either directly or through an intermediate adaptor molecule TRADD. For example, dominant negative FADD can effectively block the apoptosis mediated by these death receptors. A dominant negative FADD construct consisting of the FADD death domain was tested for the ability to modulate APOδ and APOδRP mediated apoptosis in 293T cells. Increasing amounts of dominant negative FADD led to progressive inhibition of apoptosis mediated by DR3, which has previously been shown to mediate cell death through FADD. As shown in Figure 5B, progressive inhibition of apoptosis was also observed for the hAP08 and hAPOδRP receptors, with almost complete inhibition of apoptosis at a receptor to dominant negative FADD DNA ratio of 1:10. Dominant negative FADD also inhibited APOδ mediated apoptosis in MCF-7 cells (Figure 5C) . These results demonstrate that both hAP08 and hAPOδRP induced apoptosis is dependent upon FADD/MORT. Thus, a variety of death domain containing receptors (TNFR-1, FAS/APO-1, DR3, hAPOδ and hAPOδRP) induce apoptosis through the FADD/MORT adaptor molecule.

Apoptotic signaling by the TNF receptors TNFR-1 and DR3 involves FADD-mediated recruitment of the ICE protease FLICE/MACHl (Caspase δ) to the aggregated death receptor complex. To test whether hAPOδ induced apoptosis also involves recruitment of FLICE/MACHl, the ability of a dominant negative FLICE mutant to block APOδ-induced apoptosis was tested. This FLICE mutant contains a cysteine to serine substitution at the catalytic site and is designated "FLICE-C360S . " As shown in Figure 5C, this FLICE-C360S mutant was able to effectively block apoptosis mediated by APOδ . In view of previous results demonstrating that FLICE interacts with FADD, these data provide further evidence that APOδ-induced apoptosis relies on a FADD-dependent pathway.

Several caspase inhibitors, z-VAD-fmk and CrmA, have previously been shown to block apoptosis of TNF death receptors. The ability of these caspase inhibitors to effect AP08 mediated apoptosis was assayed, and the results are shown in Figure 5C. These results demonstrate that z-VAD-fmk and CrmA each inhibit AP08-induced apoptosis, indicating that AP08 causes cell death through a caspase-dependent pathway.

Myc and 6X-His epitope tagged AP08 constructs were prepared by amplifying amino acids 51 to 411 of AP08 using pfu polymerase (Stratagene, La Jolla, CA) with a 5' primer containing a BamHI site and a 3 ' primer containing a Sail site. The amplified APOδ fragment was subsequently ligated to a modified pSecTag A vector (Invitrogen) containing a Myc or an MRG-δXHis epitope tag downstream of a murine lg K-chain signal peptide. The APOδ-L334N construct, which has a leucine to asparagine mutation at residue 334, was generated by mutagenesis of the 6X-His-AP08 plasmid with primers SEQ ID NO: 41 ( 5 ' -CGCTCATGAGGAAGTTGGGCAACATGGACAATGAGATAAAG-3 ' ) and SEQ ID NO: 2 (5 ' -CTTTATCTCATTGTCCATGTTGCCCAACTTCCTCATGAGCG-3 ' ) using the Quickchange kit (Stratagene) according to the manufacturer's instructions. Deletion construct AP08ΔCP lacks the 192 C-terminal residues of full-length AP08 and was constructed by creating a PCR generated Xbal site in the wild type APOδ sequence. Deletion construct APOδΔDD lacks the C-terminal 134 residues of AP08 and was prepared using a naturally occurring Smal site in the AP08 sequence. The sequences of each of the above constructs were confirmed by automated fluorescent sequencing.

Epitope-tagged versions of AP08RP (amino acids 52 to 410), DR3 (amino acids 27 to 418) and FAS/APOl (amino acids 19 to 335) were constructed as described above for the AP08 expression vector except that the 5' primer for amplifying Fas/APOl contained an EcoRI site and the resulting PCR product was cloned into the EcoRI and Sail sites of the modified pSecTag A vector.

The dominant negative FADD construct (DN-FADD) encodes amino acids δO to 20δ of human FADD in pCDNA3 (Invitrogen) and was prepared by Michael Wright of the University of Washington. The FLICE-C360S mutant, which contains a cysteine to serine substitution at residue 360, was prepared with primers SEQ ID NO: 43 ( 5 ' -GTGTTTTTTATTCAGGCTAGTCAGGGGGATAACTACCAGAA-3 ' ) and SEQ ID NO: 44 (TTCTGGTAGTTATCCCCCTGACTAGCCTGAATAAAAAACAC-3 ' ) using Stratagene 's Quickchange kit. The N-terminal TRADD deletion mutant ND-TRADD-HA, which lacks the N-terminal 102 amino acids, and RIP-HA tagged constructs were prepared using custom primers designed to generate a C-terminal HA-tag. The sequence of each of the above constructs was confirmed by automated fluorescent sequencing.

For transient transfection assays, 0.8-1 X 10⁵ MCF-7 or BHK cells were cotransfected with 1 μg of various test plasmids and 200 ng of a lacZ expression plasmid in a 24-well plate using 3 μl of Superfect (Qiagen, Santa Clarita, CA) in DMEM supplemented with 10% FCS essentially according to the manufacturer's instructions. After 3 hr, 0.5 ml of fresh DMEM supplemented with 10% FCS was added. Twenty-four to thirty-six hours later, cells were fixed with 0.05% glutaraldehyde in PBS and stained with

5-bromo-4-chloro-3-indolyl-β-D-galactosidase (X-GAL) . For transient transfection of 293T cells, 1 x 10⁵ cells were transfected by calcium phosphate precipitation in a 24-well plate essentially as described in Sambrook et al., supra , 1989. The data shown in Figure 5A represent the mean +/- the standard deviation determined from three independent experiments performed in duplicate. The percentage of apoptotic cells was calculated as the percentage of cells showing β-galactosidase activity and apoptotic morphology as indicated by a round and condensed shape as compared to the total number of blue staining cells.

For the dominant negative FADD experiments shown in Figure 5B, 150 ng of each receptor (AP08, APOδRP or DR3) was transfected into 293T cells with 150 ng, 750 ng or 1500 ng of DN-FADD DNA. The total amount of DNA in each transfection was held constant by adding control vector plasmid DNA. The results shown in Figure 5B are representative of two independent experiments performed in duplicate.

For the inhibition experiments shown in Figure 5C, MCF-7 cells were transfected with the Myc-AP08 expression construct and a control empty expression vector or an expression vector encoding DN-FADD, FLICE-C360S or CrmA. For experiments with the protease inhibitor z-VAD-fmk, cells transfected with Myc-AP08 and the control vector were treated with 20 μM z-VAD-fmk twelve hours after transfection. Cells were transfected and scored for apoptotic morphology as described above. The data shown in Figure 5C are representative of three independent experiments performed in duplicate.

AP08 and AP08KP activate NF-κB by a TRADD dependent pathway

Activation of TNFR-1 and DR3 has been shown to induce NF-κB expression, and NF- B activation can block apoptosis. To test the possibility that AP08 can regulate NF-κB expression, AP08 expression constructs were cotransfected into 293T cells with a NF-κB luciferase reporter containing four copies of an NF-κB binding site. As shown in Figure 5D, overexpression of AP08 produced significant activation of NF-κB in 293T cells, while C-terminal deletion mutants APOδΔCP and APOδΔDD and the AP06-L334N point mutant did not activate NF-κB. Similarly, overexpression of APOδRP activated NF-κB as compared to control expression vector (see Figure 5D) . These results show that APOδ activates NF-κB expression and that the APOδ death domain is required for this activation. In addition, these results show that APOδRP, in contrast to the DR4 receptor described in Pan et al., supra , 1997, also activates NF-κB expression.

NF-κB activation experiments were performed as follows using a NF-κB luciferase reporter with four NF-κB binding sites and a minimal thymidine kinase promoter (Berberich et al., J. Immunol. 153:4357-4366 (1994), which is incorporated herein by reference) . 293T cells were cotransfected in duplicate as described above with 0.5 μg of Myc-tagged or 6XHIS-tagged receptor expression construct, 0.5 μg NF-κB reporter construct and 0.2 μg lacZ expression construct. After eighteen hours, cells in one well were fixed and stained with X-GAL to determine the relative transfection efficiency. Cell extracts were prepared from the duplicate well and analyzed for luciferase activity using the Luciferase Assay system (Promega, Madison, WI) following the manufacturer's instructions. Shown in Figure 5D is the mean +/- standard deviation calculated from three independent representative experiments.

Dominant negative TRADD can block NF-κB activation by AP08, AP08RP, and DR3

As described above, the death domain of AP08 is required for APOδ-mediated apoptosis and for NF- B activation. TNFR-1 and DR3 mediated apoptosis and NF-κB activation also are mediated through the TNFR-1 and DR3 death domains, and may be a result of death domain recruitment of TRADD (Kitson et al., Nature 384:372-375

(1996); Tartaglia et al., Cell 74:845-653 (1993), each of which is incorporated herein by reference) . TRADD can activate the apoptotic pathway through FADD and can activate the NF- B pathway through RIP and TRAF2 (Hsu et al., Cell 84:299-30δ (1996); Liu et al., Cell 87:565-576, each of which is incorporated herein by reference) .

A TRADD mutant deleted for the N-terminal 102 residues (ND-TRADD) lacks a complete TRAF2-binding domain but has a complete RIP/FADD-binding death domain (Liu et al., supra , 1996). This TRADD mutant may function as a dominant negative by competing with endogenous full-length TRADD for binding to receptor death domains but failing to effectively recruit TRAF2. The ability of dominant negative TRADD (ND-TRADD) to block NF-κB activation by AP08, AP08RP and DR3 was tested in 293T cells . Briefly, 293T cells were cotransfected in duplicate with an appropriate receptor and ND-TRADD along with NF-κB (0.5 μg) and lacZ (0.2 μg) reporter constructs. The receptor-to-ND-TRADD ratio was 1:5 (0.15 μg to 0.75 μg) . The total amount of plasmid was kept constant by adding vector DNA without insert. Eighteen hours post transfection, luciferase activity was measured from one of the duplicate wells using the luciferase assay reagent (Promega) and following the manufacturer's instructions. The cells in the other well were fixed with glutaraldehyde and stained with X-gal to obtain the relative transfection efficiency. Results (mean +/- SD) are from at least four independent experiments.

The results showed that ND-TRADD effectively blocked NF-κB activation by all three death domain receptors. Weak NF-κB activation was observed with ND-TRADD transfected alone in 293T cells, possibly as a result of the ability of ND-TRADD to recruit RIP and, indirectly, TRAF2 through death domain-death domain interactions (Liu et al., supra , 1996). In sum, these results indicate that AP08 and AP08RP resemble DR3 and TNFR-1 in activating NF-κB through a TRADD-dependent pathway.

APOS and AP08RP interact directly with TRADD and RIP but not with FADD

Coprecipitation experiments were conducted to assay whether AP08 or AP08RP interacts directly with the FADD, TRADD or RIP adaptor molecules. Under conditions which supported the interaction of His epitope tagged FAS/APOl with FADD, both His tagged AP08 and AP08RP failed to coprecipitate AUl-tagged FADD (AU1-FADD) . In contrast, HA-tagged ND-TRADD or RIP were coprecipitated with AP08 or AP08RP. Furthermore, both AP08 and AP08RP co-immunoprecipitated FADD in the presence of ND-TRADD. Thus, these results show that both AP08 and AP08RP, like DR3 and TNFR-1, directly bind TRADD and RIP and indirectly bind FADD through TRADD. The results with AP08RP are contrary to the results reported in Pan et al., supra , 1997, with the DR4 receptor. In sum, FADD appears to be the common mediator of apoptosis by all known death domain-containing receptors.

Coimmunoprecipitation experiments between receptors and various adaptor molecules were performed as follows. 293T cells (2 x 10⁶) were plated in a 10 mm plate and cotransfected the following day with 5 μg of various epitope tagged receptor plasmids or control vector lacking receptor sequence; 5 μg of various adaptor plasmids; 2 μg of Crm-A plasmid; and 0.5 μg of a Green Fluorescent Protein (GFP) encoding plasmid (pEGFP-Nl, Clontech) by calcium phosphate coprecipitation. The following day, cells were examined under a fluorescent microscope to ensure equal transfection efficiency as determined by the expression of the GFP. Twenty-four to thirty-six hours post-transfection, cells were lysed in 1 ml of lysis buffer containing 1% Triton X-100, 20 mM sodium phosphate (pH 7.4), 150 mM NaCl and 1 EDTA-free protease inhibitor tablet per 10 ml (Boehringer Mannheim, Indianapolis, IN) , and the cell lysate cleared by centrifugation.

For immunoprecipitation, pre-cleared cells lysate (600 μl ) was incubated with 2 μg of anti-Myc monoclonal 9E10 (American Type Culture Collection, Rockville, MD) for 2 hr at 4°C. The lysate was subsequently incubated for 1 hour with 10 l of Protein A/G ultralink beads (Pierce, Rockford, IL) pre-coated with 1% BSA to reduce nonspecific binding. Beads were washed twice with lysis buffer, followed by two washes in buffer containing 1% Triton X-100, 20 mM sodium phosphate (pH 7.4) and 500 mM NaCl and once with a buffer containing 20 mM Tris-HCl (pH 7.4). Bound proteins were eluted by adding 70 μl of 0.1 M glycine (pH 3.5) and subsequently neutralized with lOμl of 1 M Tris-HCl (pH 8.0). Samples were separated by SDS-PAGE, transferred to a nitrocellulose membrane and analyzed by Western blot analysis. For immunoprecipitation of MRG-δXHis tagged receptors, a cocktail of three monoclonal antibodies (MRG-4XHis, Penta-His and Tetra-His; Qiagen) was used. For precipitation using Nickel-sepharose, cell lysates were incubated for 1 hour with 20μl of Ni-sepharose beads pre-coated with 1% BSA in a buffer containing 1 % Triton X-100, 20 mM sodium phosphate (pH 7.4), 500 mM NaCl and EDTA-free protease inhibitor cocktail. Beads were washed extensively with the above buffer supplemented with 20 mM imidazole, and the bound protein subsequently eluted and analyzed by SDS-PAGE and Western blot analysis as described above.

NF-κB expression protects against APOβ-mediated apoptosis

293T cells were transfected with AP08 expression vector in the presence or absence of an NF-κB expression vector. Thirty-six hours later, cells were fixed and stained and morphology observed. Apoptotic morphology was observed in about 55% of APOδ-transfected cells in the absence of NF-κB, while only about 30% of APOδ-transfected cells were apoptotic when co-transfected with NF- B. These results indicate that NF-κB protects against APOδ-mediated cell killing. Cell transfections were performed as described above using 1 x 10⁵ 293T cells and 150 ng AP08 expression vector, 750 ng NF-κB expression vector or control empty vector, and 200 ng lacZ encoding plasmid in 24 well plates. After thirty-six hours, cells were fixed and stained and the percent apoptotic cells determined as described above.

Myc-APOβRP is expressed on the cell surface and binds TRAIL ligand

The cytotoxic ligand TRAIL has been shown to bind the DR4 receptor based on the ability of a soluble DR4 construct to coimmunoprecipitate TRAIL and to block TRAIL-induced apoptosis in MCF-7 cells. Direct binding of APOδRP to TRAIL was assayed by preparing a stable BHK cell line expressing Myc-epitope tagged APOδRP.

Cell-surface expression of APOδRP was confirmed by immunofluorescence staining with an anti-Myc antibody using FACS analysis. Subsequently, the ability of FLAG-epitope tagged TRAIL (FLAG-TRAIL) to bind the AP08RP transformant was tested using immunofluorescence staining with an anti-FLAG antibody. The results demonstrated that FLAG-TRAIL binds Myc-AP08RP. These results demonstrate that AP08RP, like DR4 , binds the TRAIL ligand.

Epitope tagged TRAIL protein was prepared as follows. A PCR fragment encoding amino acids 96 to 281 of TRAIL was cloned into a modified pET28 (b) vector (Novagen Madison, WI) , which has a FLAG epitope downstream of the His epitope, and the sequence confirmed by automated fluorescent sequencing. The His-FLAG-TRAIL protein was purified by Nickel-affinity chromatography according to the manufacturer's instructions. Stable cell lines were generated by cotransfecting 2 x 10⁵ BHK cells with 1.5 μg of a Myc-epitope tagged AP08RP expression vector or control vector lacking the receptor coding sequence and 300 ng of an expression vector encoding human DHFR gene using Superfect (Qiagen) according to the manufacture's instructions. Stable transformants were selected in 1 μM methotrexate (Sigma, St. Louis, MO) in DMEM supplemented with 5% dialyzed fetal calf serum. After 10 to 12 days colonies were pooled to generate a mass culture.

Expression of the Myc-AP08RP construct was analyzed by FACS after indirect immunofluorescence labeling using the anti-Myc epitope antibody mAb 9E10 obtained from American Type Culture Collection or a control monoclonal antibody, followed by R-phycoerythrin (R-PE) labeled goat anti-mouse antibody (Fisher) . Binding of FLAG-TRAIL (2 μg) to the Myc-APOδRP expressing stable cell lines was detected similarly using the anti-FLAG M2 antibody (IBI Kodak, New Haven, CT) as the primary antibody and R-PE-labeled goat anti-mouse antibody as the secondary antibody.

EXAMPLE III Identification and Characterization of APQ9

This example describes the identification and characterization of alternatively spliced forms of AP09.

AP09 is a human cDNA related to IMAGE consortium clones 470799, 504745, 110226 and 129137, which are clones with homology to human Fas antigen. The partial amino acid sequence of hAP09S encodes a signal peptide at the 5' end and a hydrophobic transmembrane region at the 3' end as shown in Figure 6. In addition, the hAP09S sequence has a stop codon immediately after the transmembrane region. Thus, the hAP09S cDNA can represent an alternatively spliced non-functional receptor isoform that competes with the full-length receptor for ligand binding.

In its extracellular domain, the hAP09S amino acid sequence demonstrates significant homology to the extracellular domains of several members of the TNFR family. The greatest degree of homology is seen with the DR4 receptor, with which it shares 57% amino acid sequence identity and 71% amino acid sequence similarity (see Figure 1 and Table 1) . Based on this homology, the AP09S cDNA is classified as a new member of the TNFR family.

EXAMPLE IV Identification and Characterization of APQ4 polypeptides

This example describes the identification and characterization of nucleic acid sequences encoding the short and long isoforms of murine, rat and human AP04-α and murine AP04-β. In addition, the ability of hAP04 to induce cell death through activation of the JNK pathway and the ability of hAP04 to activate NF- B expression is demonstrated.

Characterization of marine AP04-a and mAP04-γ

mAP04 are murine cDNAs related to IMAGE consortium EST clones 472300 and 427152, which are mouse embryo-derived sequences with homology to the human DR3 receptor. These clones were identified by searching the EST database (dbEST) for homology to the extracellular domain of human DR3 using the TBLASTN algorithm. The mAP04-αS cDNA is represented by IMAGE consortium EST clone 472300 and has an open reading frame of 214 amino acids (Figure 7B) , including an amino-terminal signal-peptide; cysteine-rich pseudo- repeats with significant homology to other members of

TNFR family (20-24% sequence identity and 35-46% sequence similarity) ; a highly hydrophobic stretch of amino-acids representing the transmembrane region; and a short cytoplasmic tail.

The mAP04-γ cDNA is represented by the IMAGE consortium EST clone 427152 and has an open leading frame of 150 amino acids. The amino acid sequence of mAP04-γ is identical to the mAP04-α form with the N-terminal 149 amino acids containing the signal peptide and the cysteine-rich pseudo repeats representing the majority of the ligand binding domain. However, mAP04-γ has a stop codon after amino acid 150 and thus represents a soluble receptor lacking a transmembrane domain.

No polyadenylation signal was observed at the 3' end of mAP04-αS, indicating that the stop codon present in the 3' end can be the result of alternative splicing. 3' Rapid Amplification of cDNA Ends (RACE) was used on murine spleen marathon-ready cDNA to isolate the full length cDNA. The full length cDNA has an open reading frame of 416 amino acids and a unique cytoplasmic tail with no significant homology to other members of TNF receptor family (Figure 7A) . Thus, mAP04-α is a type I membrane protein, having short and long forms. The long mAP04-α form can have an important role in the regulation of the immune system, while the short form of the receptor is a decoy receptor which can compete with the long (full-length) form for ligand binding, thereby modulating signal-transduction through the full-length receptor.

The full-length mAP04-α (mAP04-αL) sequence was obtained using 3' RACE and murine spleen Marathon-ready cDNA (Clontech) according to the manufacturer's instructions. Primer SEQ ID NO: 45

(5'-CCCACCTCCTCCCTACGAACCACACTG-3' ) was used with API primer SEQ ID NO: 34 in a first round of PCR. Primer SEQ ID NO: 46 ' -GGCTGCTCGCCCTGCTCATCCTGTGTGT-3 ' ) and AP2 primer SEQ ID NO: 36 were used in a subsequent round of nested PCR. RACE fragments were cloned into the PCR2.1 vector using the Original TA Cloning kit (Invitrogen). Inserts from eight or more individual clones were PCR amplified using flanking vector primers Vn26 (SEQ ID NO: 39; 5 ' -TTTCCCAGTCACGACGTTGTA-3 ' ) and Vn27 (SEQ ID NO:40), and the amplified fragments purified using the Wizard PCR Preps kit (Promega) . The inserts were sequenced on an ABI 373 automated fluorescent sequencer with dye terminators using the purified PCR products or plasmid DNA as template with the Vn26 (SEQ ID NO: 39) and Vn27 (SEQ ID NO: 40) primers as sequencing primers.

mAP04-σL is a cell surface receptor

To demonstrate that mAP04-αL is a cell surface receptor, Myc-AUl tagged mAP04-αL was expressed in BHK cells. Cell surface expression was confirmed by immunofluorescence labeling with the Myc monoclonal antibody 9E10 (American Type Culture Collection) . These results show that mAP04-αL is a cell surface receptor.

The Myc-AUl tagged mAP04-αL construct was prepared by PCR amplifying the protein coding region of the mature AP04-αL polypeptide with a 5' primer containing a BamHI site and encoding the AU1 tag and a 3' primer containing a Xhol site using pfu polymerase (Stratagene). The 5' primer was SEQ ID NO:47 ( 5 ' -GGATCCAGACACATACCGCTACATTGCATGTAAAGTGAGTTGCGAAACC-3 ' , and the 3' primer was SEQ ID NO:4δ (5'-GAAGCTCGAGGCTGAAAGTATGGAAGTGCTG-3' ) . The resulting amplified product was digested with BamHI and Xhol and ligated to a BamHI/XhoI digested pSecTag A vector modified to include a Myc tag as described above.

Characterization of human AP04-α

Human AP04-α (hAP04-α) is encoded by a cDNA sequence related to IMAGE consortium clone 340844, which was identified by searching the EST database (dbEST) for sequence homology to the cytoplasmic tail of full-length mAP04-α protein using the TBLASTN algorithm. The full-length nucleotide sequence encoding hAP04-α is shown in Figure 7A. This hAP04-α nucleotide sequence (Figure 7C) encodes a hAP04-α polypeptide of 423 amino acids having 68.4% amino acid identity and 79.2% amino acid sequence similarity with mAP04-α.

The full-length hAP04-α sequence was obtained using 5' RACE and human fetal spleen Marathon-ready cDNA

(Clontech) according to the manufacturer's instructions.

Primer SEQ ID NO: 49 (5 ' -TAGCTGGCTTCTCATAGTTAGTGC-3 ' ) and

API primer SEQ ID NO: 34 were used for the first round of PCR. Primer SEQ ID NO: 50

(5'-CCTGAGTTGATGCTGATTCTACCA-3' ) and AP2 primer SEQ ID NO: 36 were used for a subsequent round of nested PCR. Inserts from eight or more individual clones were PCR amplified and sequenced essentially as described above. Characterization of rat AP04-&

Rat AP04-α (mAP04-α) is another AP04 polypeptide; the rAP04-α cDNA is related to IMAGE consortium clone 319945 which was identified by searching the EST database (dbEST) for sequences with homology to the cytoplasmic tail of mAP04-α protein using the TBLASTN algorithm. The partial cDNA sequence, shown in Figure 7D, lacks the 5' region. The partial rAP04-α nucleotide sequence shares 95% amino acid sequence identity and about 61% nucleotide sequence identity with the cytoplasmic domain of mAP04-α.

Characterization of murine APO4-

Murine AP04-β (mAP04-β) is another AP04 polypeptide; the mAP04-β cDNA is related to IMAGE consortium clone 635416, derived from a Knowles Solter mouse blastocyst library. The IMAGE consortium clone was identified as described above. The partial cDNA sequence, shown in Figure 8, represents the carboxy-terminal 54 residues of the polypeptide. The encoded polypeptide fragment bears about 77% amino acid sequence identity and about 81% amino acid similarity with mAP04-α. The mAP04-β fragment also bears about 74% amino acid identity and about 80% amino acid similarity with rAP04-α. AP04 Expression in Adult and Embryonic Tissues

Expression of AP04 in adult human tissues was studied by northern analysis using a multiple tissue northern blot obtained from Clontech (7759-1) following the manufacturer's instructions. The protein coding region of hAP04 cDNA was radiolabeled with P³² and used as a probe. High level AP04 expression was seen in the prostate gland, with very low expression seen in other tissues including spleen, thymus, testis, uterus, small intestine, colon and peripheral blood leukocytes.

The prostate gland consists primarily of fibroblast-like stromal cells and glandular epithelial cells, with the latter being the site of origin of prostatic carcinoma. Reverse-transcriptase/polymerase chain reaction (RT-PCR) was used to test the expression of hAP04 in a prostate carcinoma derived epithelial cell line, LNCaP (ATCC, Bethesda, MD) . Total RNA isolated from LNCaP cells was used for the first strand cDNA preparation using random hexamer primers and Superscript II Reverse Transcriptase (Life Technologies) . The RT-PCR procedure was performed following the manufacturer's instruction with forward primer SEQ ID NO: 57 (5'-GTCATGTAAAGTGACTTGTG-3' ) and reverse primer SEQ ID NO: 58 (5'-GAGCCGTTGTACTGAATGTCCTG-3' ) . A specific band corresponding to hAP04 was detected in RNA from LNCaP cells, indicating that AP04 is expressed in epithelial derived prostate cancer cell lines. Essentially similar results were obtained in another prostate cancer cell line, P65.

AP04 expression during embryonic development was assayed by northern analysis of a multiple tissue northern blot obtained from Clontech (7763-1) , which contained polyA RNA from day 7, 11, 15 and 17 mouse embryos. The protein coding region of mAP04 cDNA was radiolabeled with P³² and used as a probe. A strong signal was detected in samples containing RNA from day 11, 15 and 17 embryos, indicating that AP04 is expressed during early embryonic development.

hAP04 induces apoptosis

The ability of hAP04 to induce apoptosis was assayed following transient transfection into the human embryonic kidney 293T cell line. Plasmids encoding various inhibitors were cotransfected to test for their ability to modulate hAP04 mediated apoptosis in 293T cells .

A mammalian expression construct encoding human

AP04 was constructed by amplifying the sequence encoding amino acids 23 to 423 of hAP04 with a 5' primer containing a Bgl II site and a 3' primer containing a stop codon followed by a Sal I site, as described previously for the AP08 construct. Following digestion of the PCR product with Bgl II and Sal I, the resulting product was inserted into a modified pSecTag A vector as described previously.

Expression of the AP04 polypeptide described above was confirmed by transfecting the above construct into 293T cells using calcium phosphate transfection. After 24 hours, cell lysates were prepared, the proteins resolved by SDS-PAGE, and MYC-tagged hAP04 detected by western blot analysis using rabbit polyclonal antibody against the MYC epitope tag (Santa Cruz Biotechnology, Inc., Santa Cruz, California) following the manufacturer's instructions. A single band of approximately 55 kDa was detected in cells transfected with the MYC-AP04 construct.

Constructs encoding various inhibitor plasmids were prepared as follows. A K13-0RF (GenBank U90534) was prepared by PCR amplification of the desired coding sequence from a human genomic DNA sample containing KSHV/HHV8 (human herpesvirus 8) genomic DNA (obtained from Dr. Tim Rose; University of Washington, Seattle, WA) . Primers for PCR amplification were based on the published sequence of K13-ORF with additional 5' sequence for subsequent restriction digestion and cloning of the amplified insert. Constructs encoding p35 were prepared by using a baculovirus vector pFastBac HTa (Life Technologies, Inc.) as the template. DN-IκBα or IκBα-ΔN (missing the N-terminal 36 amino acids) and IκBα-S32/36A (Brockman et al., Mol. Cell. Biol.. 15: 2809-18, 1995); NFKB driven luciferase reporter construct (Berberch et al., J. Immunol . , 153:4357-66, 1994); an active site mutant of full-length rat MEKK1 (MEKK1-D1369A) (Xu et al., Proc. Natl. Acad. Sci. USA, 93:5291-5295) and the JNK binding domain of JIP-1 have been described previously (Dickens et al., Science 277:693-696, 1997). DN-mTRAF2, which encodes a TRAF2 polypeptide lacking the first 87 amino acids, was generated by amplifying the DNA encoding amino acids 86 to 501 of mTRAF2 using the IMAGE consortium EST clone 439083 as a template and incorporating a start site (i.e. methionine residue) at the N-terminus.

Cell transfection was performed using calcium phosphate coprecipitation (Sambrook et al., supra , 1989). Briefly, 293T cells (1.2 x 10⁵ cells) were cotransfected with 100 ng/well hAP04 expression plasmid or control plasmid along with lacZ reporter plasmid and 750 ng/well empty vector or one of various inhibitor plasmids (p35, Caspase 8 C360S, dominant-negative FADD (DN-FADD), MRITαl, K13-ORF, DN-IκBα, JNK binding domain (JBD) of JIP-1, BCLxL or the p65 subunit of NF-κB) and lacZ reporter plasmid and were assayed for hAP04-induced apoptosis. Thirty-six hours after transfection, cells were fixed and stained with X-gal, and the percentage apoptotic cells determined as described previously.

The results shown in Figure 14A indicate that hAP04 induces cell death and that the hAP04-induced apoptosis is activated independently of the FADD-Caspase pathway. In particular, several inhibitors of the FADD-Caspase pathway (caspase 8 C360S, DN-FADD, p35, K13-ORF and MRITαl), when cotransfected with hAP04, failed to block AP04-mediated cell death. These results demonstrate that, unlike a variety of receptors of the TNF family, AP04 can mediate apoptosis independently of the FADD-caspase pathway.

hAPθ4 activates the Jun N-teizninal kinase (JNK) pathway

Activation of the JNK pathway by hAP04 was measured using the PathDetect c-Jun Trans-Reporting system (Stratagene, La Jolla, CA) . Briefly, 1.2 x 10⁵ 293T EBNA cells were cotransfected in duplicate with 250 ng/well of hAP04 expression vector and 750 ng/well of empty vector or inhibitor plasmid (JBD of JIP-1, MEKK1-D1269A, or DN-TRAF2) . A fusion-transactivator plasmid containing the GAL4 DNA-binding domain fused to the c-Jun transcription factor (pFA-cJun, 50 ng) , a reporter plasmid encoding the luciferase gene downstream of the GAL4 Upstream Activating sequence (pFR-luc, 500 ng) and a β-galactosidase (pRSV/LacZ) reporter construct (75 ng) were also included in the cotransfection. Forty hours after transfection, cell extracts were prepared and luciferase and β-galactosidase activities measured. The luciferase activity was normalized relative to the β-galactosidase activity to control for differences in transfection efficiency.

As shown in Figure 14B, expression of hAP04 produced significant activation of c-jun. Furthermore, cotransfection of hAP04 with JBD-JIP, and to a lesser extent with MEKK1-D1369A or DN-TRAF2, prevented AP04-mediated activation of the JNK pathway. These results indicate that AP04-indcued apoptosis can be mediated through activation of the JNK pathway and MEKK1.

hAP04 induces NF-κB

To test the possibility that AP04 can regulate NF-κB expression, hAP04 expression constructs were cotransfected into 293T cells with a NF- B luciferase reporter. Transfection and NF- B activation experiments were performed as described previously. Inhibitor plasmids (DN-TRAF2 or IκBα-S32/36A) were also cotransfected to determine whether inhibition of the TRAF2 or JNK pathway affects NF- B activation by hAP04. As shown in Figure 14C, hAP04 induced NF-κB expression, and this activation was not blocked by dominant-negative TRAF2. Also as shown in Figure 14C, hAP04 failed to activate NF-κB in the presence of IκB-S32/36A, an IκB mutant that resists degradation in response to activators of NF-κB. These results demonstrate that AP04 can function, at least in part, through activation of NF-κB. Given the multiple roles of NF-κB and the high level of AP04 expression in embryonic development, these results indicate that AP04 can stimulate growth in certain contexts, in addition to its pro-apoptotic role described above .

Deletion analysis of AP04

To determine the domains of AP04 which are responsible for JNK activation and apoptosis, deletion analysis was carried out using the following constructs: pSecTag-MYC-AP04 encoding amino acid 23 to 423; pCDNA3.1 HisA-AP04.CP encoding the cytoplasmic domain of AP04 (amino acids 194 to 423) ; pSecTag-MYC-AP04-C377 encoding amino acids 23 to 377; pSecTag-MYC-AP04-C355 encoding amino acids 23 to 355; pSecTag-MYC-AP04-C286 encoding amino acids 23 to 266; pSecTag-MYC-AP04-C172 encoding the extracellular domain (amino acids 23 to 172).

These constructs were prepared by PCR amplification of the nucleic acid fragment encoding the designated amino acids using custom primers with appropriate restriction enzyme containing sites and by using human AP04 cDNA as template. The desired PCR fragment was subsequently cloned into the modified pSecTag vector described previously or the pCDNA3.1 HisA vector (Invitrogen).

The constructs were subsequently tested for their ability to activate the JNK pathway in 293EBNA cells as described above. The results indicated that, while the full-length AP04 and its cytoplasmic domain were able to activate the JNK pthway, the construct encoding the extracellular domain failed to do so. Furthermore, the pSecTag MYC-AP04-C266 failed to significantly activate the JNK pathway although the pSecTag MYC-AP04-C377 and C355 constructs could do so. could.

The above constructs were also tested for their ability to induce cell death in the 293T cells using the assay decribed before. While the full-length AP04 polypeptide, its cytoplasmic domain, C377, and C355 constructs were able to effectively induce cell death, the C2δ6 and the C172 constructs were inactive. These results indicate that the C-terminal 68 amino acids are not essential for the AP04-induced activation of the JNK pathway or apoptosis and that these activities reside among the amino acids 194 to 355.

EXAMPLE V Identification and Characterization of APOδ

This example describes the identification and characterization of a nucleic acid sequence encoding human APOδ.

Human AP06 (hAPOδ) is a cDNA related to IMAGE consortium clones 366305, 592256, 343204 and 591875, which have some similarity with the extracellular domain of human TNFR-2. These clones were identified by searching the EST database (dbEST) for sequences with homology to the extracellular domain of human TNFR-2 protein using the TBLASTN algorithm. The partial nucleotide sequence, lacking both 5' and 3' sequence, is shown in Figure 9. The partial hAPOδ sequence shows significant homology to the extracellular domains of TNFR-2, TNFR-1, and other members of the TNFR family (about 16-33% amino acid sequence identity and about 30- 50% amino acid sequence similarity) (Figure 1 and Table 1) . Based on this homology, hAP06 is classified as a new member of the TNFR family.

EXAMPLE VI Identification and Characterization of Tumor Necrosis Factor Related Ligands

This example describes the identification and characterization of nucleic acid sequences encoding human and murine TNRLl-α and TNRLl-β. This example also demonstrates that human TNRLl-α is a cytotoxic ligand.

Characterization of human TNRLl-α

Human tumor necrosis factor related ligand 1-α (hTNRLl-α) is encoded by a cDNA clone related to IMAGE consortium clones 593690, 115371 and 129696, which are clones with homology to human TNF-α. The full length cDNA encodes a predicted protein of 285 amino acids with significant sequence homology to TNF-α as well as other members of the TNF family (Figures 10 and 11A) .

The 5' end of the human TNRL-α sequence was obtained using 5' RACE on Marathon Ready cDNA derived from human leukocytes (Clontech) as described previously. The first round of RACE was performed using API primer SEQ ID NO: 34 as the forward primer and SEQ ID NO: 59 (5'- GTTGGTGTTTCACTGTCTGCAATCAG-3' ) as the reverse primer. The race products were diluted 1:50 in water and used for a second round of RACE using forward primer SEQ ID NO: 36 and SEQ ID NO: 60 (5 ' -GTTCTGACTGGAGTTGCCTTCTCCTG-3 ' ) reverse primer. As in other members of the TNRL family, the 5' sequence encodes a membrane-anchoring domain. This factor, hTNRLl-α, is designated a new member of the TNF family which can play an important role in the modulation of the immune and nervous systems.

Human TNRLl-a is a cytotoxic ligand

His-FLAG epitope-tagged extracellular portions of hTNRLl-α, hTNRL3 or cytotoxic TRAIL ligand were produced in bacteria. Western analysis of bacterial lysates with an anti-FLAG antibody demonstrated that hTNRLl-α, hTNRL3 and TRAIL extracellular polypeptide portions were produced. BJAB cells were treated with ligand, and cell survival measured after twenty-four hours using the MTT assay in wells pre-coated with anti-FLAG monoclonal antibody. The relative survival of BJAB cells treated with hTNRLl-α, hTNRL3 or TRAIL was significantly reduced as compared to control cells. These results indicate that hTNRLl-α, like TRAIL, is a cytotoxic ligand.

Epitope tagged hTNRLl-α, hTNRL3 and TRAIL polypeptide fragments were prepared in a modified pET28 (b) vector (Novagen Madison, WI), which has a FLAG epitope downstream of the His epitope, and the sequences confirmed by automated fluorescent sequencing. The His-FLAG-tagged polypeptide fragments were expressed in bacteria and purified by nickel-affinity chromatography (Qiagen) according to the manufacturer's instructions.

For cell survival assays, a 96-well plate was pre-treated by incubation with 100 μl/well 10 μg/μl anti-FLAG monoclonal antibody for two hours at 37°C. BJAB cells were plated at 2 x 10⁴ cells/well and treated with 5 μl purified ligand. Cell survival was measured after 24 hours using the MTT assay (SIGMA) according to the manufacturer's instructions. Characterization of murine TNRLl-α

Murine tumor necrosis factor related ligand 1-α (mTNRLl-α) is encoded by a cDNA related to IMAGE consortium clones 722549, 803594 and 791670. These clones were identified by searching the dbEST database (NCBI) for sequences with homology to hTNRLl-α polypeptide (SEQ ID NO: 20) using the TBLASTN algorithm. Murine TNRLl-α sequence was obtained using 5' RACE on murine spleen Marathon-ready cDNA (Clontech) according to the manufacturer's instructions. Primer SEQ ID NO: 51 (5'-GTCTCCGTTGCGTGAAATCTGTGC-3' ) and API primer SEQ ID NO: 34 were used for the first round of PCR. Primer SEQ ID NO:52 ( 5 * -AGGAATTGTTGGGCAGTGTTTTGG-3 ' ) and AP2 primer SEQ ID NO: 36 were used for a subsequent round of nested PCR. The RACE products were cloned into vector PCR2.1 using the TA cloning kit (Invitrogen) and sequenced on an automated fluorescent sequencer (ABI 373) essentially as described before.

The available mTNRLl-α sequence is shown in Figure 11B. The predicted mTNRLl-α sequence is highly homologous to hTNRLl-α at both the nucleotide and protein levels and indicates a type II membrane protein with a putative N-terminal cytoplasmic domain (amino acids 1-46, a transmembrane domain (amino acids 47-74) and a large extracellular receptor binding domain (amino acids 75- 290) . Characterization of human TNRLl-β

Human tumor necrosis factor related ligand 1-β (hTNRLl-β) is encoded by a cDNA related to IMAGE consortium clones 306307, 489038 and 306064, identified as described above, and which exhibit homology to

TNRLl-α. The hTNRLl-β sequence, shown in Figure 12A, encodes a predicted polypeptide of 250 amino acids with sequence homology to TNRLl-α and TNF-α as well as other members of TNF family (see Figure 10) . The sequence indicates a type II membrane protein with a putative N- terminal cytoplasmic domain (amino acids 1-25) , a transmembrane hydrophobic domain (26-50) and a large extracellular receptor binding domain (amino acids 51- 250) .

Characterization of murine TNRLl-β

Murine tumor necrosis factor related ligand 1-β (mTNRLl-β) is encoded by a cDNA related to IMAGE consortium clone 876879. This clone was identified by searching the dbEST database (NCBI) for sequences with homology to hTNRL-β polypeptide (SEQ ID NO:24) using the TBLASTN algorithm. The full-length mTNRLl-β cDNA sequence is shown in Figure 12B. The sequence indicates a type II membrane protein with a putative N-terminal cytoplasmic domain (amino acids 1-16) , a transmembrane hydrophobic domain (amino acids 17-42) and a large extracellular receptor binding domain (amino acids 43-241) . EXAMPLE VII Identification and Characterization of TNRL3

This example describes the identification and characterization of nucleic acid sequences encoding human and murine TNRL3. This example also demonstrates that human TNRL3 is a cytotoxic ligand.

Characterization of human TNRL3

Human tumor necrosis factor related ligand 3 (hTNRL3) is related to IMAGE consortium clones 154742 and 271670 and Genbank clone C00994. These sequences were identified by searching the dbEST database (NCBI) for sequences with homology to human TRAIL ligand protein using the TBLASTN olgorithym. 5' RACE with human fetal spleen marathon ready cDNA (Clontech) was performed according to the manufacturer's instructions to obtain additional 5' sequence. Primer SEQ ID NO: 53 (5'-CGAAGTAGGTGAGGAAGGGGGCAG-3' ) and API primer SEQ ID NO: 34 were used for the first round of PCR. Primer SEQ ID NO:54 (5 ' -GTAGACAGCCTTCCCCTCATCAAA-3 ' ) and AP2 primer SEQ ID NO: 36 were used for a subsequent round of nested PCR.

The partial hTNRL3 sequence shown in Figure 13A lacks sequence at the 5 ' end of the molecule and encodes the ligand-binding extracellular domain. The missing 5' sequence can encode a membrane anchoring sequence. The partial sequence shown in Figure 13A exhibits homology to TRAIL (TNF Related Apoptosis Inducing Ligand) and other members of the TNF family (see Figure 10) . Human TNRL3 is a cytotoxic ligand

BJAB cells were treated with epitope-tagged extracellular portions of hTNRLl-α, hTNRL3 or TRAIL, and cell survival measured after twenty-four hours using the MTT assay as described above. The relative survival of BJAB cells treated with epitope-tagged hTNRLl-α, hTNRL3 or TRAIL extracellular fragments was significantly reduced as compared to control cells. These results indicate that hTNRL3 functions as a cytotoxic ligand.

Characterization of murine TNRL3

Murine tumor necrosis factor related ligand 3 (TNRL3) is encoded by a cDNA related to IMAGE consortium clone 696209. This clone was identified by searching the dbEST database for sequences with homology to hTNRL3 protein (SEQ ID NO: 28) using the TBLASTN algorithm. The available sequence of clone 696209 is incomplete at the 5' end. Additional murine TNRL3 sequence was obtained using 5' RACE on murine spleen Marathon-ready cDNA (Clontech) according to the manufacturer's instructions. Primer SEQ ID NO:55 (5 ' -CACACCGTTCACCAGCAAGTCCAG-3 ' ) and API primer SEQ ID NO: 34 were used for the first round of PCR. Primer SEQ ID NO: 56

(5'-GGTAGACAGCCTTTCCCTCATCAA-3' ) and AP2 primer SEQ ID NO: 36 were used for a subsequent round of nested PCR. The RACE products were cloned into vector PCR2.1 using the TA cloning kit (Invitrogen) and sequenced on an automated fluorescent sequencer (ABI 373) .

The partial mTNRL3 sequence, lacking the 5' end, is shown in Figure 13B. The mTNRL3 cDNA encodes a polypeptide with significant sequence homology to hTNRL3. All journal article, reference, and patent citations provided above, in parentheses or otherwise, whether previously stated or not, are incorporated herein by reference.

Although the invention has been described with reference to the disclosed embodiments, those skilled in the art will readily appreciate that the specific experiments detailed are only illustrative of the invention. It should be understood that various modifications can be made without departing from the spirit of the invention. Accordingly, the invention is limited only by the following claims.

Claims

What is claimed is:

1. An isolated AP04 polypeptide, comprising substantially the same amino acid sequence as an AP04 polypeptide, or an active fragment thereof.

2. The isolated polypeptide of claim 1, comprising substantially the same amino acid sequence as an amino acid sequence selected from the group consisting of mAP04-αL (SEQ ID NO: 8) or an active fragment thereof, mAP04-αS (SEQ ID NO: 10) or an active fragment thereof, hAP04-α (SEQ ID NO: 12) or an active fragment thereof, and rAP04-α (SEQ ID NO: 14) or an active fragment thereof.

3. The isolated polypeptide of claim 1, comprising substantially the same amino acid sequence as mAP04-β (SEQ ID NO: 16).

4. The isolated polypeptide of claim 1, wherein said active fragment comprises a soluble form of an AP04 polypeptide having an AP04 polypeptide ligand binding domain.

5. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding substantially the AP04 polypeptide of claim 1, or an active segment thereof.

6. An AP04 selective binding agent, said agent comprising the activity of selectively binding to the AP04 polypeptide of claim 1.

7. A method of diagnosing prostate cancer in an individual, comprising determining the level of AP04 in said individual.

8. A method of treating prostate cancer, comprising administering to an individual having prostate cancer a conjugate comprising an AP04 selective binding agent linked to a therapeutic moiety.

9. A method of identifying an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity, comprising the steps of:

a) contacting an AP04 polypeptide or active fragment thereof with an agent, and

b) determining selective binding of said agent to said AP04 polypeptide or active fragment thereof,

wherein said selective binding indicates that said agent is an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity.

10. The method of claim 9, wherein said AP04 polypeptide or active fragment thereof is an AP04 extracellular ligand binding domain.

11. The method of claim 9, wherein said AP04 polypeptide or active fragment thereof is expressed in a cell or on the surface of a cell.

12. The method of claim 9, wherein said AP04 polypeptide or active fragment thereof is contacted with an agent in vi tro .

13. A method of screening for an AP04 agonist useful in treating prostate cancer, comprising the steps of: a) contacting a cell expressing an AP04 polypeptide or active fragment thereof with an agent;

b) assaying for increased AP04 activity,

wherein increased AP04 activity indicates that said agent is an AP04 agonist useful in treating prostate cancer.

14. A method of screening for an AP04 antagonist, comprising the steps of:

a) contacting a cell expressing an AP04 polypeptide or active fragment thereof with an agent;

b) assaying for decreased AP04 activity,

wherein decreased AP04 activity indicates that said agent is an AP04 antagonist.

15. A method of identifying an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity, comprising the steps of:

a) forming a mixture comprising an AP04 polypeptide or active fragment thereof, an AP04 signal transducer molecule that specifically interacts with a cytoplasmic domain of said AP04 polypeptide or active fragment thereof, and an agent;

b) detecting the level of AP04 activity in the presence of said agent,

wherein an alteration in said AP04 activity relative to control activity indicates that said agent is an effective pharmacological agent useful in the diagnosis or treatment of a disease associated with AP04 activity.

16. A method of claim 15, wherein said AP04 activity is measured in vivo .

17. A method of claim 15, wherein said AP04 activity is measured in vi tro .

16. An isolated APOδ polypeptide, comprising substantially the same amino acid sequence as APOδ, or an active fragment thereof.

19. The isolated polypeptide of claim 18, comprising substantially the same amino acid sequence as hAPOδ (SEQ ID N0:2), or an active fragment thereof.

20. The isolated polypeptide of claim 18, wherein said active fragment comprises a soluble form of AP08 having an APOδ ligand binding domain.

21. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding substantially the APOδ polypeptide of claim 16, or an active segment thereof.

22. An AP08 selective binding agent, said agent comprising the activity of selectively binding to the AP08 polypeptide of claim 18.

23. An isolated AP09 polypeptide, comprising substantially the same amino acid sequence as AP09, or an active fragment thereof.

24. The isolated polypeptide of claim 23, comprising substantially the same amino acid sequence as hAP09 (SEQ ID NO: 6), or an active fragment thereof.

25. The isolated polypeptide of claim 23, wherein said active fragment comprises a soluble form of AP09 having an AP09 ligand binding domain.

26. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding substantially the AP09 polypeptide of claim 23, or an active segment thereof.

27. An AP09 selective binding agent, said agent comprising the activity of selectively binding to the AP09 polypeptide of claim 23.

28. An isolated APOδ polypeptide, comprising substantially the same amino acid sequence as APOδ, or an active fragment thereof.

29. The isolated AP06 polypeptide of claim 26, comprising substantially the same amino acid sequence as hAPOδ (SEQ ID NO: 18), or an active fragment thereof.

30. The isolated polypeptide of claim 28, wherein said active fragment comprises a soluble form of APOδ having an APOδ ligand binding domain.

31. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding substantially the APOδ polypeptide of claim 28, or an active segment thereof.

32. An AP06 selective binding agent, said agent comprising the activity of selectively binding to the APOδ polypeptide of claim 28.

33. An isolated tumor necrosis factor related ligand 1 (TNRLl) polypeptide, comprising substantially the same amino acid sequence as TNRLl, or an active fragment thereof.

34. The isolated TNRLl polypeptide of claim 33, comprising substantially the same amino acid sequence as an amino acid sequence selected from the group consisting of hTNRLl-α (SEQ ID NO:20), or an active fragment thereof, and mTNRLl-α (SEQ ID NO:22), or an active fragment thereof.

35. The isolated TNRLl polypeptide of claim 33, comprising substantially the same amino acid sequence as an amino acid sequence selected from the group consisting of hTNRLl-β (SEQ ID NO:24), or an active fragment thereof, and mTNRLl-β (SEQ ID NO:26), or an active fragment thereof.

36. The isolated polypeptide of claim 33, wherein said active fragment comprises a soluble form of TNRLl having a TNRLl receptor binding domain.

37. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding substantially the TNRLl polypeptide of claim 33, or an active segment thereof.

38. A TNRLl selective binding agent, said agent comprising the activity of selectively binding to the TNRLl polypeptide of claim 33.

39. An isolated TNRL3 polypeptide, comprising substantially the same amino acid sequence as TNRL3, or an active fragment thereof.

40. The isolated TNRL3 polypeptide of claim 39, comprising substantially the same amino acid sequence as an amino acid sequence selected from the group consisting of hTNRL3 (SEQ ID NO:28), or an active fragment thereof, and mTNRL3 (SEQ ID NO: 30), or an active fragment thereof.

41. The isolated polypeptide of claim 39, wherein said active fragment comprises a soluble form of TNRL3 having a TNRL3 receptor binding domain.

42. An isolated nucleic acid molecule, comprising a nucleotide sequence encoding substantially the TNRL3 polypeptide of claim 39, or an active segment thereof.

43. A TNRL3 selective binding agent, said agent comprising the activity of selectively binding to the TNRL3 polypeptide of claim 39.