APO-2LI AND APO-3 APOPTOSIS POLYPEPTIDES

Abstract: Novel polypeptides, designated Apo-3 and Apo-2LI, involved in apoptosis are provided. Compositions including chimeric molecules, nucleic acids, and antibodies are also provided.

Description: AP0-2LI AND APO-3 APOPTOSIS POLYPEPTIDES FIELD OF THE INVENTION The present invention relates generally to the identification, isolation, and recombinant production of novel polypeptides involved in mammalian cell apoptosis In particular, polypeptides designated herein as "Apo-3" and certain forms thereof designated herein as "Apo-2LI" are disclosed Methods of emploving the polypeptides of the invention are also disclosed BACKGROUND OF THE INVENTION Control of cell numbers in mammals is believed to be determined, in part, by a balance between cell proliferauon and cell death One form of cell death, sometimes referred to as necrotic cell death, is tΛpicallv characterized as a pathologic form of cell death resulting from some trauma or cellular injury In contrast, there is another, "phvsiologic" form of cell death which usually proceeds in an orderly or controlled manner This orderl y or controlled form of cell death is often referred to as "apoptosis" [see, e g , Barr et al . Bιo/Technolθ j ?v. 12 487-493 ( 1994)] Apoptotic cell death naturally occurs in many physiological processes, including embryonic development and clonal selection in the immune system fltoh et al , Cell. 66 233-243 ( 1991 )] Decreased levels of apoptotic cell death have been associated with a variety of pathological conditions, including cancer, lupus, and herpes virus infecuon [Thompson, cience. 267 1456- 1462 ( 1995)] Increased levels of apoptotic cell death be associated with a variety of other pathological conditions, including AIDS, Alzheimer s disease, Parkinson s disease, amyotrophic lateral sclerosis, multiple sclerosis, retinitis pigmentosa, cerebellar degeneration, aplastic anemia, myocardial lnfarcuon, stroke, reperfusion injury, and toxin-induced liver disease [see, Thompson, supra [ Apopto c cell death is typically accompanied bv one or more characteristic morphological and biochemical changes in cells, such as condensation of cytoplasm, loss of plasma membrane microvilli, segmentation of the nucleus, degradation of chromosomal DNA or loss of mitochondπal function A vaπetv of extrinsic and intrinsic signals are believed to trigger or induce such morphological and biochemical cellular changes [Raff. Nature. 356 397-400 f 1992). Steller, Science. 267 1445-1449 (1995), Sachs et al , Blood. 82 1 (1993)] For instance, they can be triggered by hormonal stunuli, such as glucocorticoid hormones for immature thymoevtes. as well as withdrawal of certain growth factors j Watanabe-Fukunaga et al . Nature. 356 314-317 ( 1992)] Also, some identified oncogenes such as myc. rel. and El A, and tumor suppressors, like p53 have been reported to have a role in inducing apoptosis Certain chemotherapy drugs and some forms of radiation have likewise been observed to have apoptosis-inducing activity [Thompson, supra) Various molecules, such as tumor necrosis factor-α ("TNF-α "), tumor necrosis factor- β ("TNF- β" or "lymphotoxin"), CD30 ligand. CD27 ligand, CD40 ligand, OX-40 ligand, 4- 1 BB ligand, Apo- 1 ligand (also referred to as Fas ligand or CD95 ligand), TRAIL, and Apo-2 ligand have been identified as members of the tumor necrosis factor ("TNF") family oi ' cytokines [See, e g , Gruss and Dower, Blood. 85.3378-3404 ( 1995), Wiley et al , Immunit y 3 673-682 ( 1995), Pitti et al , J Biol Chem . 271 12687- 12690 ( 1996)] Among these molecules , TNF-α, TNF-β, CD30 ligand, 4- IBB ligand, Apo-1 ligand, TRAIL, and Apo-2 ligand have been repoπed to be involved in apoptotic cell death Both TNF-α and TNF-β have been reported to induce apoptotic death in susceptible tumor cells [Schmid et al., Proc. Natl. Acad Sci .. § .1881 (1986), Dealtry et al , Eur J Immunol 12689 ( 1987)] Zheng et al. have reported that TNF-α is involved in post-stimulation apoptosis of CD8-posιtιve T cells [Zheng et al , Nature. 377 348-351 ( 1995)] Other investigators have reported that CD30 ligand may be involved in deletion of self-reactive T cells in the thvmus [Amakawa et al , Cold Spring Harbor Laboratory Symposium on Programmed Cell Death, Abstr No 10, ( 1995)] Mutations in the mouse Fas/Apo- 1 receptor or ligand genes (called Ipr and gld. respectively) have been associated with some autoimmune disorders, indicating that Apo- 1 ligand mav plav a role in regulating the clonal deleUon of self-reactive lymphocytes in the periphery (Krammer et al , Curr Op Immunol . 6 279-289 ( 1994) Nagata et al . Science. 267 1449-1456 ( 1995)] Apo- 1 ligand is also reported to induce post-stimulation apoptosis in CD4-posιtιve 1 lymphocytes and m B lymphocvtes, and mav be involved in the elimination ot activated lvmphocvtes when their function is no longer needed [Krammer et al , supra. Nagata et al , supra] Agonist mouse monoclonal antibodies specifically binding to the Apo- 1 receptor have been reported to exhibit cell killing activity that is comparable to or similar to that of TNF-α [Yonehara et al , J Exp Med . 169 1747- 1756 ( 1989)] Induction of various cellular responses mediated such TNF family cytokines is believed to be initiated by their binding to specific cell receptors Two distinct TNF receptors of approximately 55-kDa (TNFRI) and 75-kDa (TNFR2) have been identified [Hohman et al , J Biol Chem . 264 14927- 14934 ( 1989) Brockhaus et al . Proc Nat] Acad Sci . 87 3 127-3 1 1 ( 1990). EP 417,563, published March 20 1991 ] and human and mouse cDNAs corresponding to both receptor types have been isolated and characterized [Loetscher et al , CeJl, 6J. 35 l ( 1990), Schall et al , Ceil, 6J. 361 ( 1990), Smith et al , ≤cjej_ce, 248. 1019- 1023 ( 1990), Lewis et al , Proc Natl. Acad Sci.. M 2830-2834 ( 1991 ). Goodwin et al , Mo Cell. Biol . JJ. 3020-3026 ( 1 91 )] Extensive polymorphisms have been associated with both TNF receptor genes [see, e g , Takao et al . Immunogenetics. 37 199-203 (1993)] Both TNFRs share the tvpicai structure of cell surtace receptors including extracellular transmembrane and intracellular regions The extracellular portions of both receptors are found naturallv also as soluble TNF-binding proteins [Nophar. Y et al , EMBO J . 9 3269 ( 1 90), and Kohno. T et al Proc Natl Acad Sci U S. . 87 8331 ( 1990 " )] More recently, the cloning of recombmant soluble TNF receptors was reported bv Hale et al [J Cell Biochem. Supplement 15F. 1991 , p 1 13 (P424)] The extracellular portion of type 1 and tvpe 2 TNFRs (TNFR 1 and TNFR2) contains a repetitive amino acid sequence pattern of four cvsteine-πch domains (CRDs) designated 1 through 4, starting from the NH 2 - terminus Each CRD is about 40 amino acids long and contains 4 to 6 cvsteine residues at positions which are well conserved [Schall et al , supra. Loetscher et al , supra. Smith et al , supra. Nophar et al , supra. Kohno et al supra] In TNFR 1 , the approximate boundaries of the four CRDs are as follows CRD 1 - ammo acids 14 to about 53, CRD2- ammo acids from about 54 to about 97. CRD3- ammo acids from about 98 to about 138, CRD4- a ino acids from about 139 to about 167 In TNFR2, CRD 1 includes amino acids 17 to about 54, CRD2- ammo acids from about 55 to about 97. CRD3- ammo acids from about 98 to about 140. and CRD4- ammo acids from about 141 to about 1 9 [Banner et al , £≤U, 73 431 -435 ( 1993)] The potential role of the CRDs in ligand binding is also descπbed by Banner et al , supra A similar repeuuve pattern of CRDs exists in several other cell-surface proteins, including the p7 nerve growth factor receptor (NGFR) [Johnson et al , Cell, 42 545 ( 1986) Radeke et al , Nature. 325 593 (1987)], the B cell anϋgen CD40 [Stamenkovic et al , EMBO J . 8 1403 ( 1989)]. the T cell antigen OX40 [Mallet et al , EMBO J .2 1063 (1990)j and the Fas antigen [Yonehara et al , supra and Itoh et al , supra] CRDs are also found in the soluble TNFR (sTNFR)-lιke T2 proteins of the Shope and mvxoma poxviruses [Upton et al , Virology. 160 20-29 (1987), Smith et al , Bioche Biophvs Res Commun,. 126 335 ( 1991 ). Upton et al , Virology . 184 370 ( 1991 )] Optimal alignment of these sequences indicates that the positions of the cvsteine residues are well conserved These receptors are sometimes collectively referred to as members of the TNF/NGF receptor superfamily Recent studies on p75NGFR showed that the deletion of CRD 1 [Welcher. A A et al , Proc Natl. Acad Sci USA. 88 159-163 (1991 )] or a 5-arruno acid insertion in this domain [Yan. H and Chao, M V . J Biol Chem . 266 12099-12104 (1991)] had little or no effect on NGF binding [Yan, H and Chao. M V , supral p75 NGFR contains a proline-πch stretch of about 60 ammo acids, between its CRD4 and transme brane region, which is not involved in NGF binding [Peetre, C et al , Eur J Hematol . 4J. 414-419 ( 1988), Seckinger, P et al , J Biol Chem .264 1 1966-1 1973 (1989). Yan, H and Chao. M V . supra] A similar proline-πch region is found Itoh et al disclose that the Apo- 1 receptor can signal an apoptotic cell death similar to that signaled by the 55-k a TNFR 1 [Itoh et al , supra] Expression of the Apo- 1 antigen has also been reported to be down-regulated aiong with that of TNFR 1 when cells are treated with either TNF-α or antι-Apo- 1 mouse monoclonal antibody [Krammer et al , supra. Nagata et al , supra] Accordingly, some investigators have hypothesized that cell lines that co-express both Apo- 1 and TNFR 1 receptors may mediate cell killing through common signaling pa ways [Id ] The TNF family gands identified to date, with the exception of lvmphotoxin-α, are type II transmembrane proteins, whose C-terminus is extracellular In contrast, the receptors in the TNF receptor (TNFR) familv identified to date are type I transmembrane proteins In both the TNF ligand and receptor families, however, homoiogv identified between family members has been found mamlv in the extracellular do am ("ECD") Several of the TNF family cytokiπes, including TNF-α, Apo- 1 ligand and CD40 ligand, are cleaved proteolyticallv at the cell surface, the resulting protein in each case typically forms a homotπmeπc molecule that functions as a soluble cytokine TNF receptor family proteins are also usually cleaved proteolvticallv to release soluble receptor ECDs that can function as inhibitors of the cognate cytol nes Two of the TNFR familv members, TNFR 1 and Fas/Apol (CD95 can activate apoptotic cell death [Chinnaivan and Dixit, Current Biology. 6 555-562 ( 1996). Fraser and Evan, C≤ϋ, 85 781 -784 ( 1996)] TNFR1 is also known to mediate activation of the transcription factor. NF-κB [1 artag a et al . Cell. 74 845-853 ( 1993), Hsu et al , Cell. §4 299-308 ( 1996)] In addition to some ECD homoiogv, these two receptors share homoiogv in their inti acellular domain GCD) in an obgomeπzation interface known as the death domain [Tartaglia et al , supral Death domains are also found in several metazoan proteins that regulate apoptosis, namely, the Drosophila protein. Reaper, and the mammalian proteins referred to as FADD/MORT1, TRADD, and RIP [Cleaveland and Ihle, Cell. 8 .479-482 ( 1995)] Using the veast-two hvbπd system, Raven et al report the identification of protein, wsl-1, which binds to the TNFR1 death domain [Raven et al , Programmed Cell Death Meeting, September 20-24, 1995, Abstract at page 127, Raven et al , European Cvtok e Network. 7 Abstr 82 at page 210 (April-June 1996)] The wsl- l protein is described as being homologous to TNFR I (48% identity) and having a restricted ussue distribution According to Raven et al , the tissue distribution of wsl- 1 is significantly different from the TNFR] binding protein, TRADD Upon ligand binding and receptor clustering, TNFR 1 and CD95 are believed to recruit FADD into a death-inducing signalling complex CD95 purportedly binds FADD directly, while TNFR 1 binds FADD indirectly \ ιa TRADD [Chmnaivan et al , CeJl, 8J.505-512 (1995), Boldin et al , J Biol Chem . 270 387-391 ( 1 95). Hsu et al , supra: Chinnaiyan et al , J Biol Chem . 27 4961 -4965 ( 1996)] It has been reported that FADD serves as an adaptor protein which recruits the thiol protease MACHα FLICE into the death signalling complex [Boldin et al , £elL 85 803-815 ( 1996), Muzio et al , Cell, S5 817-827 (1996)] MACI lα/FLICE appears to be the trigger that sets off a cascade of apoptotic proteases, including the interleukin- 1 β converting enzyme (ICE) and CPP32/Yama, which may execute some critical aspects of the cell death programme [Fraser and Evan, supra] It was recently disclosed that programmed cell death involves the acUvin ol members ot a family of cysteine proteases related to the C elegans cell death gene, ced-3. and to the mammalian IL- 1 -converting enzyme. ICE The acuvity ot the ICE and CPP32/Yama proteases can be inhibited bv the product of the covvpos virus gene, crmA [Rav et al , Cell, 69 597-604 ( 1992), Tew an et al , Cell, 8J. 801 -809 ( 1995)] Recent studies show that CrmA can inhibit TNFR 1 - and CD95-ιnduced cell death [Enaπ et al , Nature. 375 78-81 ( 1995), Tewaπ et al . J Biol Chem . 270 3255-3260 ( 1995)] As reviewed recently by Tewaπ et al , TNFR 1. TNFR2 and CD40 modulate the expression of proinllammatorv and costimulatory cytokmes. cvtokine receptors, and cell adhesion molecules through activation of the transcπption factor. NF-κB [Tewaπ et al , Curr On Genet Develop . 6 39-44 ( 1996)] NF-κB is the prototype of a familv of dimeπc transcπption factors whose subumts contain conserved Rel regions [ Verma et al Genes Develop . 92723-2735 ( 1996), Baldwin, Ann Rev Immunol . j_4 649-681 ( 1996)] In its latent torm, NF- KB IS complexed with members of the lκB inhibitor familv, upon inactivation of the IκB in response to certain stimuli, released NF-κB translocates to the nucleus where it binds to specific DNA sequences and activates gene transcription TNFR proteins may also rcguiate the AP- 1 transcπption factor family [Kaπn, J Biol Chem . 270 16483- 16486 (1995)] AP- 1 represents a separate family of dimeπc transcπptional activators composed ol members of the Fos and Jun protein families [Kaπn, supral AP- 1 activation is believed to be mediated bv immediate-earlv induction of fos andjun through the mitogen-activated protein kinases ERK and JNK (Jun N- terminal kinase, also known as stress-activated protein kinase SAPK), as well as by JNK-dependent phosphorvlation of Jun proteins [Kaπn, supra. Kvπakis et al , J Biol Chem . 271 24313-24316 ( 1996)J Transcπptional regulation bv TNFR familv members is mediated primarily bv members of the TNF receptor associated factor (TRAF family [Rothe et al , Cell, 2S 68 ] -692 ( 1994), Hsu et al CeU, 84 299-308 ( 1996), Liu et al . Cell, SI 565-576 ( 1996)] For a review of the TNF family of cytokmes and their receptors, see Gruss and Dower, supra SUMMARY OF THE INVENTION Applicants have identified cDNA clones that encode novel polypeptides. designated in the present application as Αpo-3 " The Apo-3 polypeptide has surprisingly been found to stimulate or induce apoptouc acuvity in mammalian cells It is believed that Apo-3 is a member of the TNFR familv. full-length native sequence human Apo-3 polypeptide exhibits some similanues to some known TNFRs. including TNFRl and CD95 In particular, full-length native sequence human Apo-3 exhibits similaπtv to the TNFR family in its extracellular cvsteine-πch repeats and resembles TNFR 1 and CD95 in that it contains a cvtoplasmic death domain sequence Applicants have also identified cDNA clones that encode a polypeptide. designated "Apo-2 ligand inhibitor" or "Apo-2LI" Although not being bound to any particular theorv, it is presentl y believed that Apo-2LI compπsmg amino acid residues 1 to 181 of Figure I (SEQ ID NO 1 ) [and which correspond to amino acid residues 1 to 181 of ie sequence of Fig 4 (SEQ ID NO 6)] mav be a soluble, truncated or secreted form ol Apo-3 In one embodiment, the invention provides isolated Apo-2LI In particular, the invention provides isolated native sequence Apo-2LI, which in one embodiment, includes an amino acid sequence comprising residues 1 to 181 of Figure 1 (SEQ ID NO 1 ) In other embodiments, the isolated Apo-2LI compπses one or more cvsteiπe-πch domains of the sequence of Figure 1 , or compπses biologically active polypeptides compπsmg at least about 80% identity with native sequence Aρo-2LI shown in Figure 1 (SEQ ID NO I ) In another embodiment the invention provides chimeπc molecules compπsing Apo-2LI fused to another, heterologous polypepude or amino acid sequence An example of such a chimeπc molecule compπses an Apo-2LI am o acid sequence fused to an lmmunoglobulin constant domain sequence In another embodiment, the invenuon provides an isolated nucleic acid molecule encoding Apo- 2LI In one aspect, the nucleic acid molecule is RNA or DNA that encodes an Apo-2LI or is complementarv to a nucleic acid sequence encoding such Apo-2LI. and remains stablv bound to it under stπngent conditions In one embodiment, the nucleic acid sequence is selected from (a) the coding region of the nucleic acid sequence of Figure I that codes for residue 1 to residue 181 (i e , nucleotides 377 through 919, also provided m SEQ ID NO 5), inclusive, or (b) a sequence correspondmg to the sequence of (a) within the scope of degeneracy of the genetic code In a further embodiment the invention provides a rephcable vector compπsing the nucleic acid molecule encoding the Apo-2LI operably linked to control sequences recognized by a host cell transfected or transformed with the vector A host cell compπsmg the vector or the nucleic acid molecule is also provided A method of producing Apo-2LI which compπses cultuπng a host cell compπsing the nucleic acid molecule and recovenng the protein from the host cell culture is further provided In another embodiment, the invention provides an antibody which binds to Apo-2LI In another embodiment, the invention provides isolated Apo-3 polypeptide In particular, the invention provides isolated nauve sequence Apo-3 polypeptide, which in one embodiment, includes an ammo acid sequence compπsing residues 1 to 417 of Figure 4 (SEQ ID NO 6) In other embodiments, the isolated Apo-3 polypeptide compπses at least about 80% identity with native sequence Apo-3 polypeptide compπsing residues 1 to 417 of Figure 4 (SEQ ID NO 6) In another embodiment, the invention provides an isolated extracellular domain sequence of Apo-3 polypeptide The isolated extracellular domain sequence preferably compπses residues 1 to 198 of Fig 4 (SEQ ID NO 6) In another embodiment, the invention provides an isolated death domain sequence of Aρo-3 polypeptide The isolated death domain sequence preferably compπses residues 338 to 4 ] 7 of Fig 4 (SEQ ID NO 6) In another embodiment, the invention provides chimeπc molecules compπsing Apo-3 polypeptide fused to a heterologous polvpepϋde or amino acid sequence An example ol such a chimeπc molecule compπses an Apo-3 fused to an immunoglobulin sequence Another example compπses an extracellular domain sequence of Aρo-3 fused to a heterologous polypeptide or amino acid sequence, such as an immunoglobulin sequence In another embodiment the invention provides an isolated nucleic acid molecule encodmg Apo- 3 polypeptide In one aspect, the nucleic acid molecule is RNA or DNA that encodes an Apo-3 polypeptide or a particular domain ol Apo-3. or is complementarv to such encodmg nucleic acid sequence, and remains stably bound to it under stringent conditions In one embodiment the nucleic acid sequence is selected from (a) the coding region of the nucleic acid sequence of Figure 4 (SEQ ID NO 9) that codes toi residue 1 to residue 417 (l e , nucleotides 89-91 through 1337- 1339). inclusive or (b) the coding region of the nucleic acid sequence of Figure 4 (SEQ ID NO 9) that codes for residue I to residue 198 (l e , nucleotides 89-91 through 680-682), inclusive, (c) the coding region of the nucleic acid sequence of Figure 4 (SEQ ID NO 9) that codes for residue 338 to residue 417 (1 e . nucleotides 1 100- 1 102 through 1337- ] 339), inclusive, or (d) a sequence corresponding to the sequence of (a), (b) or (c) within the scope ot degeneracy of die genetic code In a further embodiment the invention provides a vector compπsing the nucleic acid molecule encodmg the Apo-3 polypeptide or particular domam of Apo- 3 A host cell compπsing the vector or the nucleic acid molecule is also provided A method of producing Apo-3 is further provided In another embodiment, the invention provides an antibody which binds to Apo-3 In another embodiment, the invention provides non-human, transgenic or knock-out animals A further embodiment of the invention provides articles of manufacture and kits BRIEF DESCRIPTION OF THE DRAWINGS Figure I shows the nucleotide sequence of human Apo-2LI cDNA and its derived amino acid sequence Figure 2 shows an alignment of the aπuno acid sequence encoded bv clone 1 1 ot Apo-2LI with extracellular regions of other members of the human TNF receptor family Figure 3 shows a silver-stained gel of a protein A purified Apo-2LI immunoadhesin analyzed under non-reducing (lanes 3-5) or reducing (lanes 7-9) conditions Figure 4 shows the nucleotide sequence of native sequence human Apo-3 cDN A and its deπved amino acid sequence The putative signal sequence and transmembrane domain are underlined, the death domam sequence is boxed, and the potential N-lmked glycosylation sites are marked with an asteπsk Also boxed is the alanine residue which was present in the fetal lung but not in the fetal heart cDNA clone (discussed in Example 4 below) Figure 5 shows an alignment and compaπson of the ECD sequences of native sequence human Apo-3, TNFRl and CD95 Figure 6 shows an alignment and companson of the death domain sequences of native sequence human Apo-3, TNFRl, CD95, FADD, TRADD. RIP and Drosophila Reaper Figure 7 shows a schematic alignment of Apo-3. Λpo-2LI. TNFRl . and Fas/ Apo- 1 CRD, cvsteine-πch domains, TM, transmembrane domam. DD, death domain Figure 8 shows ectopic expression of Apo- 3 m HEK 293 cells Cells were transfected with pRK5-Apo-3 plus pRK5 (5 ag each) (lane 1), pRK5 alone ( 10 ug) (lane 2), or pRK5-Apo-3 plus pRK5-CrmA (5 ug each) (lane 3) Cells were metabo cally labeled with S-Met and .S-Cys Cell Ivsates were then analyzed bv radioimmunoprecipitation using mouse antι-Apo-3 antiserum The molecular weight standards are shown on Figures 9a-j illustrate the induction of apoptosis bv ectopic expression of Apo-3 in HEK 293 cells Apoptosis was examined 36 hours after transfection. by morphological analvsis (Figs 9 a-d), by FACS analvsis (Figs 9 e-i), and by DNA laddering (Fig 9 j) Cells were transfected with pRK5 alone ( 10 ag) (Figs 9 a. e, j, lane 1 ). pRK5 plus pRK5-Apo-3 (5 ug each) (Figs 9 b, f, j, lane 2), pRK5 plus pRK5-CrτnA (5 ug each) (Figs 9 c, g,j, lane 3), orpRK5-Apo-3 lus pRK5-CrmA (5 g each) (Figs 9 d, h, j, lane 4) Cells in Figs 9 a-d were photographed at 400X magnification using Hoffmann optics-based light As measured by the total number of annexin V-positive cells, the percent apoptosis in Figs 9 e-h, respectiveh , was 37%. 66%, 36% and 26% Cells in Fig 9 t were transfected with the indicated amount of pRK5-Apo-3 or pRK5-TNFR I and the appropnate amount of pRK5 plasmid to bπng the total amount of DNA to 20 ag Figure 10 shows activation of NF-κB by ectopic expression of Apo-3 HEK 293 cells were transfected with 10 ug pRK5 (lanes 1 , 4, 7), pRK5-Apo-3 (lanes 2, 5, 7), or pRK5-TNFR l (lanes 3, 6, 9) Nuclear extracts were prepared 36 hours later and reacted with an irrelevant P-Iabelled oligonucleotide probe (lanes 1 -3), or with a 32 P-labelled NF-κB-specιfic probe alone (lanes 4-6) or in the presence of 50-foid excess unlabelled oligonucleotide of the same sequence (lanes 7-9) Figure 1 1 shows activation of Jun N-terminal kinase (JNK) by ectopic expression of Apo-3 HEK 293 cells were transfected with 10 ug pRK5, pRK5-TNFR 1 , or pRK5-Apo-3 as indicated 36 hours later, JNK activity in cell extracts was determined using a JNK SAPK activation kit, which measures JNK activity bv analyzing phosphoryiation of c-Jun Figure 12 illustrates expression of Apo- 3 mRNA in human tissues as determined bv Northern blot hvbπdi auon In the left hand panel are shown fetal brain ( 1 ), lung (2), liver (3). kidney (4) In the πght hand panel are shown adult spleen ( 1 ), thymus (2), prostate (3), testis (4), ovary (5), small intestine (6), colon (7), and peπpheral blood lymphocytes (8) The sizes of the molecular weight standards are shown on the left in kb DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS I Definitions The terms "Apo-3 polypeptide" and "Apo-3" when used herein encompass native sequence Apo- 3 and Apo-3 vaπants (each of which is defined herein) These terms encompass Apo-3 from a vanety of mammals, including humans The Apo-3 may be isolated from a vanety of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods A "native sequence Apo-3" compπses a polypeptide having the same ammo acid sequence as an Apo-3 deπved from nature Thus, a native sequence Apo-3 can have the ammo acid sequence of naturallv- occumng Apo-3 from any mammal Such native sequence Apo-3 can be isolated from nature or can be produced by recombinant or synthetic means The term "native sequence Apo-3" specifically encompasses naturally- occurring truncated or secreted forms of the Apo-3 (e g . an extracellular domain sequence), naturallv-occumng variant forms (e g , alternatively spliced forms) and naturalh -occurring allelic vaπants ol the Apo-3 A naturally- occurring vaπant form of the Apo-3 includes an Apo-3 having an a mo acid deletion at residue 236 in the ammo acid sequence shown in Figure 4 (SEQ ID NO 6) In one embodiment of the invention, the native sequence Apo-3 is a mature or full-length native sequence Apo-3 compπsing the ammo acid sequence of SEQ ID NO 6 The present definition of native sequence Apo-3 excludes known EST sequences, such as GenBank W71984 "Apo-3 \aπant" means a biologicallv active Apo-3 as defined below having less than 100% sequence identity with Apo-3 having the deduced amino acid sequence shown in Fig 4 (SEQ ID NO 6) lor a full- length native sequence human Apo-3 Such Λpo-3 vaπants include, tor instance, Apo-3 polypeptides wherein one or more amino acid residues are added at the N- or C-termmus of, or within, the sequence of SEQ ID NO 6, from about one to 24 amino acid residues are deleted (including a single amino acid deletion at residue 236 in the ammo acid sequence shown in Figure 4 (SEQ ID NO 6), or optionallv substituted bv one or more ammo acid residues, and deπvatives thereof, wherein an amino acid residue has been covalentiy modified so that the resulting product has a non-naturallv occurπng amino acid Ordinanlv. an Apo-3 vaπant will have at least about 80% sequence identity, more preferably at least about 90% sequence identity, and even more preferably at least about 95% sequence identitv with the sequence of Fig 4 (SEQ ID NO 6) The present definition of Apo-3 vaπant excludes known EST sequences, such as GenBank W71984 The terms "Apo-2 ligand inhibitor polypeptide" and "Apo-2LI" when used herein encompass native sequence Apo-2LI and Apo-2LI variants (each of which is defined herein) These terms encompass Apo- 2LI from a vaπetv of mammals, including humans The Apo-2LI mav be isolated from a vaπetv of sources, such as from human tissue types or from another source, or prepared by recombinant or synthetic methods A "native sequence Apo-2LI" compnses a polypeptide having the same amino acid sequence as an Apo-2LI denved from nature Thus, a native sequence Apo-2LI can have the ammo acid sequence of naturaliy-occurπng Apo-2LI from anv mammal Such native sequence Apo-2LI can be isolated from nature or can be produced by recombinant or synthetic means The term "native sequence Apo-2Ll" specifically encompasses naturallv-occumng truncated forms, naturallv-occumng vaπant torms (e g , alternatively spliced torms ) and naturally-occumng allelic vaπants In one embodiment of the invention, the native sequence Apo-2LI compπses the ammo acid sequence of SEQ ID NO 1 The present definition of native sequence Apo-2LI excludes known EST sequences, such as GenBank H41522, H46424, H4621 1 , H46374, H46662, H41 51 , H49675, H22502. H46378 and H19739 "Apo-2LI vaπant" means a biologically active Apo-2LI as defined below having less than 100% sequence identity with Apo-2LI having the deduced amino acid sequence shown in Fig 1 (SEQ ID NO I ) Such Apo-2LI vaπants include, for instance, Apo-2LI polypeptides wherein one or more ammo acid residues are added at the N- or C-terminus of, or within, the sequence of SEQ ID NO 1. wherein one or more amino acid residues are deleted, or opuonallv substituted by one or more amino acid residues, and deπvatives thereof wherein an amino acid residue has been covalentiy modified so that the resulting product has a non-naturally occumng ammo acid Optionally, the Apo-2LI includes one or more cvsteine-πch domains, and preferably includes one or more cysteine-nch domains compπsing amino acids 34 to 71. amino acids 72 to 1 15, ammo acids 1 16 to 163. or ammo acids 164 to 181 of Figure I Ordinarily, an Apo-2LI vaπant will have at least about 80% sequence identity, more preferably at least about 90% sequence identity, and even more preferablv at least about 95% sequence identity with the sequence of Fig 1 (SEQ ID NO 1 ) The present definition of Apo-2Ll vaπant excludes known EST sequences, such as GenBank H41522. H46424, H4621 1 , H46374. H46662, H41851. H49675, H22502. H46378 and HI 9739 The term epitope tagged" when used herein refers to a chimeπc pol y peptide compπsmg at least one of the Apo-3 or Apo-2LI polypeptides disclosed herein, or a portion thereof, fused to a "tag polypeptide" The tag polypeptide has enough residues to provide an epitope against which an antibody can be made, vet is short enough such that it does not interfere with activity of the Apo-3 or Apo-2Ll The tag polypeptide preferably also is fatrh unique so that the antibody does not substantially cross-react with other epitopes Suitable lag polypeptides generally have at least six amino acid residues and usually between about 8 to about 50 amino acid residues (preferablv. between about 10 to about 20 residues) "Isolated." when used to descπbe the vaπous polypeptides disclosed herein, means polypeptide that has been identified and separated and/or recovered from a component of its natural environment Contaminant components of its natural environment are mateπals that would typically interfere with diagnostic or therapeutic uses for the polypeptide. and mav include enzvmes, hormones, and other proteinaceous or non-proteinaceous solutes In preferred embodiments, the polypeptide will be purified ( 1 ) to a degree sufficient to obtain at least 15 residues of N-termir.al or internal amino acid sequence by use of a spinning cup sequenator. or (2) to homogeneity by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver stain Isolated polypeptide includes polypeptide in situ within recombinant cells, since at least one component of the Apo-3 or Apo-2Ll natural environment will not be present Ordmanlv. however, isolated polypeptide will be prepared by at least one punfication step An "isolated" nucleic acid molecule is a nucleic acid molecule that is identified and separated from at least one contaminant nucleic acid molecule with which it is ordinarily associated in the natural source of the nucleic acid An isolated nucleic acid molecule is other than in the form or setting in which it is found in nature Isolated nucieic acid molecules therefore are distinguished from the nucleic acid molecule as it exists in natural cells However, an isolated Apo-3 nucleic acid molecule, for instance, mcludes Apo-3 nucleic acid molecules contained in cells that ordinarily express Apo-3 where, for example, the nucleic acid molecule is in a chromosomal location different from that of natural cells The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism The control sequences that are suitable for prokaryotes. for example, include a promoter, optionally an operator sequence, and a nbosome binding site Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers Nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence For example, DNA for a presequence or secretory leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide. a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence, or a nbosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation Generally, "operably linked" means that the DNA sequences being linked are contiguous, and, in the case of a secretory leader, contiguous and m reading phase. However, enhancers do not have to be contiguous Linking is accomplished bv hgation at convenient restriction sites If such sites do not exist, the synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice The term 'antibody" is used in the broadest sense and specifically covers single monoclonal anubodies (including agonist antagonist and neutralizing antibodies) and antibody compositions with polvepitopic specificity The term "monoclonal anϋbodv" as used herein refers to an antibodv obtained tram a population of substantially homogeneous antibodies. ; e . the individual antibodies compnsing the population are identical except tor possible naturallv-occumng mutations that mav be present in minor amounts Monoclonal antibodies are highly specific, being directed against a single antigenic site Furthermore, in contrast to conventional (polvclonal) antibodv preparations which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibodv is directed against a single determinant on the antigen The monoclonal antibodies herein include hybnd and recombinant antibodies produced b\ splicing a vaπable (including hypervaπable) domain of an antι-Apo-3 antibodv or antι-Apo-2LI antibodv with a constant domam (e g "humanized" antibodies), or a light chain with a heaw chain, or a chain from one species w ith a chain from another species, or fusions with heterologous proteins, regardless ot species ot ongin or immunoglobulin class or subclass designation, as well as antibodv fragments (e g , Fab, F(ab')-, and Fv), so long as thev exhibit the desired biological activity See, e g U S Pat No 4,816,567 and Mage et al . in Monoclonal Antibody Production Techniques and Applications, pp 79-97 (Marcel Dekker. Inc New York 1987) Thus, the modifier "monoclonal" indicates the character ot the antibody as being obtained from a substantiallv homogeneous population of antibodies, and is not to be construed as requiπng production of the antibodv bv am particular method For example the monoclonal antibodies to be used m accordance with the present invention may be made by the hybndoma method first descπbed bv Kohler and Milstein, Nature. 256 495 ( 1 975), or mav be made bv recombinant DNA methods such as descπbed in U S Pat No 4,816,567 The "monoclonal antibodies" may also be isolated from phage braπes generated using the techniques descπbed in McCaffertv et al , Nature. 348 552-554 ( 1990). for example "Humanized" forms ot non-human (e muπne) antibodies are specific chimeπc immunoglobuuns, immunoglobulin chains, or fragments thereof ( such as Fv Fab, Fab', F(ab') 2 or other antigen- binding subsequences of antibodies) which contain minimal sequence deπved from non-human immunoglobulin For the most part humanized anubodies are human tmmunoglobulms (recipient antibody ) in which residues from a complementarv determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibody) such as mouse, rat or rabbit having the desired specificity, affinity, and capacity In some instances, Fv framework region (FR) residues of the human immunoglobulin are replaced by corresponding non- human residues Furthermore, the humanized antibodv mav compnse residues which are found neither in the recipient antibodv nor in the imported CDR or framework sequences These modifications are made to further refine and optimize antibody pertormance In general, the humanized antibodv will compπse substantially all of at least one, and typically two, vaπable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulin consensus sequence The humanized antibody optimally also will compπse at least a portion ot an immunoglobulin constant region or domain (Fc), typically that of a human immunoglobulin "Biologically active" and "desired biological activity" for the purposes herein mean having the ability to modulate apoptosis (either in an agonistic manner bv inducing or stimulating apoptosis, or in an antagonistic manner by reducing or inhibiting apoptosis) in at least one type of mammalian cell m vivo or ex vivo The terms "apoptosis" and "apoptotic activity" are used in a broad sense and reter to the orderly or controlled form of cell death m mammals that is typically accompanied bv one or more characteristic cell changes, including condensation of cvtoplasm, loss of plasma membrane microvilli, segmentation of the nucleus degradation of chromosomal DNA or loss of mitochondπal function This activity can be determined and measured, for instance, bv cell viability assays, FACS analysis or DNA electrophoresis. all of which are known in the art The terms "treating." "treatment," and "therapy" as used herein refer to curative therapy, prophy lactic therapy, and preventative therapy The term "mammal" as used herein reters to anv mammal classified as a mammal, including humans cows, horses, dogs and cats In a preferred embodiment of the invention, the mammal is a human II Compositions and Methods of the Invention Applicants have identified and isolated various polypeptides involved in mammalian cell apoptosis In particular, Applicants have identified and isolated various Apo-3 polypeptides and forms thereof, referred to herein as Apo-2LI The properties and characteπstics of some of these Apo-3 and Apo-2LI polypeptides, particularly human Apo-3 and human Apo-2LI. are descπbed in further detail in the Examples below Based upon the properties and characteristics of the Apo-3 and Apo-2LI polypeptides disclosed herein, it is Applicants' present belief that Apo-3 is a member of the TNFR family As discussed in Example 4 below, a native sequence human Apo-3 polv peptide was identified The predicted polypeptide precursor is 417 amino acids long (see Fig 4) Hydropathv analvsis (not shown) suggested the presence of a signal sequence (residues I - 24), followed by an extracellular domain (residues 25- 198), a transmembrane domam (residues 199 - 224), and an intracellular domain (residues 225 - 417) (Fig 4. SEQ ID NO 6) The schematic diagram shown in Figure 7 illustrates such domains, as well as the cvsteme-nch domains As discussed in Example 1 , Applicants also identified and isolated a polypeptide referred to herein as " Apo-2LI." The predicted amino acid sequence of human Apo-2LI contains 181 amino acids (as shown in Figure I ). It is piesently believed that Apo-2LI compπsing amino acid residues I to 181 of Figure 1 may be a soluble, truncated or secreted form of Apo-3 The human Apo-2LI having amino acids 1 to 181 of Figure 1 is substantially homologous (l e , having at least 80% identity) to the extracellular sequence of native sequence human Apo-3 (amino acid residues 1 to 198, as shown in Figure 4), and it is presently believed that such Apo-2LI (at least in onomeπc form) is a functional equivalent to the Apo-3 A descnption follows as to how the polypeptides of the invention, as well as chimenc molecules and antibodies, may be prepared It is contemplated that the methods and mateπals descπbed below (and in the Examples herein) may be employed to prepare Apo-2LI, Apo-2LI chimeπc molecules and antι-Apo-2LI antibodies, as well as Apo-3 polypeptides, Apo-3 chimeπc molecules and antι-Apo-3 antibodies -I I- A Preparation of Polypeptides and Nucleic Acids The descπption below relates pπmaπlv to production of the polypeptides bv cultunng cells transformed or transfected with a vector containing Apo-3 or Apo-2LI nucleic acid It is of course, contemplated that alternative methods, which are well known in the art. mav be emploved to prepare the polypeptides I Isolation of Encoding DNA The DNA encoding the polypeptides of the invention mav be obtained from anv cDN A library prepared from tissue believed to possess the polypeptide mRNA and to express it at a detectable level Accordingly, human Apo-2LI DNA can be conveniently obtained from a cDNA brarv prepared from human tissues, such as the bactenophage library of human thyrnus cDNA descπbed in Example 1 The Apo-2LI-encodιng gene may also be obtained from a genomic library or bv oligonucleotide svnthesis Human Apo-3 DNA can be conveniently obtained from a cDNA library prepared from human tissues, such as the bactenophage branes ot human fetal heart and lung cDNA descπbed in Example 4 The Apo-3 -encodmg gene mav also be obtained from a genomic library or by oligonucleotide svnthesis Libranes can be screened with probes (such as antibodies or oligonucleotides of at least about 20-80 bases ) designed to identify the gene ot interest or the protein encoded bv it Examples of oli onucleotide probes are provided in Examples 1 and 4 Screening the cDNA or genomic library ith the selected probe mav be conducted using standard procedures, such as descπbed in Sambrook et al , Molecular Cloning A Laboratory Manual (New York Cold Spπng Harbor Laboratory Press 1989) An alternative means to isolate the gene encoding, for instance, Apo-3 or Apo-2Ll is to use PCR methodology [Sambrook et al . supra Dieffenbach et al , PCR Pπmer A Laboratory Manual (Cold Spnng Harbor Laboratory Press, 1995)] A preferred method of screening emplovs selected oligonucleotide sequences to screen cDNA hbranes from vanous human tissues Examples 1 and 4 below descπbe techniques tor screening cDNA libraries with different oligonucleotide probes The oli onucleotide sequences selected as probes should be of sufficient length and sufficiently unambiguous that false positives are minimized The oligonucleotide is preferablv labeled such that it can be detected upon hvbπdization to DNA in the library being screened Methods of labeling are well known in the art. and include the use ot radiolabels like P-labeled ATP, biotinvlation or enzvme labeling Nucleic acid having all the protein coding sequence mav be obtained by screening selected cDNA or genomic libranes using the deduced amino acid sequence disclosed herein tor the first tune, and, if necessary, usmg conventional pπmer extension procedures as descπbed in Sambrook et al , supra, to detect precursors and processing intermediates of mRNA that may not have been reverse-transcπbed into cDNA Apo-3 or Apo-2Ll vaπants can be prepared bv introducing appropnate nucleotide changes into the DNA of such polypeptides, or by svnthesis of the desired polypeptide Anv combination of insertion substitution, and/or deletion can be made to amve at the final construct provided that the final construct possesses the desired activity as defined herein Those skilled in the art will appreciate that amino acid changes may altei post-translational processes of the Apo-3, such as changing the number or position of glvcosvlation sites or altering the membrane anchoπng characteπstics Vaπations in the native sequence Apo-3 or Apo-2LI as descπbed above can be made using anv of the techniques and guidelines for conservative and non-conservative mutations set forth in U S Pat No 5,364,934 These include oligonucleotide-mediatcd (site-directed) mutagenesis. alanme scanning, and PCR mutagenesis 2 Insertion of Nucleic Acid into A Replicable Vector The nucleic acid (e g . cDNA or genomic DNA) encoding Apo-3 or Apo-2LI mav be inserted into a replicable vector for further cloning (amplification of the DNA) or for expression Vaπous vectors are pubhclv available The vector components generally include, but are not limited to, one or more of the following a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter, and a transcπption termination sequence, each of which is descπbed below (0 Signal Sequence Component The polypeptides of the invention mav be produced recombinantly not onlv directly, but also as a fusion polypeptide with a heterologous polypeptide. which may be a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide In general, the signal sequence mav be a component of the vector, or it mav be a part of the DNA that is inserted into the vector The heterologous signal sequence selected preferablv is one that is recognized and processed (/ e . cleaved bv a signal peptidase) bv the host cell The signal sequence mav be a prokarvotic signal sequence selected foi example, from the group ot the alkaline phosphatase penicillmase, 1pp. or heat-stable enterotoxin II leaders For veast secretion the signal sequence may be, e g., the veast invertase leader, alpha factor leader (including Saccharomyces and Kluweromyces α-factor leaders, the latter descπbed in U S Pat No 5,010,182), or acid phosphatase leader, the C albicans giucoamylase leader (EP 362.179 published 4 Apπl 1990), or the signal descπbed in WO 90/13646 published 15 November 1990 In mammalian cell expression the native Apo-3 or Apo-2LI presequence that normally directs insertion of the polypeptide in the cell membrane of human cells m vivo is sati tactorv although other mammaii an signal sequences may be used to direct secretion of the protein, such as signal sequences from secreted polypeptides of the same or related species, as well as viral secretory leaders, for example the herpes simplex glycoprotein D signal The DNA for such precursor region is preferablv gated in reading frame to DNA encoding the polypeptide (ul Oπgm of Replication Component Both expression and cloning vectors contain a nucleic acid sequence that enables the vector to replicate in one or more selected host cells Generally, in cloning vectors this sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and mcludes origins of replication or autonomously replicating sequences Such sequences are well known for a vanetv of bacteria, veast. and viruses The oπgin of replication from the plasmid pBR322 is suitable for most Gram-negative bacteπa, the 2μ plasmid oπgm is suitable for veast, and vaπous viral oπgins (SV40, polvoma, adenovirus, VSV or BPV) are useful for cloning vectors in mammalian cells Generally, the oπgm of replication component is not needed for mammalian expression vectors (the SV40 origin may typically be used because it contains the early promoter) Most expression vectors are "shuttle" vectors, ; e , thev are capable of replication in at least one class of organisms but can be transfected into another organism for expression For example, a vector is cloned in E coli and then the same vector is transfected into yeast or mammalian cells for expression even though it is not capable of replicating independently of the host cell chromosome DNA may also be amplified by insertion into the host genome This is readily accomplished using Bacillus species as hosts, for example, by including in the vector a DNA sequence that is complementary to a sequence found in Bacillus genomic DNA Transfection of Bacillus with this vector results in homologous recombination with the genome and insertion of the polypeptidc's DNΛ However, the recovery of genomic DNA encodmg Apo-3 or Apo-2LI is more complex than that of an exogenouslv replicated vector because restriction enzvme digestion is required to excise the DNA (in) Selection Gene Component Expression and cloning vectors typicallv contain a selection gene, also termed a selectable marker This gene encodes a protein necessary for the survival or growth of transformed host cells grown in a selective culture medium Host cells not transformed with the vector containing the selection gene will not survive in the culture medium T pical selection genes encode proteins that (a) confer resistance to antibiotics or other toxins, e.g., ampicillin, neomvcin, methotrexate, or tetracycline, (b) complement auxotrophic deficiencies, or (c) supply cπtical nutπents not available from complex media, e.g., the gene encoding D-alanine racemase for Bacilli One example of a selection scheme utilizes a drug to arrest growth of a host cell Those cells that are successfully transformed with a heterologous gene produce a protem confemng drug resistance and thus survive the selection regimen Examples of such dominant selection use the drugs neomvcin [Southern et al , J_ Molec Appl Genet . 1327 (1982)], mycophenolie acid (Mulligan et al , Science. 209 1422 ( 1980)] or hygromvcin [Sugden et al , Mo Cell Biol . 5_ 410-413 (1985)] The three examples given above employ bacteπal genes under eukarvotic control to convey resistance to the appropnate drug G418 or neomvcin (geneticin), xgpt (mvcophenolic acid), or hygromvcin, respectively Another example of suitable selectable markers for mammalian cells are those that enable the identification of cells competent to take up the Apo-3 or Apo-2LI nucleic acid, such as DHFR or thymidine kinase The mammalian cell transformants are placed under selection pressure that only the transformants are uniquely adapted to survive by virtue of having taken up the marker Selection pressure is imposed bv cultuπng the transformants under conditions in which the concentration of selection agent in the medium is successively changed, thereby leading to amplification of both the selection gene and the DNA that encodes the polypeptide Amplification is the process by which genes in greater demand for the production of a protein cntical for growth are reiterated in tandem within die chromosomes of successive generations of recombinant cells Increased quantities of Apo-3 or Apo-2LI are synthesized from the amplified DNA Other examples of amplifiable genes include metallothionem-I and -II, adenosine deaminase, and orruthine decarboxylase Cells transformed with the DHFR selection gene may first be identified by cultuπng all of the transformants in a culture medium that contains methotrexate (Mtx), a competitive antagonist of DHFR An appropnate host cell when wild-type DHFR is employed is the Chinese hamster ovary (CHO) cell line deficient in DHFR activity, prepared and propagated as described by Urlaub et al , Proc Natl Acad Sci USA. 77 421 (1980) The transformed cells are then exposed to increased levels of methotrexate This leads to the synthesis of multiple copies of the DHFR gene, and, concomitantly, multiple copies of other DNA compπsing the expression vectors, such as the DNA encoding the Apo-3 or Apo-2LI. This amplification technique can be used with anv otherwise suitable host, e g., ATCC No CCL61 CHO- 1 , notwithstanding the presence of endogenous DHFR if. for example, a mutant DHFR gene that is highly resistant to Mtx is employed (EP 1 17,060) Alternatively, host cells (particularly wild-type hosts that contain endogenous DHFR) transformed or co-transformed with DNA sequences encodmg Apo-3 or Λpo-2LI. wild-type DHFR protein, and another selectable marker such as aminoglycoside 3'-phosphotransferase (APFf) can be selected cell growth in medium containing a selection agent for the selectable marker such as an aminoghcosidic antibiotic, e , kanamvcin. neomvcin, or G418 See U S Patent No 4.965, 199 A suitable selection gene for use in veast is the trp 1 gene present in the veast plasmid YRp7 [Stinchcomb et al . Nature. 282 39 (1979). Kingsman et al . Gene. 7 141 ( 1979). Tschemper et al . Ciene. 10 157 (1980)] The trp l gene provides a selection marker for a mutant strain of veast lacking the ability to grow in tryptophan. for example, ATCC No 44076 or PEP4- 1 [Jones, Genetics. 85 12 ( 1977)] The presence of the trp I lesion in the yeast host cell genome then provides an effectiv e environment for detecting transformation by growth in the absence of tryptophan Similarly, Z.<?w2-defιcient veast strains (ATCC 20.622 or 38.626) are complemented by known plasmids beaπng the Leul gene In addition, vectors deπved from the I 6 μm circular plasmid pKDi can be used for transformation of Kluyveromyces veasts fBianchi et al , Curr Genet . 12 185 ( 1987)] More recently, an expression system for large-scale production of recombinant calf chvmosin was reported for K laciis [ Van den Berg, Bio Technologv. 8 135 ( ! 990)] Stable multi-copy expression vectors for secretion of mature recombinant human serum albumin by industrial strains of luweromvces have also been disclosed [Fleer et al . Bio/Technologv. 9 968-975 (1991 )] (iv) Promoter Component Expression and cloning vectors usually contain a promoter that is recognized by the host organism and is operably linked to the Apo-3 or Apo-2LI nucleic acid sequence Promoters are untranslated sequences located upstream (5') to the start codon of a structural gene (generally within about 100 to 1000 bp) that control the transcπption and translation of particular nucleic acid sequence, such as the Apo-3 or Apo-2LI nucleic acid sequence, to which they are operably linked Such promoters t pically fall into two classes, inducible and constitutive Inducible promoters are promoters that initiate increased levels of transcπption from DNA under their control in response to some change in culture conditions, e.g . the presence or absence ot " a nutrient or a change in temperature At this time a large number of promoters recognized bv a vanety of potential host cells are well known These promoters are operably linked to Apo-3 or Apo-2LI encoding DNA bv removing the promoter from the source DNA by restriction enzyme digestion and inserting the isolated promoter sequence into the vector Both the native Apo-3 or Apo-2LI promoter sequence and many heterologous promoters mav be used to direct amplification and or expression of DNA encoding the polypeptide Promoters suitable for use with prokarvotic hosts include the β-lactamase and lactose promoter systems [Chang et al , Nature. 275 617-624 (1978), Goeddel et al , Nature. 2_S_L 544 (1979)], alkaline phosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic Acids Res . 8 4057 ( 1980). EP 36,776], and hybrid promoters such as the tac promoter [deBoer et al , Proc Natl Acad Sci USA. 80 21 -25 ( 1983)] However, other known bacteπal promoters are suitable Their nucleotide sequences have been published, thereby enabling a skilled worker operably to ligate them to DNA encoding Apo-3 or Apo-2LI [Sieben st et al., Cell. 2β:269 ( 1980)] using linkers or adaptors to supply any required restπction sites Promoters for use in bacteπal systems also will contain a Shme-Dalgarno (S D ) sequence operably linked to the DNA Promoter sequences are known for eukarvotes Virtually all eukarvotic genes have an AT-πch region located approximateh 25 to 30 bases upstream from the sue where transcription is initiated Another sequence found 70 to 80 bases upstream from the staπ of transcnption ol many genes is a CXC AAT region where X mav be any nucleotide At the 3' end of most eukarvotic genes is an AATAAA sequence that mav be the signal for addition of the poly A tail to the 3' end of the coding sequence All of these sequences are suitably inserted into eukarvotic expression vectors Examples of suitable promoting sequences for use with veast hosts include Ihe promoters for 3 -phosphoglycerate kinase [Hitzeman et al . J Biol Chem . 255 12073- 12080 ( 1980)] or other glvcolvtic enzymes [Hess et al . J Adv Enzvme Rep . 7 149 ( 1968), Holland. Biochemistry. 12 4900 ( 1 78)]. such as enolase, glyceraIdehvde-3-phosphate dehvdrogenase. hexokinase, pyruvate decarboxvlase, phosphoiructokinase. glucose-6- phosphate isomerase. 3-phosphoglycerate mutase, pyruvate kinase. tπosephosphate isomerase. phosphoglucose isomerase. and glucokinase Other yeast promoters, which are inducible promoters havmg the additional advantage of transcπption controlled by growth conditions, are the promoter regions for alcohol dehvdrogenase 2, lsocvtochrome C. acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehvdrogenase. and enzvmes responsible for maltose and galactose utilization Suitable vectors and promoters for use in yeast expression are further described in EP 73.657 Yeast enhancers also are advantageously used with yeast promoters Transcπption from vectois in mammalian host cells is controlled, for example. b\ promoters obtained from the genomes of viruses such as polyoma virus, fowlpox virus (UK 2,21 1 ,504 published 5 Julv 1989), adenovirus ( such as Adenovirus 2). bovine papilloma virus, avian sarcoma virus, cvtomegalovirus a retrovirus. hepatitis-B virus and most preferably Simian Virus 40 (SV40), from heterologous mammalian promoters, e.g.. the actin promoter or an immunoglobulin promoter, from heat-shock promoters, and from the promoter normally associated with the Apo-3 or Apo-2Ll sequence, provided such promoters are compatible with the host cell systems The early and late promoters of the SV40 virus aie conveniently obtained as an S V40 restriction fragment that also contains the S V40 viral oπgin of replication [Fiers et al , Nature. 273 1 13 ( 1 978). Mulligan and Berg. Science. 209 1422- 1427 ( 1980). Pavlakis et al . Proc Natl Acad Sci USA. 78 7398-7402 ( 1981 )1 The immediate early promoter of the human cvtomegalovirus is conveniently obtained as a Hindlll E restπclion fragment [Greenawav et al., Gene. JjS 355-360 ( 1982)] A system for expressing DNA in mammalian hosts us g the bovine papilloma virus as a vector is disclosed in U S Patent No 4,419,446 A modification of this system is descπbed m U S Patent No 4,601 ,978 [See also Gray et al , Nature. 295 503-508 ( 1982) on expressing cDNA encoding immune interferon in monkey cells. Reyes ct al , Nature. 297 598-601 ( 1982) on expression of human β-interferon cDNA m mouse cells under the control of a thymidine kinase promoter from herpes simplex virus Canaani and Bera. Proc Natl Acad Sci USA 79 5166-5 70 ( 1982) on expression of the human interferon β l gene in cultured mouse and rabbit cells, and Gorman et al , Proc Natl Acad. Sci USA. 22 6777-6781 ( 1982) on expression of bacteπal CAT sequences m C V- 1 monkey kidney cells, chicken embryo fibroblasts, Chinese hamster ovary cells, HeLa cells, and mouse NTH-3T3 cells using the Rous sarcoma virus long terminal repeat as a promoter] (v) Enhancer Element Component Transcπption of a DNA encoding the Apo-3 or Apo-2LI of this invention bv higher eukaryotes mav be increased bv inserting an enhancer sequence into the vector Enhancers are cis-acting elements of DNA, usually about from 10 to 300 bp. that act on a promoter to increase its transcπption Enhancers arc relativelv oπentation and position independent, having been found 5' [Laimins et al , Proc Natl Acad Sci USA. 78 464- 468 (1981 )] and 3' [Lusky et al . Mol Cell Bio . 3 1 108 ( 1983]) to the transcπption unit within an mtron [Banerji et al , Cell.12 729 ( 1983)], as well as within the coding sequence itself fOsbome et al , Mol Cell Bio . 4 1293 ( 1984)] Manv enhancer sequences are now known from mammalian genes (globm elastase. albumin, α- tetoprotein. and insu n) Typically, however, one will use an enhancer from a eukarvotic cell virus Examples include the SV40 enhancer on the late side of the replication ongin (bp 100-270). the cvtomegalovirus carlv promoter enhancer, the polvoma enhancer on the late side of the replication ongin. and adenovirus enhancers ee also Yaniv, Nature. 297 17- 18 ( 1982) on enhancing elements tor activation of eukarvotic promoters The enhancer mav be spliced into the vector at a position 5' or 3' to the coding sequence, but is preferablv located at a site 5 from the promoter (vi) Transcπption Termination Component Expression vectors used in eukarvotic host cells (yeast, fungi, insect, plant animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary tor the termination ot transcπption and for stabilizing the mRNA Such sequences are commonlv available from the 5' and, occasionally 3', untranslated regions of eukarvotic or viral DNAs or cDNAs These regions contain nucleotide segments transcπbed as polvadenylated fragments in the untranslated portion of the mRNA encoding Apo-3 or Apo-2LI (vn) Construction and Analvsis of Vectors Construction of suitable vectors containing one or more of the above-listed components employs standard hgation techniques Isolated plasmids or DNA fragments are cleaved, tailored, and re-hgated in the form desired to generate the plasmids required For analvsis to confirm correct sequences m plasmids constructed the hgation mixtures can be ased to transform E coli 12 strain 294 (ATCC 31 ,446) and successful transformants selected bv a picillin or tetracvcline resistance where appropriate Plasmids from the transtormants are prepared, analyzed bv restπction endonuclease digestion, and or sequenced bv the method of Messing et al , Nucleic Acids Res . 9 309 ( 1981 ) or bv the method of Maxam et al , Methods in Enzvmolopy. 65 499 ( 1980) (vin) Transient Expression Vectors Expression vectors that provide for the transient expression in mammalian cells of DNA encoding Apo-3 or Apo-2LI mav be employed In general, transient expression involves the use of an expression vector that is able to replicate efficiently in a host cell, such that the host cell accumulates many copies of the expression vector and, in tum, synthesizes high levels of a desired polypeptide encoded by the expression vector [Sambrook et al , supra] Transient expression systems, compπsing a suitable expression vector and a host cell, allow for the convenient positive identification of polypeptides encoded bv cloned DNAs, as well as for the rapid screenmg of such polypeptides for desired biological or physiological propeπies Thus, transient expression systems are particularly useful in the invention for purposes of identifying Apo-3 vaπants or Apo-2LI vanants (IX) Suitable Exemplary Vertebrate Cell Vectors Other methods, vectors, and host cells suitable for adaptation to the svnthesis ol Λpo-3 or Apo- 2LI in recombinant vertebrate cell culture are described in Gething et al . Nature. 293 620-625 ( 1981 ). Mantei et al . Nature. 281 40-46 ( 1979). EP 1 17.060, and EP 1 17.058 A particularly useful plasmid for mammalian cell culture expression of Apo-2LI is pRK7 [EP 278,776. also descπbed in Example 1 ] 3 Selection and Transformation of Host Cells Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokarvote veast or higher eukarvote cells descπbed above Suitable prokaryotes for this purpose include but are not limited to eubacteπa, such as Gram-negative or Gram-positive oi ganisms. for example, Enterobacteπaceae such as Eschenchia, e g , E colt. Enterobacter. Envima, Klebsiella Proteus, Salmonella, e g , Salmonella tvphimurium, Serratia. e g , Serratia marcescans, and Shigella. as well as Bacilli such as B subtilis and B licheniformis (e g . B licheniformis 41 P disclosed in DD 266,710 published 12 Apπl 1989), Pseudomonas such as P aeruginosa. and Streptomvces Preferablv, the host cell should secrete minimal amounts of proteolvtic enzvmes In addition to prokarvotes. eukarvotic microbes such as filamentous fungi or veast are suitable cloning or expression hosts Saccharomvces cerevisiae or common baker's yeast is the most commonlv used among lower eukarvotic host microorganisms However, a number of other genera species, and strains are commonlv av ailable and useful herein Suitable host cells for the expression of glvcosvlated polypeptides are den ved from multicellular organisms Such host cells are capable of complex processing and glvcosvlation activities In principle, anv higher eukarvotic cell culture is workable, whether from vertebrate or invertebrate culture Examples ol invertebrate cells include plant and insect cells Numerous baculoviral strains and vaπants and corresponding permissive insect host cells from hosts such as Spodoptera frugiperda (caterpillar), Aedes aegypti (mosquito), Aedes albopictus (mosquito), Dmsophila melanogaster (fruitfly), and Bo bvx mori have been identified [See, e g , Luckow et al Bio/Technolopy . 6 47-55 ( 1988), Miller et al . in Genetic Engineering. Setlow et al , eds , Vol 8 (Plenum Publishing, 1 86), pp 277-279, and Maeda et al , Nature. 3 15 592-594 ( 1985)] A vanetv of viral strains for transfection are publicly available, e g , the L- 1 vaπant ot Autographa califo tca NPV and the Bm-5 strain of Bombvx mori NPV Plant cell cultures ol cotton, com, potato, soybean, petunia, tomato, and tobacco can be utilized as hosts Typically, plant cells are transfected bv incubation with certain strains of the bacterium Agrobacterium tumefaciens During incubation of the plant cell culture with A tumefaciens, the DNA encoding the polypeptide can be transferred to the plant cell host such that it is transfected, and will, under appropnate conditions, express the DNA encoding Apo-3 or Apo-2LI In addition, regulatory and signal sequences compatible with plant cells are available, such as the nopaline svnthase promoter and polyadenvlation signal sequences [Depicker et al , J_ Mol Appl Gen..1 561 (1982)] In addition, DNA segments isolated from the upstream region of the T-DNA 780 gene are capable of activating or increasing transcπption levels of plant-expressible genes in recombinant DNA- contauung plant tissue [EP 321 , 196 published 21 June 1989] Propagation of vertebrate cells in culture (tissue culture) is also well known in the art [See, e g , 1 issue Culture. Academic Press, Kruse and Patterson, editors ( 1973)] Examples of useful mammalian host cell lines are monkev kidnev CV 1 line transformed by SV40 (COS-7, ATCC CRL 1651 ), human embryonic kidney line (293 or 293 cells subcloned for growth in suspension culture. Graham et al , J Gen Virol . 3_6 59 f 1977)) babv hamster kidney cells (BHK ATCC CCL 10), Chinese hamster ovary cell.s/-DHFR (CHO. Urlaub and Chasm. Proc. Natl Acad Sci USA. 22 4216 ( 1980)), mouse serto cells (TM4, Mather, Biol Reprod . 23 243-251 ( 1980)), monkey kidney cells (CV1 ATCC CCL 70), African green monkev kidnev cells ( VERO-76, A TCC CRL- 1587), human cervical carcinoma cells (HELA, ATCC CCL 2). canine kidney cells (MDCK, ATCC CCL 34), buffalo rat liver cells (BRL 3 A. ATCC CRL 1442), human lung cells (W 138. ATCC CCL 75), human liver cells (Hep G2. HB 8065). mouse mammarv tumor (MMT 060562, ATCC CCL51 ). TRI cells (Mather et al , Annals N Y Acad Sci . 383 44-68 ( 1982)). MRC 5 cells, and FS4 cells Host cells are transfected and preferablv transformed with the above-described expression or cloning vectors and cultured in conventional nutnent media modified as appropriate tor inducing promoters selecting transformants, or amplifying the genes encodmg the desired sequences Transfection refers to the taking up of an expression vector bv a host ceil whether or not anv coding sequences are in fact expressed Numerous methods of transfection are known to the ordmanlv skilled artisan, for example. CaP0 4 and electroporation Successful transfection is generally recognized when any indication ot the operation of this vector occurs within the host cell Transformation means introducing DNA into an organism so that the DNA is replicable, either as an extrachromosomal element or by chromosomal integrant Depending on the host cell used, transformation is done using standard techniques appropriate to such cells The calcium treatment employ g calcium chloπde, as described in Sambrook et al , supra, or electroporation is generally used for prokarvotes or other cells that contam substantial cell-wall bamers Infection with Agrobacteriiim tumefaciens is used for transformation ot certain plant cells, as descπbed by Shaw et al , Gene. 21 3 15 ( 1983) and WO 89/05859 published 29 June 1989 In addition, plants may be transfected using ultrasound treatment as descnbed in WO 91 /00358 published 10 January 1991 For mammalian cells without such cell walls, the calcium phosphate precipitation method of Graham and van der Eb, Virology. 52 456-457 (1973) is preferred General aspects of mammalian cell host system transformations have been descπbed in U S Pat No 4,399.216 Transformations into veast are typically earned out according to the method of Van Solingen et al , J Bact . 1 30 946 ( 1977) and Hsiao et al , Proc Natl Acad Sci (USA).26 3829 (1979) However, other methods for introducing DNA into cells, such as by nuclear microinjection. electroporation. bacteπal protoplast fusion with intact cells, or polycations, e g , polybrene. polyomithine, may also be used For various techniques for transforming mammalian cells, see Keown et al . Methods in Enzvmolocv. 185 527-537 ( 1990) and Mansour et al . Nature. 336 348-352 ( 1988) 4 Cultuπng the Host Cells Prokaryotic cells used to produce Apo-3 or Apo-2LI mav be cultured in suitable media as descπbed generally in Sambrook et al , supra The mammalian host cells mav be cultured in a variety of media Examples of commcrciaih available media include Ham's F10 (Sigma), Minimal Essential Medium ("MEM", Sigma), RPM1- 1640 (Sigma), and Dulbecco's Modified Eagle's Medium ("DMEM". Sigma) Anv such media mav be supplemented as necessarv with hormones and/or other growth factors (such as insulin, transfemn, or epidermal growth factor), salts (such as sodium chloπde, calcium, magnesium, and phosphate), buffers (such as HEPES). nucleosides (such as adenosine and thymidine). antibiotics (such as Gentamvcin™ drug), trace elements (defined as inorganic compounds usually present at final concentrations in the micromolar range), and glucose or an equivalent energy source Anv other necessary supplements mav also be included at appropriate concentrations that would be known to those skilled in the art The culture conditions, such as temperature, pH, and the like, are those previously used with the host cell selected for expression, and will be apparent to the ordmanlv skilled artisan In general, principles, protocols, and practical techniques for maximizing the productivity of mammalian cell cultures can be found in Mammalian Cell Biotechnology a Practical Approach. M Butler, ed tIRL Press 1991 ) The host cells referred to in this disclosure encompass cells in culture as well as cells that are within a host animal 5 Detecting Gene Amplification/Expression Gene amplification and/or expression mav be measured in a sample directly, for example, bv conventional Southern blotting, Northern blotting to quantitate the transcπption of mRNA [Thomas, Proc Natl Acad Sci USA. 22 5201 -5205 (1980)], dot blotting (DNA analysis), or in situ hvbπdization. using an appropriately labeled probe, based on the sequences provided herein Vaπous labels mav be employed, most commonlv radioisotopes, and particularly 32 P However, other techniques may also be employed, such as using biotin-modified nucleotides for introduction into a polynucleotide The biotin then serves as the site for binding to avidin or antibodies, which mav be labeled with a wide variety of labels, such as radionucleotides. tluorescers or enzvmes Alternatively, antibodies may be employed that can recognize specific duplexes, including DNA duplexes. RNA duplexes, and DNA-RNA hvbπd duplexes or DNA-protem duplexes The antibodies in turn mav be labeled and the assay may be carried out where the duplex is bound to a surface, so that upon the lormation of duplex on the surface, the presence of antibody bound to the duplex can be detected Gene expression, alternatively, mav be measured by immunological methods, such as immunohistochemical staining of cells or tissue sections and assav of cell culture or bodv fluids, to quantitate directly the expression of gene product With immunohistochemical staining techniques, a cell sample is prepared t pically bv dehydration and fixation, followed by reaction with labeled antibodies specific for the gene product coupled, where the labels are usually visualh detectable, such as enzvmaiic labels, fluorescent labels, or luminescent labels Antibodies useful for immunohistochemical staining and/or assay of sample fluids may be either monoclonal or polyclonal. and may be prepared in any mammal Conveniently, the antibodies mav be prepared against a native sequence Apo-3 polypeptide or Apo-2Ll polypeptide or against a svnthetic peptide based on the DNA sequences provided herein or against exogenous sequence fused to Apo-3 or Apo-2LI DNA and encoding a specific antibody epitope 6 Puπfication of Polypeptide Apo-2Ll preferably is recovered from the culture medium as a secreted polypeptide. although it also mav be recovered from host cell Ivsates when directly produced without a secretory signal Forms of Apo-3 may be recovered from culture medium or from host cell Ivsates If the Apo-3 or Apo-2LI is membrane-bound, it can be released from the membrane using a suitable detergent solution (e g Tπton-X 100) or its extracellular region may be released bv enzymatic cleavage When Apo-3 or Apo-2Ll is produced in a recombinant cell other than one of human oπgm. the polvpeptide is free of proteins or polypeptides of human ongin However it mav be desired to puπfv the polypeptide from recombinant cell proteins or polypeptides to obtain preparations that are substantiallv homogeneous as to Apo-3 or Apo-2Ll As a first step, the culture medium or lvsate mav be centπfuged to remove particulate cell debπs Apo-3 or Apo-2LI thereafter is puπfied from contaminant soluble proteins and polypeptides. with the following procedures being exemplary of suitable purification procedures bv fractionation on an ion-exchange column, ethanol precipitation, reverse phase HPLC. chromatography on silica or on a cation- exchange resin such as DEAE, chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using, for example, Sephadex G-75 and protein A Sepharose columns to remove contaminants such as IgG Apo-3 or Apo-2Ll vaπants in which residues have been deleted, inserted or substituted can be recovered in the same fashion as native sequence polypeptides, taking account of anv substantial changes in properties occasioned by the variation For example, preparation of an Apo-3 fusion with another protein or polypeptide. e g . a bacteπal or viral antigen, immunoglobulin sequence, or receptor sequence, mav facilitate puπfication. an lmmunoaffinity column containing antibodv to the sequence can be used to adsorb the fusion polvpeptide Other types of affinity matnees also can be used A protease inhibitor such as phenvl methvl sulfonvl fluoπde (PMSF) also mav be useful to inhibit proteolvtic degradation dunng puπfication, and antibiotics may be included to prevent the growth of adventitious contaminants One skilled in the an will appreciate that puπfication methods suitable tor the native sequence polypeptide may require modification to account for changes in the character of the polvpeptide or its v aπants upon expression in recombinant cell culture 7 Covalent Modifications of Polypeptides Covalent modifications of Apo-3 or Apo-2LI are included within the scope of this invention One type of covalent modification of these polypeptides is introduced into the molecule by reacting targeted amino acid residues with an organic deπvatizing agent that is capable of reacting with selected side chains or the N- or C- terminal residues Derivatization with bifunctional agents is useful for crosslinking the polypeptide to a water - lnsoluble suppoπ matrix or surtace for use in the method for purifying antι-Apo-3 or antι-Apo-2Ll antibodies, and vice-versa Deπvatization with one or more bifunctional agents will also be useful for crosslinking, for instance. Apo-3 molecules to generate Apo-3 dimers Such dimers may increase binding avidity and extend half-life of the molecule in vivo Commonly used crosslinking agents include, e.g , l, l -bιs(dιazoacetyl)-2-phenylethane, glutaraldehvde, N-hydroxysuccinirrude esters, for example, esters with 4-azιdosalιcylιc acid, homobifunctional lmidoesters. including disuccinimidvl esters such as 3,3'-dιthιobιs(succιnιmιdylpropιonate). and bifunctional maleimides such as bis-N-maleimido- 1 ,8-octane Deπvatizing agents such as methvl-3-[(p-azιdophenvl)- dιthιo]propιoιmιdate yield photoactivatable intermediates that are capable of forming crosslinks in the presence of light Alternatively, reactive water-msoluble matnees such as cyanogen bromide-activated carbohydrates and the reactive substrates descπbed in U S Patent Nos 3,969,287, 3,691 ,016. 4, 195,128, 4.247.642. 4,229,537, and 4.330,440 are employed for protein immobilization Other modifications include dea idation of glutamin l and asparaginyl residues to the corresponding giutamyl and aspartyl residues, respectively, hydroxylation of proline and lys e, phosphorylation of hv droxvl groups of servl or threonvl residues, methviation of the α-ammo groups ot lvsine, argirune, and lustid e side chains [T E Creighton, Proteins. Structure and Molecular Propenies. W 11 Freeman & Co , San Francisco, pp 79-86 ( 1983)], acetylation of d e N-terminal amine. and amidation of anv C-terminal carboxv l group The modified forms of the residues fall within the scope of the present invention Another type of covalent modification included within the scope of this invention compnscs altering the native glvcosylation pattern of the polypeptide "Altering the native glvcosvlation pattern" is intended for purposes herein to mean deleting one or more carbohydrate moieties tound in native sequence Apo-3 or Apo- 2LI, and/or adding one or more glycosylation sites that are not present in the native sequence Apo-3 or Apo-2LI, respectively Glvcosvlation of polypeptides is typically either N-linked or O-linkcd N-hnked refers to the attachment of the carbohvdrate moiety to the side chain of an asparagine residue The tnpeptide sequences asparagine-X-senne and asparagine-X-threonme, where X is anv amino acid except pro ne arc the recognition sequences for enzvmatic attachment of the carbohvdrate moiety to the asparagine side chain Thus, the presence of either of these tnpeptide sequences in a polypeptide creates a potential glvcosvlation site 0-lιnked glv cosv lation refers to the attachment of one of the sugars N-acevlgalactosamine, galactose, or xylose to a hvdroxv lamino acid, most commonlv seπne or threonine, although 5-hvdroxvprolιne or 5-hvdroxvlvsιne mav also be used Addition of glycosylation sites may be accomplished bv altenng the ammo acid sequence such that it contains one or more of the above-descπbed tnpeptide sequences (for N-hnked glvcosvlation sites) The alteration mav also be made the addition of, or substitution bv, one or more seπne or threonine residues to the native sequence Apo-3 or Apo-2LI (for O-linked glycosylation sites) The ammo acid sequence may opuonallv be altered through changes at the DNA level, particularly bv mutating the DNA encoding the Apo-3 or Apo-2LI polypeptide at preselected bases such that codons are generated that will translate into the desired am o acids The DNA mutatιon(s) may be made using methods descπbed above and in U S Pat No 5.364,934, supra Another means of increasing the number of carbohvdrate moieties on the polypeptide is bv chemical or enzvmatic coupling of glvcosides to the poivpeplide Depending on the coupling mode used, the sugar(s) may be attached to (a) argirune and histidine, (b) tree carboxvl groups, (c) free sulfhvdrvl groups such as those of cvsteme, (d) free hvdroxyl groups such as those of senne, direonine, or hydroxyprohne, (e) aromatic residues such as those of phenylalanine, tyrosine, or tryptophan. or (f) the amide group of glutamine These methods are descπbed in WO 87/D5330 published 1 1 September 1987, and m Aplm and Wπston, CRC Cπt Rev Bioche . pp 259-306 ( 1981 ) Removal of carbohydrate moieties present on the polypeptide may be accomplished chemicallv or enzvmaϋcally or bv mutational substitution of codons encodmg for amino acid residues that serve as targets for glvcosvlation For instance, chemical deglvcosylation by exposing the polypeptide to the compound tnfJuoromethanesulfonic acid, or an equivalent compound can result in the cleavage of most or all sugars except the linking sugar (N-acetylglucosamine or N-acetylgalaclosamine), while leaving the polypeptide intact Chemical deglycosvlation is descπbed bv Hakimuddin, et al , Arch Biochem Biophvs , 259 52 ( 1987) and by Edge et al Anal. Biochem . 1 18 131 ( 1981 ) Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved bv the use of a vanetv of endo- and exo-glycosidases as descnbed bv Thotakura et al . Meth Enzvmol . 138 350 ( 1987) Glycosylation at potential glvcosvlation sites mav be prevented bv the use of the compound tunicamycin as descπbed bv Duksm et al , J Biol Chem . 257 3 105-3 109 ( 1982) Tunicamvcin blocks the formation of protein-N-glycoside linkages Another tvpe of covalent modification compπses linking the Apo-3 or Apo-2LI polvpeptide to one of a vanety of nonproteinaceous polymers, e g , polyethylene glycol, polypropylene glycol. or polyoxvalkvlenes. in the manner set forth in U S Patent Nos 4 640,835, 4,496,689 4,301.144. 4.670.417. 4,791.192 or 4, 179,337 8 Chimeπc Molecules The present invention also provides chimeπc molecules compπsing Apo-3 or Apo-2LI fused to anoiJier. heterologous polypeptide or amino acid sequence In one embodiment, the chimeπc molecule compπses a fusion of the Apo-3 or Apo-2LI with a tag polypeptide which provides an epitope to which an anti-tag antibodv can selectively bind The epitope tag is generally placed at the ammo- or carboxvl- terminus ot the Apo-3 or Apo-2Ll. respectivelv The presence of such epitope-tagged forms can be detected using an antibodv against the tag polvpeptide Also, provision of the epitope tag enables the Apo-3 or Apo-2LI to be readily puπfied by affinity purification using an anti-tag antibodv or another type of affinity matπx that binds to the epitope tag Vaπous tag polypeptides and their respective antibodies are well know n in the art Examples include the flu HA tag polypeptide and its antibodv 12CA5 [Field et al , Mol Cell Biol . 8 2159-2165 ( 1988)], the c-myc tag and the 8F9, 3C7, 6E 10, G4. B7 and 9E 10 antibodies thereto [Evan el al . Molecular and Cellular Biology. 5 3610-3616 ( 1985)] . and the Herpes Simplex virus glycoprotein D (gD) tag and its antibody [Paborsky et al , Protein Engiπeenng. 2(6) 547-553 ( 1990)] Other tag polypeptides include the Flag-peptide [Hopp et al , B oTechnology.6 1204-1210 (1988)], the KT3 epitope peptide [Martin et al , Science, 255 192- 194 ( 1992)], an α-tubulin epitope peptide (Skinner et al . J Biol Chem.. 266 14163- 14166 ( 1991 )j, and the T7 gene 10 protein peptide tag [Lutz-Freyermuth et al , Proc Natl Acad Sci USA. 87 6393-6397 ( 1990)] Once the tag polypeptide has been selected, an antibody thereto can be generated using the techniques disclosed herein Generally, epitope-tagged- Apo-3 or Apo-2LI may be constructed and produced according to the methods descnbed above Apo-3 or Apo-2LI-tag polypeptide fusions are preferably constructed by fusing the cDNA sequence encodmg the Apo-3 or Apo-2LI portion in-frame to the tag polypeptide DNA sequence and expressing the resultant DNA fusion construct in appropnate host cells. Ordinaπly, when preparing the tag polypeptide chimeras of the present invention, nucleic acid encoding the Apo-3 or Apo-2LI will be fused at its 3' end to nucleic acid encodmg the N-terminus of the tag polypeptide, however 5' fusions are also possible For example, a polyhistidine sequence of about 5 to about 10 histidine residues may be fused at the N- terminus or the C- terminus and used as a purification handle in affinity chromatography Epitope-tagged Apo-3 or Apo-2LI can be puπfied by affinity chromatography using the anti-tag antibody The matπx to which the affinity antibody is attached mav include, for instance, agarose. controlled pore glass or poly(styτenedιvmyl)benzene The epitope-tagged polypeptide can then be eluted from the affinity column using techniques known in the art In another embodiment, the chimeric molecule compπses an Apo-3 or Apo-2LI polypeptide fused to an immunoglobulin sequence or other heterologous sequence Optionally, the chimeric molecule composes an Apo-2LI fused to an immunoglobulin constant domain or TNFR sequence The chimeric molecule mav also compπse a particular domam sequence of Λpo-3, such as the extracellular domain sequence of native Apo-3 fused to an immunoglobulin sequence This includes chimeras in monomeric, homo- or heteromultimenc, and particularly homo- or heterodimenc, or -tetrameπc forms, optionally, the chimeras mav be in dimeπc torms or homodimeπc heavy chain forms Generally, these assembled immunoglobulins will have known unit structures as represented bv the following diagrams X or A \ Cu or C, X or A \ Y 1 or C A A \ C \ Cu A v L A \ C L \ C H X A \ A X \ l \ Cu A basic four chain structural unit is the form in which IgG, IgD, and IgE exist A four chain unit is repealed in the higher molecular weight immunoglobulins, lgM generally exists as a pentamer of basic four- chain units held together by disulfide bonds IgA globulin, and occasionally IgG globulin, may also exist in a multimeπc foπn in serum In the case of multimers, each four chain unit may be the same or different The following diagrams depict some exemplary monomer, homo- and helerodimer and homo- and heteromulϋmer structures These diagrams are merely illustrative, and the chains of the multimers are believed to be disulfide bonded in the same fashion as native immunoglobulins monomer : A C, or C homodimer : A \ c or C- Cr or C / A heterodimer : A \ c or C c- or Cv / X A homotetramer . A \ c. \ Cr or C„ c- or C, / c A / A A heterotetramer : X and A X In the foregoing diagrams, "A" means an Apo-3 sequence, Apo-2LI sequence, or an Apo-3 or Apo-2LI sequence fused to a heterologous sequence. X is an additional agent, which may be the same as A or different a portion of an immunoglobulin superfamily member such as a vaπable region or a vanable region-like domain, including a native or chimeπc immunoglobulin vaπable region, a toxin such a pseudomonas exotoxin or πcin. or a sequence functionally binding to another protein, such as other cvtokmes (I e IL- 1 , mterferon-γ ) or cell surtace molecules d e , NGFR, CD40, OX40, Fas antigen, T2 proteins of Shope and mvxoma poxviruses), or a polypeptide therapeutic agent not otherwise normally associated with a constant domam, Y is a linker or another receptor sequence, and V L , V H , C L and C H represent light or heavy cham vaπable or constant domains of an immunoglobulin Structures compnsing at least one CRD of an Apo-3 or Apo-2LI sequence as ' A' and another cell-surface protein having a repetitive pattern of CRDs (such as TNFR) as "X" are specifically included It will be understood that the above diagrams are merelv exemplary ot the possible structures ot the chimeras of the present invention, and do not encompass all possibilities For example there might desirabh be several different "A"s, "X"s. or "Y"s in anv of these constructs Also, the heaw or light chain constant domains mav be oπginated from the same or different immunoglobulins All possible permutations ot the illustrated and similar structures are all within the scope of the invention herein In general, the chimeπc molecules can be constructed in a fashion similar to chimcπc antibodies which a variable domam from an antibodv ot one species is substituted for the vaπabie domam of another species See. for example, EP 0 125023. EP 173,494, Munro, Nature. 3 12 597 ( 13 December 1984). Neuberger et al . Nature. 3 12 604-608 ( 13 December 1984), Sharon et al , Nature. 309 364-367 (24 Mav 1984) Momson et al , Proc Nat'l Acad Sci USA. 81 6851 -6855 ( 1984), Momson et al , Science. 229 1202- 1207 ( 1985), Boulianne et al , Nature. 312 643-646 ( 13 December 1984), Capon et al , Nature. 337 525-53 1 ( 1989) , Traunecker et al . Nature. 339 68-70 ( 1989) Alternatively, the chimeric molecules may be constructed as follows I he DNA including a region encoding the desired sequence, such as an Apo-3. Apo-2LI and or TNFR sequence, is cleaved bv a restπction enzvme at or proximal to the 3 end ol the DNA encoding the immunoglobulin-like domain* s) and at d point at or near the DNA encoding the N-terminal end of the Apo-3, Apo-2LI, or TNFR polvpeptide (where use of a different leader is contemplated) or at or proximal to the N-terminal coding region for TNFR (where the native signal is employed) This DNA fragment then is readilv inserted proximal to DNA encoding an immunoglobulin light or heaw chain constant region and, if necessary, the resulting construct tailored bv deletional mutagenesis Preferablv, the Ig is a human immunoglobulin when the chimeπc molecule is intended for in vivo therapy tor humans DNA encodmg immunoglobulin light or heaw chain constant regions is known or readilv av ailable from cDNA libranes or is synthesized See for example, Adams et al , Biochemistry. J_9 271 1 -271 ( 1980), Gough et al . Biochemistry. 19 2702-2710 (1980). Dolbv et al . Proc Natl Acad Sci USA. 77 6027-603 1 ( 1980), Rice et al , Proc. Natl Acad Sci . 79 7862-7865 ( 1982), Falkner et al , Nature. 298 286-288 ( 1982). and Momson e al . Ann. Rev Immunol . 2 239-256 ( 1984) Further details of how to prepare such fusions are lound in publications concerning the preparation of tmmunoadhesins lmmunoadhesins in general, and CD4-Ig fusion molecules specifically are disclosed in WO 8902922, published 6 Apnl 1989) Molecules compnsing the extracellular portion of CD4, the receptor for human immunodeficiency virus (HIV), linked to IgG heavy chain constant region are known in the art and have been found to have a markedlv longer half-life and lower clearance than the soluble extracellular portion of CD4 [Capon et al , supra. Bvm et al , Nature. 344 667 (1990)] The construction ot specdic chimenc TNFR-IgG molecules is also descnbed in Ashkenazi et al Proc Natl Acad Sci.. 88 10535- 10539 ( 1991 ), Lesslauer et al [J Cell Biochem Supplement 15F. 1991 , p 1 15 (F 432)], and Peppel and Beutler, J Cell Biochem Supplement 15F. 1991. p 1 18 (P 439)] B Therapeutic and Non-therapeutic Uses lor Apo-3 and Apo-2LI Apo-3, as disclosed m the present specification, can be employed therapeutically to induce apoptosis or NF-κB or JNK mediated gene expression in mammalian cells This therapy can be accomplished for instance, usmg in vivo or ex vivo gene therapy techniques and includes the use of the death domam sequences disclosed herein The Apo-3 chimeric molecules (including the chimeπc molecules containing the extracellular domain sequence of Apo-3) compπsing immunoglobulin sequences can also be employed therapeuticallv to inhibit apoptosis or NF-κB induction or JNK activation Λpo-2LI. as disclosed in the application, can be emploved therapeuticallv to inhibit mammalian cell apoptosis in vivo or ex vivo Generally , the methods compπse exposing the cells to an effective amount of the Apo-2LI The Apo-3 and Apo-2LI of the invention also have utility in non-therapeutic applications Nucleic acid sequences encoding the Apo-3 or Apo-2LI may be used as a diagnostic for tissue-specific typing For example, procedures like in situ hybπdization, Northern and Southern blotting, and PCR analvsis mav be used to determine whether DNA and or RNA encoding the pol peptide is present in the cell tvpe(s) being evaluated Apo-3 or Apo-2LI nucleic acid will also be useful for the preparation of Apo-3 or Apo-2LI, respectively bv the recombinant techniques descπbed herein The isolated Apo-3 or Aρo-2LI mav be used m quantitative diagnostic assays as a control against which samples containing unknown quantities of Apo-3 or Apo-2LI mav be prepared Apo-3 preparations are also useful in generating antibodies, as standards in assavs for Apo-3 or Apo-2LI (e.g , by labeling Apo-3 or Apo-2LI for use as a standard in a radioimmunoassav, radioreceptor assav. or enzyme-linked immunoassay), in affinity purification techniques, and in competitive-tvpe receptor binding assavs when labeled with, for instance, radioiodme. enzymes, or tluorophores Modified forms of the Apo-3, such as the Apo-3-IgG chimeπc molecules ( lmmunoadhesins ) descπbed above, can be used as lmmunogens m producing antι-Apo-3 antibodies Nucleic acids which encode Apo-3 or its modified forms can also be used to generate either transgemc animals or "knock out" animals which, in turn, are useful in the development and screening of therapeuticallv useful reagents A transgemc animal (e g , a mouse or rat) is an animal having cells that contain a transgene, which transgene was mtroduced into the animal or an ancestor of the animal at a prenatal, e g , an embrvonic stage A transgene is a DNA which is integrated into the genome of a cell from which a transgemc animal develops In one embodiment, cDNA encoding Apo-3 or an appropriate sequence thereof can be used to clone genomic DNA encoding Apo-3 in accordance with established techniques and the genomic sequences used to generate transgemc animals that contain ceils which express DNA encoding Apo-3 Methods for generating transge c animals, particularly animals such as mice or rats, have become conventional in the art and are descπbed, for example, in U.S Patent Nos 4,736,866 and 4,870,009 Typically, particular cells would be targeted for Apo-3 transgene incorporation with tissue-specific enhancers Transgemc animals that include a copy of a transgene encoding Apo-3 mtroduced into the germ line of the animal at an embrvonic stage can be used to examine the effect of increased expression of DNA encodmg Apo-3 Such animals can be used as tester animals for reagents thought to confer protection from, for example, pathological conditions associated with excessive apoptosis In accordance with this facet of the invention, an animal is treated with the reagent and a reduced incidence of the padiological condition, compared to untreated animals beaπng the transgene. would indicate a potential therapeutic intervention for the pathological condition In another embodiment, transgemc animals that carry a soluble form of Apo-3 such as the Apo-3 ECD or an immunoglobulin chimera of such form could be constructed to test the effect of chronic neutralization of the ligand of Aρo-3 Alternatively, non-human homologues of Apo- 3 can be used to construct a Apo-3 "knock out" animal which has a defective or altered gene encoding Apo-3 as a result of homologous recombination between the endogenous gene encodmg Apo-3 and altered genomic DNA encoding Apo-3 introduced into an embrvonic cell of the animal For example, cDNA encoding Apo-3 can be used to clone genomic DNA encoding Apo-3 m accordance with established techniques A portion of the genomic DNA encoding Apo-3 can be deleted or replaced with another gene, such as a gene encoding a selectable marker which can be used to monitor integration 1 vpicallv. several kilobases of unaltered flanking DNA (both at the 5' and 3' ends) are included in the vector [see e g , Thomas and Capecchi. Cell. 51 503 ( 1987) for a description of homologous recombination vectors] The vector is introduced into an embrvonic stem cell line (e g , by electroporation) and cells in which the introduced DNA has homologouslv recombined with the endogenous DNA are selected [see e g . Li et al . Cell. 69 915 ( 1992)] The selected cells are then iniected into a blastocv t of an animal (e . a mouse or rat) to form aggregation chimeras [see e g , Bradlev, in Teratocarcinomas and Embrvonic Stem Cells A Practical Approach, E J Robertson ed (IRL, Oxford, 1987), pp 1 13- 151 ] A chimeric embryo can then be implanted into a suitable pseudopregnant female foster animal and the embrvo brought to term to create a "knock out" animal Progeny harboπng the homologouslv recombined DNA in their germ cells can be identified bv standard techniques and used to breed animals in which all cells of the animal contain the homologouslv recombined DNA Knockout animals can be characterized for instance for their abihtv to defend against certain pathological conditions and for their development ot pathological conditions due to absence ol the Apo-3 polvpeptide. including lor example, development of tumors C Antibodv Preparation The present invention further provides antι-Apo-3 antibodies and antι-Apo-2LI antibodies Such anubodies may be prepared as follows Exemplary antibodies include polyclonal. monoclonal, humanized bispecific. and heteroconjugate antibodies 1 Polvclonal Antibodies The antibodies mav compπse polyclonal antibodies Methods of preparing polyclonal antibodies are known to the skilled artisan Polvclonal antibodies can be raised in a mammal, for example, bv one or more inactions of an immumzing agent and. if desired, an ad)uvant Typically, the immunizing agent and/or adjuvant will be injected in the mammal bv multiple subcutaneous or intrapeπtoneal iniections The immunizing agent mav include the Apo-3 or Apo-2LI polypeptide or a fusion protein thereof An example ot a suitable immunizmg agent is a Apo-3-lgG fusion protein or chimeric molecule (including an Apo-3 ECD-IgG fusion protein) Cells expressing Apo-3 at their surface may also be emplovcd It may be useful to conjugate the immunizing agent to a protein known to be immunogeruc in the mammal bemg immunized Examples of such immunogenic proteins which may be employed include but are not limited to kevhole limpet hemocvamn, serum albumin, bovine thvroglobuhn, and soybean trypsin inhibitor An aggregating agent such as alum mav also be employed to enhance the mammal's immune response Examples of adjuvants which may be employed include Freund's complete adjuvant and MPL-TDM adjuvant (monophosphorvl Lipid A synthetic trehalose dicorvnomvcolate) The immunization protocol may be selected by one skilled in the art without undue experimentation The mammal can then be bled, and the serum assayed for antibodv liter If desired, the mammal can be boosted until the antibodv titer increases or plateaus 2. Monoclonal Antibodies The antibodies mav, alternatively, be monoclonal antibodies Monoclonal antibodies may be prepared using hybridoma methods, such as those descπbed by Kohler and Milstein, supra In a hybπdoma method, a mouse, hamster, or other appropnate host animal, is typically immunized (such as described above > with an immunizing agent to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the immunizing agent Alternatively, the lymphocytes may be immunized in vitro The immunizing agent will typically include the Apo-3 or Apo-2LI polvpeptide or a fusion protein thereof An example of a suitable immunizing agent is a Apo-3-IgG fusion protein or chimeπc molecule Cells expressing Apo-3 or Apo-2Ll at their surface may also be employed. Generally, either peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol. to form a hybndoma cell [Goding, Monoclonal Antibodies Pπnciples and Practice. Academic Press, ( 1986) pp. 59-103]. Immortalized cell lines are usually transformed mammalian cells, particularly myeloma cells of rodent, bovine and human ongin. Usually, rat or mouse mveloma cell lines are employed The hybridoma cells may be cultured in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused. immortalized cells For example, if the parental cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT). the culture medium for the hybπdomas typically will include hypoxanthine, aminopteπn, and thymidine ("HAT medium "), which substances prevent the growth of HGPRT-deficient cells. Preferred immortalized cell lines are those that fuse efficiently, support stable high level expression of antibody by the selected antibody-producing cells, and are sensitive to a medium such as HAT medium. More preferred immortalized cell lines are muπne myeloma lines, which can be obtained, for instance, from the Salk Institute Cell Distnbution Center, San Diego, California and the Ameπcan Type Culture Collection. Rockville. Maryland. Human myeloma and mouse-human heteromyeloma cell lines also have been described for the production of human monoclonal antibodies [Kozbor, J Immunol . 111:3001 ( 1984). Brodeur et al . Monoclonal Antibodv Production Techniques and Applications. Marcel Dekker. lnc , New York. ( 1987) pp 51 - 63]. The culture medium in which the hybridoma cells are cultured can then be assayed for the presence of monoclonal antibodies directed against Apo-3 or Apo-2LI Preferably, the binding specificity of monoclonal antibodies produced by the hybπdoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA) Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can. for example, be determined by the Scalchard analysis of Munson and Pollard, Anal. Biochem..107:220 ( 1980) After the desired hybridoma cells are identified, the clones may be subcloned bv limiting dilution procedures and grown by standard methods [Goding, supral. Suitable culture media for this purpose include, for example, Dulbecco's Modified Eagle's Medium and RPMI- 1640 medium Alternatively, the hybridoma cells mav be grown in vivo as ascites in a mammal. The monoclonal antibodies secreted by the subclones mav be isolated or purified from the culture medium or ascites fluid bv conventional immunoglobulin purification procedures such as. for example. protein A-Sepharose. hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography The monoclonal antibodies may also be made by recombinant DNA methods, such as those dcscπbed in U S Patent No 4,816,567 DNA encoding the monoclonal antibodies of the invention can be readily isolated and sequenced using conventional procedures (e g , bv usmg oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of munne antibodies) The hvbndoma cells of the invention serve as a preferred source of such DNA Once isolated, the DNA may be placed into expression vectors, which are then transfected into host cells such as simian COS cells. Chinese hamster ovary (CHO) cells, or mveloma cells that do not otherwise produce immunoglobulm protein, to obtam the synthesis of monoclonal anubodies in the recombinant host cells The DNA also may be modified, for example, bv substituting the coding sequence for human heavy and light chain constant domains in place of the homologous munne sequences [U S Patent No 4,816.567, Momson et al , supral or by covalentiy loining to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobu n polypeptide Such a non-rmmunoglobulin polvpeptide can be substituted for the constant domains of an antibody of the invention, or can be substituted for the vaπable domains ot one anti en-combining site of an antibodv of the invention to create a chimeπc bivalent antibodv The antibodies mav be monovalent antibodies Methods for prepanng monovalent antibodies are well known in the art For example, one method involves recombinant expression of immunoglobulin light chain and modified heavy chain The heaw chain is truncated generally at any point in die Fc region so as to prevent heaw cham crosslinking Alternatively, the relevant cysteme residues are substituted with another amino acid residue or are deleted so as to prevent crosslinking In vitro methods are also suitable for preparing monovalent antibodies Digestion ot antibodies to produce fragments thereof, particularly. Fab fragments, can be accomplished using routine techniques known in the art For instance, digestion can be performed using papain Examples of papain digestion are descπbed in WO 94/29348 published 12/22/94 and U S Patent No 4,342,566 Papain digestion of antibodies typically produces two identical antigen binding fragments, called Fab fragments, each with a single antigen binding site, and a residual Fc fragment Pepsin treatment yields an F(ab') 2 fragment that has two antigen combining sues and is still capable of cross-linking antigen. The Fab fragments produced in the antibody digestion also contain the constant domains of the light chain and the first constant domain (CH j ) of the heavy cham F ab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH ] domain including one or more cysteines from the antibodv hinge region Fab'-SH is the designation herein for Fab' in which the cysteme residue! s) of the constant domains bear a free thiol group F(ab') 2 antibody fragments onginaliv were produced as pairs of Fab' fragments which have h ge cysteines between them Other chemical couplings of antibody fragments are also known 3 Humanized Antibodies The antibodies of the invention may further compπse humanized antibodies or human antibodies. Humanized forms of non-human (e g , munne) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (such as Fv, Fab, Fab', F(ab') 2 or other antigen-binding subsequences of antibodies) which contain minimal sequence den ved from non-human immunoglobulm Humanized antibodies include human immunoglobulins (recipient antibodv) in which residues from a complementary determining region (CDR) of the recipient are replaced by residues from a CDR of a non-human species (donor antibodv) such as mouse, rat or rabbit having the desired specificity, affinity and capacity In some instances. Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues Humanized antibodies mav also compπse residues which are found neither in the recipient antibodv nor in the imported CDR or framework sequences In general, the humanized antibodv ill comprise substantiallv all of at least one. and tvpicallv two variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are those of a human immunoglobulm consensus sequence The humanized antibodv optimally also will compπse at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulm [Jones et al , Nature. 321 522-525 ( 1986), Riechmann et al , Nature. 332 323-327 ( 1988). and Presta, Curr Op Struct Biol . 2 593-596 ( 1992)] Methods for humanizing non-human antibodies are well known in the art Generallv, a humanized antibodv has one or more amino acid residues introduced into it from a source which is non-human These non-human ammo acid residues are often refened to as "import' 1 residues, which are tvpicallv taken from an "import ' vanable domain Humanization can be essentiallv performed following the method of Winter and co- workers [Jones et al , Nature.121.522-525 ( 1986), Riechmann et al , Nature. 332 323-327 ( 1988) Verhoeven et al , Science, 239 1534- 1536 ( 1988)], by substituting rodent CDRs or CDR sequences for the conesponding sequences of a human antibody Accordingly, such "humanized" antibodies are chimeric antibodies ( U S Patent No 4.816,567). wherein substantially less than an intact human vaπable domain has been substituted bv the corresponding sequence from a non-human species In practice, humanized antibodies are typically human antibodies in which some CDR residues and possiblv some FR residues are substituted bv residues from analogous sites in rodent antibodies The choice of human vaπable domains, both light and heavy, to be used in making the humanized antibodies is very important in order to reduce antigenicity According to the "best-fit" method, the sequence of the variable domain of a rodent antibodv is screened against the entire library of known human variable domain sequences The human sequence which is closest to that of the rodent is then accepted as the human framework (FR) for the humanized antibody [Sims et al , J Immunol . 151 2296 ( 1 93). Chothia and Lesk, J Mol Biol . 1 6 901 ( 1987)] Another method uses a particular framework deπved from the consensus sequence of all human antibodies of a particular subgroup of light or heaw chains The same framework mav be used for several different humanized antibodies [Carter et al , Proc Natl Acad Sci USA. 89 4285 ( 1992). Presta et al . J Immunol . 151 2623 (1993)] It is further important that antibodies be humanized with retention of high affinitv tor the antigen and other favorable biological properties To achieve this goal, according to a preferred method, humanized antibodies are prepared by a process of analvsis of the parental sequences and vaπous conceptual humanized products using three dimensional models of the parental and humanized sequences Three dimensional immunoglobulin models are commonly available and are familiar to those skilled in the art Computer programs are available which illustrate and display probable three-dimensional conformational structures of selected candidate immunoglobulin sequences Inspection of these displays permits analysis of the likely role of the residues in the functioning of the candidate immunoglobulm sequence.1 e . the analvsis ot residues that influence the ability of the candidate immunoglobulin to bind its antigen In ihis wav, FR residues can be selected and combined from the consensus and import sequence so that the desired antibodv characteπstic. such as increased affinity for the target antιgen(s), is achieved In general, the CDR residues are directlv and most substantially involved in influencing antigen binding [see. WO 94/04679 published 3 March 1994] Transgemc animals (e g , mice) that are capable, upon immunization, ot producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production can be employed For example, it has been descπbed that the homozygous deletion of the antibodv heaw chain loirung region (J H ) gene in chimeπc and germ-line mutant mice results in complete inhibition of endogenous antibodv production Transfer of the human germ-line immunoglobulin gene anay in such germ-line mutant mice will result in the production of human antibodies upon antigen challenge [see, e g , Jakobovits et al , Proc Natl Acad Sci USA. 90 2551 -255 ( 1993), Jakobovits et al , Nature. 362 255-258 ( 1993). Bruggemanπ et al , Year in Immune. 7 33 ( 1993)] Human antibodies can also be produced m phage displav libranes [Hoogenboom and Winter, J Mol Biol . 22-7 381 ( 19911. Marks et al . J Mol Bιol .222 581 ( 1991 )] The techniques of Cole et al and Boerner et al are also available for the preparation of human monoclonal antibodies (Cole et al , Monoclonal Antibodies and Cancer Therapy. Alan R Liss, p 77 ( 1985 ) and Boerner et al , J Immunol . 147( 1 ) 86-95 ( 1991 )] 4 Bispecific Antibodies Bispecific antibodies are monoclonal, preferably human or humanized, antibodies that have binding specificities for at least two different antigens In the present case, one of the binding specificities is for the Apo-3 or the Apo-2LI. the other one is for anv other antigen, and preferably for a cell-surtace protein or receptor or receptor subunit Methods for making bispecific antibodies are known in the art Traditionally, the recombinant production of bispecific antibodies is based on the co-expression of two immunoglobulm heaw-chain/ ght-chain pairs, where the two heaw chains have different specificities [Milstein and Cuello, Nature. 305 537-539 ( 1983)] Because of the random assortment of immunoglobulin heaw and light chains, these hvbridomas (quadromas) produce a potential mixture of ten different antibodv molecules, of which onlv one has the correct bispecific structure The purification of the conect molecule is usually accomplished bv affinitv chromatography steps Similar procedures are disclosed in WO 93/08829, published 13 May 1993. and in Traunecker et al , EMBO J . 10 3655-3659 ( 1991 ) According to a different and more prefened approach, antibodv variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulm constant domain sequences The fusion preferably is with an immunoglobulm heavy-chain constant domain, compnsing at least part of the hinge. CH2. and CH3 regions It is preferred to have the first heaw-cham constant region (CHI ) containing the site necessarv for light-chain binding present m at least one of the fusions DNAs encoding the immunoglobulm heavy-chain fusions and, if desired, the lrnmunoglobulin light chain, are inserted into separate expression vectors, and are co-transfected into a suitable host organism This provides for great flexibility in adiusting the mutual proportions of the three polypeptide fragments in embodiments when unequal ratios of the three polypeptide chains used in the construction provide the optimum yields It is, however, possible to insert the coding sequences for two or all three polypeptide chains in one expression vector when the expression of at least tw o polypeptide chains in equal ratios results in high vields or when the ratios are of no particular significance in a pretened embodiment of this approach, the bispecific antibodies are composed ol a hybrid immunoglobulm heaw chain with a first binding specificity in one arm. and a hvbrtd immunoglobulin heavy - chain/light-chain pair (providing a second binding specificity ) in the other arm It was found that this asvmmetπc structure facilitates the separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, as the presence of an immunoglobulm light chain only one half of the bispecific molecule provides for a facile wav ot separation This approach is disclosed in WO 94/04690 published 3 March 1994 For lurther details of generating bispecific antibodies see. for examp le Suresh et al , Methods in Enzvmologv. 121 210 ( 1986) 5 Heteroconiugate Antibodies Heteroconjugate antibodies are also within the scope of the present invention Heteroconjugate antibodies are composed of two covalentiy joined antibodies Such antibodies have, lor example, been proposed to target immune system cells to unwanted cells [US Patent No 4,676,980], and for treatment of HIV infection [WO 91/00360, WO 92 20373. EP 03089] It is contemplated that the antibodies mav be prepared in vitro using known methods in synthetic protein chemistry, including those involving crosslinking agents For example immunotoxins mav be constructed using a disulfide exchange reaction or by forming a thioether bond Examples of suitable reagents for this purpose include iminothiolate and methvl-4-mercaptobutvπmιdaie and those disclosed for example, in U S Pat No 4.676,980 D Antibodv Therapeutic and Non-therapeutic Uses The antibodies of the invention have therapeutic utility Agonistic Apo-3 antibodies, for instance, mav be employed to activate or stimulate apoptosis in cancer cells Alternatively, antagonistic antibodies may be used to block excessive apoptosis (for instance in neurodegenerative diseasel or to block potential autoimmune/inflammatory effects of Apo-3 resulting from NF-κB activation and/or JNK activation The antibodies may further be used in diagnostic assays For example, Apo-3 antibodies or Apo-2LI antibodies mav be used in diagnostic assavs for Apo-3 or Apo-2LI, respectivelv, e g . detecting its expression in specific cells, tissues, or serum Vaπous diagnostic assav techniques known in the art mav be used such as competitive binding assays, direct or indirect sandwich assavs and immunoprecipitation assavs conducted in either heterogeneous or homogeneous phases [Zola, Monoclonal Antibodies A Manual of Techniques. CRC Press, Inc ( 1987) pp 147- 158] The antibodies used in the diagnostic assays can be labeled with a detectable moietv The detectable moietv should be capable of producmg, either directly or indirectly, a detectable signal For example, the detectable moiety may be a radioisotope, such as 3 H, C, 32 P, 35 S, or ,25 I. a fluorescent or chemiluminescent compound, such as fluorescem isothiocvanate. rhodamine, or lucifenn, or an enzvme, such as alkaline phosphatase beta-galactosidase or horseradish peroxidase Anv method known in the art for conjugating the antibody to the detectable moiety mav be employed, including those methods descπbed bv Hunter et al , Nature. 144 945 ( 1962), David et al . Biochemistry. 13 1014 ( 1974), Pam et al . J Immunol Meth . 40 219 ( 1981 ). and Nygren, J Histoche and Cvtochem . 1Q 407 ( 1 82) Apo-3 or Apo-2LI antibodies also are useful for the affinity punfication of Apo-3 or Apo-2LI from recombinant cell culture or natural sources In this process, the antibodies are immobilized on a suitable support, such a Sephadex resin or filter paper, using methods well known in the art The immobilized antibodv then is contacted with a sample containing the Apo-3 or Apo-2LI to be purified, and thereafter the support is washed with a suitable solvent that will remove substantially all the material in the sample except the Apo-3 or Apo-2LI. which is bound to the immobilized antibody Finally, the support is washed with another suitable solvent that will release the Apo-3 or Apo-2LI from the antibody E. Kits and Articles of Manufacture In a further embodiment of the invention, there are provided articles of manufacture and kits containing Apo-3, Apo-2LI, or Apo-3 or Apo-2LI antibodies which can be used, for instance, for the therapeutic or non-therapeutic applications described above. The article of manufacture compπses a container with a label Suitable containers include, for example, bottles, vials, and test tubes The containers mav be formed from a vanety of materials such as glass or plastic The container holds a composition which mcludes an active agent that is effective for therapeutic or non-therapeutic applications, such as descπbed above The active agent in the composition is Apo-3 or Apo-2Ll, or an Apo-3 or Apo-2LI antibody. The label on the container indicates that the composition is used for a specific therapy or non-therapeutic application, and may also indicate directions for either in vivo or in vitro use, such as those descπbed above The kit of the invention will tvpicallv comprise the container descπbed above and one or more other containers compπsing mateπals desirable from a commercial and user standpoint, including buffers, diluents. filters, needles, syringes, and package inserts with instructions for use ***************************** The following examples are offered for illustrative purposes only, and are not intended to limit the scope of the present invention in any way All references cited in the present specification are hereby incorporated bv reference in their entiret EXAMPLES All restriction enzymes referred to in the examples were purchased from New England Biolabs and used according to manufacturer's instructions. All other commercially available reagents referred to in the examples were used according to manufacturer's instructions unless otherwise indicated. The source of those cells identified in the following examples, and throughout the specification, by ATCC accession numbers is the Ameπcan Type Culture Collection, Rockville, Maryland. EXAMPLE I Isolation of cDNA clones Encoding Human Apo-2LI To isolate a cDNA for Apo-2LI, a lambda gtIO bactenophage library of human thymus cDNA (about 1 10° clones) (HL 1074a. commercially available from Clontech) was screened by hybridization with synthetic oligonucleotide probes based on an EST sequence (GenBank locus H41522), which showed some degree of homology to human Fas/ Apo- 1. The EST sequence of H41522 is 433 bp and when translated in its + 1 frame. shows 20 identities to a 78 ammo acid region of human Fas/ Apo- 1. The sequence of H41522 is as follows: CTGCTGGGGGCCCGGGCCAGNGGCGGCACTCGTAGCCCCAGGTGTGACTGTGCCGGTGAC TTCCACAAGAAGATTGGTCTGTTTTGTTGCAGAGGCTGCCCAGCGGGGCAACTACCTGAA GGCCCCTTGCACGGAGCCCTGCGCAACTCCACCTGCCTTGTGTGTCCCCAAGACACCTTC TTGGCCTGGGAGAACCACCATAATTCTGAATGTGCCCGCTGCCAGGCCTGTGATGAGCAG GCCTCCCAGGTGGCGCTGGAGAACTGTTCAGCAGTGGCCGACACCCGCTGTGGCTGTAAG CAGGGCTGGTTTGTGGAGTGCCAGGGTCAGCCAA I GTGT AGC AGTTTC ACCCTTC ' I AAT GCCAACCATGCCTAGACTGCGGGGCCCTGCAACGCAACACACGGCTΛATNTGTTTCCCGC AGAGATNATTGTT (SEQ ID NO 2) The obgonucleotide probes employed m the screening were 28 bp long, with the following respective sequences CCCGCTGCCAGGCCTGTGATGAGCAGGC (SEQ ID NO 3) CAGGGCCCCGCAGTCTAGGCATGGTTGG (SEQ ID NO 4) Hybridization was conducted with a I 1 mixture of the two probes overnight at room temperature in buffer containing 20% formamide.5X SSC, 10% dextran sulfate, 0 1% NaPιP0 4 , 0 05M NaP0 4 . 0 05 mg salmon sperm DNA and 0 1 % sodium dodecvl sulfate. followed consecutively bv one wash at room temperature in 6X SSC t o washes at 37" C in 1 X SSC/0 1 % SDS, two washes at 37" C in 0 5X SSC/0 I % SDS, and two washes at 37" C in 02X SSC 0 1% SDS Four positive clones were identified in the cDNA library, and the positive clones were confirmed to be specific by PCR using the above hybπdization probes as PCR primers Single phage plaques containing each of the four positive clones were isolated by limiting dilution and the DNA was purified using a Wizard Lambda Prep DNA purification kit (commercially available from Promega) 1 he cDNA inserts from the four bactenophage clones ere excised from the vector arms bv ώgestion with EcoRJ. gel-puπfied. and subcloned into pRK7 [EP 278,776 published August 17 1988] that was predigested with EcoRI Three of the clones ( 18 1. 24 1. and 28 1 ) contained an identical open reading frame, therefore further analvsis was done with only one clone, 18 1 Clone 18 1 was approximately 1 4kb long Theenure nucleotide sequence of Apo-2LI is shown in Figure I (SEQ ID NO 5) The cDNA contained one open reading frame with a translational initiation site assigned to the ATG codon at nucleotide positions 377-379 The surrounding sequence at this site is in reasonable agreement with the proposed consensus sequence for initiation sites [Kozak, J. Cell. Biol . 1 15 887-903 (1 91)] The open reading frame ends at the termination codon TAA at nucleotide positions 919-921 The predicted ammo acid sequence of the Apo-2LI encoded bv clone 18 I contains 181 amino acids, and has a calculated molecular weight of approximately 19 3 kDa and an lsoelectnc point ol approximatelv 7 I Hvdropathv analvsis indicated the presence of a hvdrophobic signal sequence at the N-terminus ot approximately 20 ammo acids Two potential N-hnked glvcosvlation sites are located at residues 67 and 105 of the polvpeptide precursor An alignment (using the Align' computer program) of the amino acid sequence encoded by clone 18 1 with the extracellular regions of other known members of the human TNF receptor tamilv showed the following percentages of identity 30 2% identity to Fas/ Apo- 1 , 28 7% to tvpe I TNF receptor (TNFR 1 ), 22 5% to the low affinity NGF receptor (LNGFR) and to CD40, 21 8% to CD30. 21 5% to CD27. 21 4% to OX40 20 5% to type 2 TNF receptor (TNFR2). 20 1% to TNF receptor related protein (TNFRrp) (See also. Figure 2) TNF receptor familv proteins are typically characterized by the presence of multiple (usually four) cvsteine-πch domains in their extracellular regions — each cysteine-nch domam being approximately 45 amino acid long and contains approximately 6, regularly spaced, cvsteine residues Based on the crystal structure of the type I TNF receptor, the cysteines in each domam typically form three disulfide bonds in which usually cysteines 1 and 2, 3 and 5. and 4 and 6 are paired together Applicants found that the polypeptide encoded bv clone 18 1 contains three cysteine-πch domains and an apparently truncated fourth cvsteine-πch domain that contains onlv three cysteines and stops 5 amino acids C-terminally to the third cvsteinc Amino acids I to 181 of the Apo-2LI clone 18 1 shown in Figure 1 (SEQ ID NO 1 ) are identical to amino acids 1 to 181 of the Apo-3 polypeptide. as descπbed in Example 4 below, and shown in Figure 4 (SEQ ID NO 6) Compared to Apo-3 polvpeptide descπbed in Example 4 below, the polypeptide encoded bv clone 18 1 is truncated within the C-terminal region of the ECD and lacks some extracellular sequence as well as the transmembrane and cvtoplasmic sequences of Apo-3 The truncation is believed to occur bv alternative splicing of the mRNA which introduces a stop codon 5 amino acids downstream ot the third cvsteine of the fourth cvsteine- πch domain The 3' untranslated region is distinct from that of the Apo-3 clone FL8A 53 and contains a distinct polyadenvlation site, suggesting that clone 18 I represents a naturallv-occumng mRNA EXAMPLE 2 Expression of APQ-2LI Clone 18 1 A pRK7 plasmid (descπbed in Example 1 ) containing the Apo-2I.I cDNA (as descπbed in Example 1 ) in the forward onentation. or a control pRK5 plasmid [Schall et al , Cell. 61 361 -370 ( 1990), Suva, Science. 237 893-896 ( 1987)) containing the Apo-2LI cDNA in the reverse orientation, were transfected transiently into human 293 cells (ATCC CRL 1573) bv calcium phosphate precipitation After 24 hours, the medium was replaced bv serum free medium, and the cells were incubated tor an additional 48 hours The serum free conditioned media were then collected, cleared bv centπfugation. and concentrated 5-fold bv centnfugation in centπcon tubes EXAMPLE 3 Expression of Apo-2Ll Immunoadhesin An immunoadhesin was constructed that consisted of the Apo-2LI coding region (as descπbed in Example I ), including its endogenous signal sequence, fused C-terminallv to residues 183-21 1 of type I TNF receptor, which was fused in turn to the hinge and Fc regions of human IgG 1 heaw chain, as described previously by Ashkenazi et al , supra The pRK5 plasmid encoding the chimeπc Apo-2Ll immunoadhesin vv as transiently transfected into human 293 cells (descπbed tn Example 2) bv calcium phosphate precipitation After 24 hours, the medium was replaced bv serum free medium, and the cells were incubated for an additional 6 days The serum free conditioned media were then collected, cleared bv centπfugation, and puπfied by protein A affinity chromatography, as descπbed previously by Ashkenazi et al , supra Gel electrophoresis showed that the purified protein exhibited a molecular weight of approximately 1 10 kDa under non-reducing conditions (Figure 3, lanes 3-5) and approximately 55 kDa under reducing conditions ( 100 mM DTT, Figure 3, lanes 7-9). thus indicating a disulfide-bonded homodimeπc immunoadhesin structure Higher molecular weight bands observed for non- reducing conditions are believed to be due to some aggregation of the immunoadhesin dunng sample preparation EXAMPLE 4 Isolation of cDNA clones Encoding Human APO-3 Human fetal heart and human fetal lung lgt 10 bactenophage cDNA hbranes (both purchased from Clontech) were screened by hybπdization with synthetic oligonucleotide probes based on an EST (Genbank locus W71984), which showed some degree of homoiogv to the intracellular domain (ICD) of human TNFR 1 and CD95 W71984 is a 523 bp bS'l , which in its - 1 reading frame has 27 identities to a 43 amino acid long sequence in the ICD of human TNFRl The oligonucleotide probes used in the screening were 27 and 25 bp long, respectively, with the following sequences GGCGCTCTGGTGGCCCTTGCAGAAGCC [SEQ ID NO 7] and TTCGGCCGAGAAGTTGAGAAATGTC [SEQ ID NO 8] Hybπdization was done with a 1 1 mixture of the two probes overnight at room temperature in buffer containing 20% formarrude, 5X SSC, 10% dextran sulfate, 0 1 % NaPιP0 4 , 0 05 M NaPO α , 0 05 mg salmon sperm DNA, and 0 1% sodium dodecvl sulfate (SDS). followed consecutively bv one wash at room temperature in 6X SSC, two washes at 37°C in lX SSC/0 1% SDS, two washes at 37°C in 0 5X SSC/0 1 % SDS, and two washes at 37°C in 02X SSC/0 1% SDS One positive clone from each of the fetal heart (FH20A 57) and fetal lung (FL8A 53) libranes were confirmed to be specific bv PCR using the respective above hvbndtzation probes as primers Single phage plaques containing each of the positive clones were isolated by limiting dilution and the DNA was punfied using a Wizard lambda prep DNA purification kit (Promega) The cDNA inserts were excised from the lambda vector arms by digestion with EcoRI, gel- purified, and subcloned uito pRK5 that was predigested with EcoRI The clones were then sequenced in entirety' Clone FH20A 57 (also refened to as Apo 3 clone FH20 57 deposited as ATCC 55820. as indicated below) contains a single open reading frame with an apparent translational initiation site at nucleotide positions 89 - 91 and ending at the stop codon found at nucleotide positions 1340- 1342 (Fig 4. SEQ ID NO 9) [Kozak et al , supra] The cDNA clone also contains a polyadenvlation sequence at its 3' end The predicted polypeptide precursor is 417 am o acids long and has a calculated molecular weight of approximately 45 kDa and a PI of about 64 Hydropathy analysis (not shown) suggested the presence of a signal sequence < residues 1 - 24), followed by an extracellular domain (residues 25-198), a transmembrane domain (residues 199 - 224), and an intracellular domain (residues 225 - 417) (Fig 4. SEQ ID NO 6) There are two potential N-hnked glycosylation sites at amino acid positions 67 and 106 The ECD contains 4 cvsteine-πch repeals which resemble the conespondmg regions of human TNFR l (4 repeals), of human CD95 (3 repeats) (Fig 5) and of the other known TNFR familv members (not shown) The ICD contains a death domain sequence that resembles the death domains found in the [CD of TNFR I and CD95 and in cytoplasmic death signalling proteins such as human FADD/MORTI , TRADD RIP, and Drosophila Reaper (Fig. 6) Both globally and in individual regions, Apo-3 is related more closely lo TNFR 1 than to CD95, the respective amino acid identities are 29 3% and 22 8% overall, 28 2% and 24 7% in the ECD. 31 6% and 18 3% in the ICD, and 47 5% and 20% in the death domain The fetal lung cDNA clone, clone 5L8A 53, was identical to the fetal heart clone, with the following two exceptions ( I ) it is 172 bp shorter at the 5' region; and (2) it lacks the Ala residue at position 236. possibly due to differential mRNA splicing via two consecutive splice acceptor consensus sites (Fig 6) As mentioned in Example I above, amino acids 1 to 181 of the Apo-2LI clone 18 1 shown in Figure 1 (SEQ ED NO' I) are identical to amino acids 1 to 181 of the Apo-3 polypeptide. shown in Figure 4 (SEQ ID NO 6) EXAMPLE 5 Expression of Apo-3 A pRK5 mammalian expression plasmid (descnbed in Example 2) carrying clone FH20A 57 (referred to in Example 4) was transfected transiently into HEK293 cells (referred to in the Examples above) bv calcium phosphate precipitation and into HeLa-S3 cells (ATCC No CCL 2 2) by standard electroporation techniques Lvsates of metabo callv labeled transfected 293 ceils were analyzed bv immunoprecipitation with a mouse antiserum raised against an Apo-2LI-IgG fusion protein Transfected cells ( 5 x 10 per lane) were labeled metaboϋcally by addition of 50 wCi 35 S-Met and -"S-Cys to the growth media 24 hours after transfection After a 6 hour incubation, the cells were washed several tunes with PBS, lvsed and sub j ected to immunoprecipitation by antι-Apo-3 antiserum as descπbed in Marsters et al , Proc Natl Acad Sci . 92 5401 - 5405 ( 1995) The anti-Apo-3 antiserum was raised in mice against a fusion protem containing the Apo-2LI ECD (as descπbed in Example 3) A predominant radioactive band with a relative molecular weight of about 47 kDa was observed in the pRK5-Apo-3-transfected cells, but not in the cells transfected with pRK5 alone (control) (See Fig 8, lanes 1 , 2) Given the potential glycosylation sites of Apo-3, die observed size is consistent with the size ot approximately 45 kDa predicted for the Apo-3 polypeptide precursor EXAMPLE 6 Apoptotic Activity of Apo- 3 The transiently transfected HEK293 and HeLa cells descπbed in Example 5 were tested and analvzed for apoptotic activity 36 hours after transfection Apoptosis was assessed morphologically or quantitated by FACS analysis of cells stained with fluoresceinisothiocvanate (FΙTC)-conjugated annexm V (Brand Applications) and propidium iodide (PI) The FACS analysis was conducted, using established cnteπa for apoptotic cell death, namely, the relation of fluorescence staining of the cells with two markers (a) propidium iodide (PI) which stains the apoptotic cells but not the live ceils, and (b) a fluorescent denvative of the protein, annexin V, which binds to the exposed phosphatidylsenne found on the surface of apoptotic cells, but not on live cells [Darsynkie icz et al , Methods in Cell. Biol . 4J. 15-38 ( 1994), Fadok et al . J Immunol . 148 2207-2214 ( 1992). Koopman et al.. Blood. 84 1415-1420 ( 1994)] The annexin V-positive/PI negative cells are in earlv stages of apoptosis and double-positive cells are in late apoptosis, while annexin V-negative/PI-posilive cells are necrotic Apoptosis was also assessed by DNA fragmentation testing Microscopic examination of the HEK 293 cells transfected with the pRK5-Apo-3 expression plasmid (see Example 5) showed a substantial loss of cell viability as compared to control cells transfected with pRK5 alone, many of the Apo-3 transfected cells exhibited a charactenstic apoptotic morphology of membrane blebbing and loss of cell volume (Figs 9 a and b), suggesting cell death bv apoptosis [Cohen. Advances in Immunolo y. 50 55-85 (1990)1 The FACS analysis also revealed that the Apo-3 -transfected cells died by apoptosis, by virtue of the presence of exposed phosphatidylsenne on their surface (Figs 9 e-i) It was found that the transient transfection efficiency of the HEK 293 cells was 60-70%, therefore, to target FACS analysis to cells that had taken up the plasmid DNA, the 293 cells were co-transfected with a pRK5-CD4 expression vector (3 ug) as a marker and gated on CD4-posιtιve cells (using phvcoervtfiπn-coniugated antι-CD4 antibody) for analvsis For the co- transfection. the total amount of plasmid DNA was kept constant but divided between different plasmids The Apo-3-transtected cells showed a marked increase in PI and annexin V-FITC staining as compared to pRK5- transtected control cells indicating induction of apoptosis bv Apo-3 (Figs 9 e and f) The effect of the dose of plasmid on apoptosis was also tested in the FACS assav (Fig 90 Tr anstection of 293 cells with either Apo-3 or TNFR 1 expression plasmids was associated ith a dose-dependent increase in apoptosis. the effect of Apo-3 was more pronounced than that of TNFR 1 (Fig 9ι ) Similar results were obtained upon Apo-3 transfection of the HeLa cells (data not shown) Apoptosis was also assaved bv extraction of DNA from the cells, terminal translerase-mediated 2 P-labellιng of 3' ends of DNA and 1 5% agarose gel electrophoresis as descnbed bv Moore et al Cvtotechnologv. 17 1 - 1 1 (1995) Analvsis ol the cellular DNA revealed that the Apo-3-transtected cells showed a marked increase in DNA fragmentation as compared to controls (Fig 9j, lanes 1. 2) The fragmented DNA migrated on agarose gels as a ladder of bands, indicating internucleosomal DNA cleav age, an indication of programmed cell death [Cohen, supral EXAMPLE 7 Inhibition Assav Using CrmA To investigate whether proteases such as ICE and CPP32/Yama plav a role in apoptosis- lnduclion by Apo-3. an assay was conducted to determine if CrmA inhibits Apo-3 function Co-transfection of HEK293 cells by a pRK5-CrmA expression plasmid (CrmA sequence reported in Ray et al , supra) and pRK5-Apo-3 did not affect the apparent levels of Apo- 3 expressed by the cells (Fig 8, lane 3) CrmA, however, blocked Apo-3 associated apoptosis as analyzed by morphological examination (Figs 9 c and d), FACS (Figs 9 g and h) and DNA fragmentation (Fig 9j, lanes 3,4) methods descπbed in Example 6 A similar inhibitory effect of CrmA was observed in Apo-3-transfected HeLa cells (data not shown) CrmA a pox us-denved inhibitor of the death proteases ICE and CPP32/Yama. blocks death signalling bv TNFR l and CD95 Accordingly, the assay results suggest that Apo-3, TNFR l and CD95 engage a common signalling pathway to activate apoptotic cell death In particular, the results suggest that proteases such as ICE and CPP32/Yama mav be required lor Apo-3 induced apoptosis EXAMPLE 8 Activation of NF-KB bv Apo-3 An assay was conducted to determine whether Apo-3 activates NF-κB HEK 293 cells were harvested 36 hours after transfection (see Example 5) and nuclear extracts were prepared and 1 ug of nuclear protein was reacted with a 32 P-labelled NF-κB-specιfic synthetic oligonucleotide probe ATCAGGGACTTTCCGCTGGGGACTTTCCG (SEQ ID NO 10) [see, also. MacKav et al , J Immunol 153 5274-5284 ( 1994)], alone or together with a 50-fold excess of unlabelled probe, or with an irrelevant 32 P-labeIled synthetic oligonucleotide AGGATGGGAAGTGTGTGATATATCCTTGAT (SEQ ID NO 1 1 ) DNA binding was analyzed bv an electrophoretic mobility shift assay as described bv Hsu et al , supra. Marsters et al , supra, and MacKav et al , supra The results are shown in Fig 10 The radioactive band at the bottom of the gel in all lanes is the free labelled probe, the two other radioactive bands seen in lanes 1 -3 represent non-specific interaction, as does the band common to lanes 1 -3 and lanes 4-6 The top radioactive band in lanes 4-6 represents the labelled NF-κB probe, hose migration is delayed bv specific interaction with activated NF-κB protein in the nuclear extracts Apo-3 transfected cells showed a significant increase in NF-κB-specιfιc DNA binding activity relative to pRK5-transfected controls TNFR 1 -transfected cells sho ed NF-κB activation as well, this activation appeared to be enhanced as compared to the Apo- 3 -transfected cells The data thus shows that Aρo-3 is capable of regulating transcπption of inflammatory response genes and in particular, mav be linked to a NF-κB activation pathway EXAMPLE 9 Activation of .INK by Apo- 3 Ai assav as conducted to determine whether Apo-3 activates c-Jun N-terminal kinase (JNK) HEK 293 cells were harvested 36 hours after transfection (see Example 5) and JNK activation was determined bv analyzing phosphorylation of c-Jun with a SAPK/JNK assay kit (New England Biolabs) accordmg to manufacturer instructions Cell Ivsates were prepared from HEK 293 cells transfected with 10 ug pRK5. ρRK5-TNFR 1 , or pRK5-Apo-3 JNK was precipitated with a GST-c-Jun fusion protein bound to glutathione-sepharose beads Alter washing, the kinase reaction was allowed to proceed in the presence of ATP. and was resolved bv SDS- PAGE Phospho-c-Jun was detected bv immunoblot with antibodv specific for c-Jun phosphorvlated on Ser63 a site important for transcπptional activity, using chemiluminescence detection The results are shown in Figure I I Apo-3 transfected cells showed a significant level of JNK activation as compared to pRK5 transfected controls TNFR l transfected cells showed JNK activation as well this activation appeared to be reduced relative to that seen in Apo-3 transfected cells The data thus shows that Apo-3 is capable of regulating the stress-response signaling pathway which is also known to be regulated by stimuli such as U V inadiation and vaπous cvtokmes EXAMPLE 10 Northern Blot Analvsis Expression of Apo- 3 mRNA in human tissues was examined bv Northern blot analvsis Human RNA blots were hybπdized lo a 206 bp P-labelled DNA probe based on the 3' untranslated region ot Apo-3 the probe was generated by PCR with the 27 and 25 bp probes (descπbed in Example 4) as PCR pnmers Human fetal RNA blot MTN (Clontech) and human adult RNA blot MTN-II (Clontech) were incubated with the DNA probes Blots were incubated with the probes in hybπdization buffer (5X SSPE. 2X Denhardt's solution. 100 mg/mL denatured sheared salmon sperm DNA. 50% formamide, 2% SDS) for 60 hours at 42°C The blots were washed several times in 2X SSC, 005% SDS for 1 hour at room temperature, followed bv a 30 minute wash in 0 I X SSC, 0 1 % SDS at 50°C The blots were developed after overnight exposure As shown in Fig 12, a predominant mRNA transcnpt of approximately 4kb was detected in adult spleen, thy us, and peπpheral blood lymphocytes, and less abundantly in small intestine, colon, fetal lung, and fetal kidney Additional transcπpts of approximately 7 and 9kb were seen mainlv in fetal brain, lung and kidnev. and in adult spleen and ovarv These results suggest that Apo-3 mRNA is expressed in several types ol tissues, including both Ivmphoid and non-lvmphoid tissues EXAMPLE 1 1 Chromosomal Localization of the Apo-3 gene Chromosomal localization of ihe Apo-3 gene was examined bv fluorescence in situ hybridization ("FISH") to normal human Ivmphocvte chromosomes Initial testing by direct hvbπdization with the Apo-2LI (clone 18 1 ) cDNA (see Example 1 and Fig 1 ) as a probe gave a relatively poor signal to background ratio (data not shown) but suggested that the gene is located on chromosome I p36 Further testing was conducted usmg the Apo-3 cDNA probe and FISH mapping [as descnbed by Lichter et al , Science. 247 64-69 ( 1990)] of a human genomic p 1 -den ved artificial chromosome (PAC) library (obtained from Dr L C T sui, University of Toronto Toronto. Canada) The Apo-3 probes were biotinvlated and detected with avidin-FFI C The normal human Ivmphocvte chromosomes w ere counierstamed with PI and DAPI [Heng and Tsui, Chromosome. 102 325-332 ( 1993)] In addition to the "direct" FISI I using the Apo-3 cDNA as a probe, the probe was used to identify clones in the genomic PAC library that contain the Apo-3 gene, and the PACs were used as confirmatory probes in FISH The regional assignment of the genomic probe was determined bv the analvsis of 20 well-spread metaphases A positiv e PAC clone was mapped bv FISH to the short arm of chromosome 1 at position l p36 3 A second Apo-3-posιtιve genomic PAC was mapped to the same position (data not shown ) Positive hvbπdization signals at I p36 3 were noted at >95% of the cells Signals were seen in both chromosome I homologues in >90% of the positive spreads Recent reports disclose that a genomic region which is deleted in certain human neuroblastomas maps withm lp362- lp36 3. indicating that a tumor suppressor gene mav be present at this locus Four additional TNFR gene familv members, TNFR2, CD30, 1 BB and OX40, reside in I p36 [see Gruss and Dower, supra] but are outside the deleted region [White et al , Proc Natl Acad Sci . 92 5520-5524 ( 1995)] * * » * * Deposit of Mateπal The following matenals have been deposited with the Ameπcan 1 vpe Culture Collection. 12301 Parklawn Drive. Rockville, MD. USA (ATCC) Matenal ATCC Pep No Deposit Date Apo-2LI clone 18 1 97493 Mar 27. 1996 Apo-3 clone FH20 57 55820 Sept 5, 1996 This deposit was made under the provisions of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for the Purpose of Patent Procedure and the Regulations thereunder (Budapest Treaty ) This assures maintenance of a viable culture of the deposit for 30 vears from the date of deposit The deposit will be made available bv ATCC under the terms of the Budapest I reatv and sub j ect to an agreement between Genentech, Inc and ATCC, which assures permanent and unrestincted availability of the progeny of the culture of the deposit to the public upon issuance of the pertinent U S patent or upon laving open to the public of anv U S or foreign patent application, whichever comes first, and assures availability of the progeny to one determined by the U S Commissioner of Patents and Trademarks to be entitled thereto according The assignee of the present application has agreed that if a culture of the materials on deposit should die or be lost or destroyed when cultivated under suitable conditions, the mateπals will be promptly replaced on notification with another of the same Availability of the deposited matenal is not to be construed as a license to practice the invention in contravention of the nghts granted under the authoπtv of anv government in accordance with its patent laws The foregoing wπtlen specification is considered to be sufficient to enable one skilled in the art lo practice the invention The present invention is not to be limited in scope bv the construct deposited, since the deposited embodiment is intended as a single illustration of certain aspects of the invention and anv constructs that are functionally equivalent are within the scope of this invention The deposit of matenal herein does not constitute an admission that the wπtten descπption herein contained is inadequate to enable the practice ot anv aspect ot the invention, including the best mode thereof, nor is it to be construed as limiting the scope of the claims to the specific illustrations that it represents Indeed, vaπous modifications of the invention in addition to those shown and descπbed herein will become apparent to those skilled in the art from the foregoing descπption and fall within the scope of the appended claims SEQUENCE LISTING (1) GENERAL INFORMATION: (1) APPLICANT. Genentech, Inc. (11) TITLE OF INVENTION. Apo-2 LI AND Apo-3 POLYPEPTIDES (in) NUMBER OF SEQUENCES: 11 (iv) CORRESPONDENCE ADDRESS: (A) ADDRESSEE: Genentech, Inc. (B) STREET: 460 Point San Bruno Blvd (C) CITY: South San Francisco (D) STATE: California (E) COUNTRY: USA (F) ZIP: 94080 (v) COMPUTER READABLE FORM: (A) MEDIUM TYPE: 3.5 inch, 1.44 Mb floppy disk (B) COMPUTER: IBM PC compatible (C) OPERATING SYSTEM: PC-DOS/MS-DOS (D) SOFTWARE: WmPatm (Genentech) (vi) CURRENT APPLICATION DATA: (A) APPLICATION NUMBER: (B) FILING DATE: (C) CLASSIFICATION: (vn) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/625328 (B) FILING DATE: 1-Apr-1996 (vii) PRIOR APPLICATION DATA: (A) APPLICATION NUMBER: 08/710802 (B) FILING DATE: 23-Sep-1996 (vin) ATTORNEY/AGENT INFORMATION: (A) NAME: Marschang, Diane L. (B) REGISTRATION NUMBER: 35,600 (C) REFERENCE/DOCKET NUMBER: P1007P1PCT (ix) TELECOMMUNICATION INFORMATION. (A) TELEPHONE: 415/225-5416 (B) TELEFAX: 415/952-9881 (C) TELEX: 910/371-7168 (2) INFORMATION FOR SEQ ID NO:l: (l) SEQUENCE CHARACTERISTICS: (A) LENGTH: 181 ammo acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:l: Met Glu Gin Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu 1 5 10 15 Leu Leu Val Leu Leu Gly Ala Arg Ala Gin Gly Gly Thr Arg Ser 20 25 30 Pro Arg Cys Asp Cys Ala Gly Asp Phe His Lys Lys lie Gly Leu 35 40 45 Phe Cys Cys Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro 50 55 60 Cys Thr Glu Pro Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gin 65 70 75 Asp Thr Phe Leu Ala Trp Glu Asn His His Asn Ser Glu Cys Ala 80 85 90 Arg Cys Gin Ala Cys Asp Glu Gin Ala Ser Gin Val Ala Leu Glu 95 100 105 Asn Cys Ser Ala Val Ala Asp Thr Arg Cys Gly Cys Lys Pro Gly 110 115 120 Trp Phe Val Glu Cys Gin Val Ser Gin Cys Val Ser Ser Ser Pro 125 130 135 Phe Tyr Cys Gin Pro Cys Leu Asp Cys Gly Ala Leu His Arg His 140 145 150 Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr Asp Cys Gly Thr Cys 155 160 165 Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys Val Ser Cys Pro 170 175 180 Thr 181 (2) INFORMATION FOR SEQ ID NO:2 : (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 433 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: CTGCTGGGGG CCCGGGCCAG NGGCGGCACT CGTAGCCCCA GGTGTGACTG 50 TGCCGGTGAC TTCCACAAGA AGATTGGTCT GTTTTGTTGC AGAGGCTGCC 100 CAGCGGGGCA ACTACCTGAA GGCCCCTTGC ACGGAGCCCT GCGCAACTCC 150 ACCTGCCTTG TGTGTCCCCA AGACACCTTC TTGGCCTGGG AGAACCACCA 200 TAATTCTGAA TGTGCCCGCT GCCAGGCCTG TGATGAGCAG GCCTCCCAGG 250 TGGCGCTGGA GAACTGTTCA GCAGTGGCCG ACACCCGCTG TGGCTGTAAG 300 CAGGGCTGGT TTGTGGAGTG CCAGGGTCAG CCAATGTGTC AGCAGTTTCA 350 CCCTTCTAAT GCCAACCATG CCTAGACTGC GGGGCCCTGC AACGCAACAC 400 ACGGCTAATN TGTTTCCCGC AGAGATNATT GTT 433 (2) INFORMATION FOR SEQ ID NO:3: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: CCCGCTGCCA GGCCTGTGAT GAGCAGGC 28 (2) INFORMATION FOR SEQ ID NO:4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 28 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: CAGGGCCCCG CAGTCTAGGC ATGGTTGG 28 (2) INFORMATION FOR SEQ ID NO:5: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1438 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:5 : GAATTCCGGC GCGGAGGCCG AGAGAGAAGT CACTTGCCCT GGCTCTACCT 50 TGAAGTGGTT CTCAGGGTTG GGGCGAGAGT CGGGGTGGGG ACCGAGATGC 100 AGCTCTATCC TGTGCCCCTG GTCGCAGCAG GCAGCCCAGC GCTTCGCGTG 150 TTCTACTTGG CCTGTCCGCT GCCGCCTAAT GAGCTCAGGT CTAGGCCGAG 200 CAGAGGGGGC ACCTGGTCGG ACTCGGTTGG GCTCGGGCGG CCCCGCCTCC 250 CCCCGCCCGC CAGGCGGGCC CTTCTCGACG GCGCGGGGCG GGCCCTGCGG 300 GCGCGGGGCT GAAGGCGGAA CCACGACGGG CAGAGAGCAC GGAGCCGGGA 350 AGCCCCTGGG CGCCCGTCGG AGGGCTATGG AGCAGCGGCC GCGGGGCTGC 400 GCGGCGGTGG CGGCGGCGCT CCTCCTGGTG CTGCTGGGGG CCCGGGCCCA 450 GGGCGGCACT CGTAGCCCCA GGTGTGACTG TGCCGGTGAC TTCCACAAGA 500 AGATTGGTCT GTTTTGTTGC AGAGGCTGCC CAGCGGGGCA CTACCTGAAG 550 GCCCCTTGCA CGGAGCCCTG CGGCAACTCC ACCTGCCTTG TGTGTCCCCA 600 AGACACCTTC TTGGCCTGGG AGAACCACCA TAATTCTGAA TGTGCCCGCT 650 GCCAGGCCTG TGATGAGCAG GCCTCCCAGG TGGCGCTGGA GAACTGTTCA 700 GCAGTGGCCG ACACCCGCTG TGGCTGTAAG CCAGGCTGGT TTGTGGAGTG 750 CCAGGTCAGC CAATGTGTCA GCAGTTCACC CTTCTACTGC CAACCATGCC 800 TAGACTGCGG GGCCCTGCAC CGCCACACAC GGCTACTCTG TTCCCGCAGA 850 GATACTGACT GTGGGACCTG CCTGCCTGGC TTCTATGAAC ATGGCGATGG 900 CTGCGTGTCC TGCCCCACGT AATTCCTAGC TGTCGTGGGA TGGAGGGAAG 950 GGCGGCTGGG AGCAGAGCAG GGGCCTGGGG TGGGGCAGGT GCTGCTGGTT 1000 CAGGAATAGG AAGAGGGGAT AGGGAGGAGG GAGCCTTGGC CCTGTGATGG 1050 GTGGGCCCCA CTTCAGGCAA ACTTAGATGG CAAAAGAGCA ATCTGGATCC 1100 GCCTTAGCCA GATACATAAG GGTATTTGCC TTCACTT CA GCCAGCATTC 1150 CCCCCAGCGA TCCTAGCCAG ATATTACAGA TGATTTGTCA CTTACACAGA 1200 GAGTCACATT GATATAGCTT TAAAACTTGG GCTGAAGGAG GTTGAGGCTG 1250 CAGTGAGCTA TGATCGTGCC ACTGCACTTC AGCCTGGGCA ACAGAGCGAG 1300 ACCTATTAAA TAAATAAATA AATATTAAAT CTATTAAATA TTAAATATTA 1350 AATCTATTAA ATAAATAAAT ACAAAGGGCT GAGAGTCAGG ACTGTGCTGC 1400 TAGTTCTCTA GGGGATCTTG GGCAAGTGCA GAGAATTC 1438 (2) INFORMATION FOR SEQ ID NO:6: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 417 amino acids (B) TYPE: Amino Acid (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: Met Glu Gin Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu 1 5 10 15 Leu Leu Val Leu Leu Gly Ala Arg Ala Gin Gly Gly Thr Arg Ser 20 25 30 Pro Arg Cys Asp Cys Ala Gly Asp Phe His Lys Lys lie Gly Leu 35 40 45 Phe Cys Cys Arg Gly Cys Pro Ala Gly His Tyr Leu Lys Ala Pro 50 55 60 Cys Thr Glu Pro Cys Gly Asn Ser Thr Cys Leu Val Cys Pro Gin 65 70 75 Asp Thr Phe Leu Ala Trp Glu Asn His His Asn Ser Glu Cys Ala 80 85 90 Arg Cys Gin Ala Cys Asp Glu Gin Ala Ser Gin Val Ala Leu Glu 95 100 105 Asn Cys Ser Ala Val Ala Asp Thr Arg Cys Gly Cys Lys Pro Gly 110 115 120 Trp Phe Val Glu Cys Gin Val Ser Gin Cys Val Ser Ser Ser Pro 125 130 135 Phe Tyr Cys Gin Pro Cys Leu Asp Cys Gly Ala Leu His Arg His 140 145 150 Thr Arg Leu Leu Cys Ser Arg Arg Asp Thr Asp Cys Gly Thr Cys 155 160 165 Leu Pro Gly Phe Tyr Glu His Gly Asp Gly Cys Val Ser Cys Pro 170 175 180 Thr Ser Thr Leu Gly Ser Cys Pro Glu Arg Cys Ala Ala Val Cys 185 190 195 Gly Trp Arg Gin Met Phe Trp Val Gin Val Leu Leu Ala Gly Leu 200 205 210 Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr Tyr Arg 215 220 225 His Cys Trp Pro His Lys Pro Leu Val Thr Ala Asp Glu Ala Gly 230 235 240 Met Glu Ala Leu Thr Pro Pro Pro Ala Thr His Leu Ser Pro Leu 245 250 255 Asp Ser Ala His Thr Leu Leu Ala Pro Pro Asp Ser Ser Glu Lys 260 265 270 lie Cys Thr Val Gin Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr 275 280 285 Pro Glu Thr Gin Glu Ala Leu Cys Pro Gin Val Thr Trp Ser Trp 290 295 300 Asp Gin Leu Pro Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro Thr 305 310 315 Leu Ser Pro Glu Ser Pro Ala Gly Ser Pro Ala Met Met Leu Gin 320 325 330 Pro Gly Pro Gin Leu Tyr Asp Val Met Asp Ala Val Pro Ala Arg 335 340 345 Arg Trp Lys Glu Phe Val Arg Thr Leu Gly Leu Arg Glu Ala Glu 350 355 360 He Glu Ala Val Glu Val Glu He Gly Arg Phe Arg Asp Gin Gin 365 370 375 Tyr Glu Met Leu Lys Arg Trp Arg Gin Gin Gin Pro Ala Gly Leu 380 385 390 Gly Ala Val Tyr Ala Ala Leu Glu Arg Met Gly Leu Asp Gly Cys 395 400 405 Val Glu Asp Leu Arg Ser Arg Leu Gin Arg Gly Pro 410 415 417 (2) INFORMATION FOR SEQ ID NO:7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 27 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: GGCGCTCTGG TGGCCCTTGC AGAAGCC 27 (2) INFORMATION FOR SEQ ID NO:8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 25 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: TTCGGCCGAG AAGTTGAGAA ATGTC 25 (2) INFORMATION FOR SEQ ID NO:9: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1634 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: CGGGCCCTGC GGGCGCGGGG CTGAAGGCGG AACCACGACG GGCAGAGAGC 50 ACGGAGCCGG GAAGCCCCTG GGCGCCCGTC GGAGGGCT ATG GAG 94 Met Glu 1 CAG CGG CCG CGG GGC TGC GCG GCG GTG GCG GCG GCG CTC 133 Gin Arg Pro Arg Gly Cys Ala Ala Val Ala Ala Ala Leu 5 10 15 CTC CTG GTG CTG CTG GGG GCC CGG GCC CAG GGC GGC ACT 172 Leu Leu Val Leu Leu Gly Ala Arg Ala Gin Gly Gly Thr 20 25 CGT AGC CCC AGG TGT GAC TGT GCC GGT GAC TTC CAC AAG 211 Arg Ser Pro Arg Cys Asp Cys Ala Gly Asp Phe His Lys 30 35 40 AAG ATT GGT CTG TTT TGT TGC AGA GGC TGC CCA GCG GGG 250 Lys He Gly Leu Phe Cys Cys Arg Gly Cys Pro Ala Gly 45 50 CAC TAC CTG AAG GCC CCT TGC ACG GAG CCC TGC GGC AAC 289 His Tyr Leu Lys Ala Pro Cys Thr Glu Pro Cys Gly Asn 55 60 65 TCC ACC TGC CTT GTG TGT CCC CAA GAC ACC TTC TTG GCC 328 Ser Thr Cys Leu Val Cys Pro Gin Asp Thr Phe Leu Ala 70 75 80 TGG GAG AAC CAC CAT AAT TCT GAA TGT GCC CGC TGC CAG 367 Trp Glu Asn His His Asn Ser Glu Cys Ala Arg Cys Gin 85 90 GCC TGT GAT GAG CAG GCC TCC CAG GTG GCG CTG GAG AAC 406 Ala Cys Asp Glu Gin Ala Ser Gin Val Ala Leu Glu Asn 95 100 105 TGT TCA GCA GTG GCC GAC ACC CGC TGT GGC TGT AAG CCA 445 Cys Ser Ala Val Ala Asp Thr Arg Cys Gly Cys Lys Pro 110 115 GGC TGG TTT GTG GAG TGC CAG GTC AGC CAA TGT GTC AGC 484 Gly Trp Phe Val Glu Cys Gin Val Ser Gin Cys Val Ser 120 125 130 AGT TCA CCC TTC TAC TGC CAA CCA TGC CTA GAC TGC GGG 523 Ser Ser Pro Phe Tyr Cys Gin Pro Cys Leu Asp Cys Gly 135 140 145 GCC CTG CAC CGC CAC ACA CGG CTA CTC TGT TCC CGC AGA 562 Ala Leu His Arg His Thr Arg Leu Leu Cys Ser Arg Arg 150 155 GAT ACT GAC TGT GGG ACC TGC CTG CCT GGC TTC TAT GAA 601 Asp Thr Asp Cys Gly Thr Cys Leu Pro Gly Phe Tyr Glu 160 165 170 CAT GGC GAT GGC TGC GTG TCC TGC CCC ACG AGC ACC CTG 640 His Gly Asp Gly Cys Val Ser Cys Pro Thr Ser Thr Leu 175 180 GGG AGC TGT CCA GAG CGC TGT GCC GCT GTC TGT GGC TGG 679 Gly Ser Cys Pro Glu Arg Cys Ala Ala Val Cys Gly Trp 185 190 195 AGG CAG ATG TTC TGG GTC CAG GTG CTC CTG GCT GGC CTT 718 Arg Gin Met Phe Trp Val Gin Val Leu Leu Ala Gly Leu 200 205 210 GTG GTC CCC CTC CTG CTT GGG GCC ACC CTG ACC TAC ACA 757 Val Val Pro Leu Leu Leu Gly Ala Thr Leu Thr Tyr Thr 215 220 TAC CGC CAC TGC TGG CCT CAC AAG CCC CTG GTT ACT GCA 796 Tyr Arg His Cys Trp Pro His Lys Pro Leu Val Thr Ala 225 230 235 GAT GAA GCT GGG ATG GAG GCT CTG ACC CCA CCA CCG GCC 835 Asp Glu Ala Gly Met Glu Ala Leu Thr Pro Pro Pro Ala 240 245 ACC CAT CTG TCA CCC TTG GAC AGC GCC CAC ACC CTT CTA 874 Thr His Leu Ser Pro Leu Asp Ser Ala His Thr Leu Leu 250 255 260 GCA CCT CCT GAC AGC AGT GAG AAG ATC TGC ACC GTC CAG 913 Ala Pro Pro Asp Ser Ser Glu Lys He Cys Thr Val Gin 265 270 275 TTG GTG GGT AAC AGC TGG ACC CCT GGC TAC CCC GAG ACC 952 Leu Val Gly Asn Ser Trp Thr Pro Gly Tyr Pro Glu Thr 280 285 CAG GAG GCG CTC TGC CCG CAG GTG ACA TGG TCC TGG GAC 991 Gin Glu Ala Leu Cys Pro Gin Val Thr Trp Ser Trp Asp 290 295 300 CAG TTG CCC AGC AGA GCT CTT GGC CCC GCT GCT GCG CCC 1030 Gin Leu Pro Ser Arg Ala Leu Gly Pro Ala Ala Ala Pro 305 310 ACA CTC TCG CCA GAG TCC CCA GCC GGC TCG CCA GCC ATG 1069 Thr Leu Ser Pro Glu Ser Pro Ala Gly Ser Pro Ala Met 315 320 325 ATG CTG CAG CCG GGC CCG CAG CTC TAC GAC GTG ATG GAC 1108 Met Leu Gin Pro Gly Pro Gin Leu Tyr Asp Val Met Asp 330 335 340 GCG GTC CCA GCG CGG CGC TGG AAG GAG TTC GTG CGC ACG 1147 Ala Val Pro Ala Arg Arg Trp Lys Glu Phe Val Arg Thr 345 350 CTG GGG CTG CGC GAG GCA GAG ATC GAA GCC GTG GAG GTG 1186 Leu Gly Leu Arg Glu Ala Glu He Glu Ala Val Glu Val 355 360 365 GAG ATC GGC CGC TTC CGA GAC CAG CAG TAC GAG ATG CTC 1225 Glu He Gly Arg Phe Arg Asp Gin Gin Tyr Glu Met Leu 370 375 AAG CGC TGG CGC CAG CAG CAG CCC GCG GGC CTC GGA GCC 1264 Lys Arg Trp Arg Gin Gin Gin Pro Ala Gly Leu Gly Ala 380 385 390 GTT TAC GCG GCC CTG GAG CGC ATG GGG CTG GAC GGC TGC 1303 Val Tyr Ala Ala Leu Glu Arg Met Gly Leu Asp Gly Cys 395 400 405 GTG GAA GAC TTG CGC AGC CGC CTG CAG CGC GGC CCG T 1340 Val Glu Asp Leu Arg Ser Arg Leu Gin Arg Gly Pro 410 415 417 GACACGGCGC CCACTTGCCA CCTAGGCGCT CTGGTGGCCC TTGCAGAAGC 1390 CCTAAGTACG GTTACTTATG CGTGTAGACA TTTTATGTCA CTTATTAAGC 1440 CGCTGGCACG GCCCTGCGTA GCAGCACCAG CCGGCCCCAC CCCTGCTCGC 1490 CCCTATCGCT CCAGCCAAGG CGAAGAAGCA CGAACGAATG TCGAGAGGGG 1540 GTGAAGACAT TTCTCAACTT CTCGGCCGGA GTTTGGCTGA GATCGCGGTA 1590 TTAAATCTGT GAAAGAAAAC AAAAAAAAAA AAAAAAAAAA AAAA 1634 (2) INFORMATION FOR SEQ ID NO:10: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 29 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:10: ATCAGGGACT TTCCGCTGGG GACTTTCCG 29 (2) INFORMATION FOR SEQ ID NO:11: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 30 base pairs (B) TYPE: Nucleic Acid (C) STRANDEDNESS: Single (D) TOPOLOGY: Linear (xi) SEQUENCE DESCRIPTION: SEQ ID NO:ll: AGGATGGGAA GTGTGTGATA TATCCTTGAT 30

Claims: WHAT IS CLAIMED IS 1 Isolated biologically active Apo-2LI having at least about 80% sequence identity with native sequence Aρo-2LI having ammo acid residues 1 to 181 of SEQ ID NO I 2 I he Apo-2LI of claim 1 wherein said Apo-2LI has at least about 90% sequence identity 3 I he Apo-2LI of claim 2 wherein said Apo-2LI has at least about 95% sequence identity 4 Isolated Λpo-2LI compπsing amino acid residues 1 to 181 of SEQ ID NO 1 5 A chimeric molecule compπsing the Apo-2LI of claim I or claim 4 fused to a heterologous ammo acid sequence 6 fhe chimeπc molecule of claim 5 wherein said heterologous ammo acid sequence is an epitope tag sequence 7 The chi enc molecule of claim 5 wherem said heterologous amino acid sequence is an immunoglobulm sequence 8 The chimeπc molecule of claim 7 wherem said immunoglobulin sequence is an IgG 9 A dimer molecule compπsing a first Apo-2LI and a second Apo-2LI 10 An antibodv which binds to Apo-2LI 1 1 The antibodv of claim 10 wherein said antibodv is a monoclonal antibodv 12 Isolated nucleic acid encoding Apo-2LI 13 The nucleic acid of claim 12 wherem said nucleic acid encodes an Apo-2I.I compπsing ammo acid residues 1 to 181 of SEQ ID NO I 14 A vector compπsing the nucleic acid of claim 12 15 A host cell compnsing the vector of claim 14 16 A method of producing Apo-2LI compnsing cultunng the host cell of claim 15 and recoveπng the Apo-2LI from the host cell culture 17 An article of manufacture, compπsing a container, a label on said container, and a composition contained within said container, said composition compnsing Apo-2LI 18 The article of manufacture of claim 17 further compnsing instructions for usmg the Λpo-2LI 19 Isolated biologically active Apo-3 polypeptide having at least about 80% sequence identity with native sequence Apo-3 having ammo acid residues I to 417 of SEQ ID NO 6 20 The Apo-3 of claim 19 wherem said Apo-3 has at least about 90% sequence identity 21 The Apo-3 of claim 20 wherem said Apo-3 has at least about 95% sequence identity 22 Isolated native sequence Apo-3 compπsmg ammo acid residues 1 to 417 of SEQ ID NO 6 23 Isolated biologically active polypeptide havmg at least about 80% sequence identity with the extracellular domain sequence of Apo- 3 having ammo acid residues 1 to 198 of SEQ ID NO 6 24 The polypeptide of claim 23 wherein said polypeptide has at least about 90% sequence identity 25 The polvpeptide of claim 24 wherein said polypeptide is Apo-2LI 26 Isolated extracellular domam sequence of Apo-3 compnsing ammo acid residues 1 to 198 of SEQ ID NO 6 27 Isolated death domain sequence of Apo-3 compπsmg ammo acid residues 338 to 417 of SEQ ID NO 6 28 A chimeπc molecule compπsmg the Apo-3 of claun 22 or the extracellular domain sequence of claun 23 fused to a heterologous ammo acid sequence 29 The chimeπc molecule of claim 28 wherein said heterologous amino acid sequence is an epitope tag sequence 30 The chimeric molecule of claim 28 wherein said heterologous ammo acid sequence is an immunoglobulin sequence 31 The chimeπc molecule of claim 30 wherein said immunoglobulin sequence is an IgG 32 An antibodv which binds to Apo-3 or to the extracellular domain sequence of claim 23 33 The antibody of claim 32 wherein said antibody is a monoclonal antibody 34 Isolated nucleic acid encodmg the Aρo-3 of claim 22, the extracellular domain sequence of claim 23 or the death domain sequence of claim 27 35 The nucleic acid of claim 34 wherein said nucleic acid encodes native sequence Apo-3 compπsmg amino acid residues 1 to 417 of SEQ ID NO 6 36 A vector compπsmg the nucleic acid of claim 34 37 The vector of claun 36 operably linked to control sequences recognized by a host cell transformed with the vector 38 A host cell compπsing the vector of claim 36 39 A process of usmg a nucleic acid molecule encodmg Apo-3 to effect production of Apo-3 compπsing cultunng the host cell of claim 38 40 A non-human, transgemc animal which contains cells that express nucleic acid encoding Apo-3 41 The animal of claim 40 which is a mouse or rat 42 A non-human, knockout animal which conta s cells having an altered gene encoding Apo-3 43 The animal of claim 42 which is a mouse or rat 44 An article of manufacture, compπsing a container and a composition conta ed within said container, wherem the composition mcludes Apo-3 polypeptide or Apo-3 antibodies 45 The article of manufacture of claim 44 further compπsing instructions for using the Apo-3 polypeptide or Apo-3 antibodies in vivo or ex vivo

Inventors: ASHKENAZI AVI J, ASHKENAZI, AVI, J.
Applicants: GENENTECH INC, GENENTECH, INC.
Application year: 1997
Main classification: C12N15
IPC codes: A01K67, A61K31, A61K38, A61K39, A61K48, A61P35, A61P43, C07K14, C07K16, C07K19, C12N1, C12N5, C12N15, C12P21