MXPA00000578A - Trail receptor - Google Patents

Abstract

A protein designated TRAIL receptor binds the protein known as TNF-Related Apoptosis-Inducing Ligand (TRAIL). The TRAIL receptor finds use in purifying TRAIL or inhibiting activities thereof. Isolated DNA sequences encoding TRAIL-R polypeptides are provided, along with expression vectors containing the DNA sequences, and host cells transformed with such recombinant expression vectors. Antibodies that are immunoreactive with TRAIL-R are also provided.

Description

LIGANDO INDUCTOR RECEIVER OF TNF-RELATED APOPTOSIS BACKGROUND OF THE INVENTION A protein known as the apoptosis-inducing ligand related to tumor necrosis factor (TNF) TRIAL is a member of the family of tumor necrosis factor ligands (TNF). Wiley et al., Immunity, 3: 673-682, 1995). TRAIL has demonstrated the ability to induce apoptosis of certain transformed cells, including a number of different types of cancer cells as well as virally infected cells (PCT application WO 97/01633 and Wiley et al., Supra). The identification of protein or receptor proteins that bind to TRAIL would prove useful in further study of the biological activities of TRAIL. COMPENDIUM OF THE INVENTION The present invention is directed to a novel protein designated TRAIL receptor (TRAIL-R), which binds to a protein known as the TNF-related apoptosis-inducing ligand (TRAIL). The DNA encoding TRAIL-R, and the expression vectors comprising such DNA are provided. One method for producing TRAIL-R polypeptides comprises culturing transformed host cells with a recombinant expression vector encoding TRAIL-R, under conditions that promote the expression of TRAIL-R, then recovering the expressed TRAIL-R polypeptides from the culture. Antibodies that are immunoreactive with TRAIL-R are also provided. BRIEF DESCRIPTION OF THE FIGURES Figures 1A and 1B present the nucleotide sequence of a human TRAIL receptor cDNA, as well as the amino acid sequence encoded thereby. Figures 2A and 2B present the nucleotide and amino acid sequences encoded by a second human TRAIL cDNA receptor clone. The amino acid sequence of Figures 2A-2B differs in two positions from the sequence presented in the FIGS. 1 A-1 B. DETAILED DESCRIPTION OF THE INVENTION A novel protein designated TRAIL receptor (TRAIL-R) is provided herein. TRAIL-R binds to the cytosine designated TNF-related apoptosis-inducing ligand (TRAIL). Certain uses of TRAIL-R flow of this ability to join the TRAIL, as discussed below. TRAIL-R finds use in the inhibition of biological activities of TRAIL, or for example, in the purification of TRAIL by the affinity of chromatography. The TRAIL-R protein or immunogenic fragments thereof can be used as immunogens to generate antibodies that are immunoreactive therewith. In one embodiment of the invention, the antibodies are monoclonal antibodies. The nucleotide sequence of a human TRAIL receptor cDNA is presented in Figures 1A to 1B, along with the amino acid sequence encoded by the cDNA (SEQ ID NO: 1 and SEQ ID NO: 2). Figures 2A to 2B (SEQ ID NO: 3 and SEQ ID NO: 4) present the nucleotide sequence of a second human TRAIL receptor cDNA clone, and the amino acid sequence encoded by it. The nucleotide sequences of Figures 1 and 2 differ in two positions. The nucleotide at position 145 is a C in the sequence of Figure 1 (SEQ ID NO: 1), since nucleotide 145 is a T in Figure 2 (SEQ ID NO: 3); the nucleotide at position 91 is a C in Figure 1 (SEQ ID NO: 1), but in a T in Figure 2 (SEQ ID NO: 3). The amino acid sequences also differ in two positions. Residue 35 is Pro in Figure 1 (SEQ ID NO: 2) and Ser in Figure 2 (SEQ ID NO: 4); residue 310 is Ser in Figure 1 (SEQ ID NO: 2) and Leu in Figure 2 (SC ID NO: 4). One possible explanation is that the TRAIL receivers of Figures 1 and 2 are allelic variants. The sequence information presented in Figures 1 and 2 identifies the TRAIL receptor protein as a member of the receptor necrosis tumor (TNF-R) family of receptors (summarized in Smith et al., Cell 76: 959-962 , 1994). The TRAIL-R proteins include certain aspects of other proteins in this family, which include cysteine-rich repeats in the extracellular domain, as discussed below. However, TRAIL-R lacks a "dead domain", so-called, which is found in the cytoplasm region of certain receptor proteins. It has been reported that these domains are associated with the transduction of apoptotic signals, that is, they have a role in initiating cascades of intracellular apoptotic signaling. Dead cytoplasmic domains have been identified in the Fas antigen (Itoh and Nagata, J. Biol. Chem. 268: 10931, 1993), the TNF receptor type I (Tartaglia et al., Cell 74: 845, 1993), DR3 (Chinnaiyan et al., Science 274: 990-992, 1996), and CAR-1 (Brojatsch et al., Cell 87: 845-855, 1996). The TRAIL-R proteins of Figure 1 (SEQ ID NO: 2) and Figure 2 (SEQ ID NO: 4) include an N-terminal hydrophobic region that functions as a signal peptide, followed by an extracellular domain, an transmembrane region comprising amino acids 212 through 232, and a C-terminal cytoplasmic domain comprising 23 to 386 amino acids. Computer analysis predicts that the signaling peptide is likely to be separated after residue 55. The separation of signaling peptides will therefore produce a mature protein comprising 56 to 386 amino acids. The calculated molecular weight for a mature protein having the amino acid sequence of residues 56 to 386 of Figure 1 is about 36 klodaltons. The isoelectric point (pl) is predicted to be around 5.27. Skilled artisans will recognize that the particular molecular weight of TRAIL-R protein preparations may differ, in accordance with such factors as the degree of glycosylation. The glycosylation pattern of a particular preparation of TRAIL-R may vary according to the type of cells in which the proteins are expressed, for example, and a given preparation may include differentially glycosylated multiple species of the protein. TRAIL-R proteins with or without glycosylation of associated native pattern are provided herein. The expression of TRAIL-R in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. In addition, the N-glycosylation sites in the native protein can be deactivated, as discussed below. The present invention includes TRAIL-R in various forms, including those that occur naturally or are produced through various techniques such as procedures involving recombinant DNA technology. Such forms of TRAIL-R include, but are not limited to, fragments, derivatives, variants, and oligomers of TRAIL-R as well as fusion proteins containing TRAIL-R or fragments thereof. TRIAL-R can be modified to create derivatives thereof by forming aggregation or covalent conjugates with other chemical proportions, such as glycosyl groups, lipids, phosphates, acetyl groups and the like. The covalent derivatives of TRAIL-R can be prepared by linking the chemical ratios to functional groups on amino acid side chains of TRAIL-R or on the N-terminus or C-terminus of a TRAIL-R polypeptide. Conjugates comprising diagnostic (detectable) or therapeutic agents linked to TRAIL-R are contemplated herein, as discussed in more detail below.

Other TRAIL-R derivatives within the scope of this invention include aggregation or covalent conjugates of TRAIL-R polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion proteins are discussed below in connection with TRAIL-R oligomers. In addition, TRAIL-R containing fusion proteins can comprise aggregated peptides to facilitate the purification and identification of TRA1L-R. Such peptides include, for example, poly-His or the antigenic identification peptides described in the U.S. Patent. No. 5,011,912 and in Hopp et al., Bio / Technology 6: 1204.1988. Such a peptide is the peptide, Flag®Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO: 5), which is highly antigenic and provides a reversibly epitope binding by a specific monoclonal antibody, allowing the rapid analysis and easy purification of expressed recombinant protein. A murine hybridoma designated 4E11 produces a monoclonal antibody that binds to the Flag® peptide in the presence of certain divalent metal cations, as described in U.S. Pat. 5,011,912, incorporated herein by reference. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under access no. HB 9259. The monoclonal antibodies that bind the Flag® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.

Both forms of membrane-bound and soluble (secreted) cell of TRAIL-R are provided herein. The soluble TRAIL-R can be identified (and distinguished from non-soluble membrane-bound counterparts) by separating the intact cells expressing a TRAIL-R polypeptide from a culture medium, for example, by centrifugation, and analyzing the medium (supernatant) for the presence of the desired protein. The presence of TRAIL-R in the medium indicates that the protein was secreted from the cells and therefore is a soluble form of the desired protein. Soluble forms of the receptor proteins usually lack the transmembrane region that could cause retention of the cell surface protein. In one embodiment of the invention, a soluble TRAIL-R polypeptide comprises the extracellular domain of the protein. In certain other embodiments, the soluble TRAIL-R polypeptides are extracellular domain fragments. A TRAIL-R polypeptide includes the cytoplasmic domain, or a portion thereof, while the polypeptide is secreted from the cell in which it is produced. Examples of soluble TRAIL-R include, but are not limited to, polypeptides comprising amino acids 56 through 211 of Figure 1 or 2 (the extracellular domain) or amino acids 56 through 208 of Figure 1 or 2 (a fragment of the extracellular domain). ). An expression vector encoding a soluble TRAIL-R polypeptide comprising amino acids 1 to 208 of Figures 1 or 2, fused to an antibody derived from Fc polypeptide, is described in Example 3 below. In addition, examples include, but are not limited to, fragments of the extracellular domain that includes the cysteine-rich repeats of TRAIL-R, as described below. The soluble forms of TRAIL-R have certain advantages over the form of the membrane-bound protein. Purification of the protein from recombinant host cells is facilitated, whereas soluble proteins are secreted from the cells. In addition, the proteins are generally more suitable for certain applications, for example, for intravenous administration. In the present fragments of TRAIL-R are provided. Such fragments can be prepared by any of a number of conventional techniques. The desired peptide fragments can be chemically synthesized. An alternative involves the generation of TRAIL-R fragments by enzymatic digestion, for example, by treating the protein with a known enzyme to separate proteins at sites defined by particular amino acid residues. A TRAIL-R DNA can be digested with adequate restriction enzymes, to derive a DNA fragment encoding a desired polypeptide fragment. Yet another suitable technique involves the isolation and amplification of a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). The oligonucleotides defining the desired term of the DNA fragment are used as 5 'and 3' primers in the PCR.

The fragments of TRAIL-R polypeptides can be used as immunogens in the generation of antibodies. Particular modalities are directed to fragments of TRAIL-R polypeptides that retain the ability to bind TRAIL. Such a fragment may be a soluble TRAIL-R polypeptide, as described above. In particular embodiments, the TRAIL-R fragments include repeat cysteine-rich motifs found in the extracellular domain. The human TRAIL-R proteins of Figures 1 and 2 contain two such cysteine-rich repeats, the first including residues 98 to 139, and the second including residues 140 to 181. The receptors of the TNF-R family contain such cysteine-rich repeats in its extracellular domain (Masters et al., J. Biol. Chem. 267: 5747-5750, 1992, Smith et al., Cell 76: 76: 959-962, 1994). These repeats are believed to be important for the binding ligand. As an example, Masters et al., Supra, report that soluble type 1 TNF-R polypeptides lack one of the repeats exhibited a tenfold reduction in binding affinity for TNFα and TNFβ; the deletion of the second repeat resulted in a complete loss of the detectable binding of the ligands. Residues 182 to 211 of Figures 1 and 2 constitute a separation region. This separation is the C-terminal portion of the extracellular domain, positioned between the cysteine-rich repeats and the transmembrane region. Such regions of separation have been identified in certain other proteins of the TNF-R family, and reportedly are not critical for the binding of the ligands. Fragments of TRAIL-R lacking the region of separation are provided herein. Of course, variants of the TRAIL.-R protein of FIGS. 1 and 2 are provided herein. Such variants include, for example, proteins that result from alternating mRNA spliced events or the proteolytic separation of the TRAIL-R protein. The alternating impaling of the mRNA can, for example, produce a truncated but biologically active TRAIL-R protein, such as a soluble form that naturally occurs from the protein. Variations that are attributed to proteolysis include, for example, differences in N or C endings by expression in different host cell types, due to the proteolytic removal of one or more terminal amino acids of the TRAIL-R protein (generally from 1-5 terminal amino acids). Proteins in which differences in amino acid sequence are attributed to genetic polymorphism (allelic variation between individuals that produce the protein) are also contemplated herein. Skilled artisans will also recognize that the positions at which peptide signals are separated may differ from those predicted by a computer program, and may vary according to such factors as the type of host cells employed in expressing a TRAIL polypeptide. -R recombinant. A protein preparation can include a mixture of protein molecules having different N-terminal amino acids, which result from the separation of the signaling peptide at more than one site. With respect to the discussion in the present of several protein domains of TRAIL-R, the experts will recognize that the above-described linkages of such regions of the protein are approximate. For illustration, the boundaries of the transmembrane region (which can be predicted using computer programs available for that purpose) may differ from those described above. Therefore, the soluble TRAIL-R polypeptides in which the C-terminus of the extracellular domain differs from the residues thus identified above are contemplated herein. Other TRAIL-R DNAs and polypeptides present in nature include those derived from non-human species. The homologs of the human TRAIL-R of Figures 1 and 2, for example of other mammalian species, are contemplated herein. Tests based on the human DNA sequence of Figure 1 and 2 can be used to screen cDNA libraries derived from other mammalian species, using cross-species hybridization techniques. The DNA sequences of TRAIL-R may vary from the native sequences described herein. Due to the known degeneracy of the genetic codon, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in Figure 1 (SEQ ID NO: 1) or Figure 2 (SEQ ID NO: 3) and still encode a TRAIL-R protein having the amino acid sequence presented in the Figures (SEQ ID NO: 2 or 4 respectively). Such variant DNA sequences can result from silent mutations (e.g., occurring during PCR amplification), or they can be the product of deliberate mutagenesis of a native sequence. Therefore, among the DNA sequences provided herein, there are native TRAIL-R sequences (e.g., cDNA comprising the nucleotide sequence presented in Figures 1 and 2) and DNA that degenerates as a result of the codon. to a DNA sequence of native TRAIL-R. Among the TRAIL-R polypeptides provided herein are variants of TRAIL-R polypeptides that retain a biological activity of a TRAIL-R. Such variants include polypeptides that are substantially homologous to native TRAIL-R, but that have an amino acid sequence different from a native TRAIL-R due to one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, TRAIL-R polypeptides comprising one to ten deletions, insertions or substitutions, but which encodes a biologically active TRAIL-R polypeptide. A biological activity of TRAIL-R is the ability to bind TRAIL. Nucleic acid molecules capable of hybridizing to the DNA of Figures 1 and 2 under moderately stringent or highly stringent conditions, and which encode a biologically active TRAIL-R, are provided herein. Such hybridization nucleic acids include, but are not limited to, variant DNA sequences and DNAs derived from non-human species, for example, non-human mammals. Moderately stringent conditions include conditions described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed, Vol. 1, pp. 1101-104, Cold Spring Harbor Laboratory Press, 1989. Conditions of moderate restriction, such as defined by Sambrook et al., include the use of a 5X SSC prewash solution, 0.5% SDS, 1.0 mM EDTA (pH 8.0) and hybridization conditions of about 55 ° C, 5X SSC, overnight. Highly stringent conditions include higher hybridization and washing temperatures. One embodiment of the invention is directed to DNA sequences that will hybridize to the DNA of Figures 1 or 2 under highly stringent conditions, wherein said conditions include hybridization at 68 ° C followed by washing in 0.1X SSC / 0.1% SDS a 63-68 ° C. Certain DNA and polypeptides provided herein comprise nucleotide or amino acid sequences, respectively, which are at least 80% identical to a native TRAIL-R sequence. Also contemplated are embodiments in which a TRAIL-R DNA or polypeptide comprises a sequence that is at least 90% identical, at least 95% identical, or at least 98% identical to a native TRAIL-R sequence. . The percentage identity can be determined, for example, by comparing the sequence information using the GAP computer program, version 6.0, described by Devereux et al. (Nucí.Aids Res. 12: 387, 1984) and available from University of Wisconsin Genetics Computing. Group (UGCGW). The preferred error parameters for the GAP program include: a unit comparison matrix (containing a value of 1 for identities and 0 for: without identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucí. Acids Res. 14: 6745, 1986, as described by Schwartz and Dayhoff, eds, Atlas of Protein Sequence and Structure, National Biomedical Research Foundation, pp 353-358, 1979; (2) a penalty of 3.0 for each opening and a fine of 0.10 additional for each symbol in each opening; and (3) without penalty for extreme openings. For fragments, the percent identity is calculated by comparing the sequence of the fragment with the corresponding portion of a native TRAIL-R. In particular embodiments of the invention, a variant TRAIL-R polypeptide differs in amino acid sequence from a native TRAIL-R, but is substantially equivalent to a TRAIL-R native in a biological activity. An example is a TRAIL-R variant that binds TRAIL with essentially the same binding affinity as does a native TRAIL-R. The binding affinity can be measured by conventional methods, for example, as described in the U.S. Patent. do not. 5,512,457. The variant amino acid sequences may comprise conservative substitution (s), meaning that one or more amino acid residues of a native TRAIL-R are replaced by a different residue, but that the natively substituted TRAIL-R polypeptide retains a biological activity of the native protein (for example, the ability to bind TRAIL). A given amino acid can be replaced by a residue that has similar physicochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Lie, Val, Leu, or Alpha for another, or substitutions of one polar residue for another, such as between Lis and Arg; Glu and Asp; or Gln and Asn. Other conservative substitutions, for example, involving substitutions of entire regions that have similar hydrophobicity characteristics, are well known. In another example of variants, the sequences encoding Cis residues that are not essential for biological activity can be altered to cause the Cis residues to be suppressed or replaced with other amino acids, preventing the formation of unduly intramolecular disulfide bridges by renaturation. The cysteine residues within the cysteine-rich repeat domains described above are advantageously unchanged in TRAIL-R variants, when retention of the binding activity of TRAIL-R is desired.

Other variants are prepared by modifying adjacent dibasic amino acid residues, to increase expression in fermentation systems in which the KEX2 protease activity is present. EP 212,914 describes the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. The protease processing sites of KEX2 are deactivated by deletion, aggregation or substitution of residues to alter pairs of Arg-Arg, Arg-Lis, and Lis-Arg in order to eliminate the presentation of these adjacent basic residues. The human TRAIL-R contains such pairs of adjacent basic residues at amino acids 75-76, 233-234, 260-261, 261-262, 328-329, and 329-330 of Figure 1 and 2. The pairs of Lis -Lis are considerably less susceptible to the separation of KEX2, and the conversion of Arg-Lis or Lis-Arg to Lis-Lis represents a conservative and preferred feature for KEX2 deactivation sites. In yet other variants, the N-glycosylation sites are deactivated in a native TRAIL-R. The N-glycosylation sites can be modified to prevent glycosylation, allowing the expression of a more homogeneous reduced carbohydrate analog, in mammals and yeast expression systems. The N-glycosylation sites in eukaryotic polypeptides are characterized by a triplet of amino acids Asn-X-Y, wherein X is any amino acid except Pro, and Y is Ser or Thr. The TRAIL-R protein of Figures 1 and 2 comprises three such triplets, at amino acids 127-129, 171-173, and 182-184. Appropriate substitutions, additions or deletions of the nucleotide sequence encoding these triplets will result in the prediction of binding of the carbohydrate residues to the Asn side chain. The alteration of a single nucleotide, chosen in such a way that the Asn is replaced by a different amino acid, for example, is sufficient to deactivate an N-glycosylation site. Known methods for the deactivation of N-glycosylation sites in proteins include those described in the U.S. Patent. 5, 071,972 and EP 276,846, incorporated herein by reference. The TRAIL-R polypeptides, which include variants and fragments thereof, can be tested for biological activity in any suitable assay. The ability of a TRAIL-R polypeptide to bind TRAIL can be confirmed in conventional binding assays, examples, which are described below. Expression Systems The present invention also provides recombinant cloning and expression vectors containing TRAIL-R DNA, as well as host cells containing the recombinant vectors. Expression vectors comprising TRAIL-R DNA can be used to prepare TRAIL-R polypeptides encoded by DNA. A method for producing TRAIL-R polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding TRAIL-R, under conditions that promote expression of TRAIL-R, then recovering the expressed TRAIL-R polypeptides. of the crop. The experts will recognize that the method for purifying the expressed TRAIL-R will vary according to such factors as the type of host cells employed, and whether the TRAIL-R is a membrane bound or soluble form that is secreted from the host cell. Any suitable expression system can be used. The vectors include a DNA encoding a TRAIL-R polypeptide, operably linked to transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammal, microbe, virus, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, a ribosomal binding site of mRNA, appropriate sequences which control transcription and initiation of translation and termination. The nucleotide sequences are incoperably linked when the functionally regulatory sequence refers to the DNA sequence of TRAIL-R. Thus, a promoter nucleotide sequence is operably linked to a TRAIL-R DNA sequence if the promoter nucleotide sequence controls the transcription of the TRAIL-R DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene is identified by which transformants are generally incorporated into the expression vector.

In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into the expression vectors. A DNA sequence for a signal peptide (secretion leader) can be fused in structures to the TRAIL-R sequence such that the peptide that is functional in the target host cells promotes the extracellular secretion of the TRAIL-R polypeptide. The signal peptide is separated from the TRAIL-R polypeptide to the secretion of TRAIL-R from the cell. Suitable host cells for the expression of TRAIL-R polypeptides include prokaryotes, yeasts or major eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, and fermentation cell hosts are described, for example, in Pouweis et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems could also be used to produce TRAIL-R polypeptides using RNAs derived from DNA constructs described herein. Prokaryotes include gram positive or gram negative organisms, for example, E. coli or Bacilli. Prokaryotic host cells suitable for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and several other species within the genus Pseudomonoas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a TRAIL-R polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. N-terminal Met can be separated from the expressed recombinant TRAIL-R polypeptide. Expression vectors for use in prokaryotic host cells comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene that encodes a protein that confers resistance to antibiotics or that provides an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and therefore provides simple means to identify transformed cells. An appropriate promoter and a TRAIL-R DNA sequence are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, Wl, E.U.A.). Promoter sequences commonly used for expression vectors of recombinant prokaryotic host cells include β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275: 615, 1978, and Goeddel et al., Nature 281: 544, 1979) , tryptophan (trp) promoter system (Maniatis, Molecular Clonng: A Laboratory Manual, Cold Spring Habor Laboratory, p.412, 1982). A particularly useful prokaryotic cell expression system employs a phage? P promoter and a thermolabile repressor sequence cl857ts. The plasmid vectors available from the American Type Culture Collection which incorporates the promoter derivatives? P include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082). TRAIL-R can alternatively be expressed in fermentation host cells, preferably of the Saccharomyces class (eg, S, cerevisiae). Another type of yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will frequently contain a replication sequence origin of a 2μ yeast plasmid, an autonomously replicating sequence (SRA), a promoter region, sequence for polyadenylation, sequences for transcription of termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionine, 3-phosphoglycerate quinaza (Hitzeman et al., J. Biol. Chem. 225: 2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968; and Holland and others, Biochem. 17: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, sucrose-6-phosphate, 3-phosphoglycerate mutase, pyruvate quinaza, triosephosphate isomerase, shampoo of phospho-glucose, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the repressible glucose ADH2 promoter described by Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). Shuttle vectors replicable in both yeast and E. coli can be constructed by inserting the DNA sequences of pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) in the yeast vectors described above. The leader sequence of yeast a-factor can be used for direct secretion of the TRAIL polypeptide. The leader sequence of a-factor is frequently inserted between the promoter sequence and the structural gene sequence. See, for example, Kurjan et al., Cell 30: 933, 1982 and Bitter et al., Proc. Nati Acad. Sci. E.U.A. 81: 5330, 1984. Other suitable leader sequences for facilitating the secretion of recombinant polypeptides from known yeast hosts are known to those skilled in the art. A leader sequence can be modified near its 3 'end to contain one or more restriction sites. This will facilitate the fusion of the leader sequence to the structural gene. Yeast transformation protocols are known to those skilled in the art. A protocol is described by Hinnen and others. Proc. Nati Acad. Sci. E.U.A. 75: 1929, 1978. The Hinnen et al. Protocol selects for TRP + transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acid, 2% glucose, 10 μg / ml adenine and 20 μg / ml uracil. The yeast host cells transformed by vectors containing an ADH2 promoter sequence can grow by expression of induction in a "rich" medium. An example of a rich medium is that consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg / ml adenine and 80 μg / ml uracil. The repression of the ADH2 promoter occurs when the glucose is exhausted from the medium. Mammalian or insect host cell culture systems can also be used to express recombinant TRAIL-R polypeptides. Baculovirus systems for the production of heterologous proteins in insect cells are reviewed by Luckov and Summers, Bio / Technology 6:47 (1988). The established cell lines of mammalian origin can also be used. Examples of suitable mammalian host cell lines include COS-7 monkey kidney cell lines (ATCC CRL 1651) (Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary cells (CHO), HeLa cells, and BHK cell lines (ATCC CRL 10), and the CV1 / EBNA cell lines (ATCC CRL 10478) derived from the monkey kidney cell line African green (CV1 b (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).h.

Transcriptional and translational control sequences for expression vectors of mammalian host cells can be excised from viral genomes. Commonly the promoter sequences and enhancer sequences used are derived from polyoma virus, Adenovirus 2, Simian Virus 40 (VS40), and human cytomegalovirus. The DNA sequences derived from the VS40 viral genome, for example, can be used sites of origin of VS40, early or late promoter, enhancer, separator and polyadenylation to provide other genetic elements for the expression of a structural gene sequence in a cell mammalian host. Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 273: 113, 1978). Smaller or longer VS40 fragments can also be used, as long as the approximately 250 bp sequence extending from the Hind III site to the Bgl site located in the VS40 viral origin of the replication site is included. Expression vectors for use in mammalian host cells can be constructed, for example, as described by Okayama and Berg (Mol Cell Biol. 3: 280, 1983). A useful system for high level of stable expression of mammalian cDNA in C127 epithelial mammary murine cells can be constructed substantially as described in EP-A-0367566, and in WO 91/18982. As an alternative, the vector can be derived from a retrovirus. With respect to the signal peptides that can be employed in the production of TRAIL-R, the native TRAIL-R signal peptide can be replaced by a heterologous signal peptide or leader sequence, if desired. The choice of signal peptide or leader depends on factors such as the type of host cells in which the recombinant TRAIL-R is to be produced. To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in U.S. Patent 4,965,195, the signal sequence for interleukin-2. described in Costman et al., Nature 312: 768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; type I interleukin-1 receptor signal peptide described in the patent of E.U.A. 4,968,607; and type II interleukin-1 receptor signal peptide described in EP 460,846: another example is a leader peptide derived from cytomegalovirus, as described in WO 97/01633, incorporated herein by reference. Purified Protein The TRAIL-R polypeptides of the present invention can be produced by recombinant expression systems as described above, or purified from naturally occurring cells. TRAIL-R can be purified by any of a number of suitable methods, which can employ conventional protein purification techniques. As is known to the experts, the methods for purifying a given protein are chosen according to such factors as the types of contaminants to be removed, which may vary according to the particular cells in which the TRAIL is expressed. R. For recombinant proteins, other considerations include the particular expression systems employed and whether the desired protein is secreted or not in the culture medium. In one method, the cells expressing the proteins are disrupted by any of the numerous known techniques, including the cold dissolution cycle, sound treatment, mechanical disruption, or by the use of cell lysine dissolving agents. Alternatively, a TRAIL-R can be expressed and secreted from the cell. The subsequent purification process may include chromatography affinity, for example, employing a chromatography matrix containing TRAIL. The chromatography matrix can instead comprise an antibody that binds TRAIL-R. TRAIL-R polypeptides can be recovered from a chromatography column affinity using conventional techniques (e.g., elution in a high pH regulating salt), then dialyzed in a lower salt pH regulator for use. An example of a suitable chromatography matrix affinity is a Flag®-TRAIL-R Affi-gel column (10 mg of recombinant protein coupled to 1 ml of Affi-gel beads). The Affi-gel support is an N-hydroxysuccinimide ester of a derivatized crosslinked agarose gel bead (available from Biorad Laboratories, Richmond, CA). As discussed above, the Flag® peptide, Asp-Tir-Lis-Asp-Asp-Asp-Asp-Lis (SEQ ID NO: 5), provides a reversible binding of epitope by specific monoclonal antibodies, which allows rapid analysis and easy purification of expressed recombinant protein. The preparation of Flag®-TRAIL fusion proteins (comprising Flag® fused to a soluble TRAIL polypeptide) is further described in the PCT application WO 97/01633, incorporated by reference herein. The Flag®-TRAIL fusion protein binds to the Affi-gel beads by conventional techniques. In another aspect, when an expression system that secretes the recombinant protein is used, the culture medium can first be concentrated using a commercially available protein concentration filter, for example, an Amicon or Milipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration matrix. Alternatively, an anion exchange resin may be employed, for example, a matrix or substrate having pendant diethylaminoethyl groups (DEAE). The matrices can be, acrylamide, agarose, dextran, cellulose or other support materials commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include several insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. In addition, one or more steps of reverse phase high performance liquid chromatography (CLAR-FI) can be employed using hydrophobic CLAR-FI media (eg, silicon gel having pendant methyl groups or other aliphatic). Some or other of the above purification steps may be employed in various combinations. The recombinant protein produced in bacterial culture can be isolated by initial disruption of host cells, centrifugation, cell pellet extraction if it is an insoluble polypeptide, or from the supernatant fluid if it is a soluble polypeptide, followed by one or more of the concentration steps , salinization, ion exchange, purification or size exclusion chromatography. Finally, CLAR-FI can be used for the final purification steps. Microbial cells can be disrupted by any convenient method, including cold dissolution cycle, sound treatment, mechanical disruption or use of cell lysing agents. In yeast host cells, TRAIL-R is preferably expressed as a secreted polypeptide, to simplify purification. The recombinant polypeptides secreted from a fermentation of yeast host cells can be purified by methods analogous to those described by Urdal et al. (J. Chromatog, 296: 171, 1984). Urdal et al. Describe two reverse phase, sequential CLAR steps for the purification of recombinant human IL-2 on a preparative HPLC column. The desired degree of purity depends on the intended use of the protein. A relatively high degree of purity is desired when the protein is to be administered in vivo, for example. Advantageously, the TRAIL-R polypeptides are purified in such a way that no protein bands corresponding to other proteins (no.TRAIL-R) are detected when analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). Those of skill in the relevant field will recognize that multiple bands corresponding to the TRAIL-R protein can be visualized by SDS-PAGE, due to differential glycosylation, differential post-translational process, and the like. TRAIL-R more preferably is purified for substantial homogeneity, as indicated by a single protein band in the SDS-PAGE analysis. The protein band can be visualized by tinsion with silver. Tinsion of Coomasie blue, or (if the protein is radiolabelled) by autoradiography. Oligomeric Forms of TRAIL-R Included by the present invention are oligomers containing TRAIL-R polypeptides. The TRAIL-R oligomers may be in the form of covalently bound or non-covalently linked dimers, trimers or oligomers. One embodiment of the invention is directed to oligomers comprising multiple TRAIL-R polypeptides joined via covalent or non-covalent interactions between proportions of peptides fused to the TRAIL-R polypeptides. Such peptides may be peptide crosslinkers (separators), or peptides having the property of promoting oligomerization. Leucine closures and certain polypeptides derived from antibodies are among the peptides that can promote the oligomerization of TRAIL-R polypeptides attached thereto, as described in more detail below. In particular embodiments, the oligomers comprise from two to four TRAIL-R polypeptides. The proportions of TRAIL-R of the oligomer may be soluble polypeptides, as described above. As an alternative, a TRAIL-R oligomer is prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising certain heterologous polypeptides fused to several portions of antibodies derived from polypeptides (including the Fc domain) has been described, for example, by Ashkenazi et al. (PNAS E.U.A. 88: 10535.1991); Byrn et al. (Nature 344: 677, 1990); Hollebnbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl 4, pages 10.19.1-10.19.11, 1991); Smith et al (Cell 73: 1349-1360, 1993); and Fanslow et al (J. Immunol. 149: 655-660, 1992). One embodiment of the present invention is directed to a TRAIL-R dimer comprising two fusion proteins created by fusing the TRAIL-R to the Fc region of an antibody. A fusion of the gene encoding the TRAIL-R / Fc fusion protein is inserted into an appropriate expression vector. The TRAIL-R / Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble very similar antibody molecules, where the interchain disulfide bonds are formed between the Fc ratios to produce TRAIL -R divalent. Provided herein are fusion proteins comprising a polypeptide fused to an Fc polypeptide derived from an antibody. DNA encoding such fusion proteins, as well as dimers containing two fusion proteins linked via disulfide linkages between the Fc ratios thereof, are also provided. The term "Fc polypeptides" as used herein includes forms of native and mutein polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides that contain a hinge region that promotes dimerization are also included. A suitable Fc polypeptide, described in the application of TCP WO 93/10151 (hereby incorporated by reference), is a single chain polypeptide extending from the hinge region from N-terminal to C-terminus native to the region. Fc of a human IgG1 antibody. Another useful Fc polypeptide is the Fc mutein described in the U.S. Patent. 5,457,035 and in Baum et al. (EMBO J. 13: 3992-4001, 1994). The amino acid sequence of this mutein is identical to the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acids 22 ha been changed from Gli to Ala. The mutein exhibits reduced affinity for Fc receptors. In another embodiment, TRAIL-R may be substituted for the variable portion of a heavy or light chain antibody. If the fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a TRAIL-R oligomer with as many as four extracellular TRAIL-R regions. Alternatively, the oligomer is a fusion protein comprising multiple TRAIL-R polypeptides, with or without peptide crosslinkers (spacer peptides). Suitable peptide crosslinkers are those described in US Patents. 4,751,180 and 4,935,233, which are by reference incorporated by reference. A DNA sequence encoding a desired peptide ligand can be inserted between, and in the same reading frame as, the DNA sequences encoding TRAIL-R, using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding the interleaver can be ligated between the sequences encoding TRAIL-R. In particular embodiments, a fusion protein comprises from two to four soluble TRAIL-R polypeptides, separated by peptide crosslinkers. Another method to prepare oligomeric TRAIL-R involves the use of a leucine lock. The closing domains of leucine are peptides that promote the oligomerization of the proteins in which they are found. The Leucine closures were originally identified in several DNA binding proteins (Landschulz et al., Science 240: 1759, 1988), and have since been found in a variety of different proteins. Among the known leucine closures are naturally occurring peptides and derivatives thereof that are dimerized or trimerized. Examples of leucine zipper domains suitable for the production of soluble oligomeric proteins are described in the TCP application WO 94/10308, and the derivative leucine closure that allows stable trimerization of a heterologous protein fused thereto is described in Fanslow et al. (Semin. Immunol., 6: 267-278, 1994). Recombinant fusion proteins comprising a soluble TRAIL-R polypeptide fused to a leucine-closing peptide are expressed in suitable host cells, and the soluble oligomeric TRAIL-R that forms is recovered from the supernatant culture.

The Oligomeric TRAIL-R has the property of bivalent, trivalent binding sites, etc., for TRAIL. The fusion proteins described above comprising Fc proportions (and oligomers formed thereof) offer the advantage of easy purification by affinity chromatography on the Protein A or Protein G columns. The DNA sequences encoding the oligomeric TRAIL-R, or which encode fusion proteins useful in the preparation of TRAIL-R oligomers, are provided herein. Analysis TRAIL-R proteins (which include fragments, variants, oligomers and other forms of TRAIL-R) can be tested for the ability to bind TRAIL in any suitable assay. To illustrate, TRAIL-R may be labeled with a detectable reagent (eg, a radionuclide, chromophore, enzymes that catalyze a colorimetric or fluorometric reaction, and the like). The labeled TRAIL-R is contacted with the cells expressing TRAIL. The cells are then washed to remove unbound labeled TRAIL-R, and the presence of cell binding label is determined by a suitable technique, chosen according to the nature of the label. An example of a joining analysis procedure is as follows. A recombinant expression vector containing TRAIL cDNA is constructed, for example, as described in the PCT application WO 97/01633, herein incorporated by reference. The DNA and amino acid sequence information for human and mouse TRAIL is presented in WO 97/01633. TRAIL comprises a cytoplasmic N-terminal domain, a transmembrane region, and an extracellular C-terminal domain. Cells CV1-EBNA-1 (ATCC CRL 10478) constitutively expresses EBV-driven nuclear antigen-promoter / immediately increased-near CMV. CV1-EBNA-1 was derived from the African Green Monkey CV-1 kidney cell line (ATCC CCL 70), as described by McMahan et al. (EMBO J. 10: 2821, 1991).

The transfected cells are cultured for 24 hours, and the cells in each dish are subsequently separated into a good plate 24. After culturing an additional 48 hours, the transfected cells (about 4 × 10 4 cells / well) are washed with BM-NFDM, the which is binding medium (RPMI 1640 containing 25 mg / ml bovine serum albumin, 2 mg / ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg / ml of defatted dry milk have been added. The cells are subsequently incubated for 1 hour at 37 ° C with various concentrations of a soluble TRAIL-R / Fc fusion protein. The cells are then washed and incubated with a constant saturation concentration of a 125-human anti-human IgG in binding medium, with constant agitation for one hour at 37 ° C. After extensive washing, the cells are released via trypsinization. The mouse anti-human IgG employed is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA. The antibody is radioiodinated using the normal chloramine-T method. The antibody will bind to the Fc portion of any TRAIL-R / Fc protein that has bound to the cells. In all analyzes, the non-specific binding of antibody 1 5l is assayed in the absence of TRAIL-R / Fc, as well as in the presence of TRAIL-R / Fc and a 200-fold molar excess of anti-human IgG antibodies of mouse not labeled. The cell 125l antibody bound on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N.Y. Acad.

Sci. 51: 660,1949) are generated on RS / 1 (BBn Software, Boston, MA) operates on a Microvax computer. Another type of suitable binding analysis is a competitive binding analysis. To illustrate, the biological activity of a variant of TRAIL-R can be determined by analyzing for the ability of the variant to compete with a native TRAIL-R to bind TRAIL. Competitive union analysis can be performed by conventional methodology. Reagents that can be used in competitive binding analysis include radiolabeled TRAIL-R and intact cells expressing TRAIL (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble TRAIL-R fragment can be used to compete with a soluble TRAIL-R variant to bind TRAIL to the cell surface. Instead of intact cells, one could substitute a soluble TRAIL-R fusion protein bound to a solid phase through the interaction of Protein A or Protein G (on the solid phase) with the Fc portion. Chromatography columns containing Protein A and Protein G include those available from Pharmacia Biotech, Inc., Piscataway, NJ. Another type of competitive binding assay utilizes radiolabeled soluble TRAIL, such as a TRAIL / Fc fusion protein, and intact cells expressing TRAIL-R. Qualitative results can be obtained by competitive autoradiographic plate binding analysis, while Scatchard graphs (Scatchard, Ann. N.Y. Acad. Sci. 51: 660, 1949) can be used to generate quantitative results.

Another type of analysis for biological activities involves testing a TRAIL-R polypeptide for the ability to block TRAIL-mediated apoptosis of target cells, such as the human leukemic T-cell line known as Jurkat cells, for example, apoptosis mediated by TRAIL of the cell line designated clone E6-1 of Jurkat (ATCC TIB 152) is demonstrated in assay methods described in the application of TCP WO 97/01633, incorporated herein by reference. Uses of TRAIL-R. Uses of TRAIL-R include, but are not limited to the following.

Certain of these TRAIL-R flow uses your ability to join TRAIL. TRAIL-R finds use as a protein purification reagent. The TRAIL-R polypeptides can be attached to a solid support material and used to purify TRAIL proteins by affinity chromatography. In particular embodiments, a TRAIL-R polypeptide (in any form described herein that is capable of binding TRAIL) is attached to a solid support by standard procedures. As an example, chromatography columns containing functional groups that react with functional groups on amino acid side chains of proteins are available (Pharmacia Biotech, Inc., Piscataway, NJ). As an alternative, a TRAIL-R / Fc protein binds to a Protein A or Protein G containing chromatography columns through interaction with the Fc portion.

The TRAIL-R proteins also find use in the measurement of the biological activity of TRAIL proteins in terms of their binding affinity for TRAIL-R. The TRAIL-R proteins can therefore be used by those who perform "quality assurance" studies, for example, they monitor the shelf life and stability of the TRAIL protein under different conditions. To illustrate, TRAIL-R can be used in a binding affinity study to measure the biological activity of a TRAIL protein that has been stored at different temperatures, or produced in different cell types. TRAIL-R can also be used to determine whether biological activity is retained or not after modification of a TRAIL protein (eg, truncation modification, chemical mutation, etc.). The binding affinity of the TRAIL protein modified by TRAIL-R will be purchased with the unmodified TRAIL protein to detect any adverse impact of the modifications on the biological activity of TRAIL. Therefore, the biological activity of a TRAIL protein can be specified before it is used in a research study, for example. TRAIL-R also finds use in purification or identification of cells expressing TRAIL on the surface of the cell. The TRAIL polypeptides are attached to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, the magnetic microspheres can be coated with TRAIL-R and contained in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing cells expressing TRAIL are contacted with the solid phase having TRAIL thereon. Cells expressing TRAIL on the cell surface bind to fixed TRAIL-R, and unbound cells are subsequently washed. Alternatively, TRAIL-R can be conjugated to a detectable position, then incubated with cells to be tested for TRAIL expression. After incubation, the unbound labeled TRAIL-R is removed and determined in the presence or absence of the detectable portion on the cells. In a further alternative, mixtures of cells suspected of containing TRAIL cells are incubated with biotinylated TRAIL-R. Incubation periods are usually at least one hour in duration to ensure sufficient binding. The resulting mixture is then passed through a column packed with beads coated with avidin, whereby the high affinity of biotin to avidin provides for the binding of the desired cell to the beads. The procedures for using avidin-coated beads are known (see Berenson et al. J. Cel. Biochem., 10D: 239, 1986). Washing to remove unbound material, and release of bound cells, is done using conventional methods. The TRAIL-R polypeptides also find use as carriers for delivery agents linked to the same cells that carry TRAIL. Cells expressing TRAIL include those identified in Wiley et al. (Immunity, 3: 673-682, 1995). The TRAIL proteins can therefore be used to deliver diagnostic or therapeutic agents to such cells (or other types of cells found to express TRAIL on the surface of the cell) in in vitro or in vivo procedures. Detectable (diagnostic) and therapeutic agents that can bind to a TRAIL-R polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores. Enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent that is chosen according to the intended application. Among the toxins are castor bean, abrin, diphtheria toxin, Pseudomonas aeruginosa A exotoxin, ribosomal deactivation proteins, mycotoxins such as trichothecenes, and derivatives and fragments (eg, single chains) thereof. Radionuclides suitable for use in dentistry, but are not limited to 112Z3d, 1, 3J1, i1, 9 M9Mmm-Trc ", 1 '1" 1, 76, diagnosis, and' Br. Examples of suitable radionuclides for therapeutic use are 131l, 211At, 77Br, 186Re, 188Re, 212Pb, 212Bi, 109Pd, 64Cu, and 67Cu Such agents can be linked to TRAIL-R by any suitable conventional procedure.Trail-R, which is a protein, comprises functional groups on amino acid side chains that can react with functional groups on a desired agent to form covalent bonds, for example, Alternatively, the protein or agent can be derivatized to generate or bind a desired reactive functional group. One of the bifunctional coupling reagents available for joining various molecules to proteins (Pierce Chemical Company, Rockford, Ill.) A number of techniques for radiolabelling proteins are known. can join TRAIL-R using a suitable bifunctional chelating agent, for example. Therefore, conjugates comprising TRAIL-R and a suitable diagnostic or therapeutic agent (preferably covalently linked) are prepared. The conjugates are administered or in some way employed in an amount appropriate for the particular application. The DNA of TRAIL-R and polypeptides of the present invention can be used to develop treatments for any disorder mediated (directly or indirectly) by defect, or in sufficient amounts of TRAIL-R. The TRAIL-R polypeptides can be administered to a mammal suffering from such a disorder. Alternatively, any aspect of gene therapy can be taken. The description herein of nucleotide sequences of native TRAIL-R allows the detection of defective TRAIL-R genes, and the replacement thereof with that of a TRAIL-R gene derived from a person suspected of having a defect in TRAIL-R. this gene. Another use of the protein of the present invention is as a research tool to study the biological effects that result from the inhibition of TRAIL / TRAIL-R interactions on different cell types. The TRAIL-R polypeptides can also be used in in vitro assays to detect TRAIL or TRAIL-R interactions thereof. A purified TRAIL-R polypeptide can be used to bind TRAIL, thereby inhibiting the binding of TRAIL to endogenous cell surface TRAIL receptors. Such TRAIL receptors include the TRAIL-R described herein. As well as TRAIL binding proteins that are distinct from the TRAIL-R of the present invention. It has been reported that certain ligands of the TNF family (of which TRAIL is a member) bind to more than one different cell surface receptor protein. TRAIL can also bind multiple cell surface proteins. A DR4 probe receptor protein reported to bind TRAIL, but is distinct from the TRAIL-R of the present invention, is described in Pan et al. (Science 276: 111-113, 1997; in the present incorporated by reference). TRAIL-R can be used to inhibit a biological activity of TRAIL, in in vitro or in vivo procedures. Inhibiting the binding of TRAIL to cell surface receptors, TRAIL-R consequently inhibits biological effects resulting from the binding of TRAIL to endogenous receptors. Various forms of TRAIL-R can be employed, including, for example, the fragments, oligomers, derivatives and variants of TRAIL-R described above which are capable of binding to TRAIL. In a preferred embodiment, a soluble TRAIL-R is employed to inhibit a biological activity of TRAIL, for example to inhibit TRAIL-mediated apoptosis of cells susceptible to said apoptosis. TRAIL-R can be administered to a mammal to treat a disorder mediated by TRAIL. Such disorders mediated by TRAIL include conditions caused (directly or indirectly) or exacerbated by TRAIL. TRAIL-R may be useful to treat thrombotic microangiopathies. One such disorder is thrombotic thrombocytopenic purpura (PTT) (Keaan, H.C., Semin.Hetmatol, 24:71, 1987, Thompson et al., Blood, 80: 1890, 1992). Increasing the mortality regimes associated with PTT have been reported by the Centers for Disease Control of E.U.A. (Torok et al., Am. J. Hematol 50: 84,1995). The plasma of patients affected with PTT (including HIV + and HIV patients ") induces apoptosis of human endothelial cells of dermal microvascular origin, but no longer than the vessel of origin (Laurence et al., Blood, 87: 3245, 15 April 1996) Plasma of PTT patients is therefore through to contain one or more factors that directly or indirectly produce apoptosis As described in the application of TCP WO 97/01633 (hereby incorporated by reference ), TRAIL is present in the serum of PTT patients, and may have a role in the induction of microvascular endothelial cell apoptosis.

Another thrombotic microangiopathy is haemolytic-uremic syndrome (HUS) (Moake, JL Lancet, 343: 393, 1994, Melnyk et al., (Arch. Intern. Med., 155: 2077, 1995, Thompson et al., Supra). of the invention is directed to the use of TRAIL-R to treat the condition that is frequently referred to as "adult HUS" (although this may also strike children) A disorder known as HUS associated with childhood / diarrhea differs in etiology from HUS adult Other conditions characterized by accumulation of small blood vessels can be treated using TRAIL-R, such conditions include but are not limited to the following: Heart problems observed in about 5-10% of AIDS patients are thought to be involved in the grouping of small blood vessels The rupture of the microvasculature in the heart has been reported in patients with multiple sclerosis As a further example, the treatment of lupus erythromatosis is contemplated Stressed (SLE) In one modality, the patient's blood or plasma is contacted with TRAIL-R ex vivo. TRAIL can be attached to a suitable chromatography matrix by conventional methods. The patient's blood or plasma flows through a chromatography column containing TRAIL-R attached to the matrix, before it is returned to the patient. The immobilized receptor binds TRAIL, thus removing the TRAIL protein from the patient's blood.

Alternatively, TRAIL-R can be administered in vivo to a patient affected with a thrombotic microangiopathy. In one embodiment, a soluble form of TRAIL-R is administered to the patient. The present invention therefore provides a method for treating a thrombotic microangiopathy, which involves the use of an effective amount of TRAIL-R. A TRAIL-R polypeptide can be employed in in vivo or ex vivo procedures to inhibit TRAIL-mediated damage to (e.g., apoptosis of) microvascular endothelial cells. TRAIL-R can be used in conjunction with other agents useful in the treatment of a particular disorder. In an in vitro study reported by Laurence et al. (Blood 87: 3245, 1996), some plasma-mediated apoptosis reduction of TTP from microvascular endothelial cells was carried out using an anti-Fas antibody, aurintricarboxylic acid, or normal plasma depleted of cryoprecipitate. Therefore, a patient can be treated with an agent that inhibits apoptosis mediated by Fas ligands of endothelial cells, in combination with an agent that inhibits TRAIL-mediated apoptosis of endothelial cells. In one embodiment, TRAIL-R and an anti-FAS blocking antibody and both are administered to a patient affected with a disorder characterized by thrombotic microangiopathy, such as PTT or HUS. Examples of blocking monoclonal antibodies directed against the Fas antigen (CD95) are described in the PCT application publication number WO 95/10540, incorporated herein by reference. Another embodiment of the present invention is directed to the use of TRAIL-R to reduce TRAIL-mediated death of T cells in patients infected with HIV. The role of T cell apoptosis in the development of AIDS has been the goal of a number of studies (see, for example, Meyaard et al. Science 257: 217-219, 1992; Groux et al., J. Exp. Med. , 175: 331, 1992, and Oyaizu et al., In Cell Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-104). Some researchers have studied the role of Fas-mediated apoptosis; the involvement of interleukin-1ß-converting enzyme (ICE) has also been explored (Estaquier et al., Blood 87: 4959-4966, 1996; Mitra et al., Immunology 87: 581-585, 1996; Katsikis et al., J; Exp. Med. 181: 2029-2036,1995). It is possible that T-cell apoptosis occurs through multiple mechanisms. At least some of the T cell deaths observed in HIV patients are believed to be measured by TRAIL. Until you want to join by theory, such T-cell death mediated by TRAIL is thought to occur through the mechanism known as cell death induced by activation (AICD). Activated human T cells are induced to undergo programmed cell death (apoptosis) by cycling through the CD3 / T cell complex receptor, a completed process of cell death induced by activation (AICD). Cells isolated from asymptomatic individual CD4'T AICDs infected with HIV have been reported. (Groux and others, supra). Therefore, AICD may play a role in the depletion of CD4 'T cells and progression to AIDS in individuals infected with HIV. The present invention provides a method of inhibiting T cell death mediated by TRAIL in HIV patients, comprising the administration of TRAIL-R (preferably, a soluble TRAIL-R polypeptide) to patients. In one modality, the patient is asymptomatic when treatment with TRAIL-R begins. If desired, prior to treatment, peripheral blood T cells can be extracted from an HIV patient, and tested for susceptibility to cell death mediated by TRAIL by standard procedures. In one embodiment, the blood or plasma of a patient is contacted with TRAIL-R ex vivo. TRAIL-R can be attached to a suitable chromatography matrix by suitable methods. The patient's blood or plasma flows through a chromatography column containing TRAIL-R attached to the matrix, before it is returned to the patient. The immobilized TRAIL-R binds TRAIL, therefore it removes the TRAIL protein from the patient's blood. In the treatment of HIV + patients, TRAIL-R can be used in combination with other inhibitors of T cell apoptosis. Fas-mediated apoptosis has also been implicated in the loss of T cells in HIV + individuals (Katsikis et al. J. Exp. Med. 181: 2029-2036, 1995). Therefore, a patient susceptible to both T cell death mediated by Fas (L-Fas) and TRAIL-mediated ligands can be treated with both agents that block TRAIL / TRAIL receptor interactions and an agent that blocks interactions. Fas-L / Fas. Suitable agents to block the binding of Fas-L to Fas include, but are not limited to, soluble Fas polypeptides.; oligomeric forms of soluble Fas polypeptides (e.g., sFas / Fc dimers); the anti-Fas antibodies that bind the Fas without transducing the biological signal that results in apoptosis; anti-Fas-L antibodies that block the binding of Fs-L to Fas; and Fas-L muteins that bind Fas but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies used in the method with monoclonal antibodies. Examples of agents suitable for blocking Fas-L / Fas interactions, including blocking of anti-Fas monoclonal antibodies, are described in WO 95/10540, incorporated herein by reference. Compositions comprising an effective amount of a TRAIL-R polypeptide of the present invention, in combination with other components such as a physiologically acceptable diluent, carrier or excipient are provided herein. TRAIL-R can be formulated according to known methods used to prepare pharmaceutically useful compositions. TRAIL-R may be combined in combination, either from the single active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (eg, saline, Tris-HCl, pH buffer solutions of acetate, and phosphate) , preservatives (for example, thimerosal, benzyl alcohol, paraben), emulsifiers, solubilizers, adjuvants and / or carriers. Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton, PA. In addition, such compositions may contain TRAIL-R complex with polyethylene glycol (GPE), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles , vesicles of a lamina or of multiple laminae, ghosts of erythrocytes or esfereoblasts. Such compositions will influence the physical state, solubility, stability, in vivo release regimen, and in vivo clearance regimen of TRAIL-R, and are therefore chosen in accordance with the intended application. TRAIL-R expressed on the surface of a cell can find use, too. The compositions of the present invention may contain a TRAIL-R polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers and biologically active fragments. In particular embodiments, the composition comprises a soluble TRAIL-R polypeptide or an oligomer comprising soluble TRAIL-R polypeptides.

TRAIL-R can be administered in any suitable manner, for example, topically, parenterally, or by inhalation. The term "parenteral" includes injection, for example by subcutaneous, intravenous, or intramuscular routes, also including localized administration. The sustained release of implants is also contemplated. One of skill in the pertinent art will recognize that adequate dosages will vary, depending on such factors as the nature of the disorder being treated, the patient's body weight, age, and general condition, and the route of dose administration for human administration are displayed. according to the practices accepted by the technique. Compositions comprising the TRAIL-R nucleic acids in physiologically acceptable formulations are also contemplated. The TRAIL-R DNA can be formulated by injection, for example. Antibodies The antibodies that bind the TRAIL-R polypeptides are provided herein. The TRAIL-R protein of Figure 1 or 2 (SEQ ID NO: 2 or 4) can be used as an immunogen in production of immunoreactive antibodies therewith. Alternatively, another form of TRAIL-R, such as a fragment or fusion protein, is employed as the immunogen. The present invention therefore provides antibodies obtained by immunizing an animal with the TRAIL-R of Figure 1 or 2, or an immunogenic fragment thereof. A method for producing antibodies directed against TRAIL-R is generated in said animal. The desired antibodies can be purified, for example, from the serum of the animal, by means of conventional techniques. Among the procedures for preparing polyclonal and monoclonal antibodies are described in Monoclonal Antibodies, Hybridomas: A new Dimension in Biological! Analyzes, Kennet et al. (Eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). The production of monoclonal antibodies directed against TRAIL-R is further illustrated in Example 4. Antigen binding fragments of such antibodies, which can be produced by conventional techniques, are also included in the present invention. Examples of such fragments include, but are not limited to, Fab and F (ab ') 2 fragments. Antibody fragments and derivatives produced by genetic engineering techniques are also provided. The monoclonal antibodies of the present invention include chimeric antibodies, for example, humanized versions of murine monoclonal antibodies. Such humanized antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody and a variant region fragment (lacking the antigen binding site) derived from a human antibody. Methods for the production of chimeric and additional engineered monoclonal antibodies include those described in Riechmann et al. (Nature 332: 323, 1988), Liu et al. (PNAS 84: 3439.1987), Larrick et al. (Bio / technology 7: 934). , 1989), and Winter and Harris (Tips 14: 139, May 1993). In one embodiment, the antibodies are specific for the TRAIL-R of the present invention, and do not react to cross with other proteins (non-TRAIL-R). Screening procedures by which such antibodies can be identified are well known, and may, for example, involve immunoaffinity chromatography. Hybridoma cell lines that produce monoclonal antibodies specific for TRAIL-R are also contemplated herein. Such hybridomas can be produced and identified by conventional techniques. One method to produce such a hybridoma cell line comprises immunizing an animal with a TRAIL-R; harvesting spleen cells from the immunized animal; fusing said spleen cells to a myeloma cell line, thereby generating hybridoma cells; and identify a hybridoma cell line can be recovered by conventional techniques. Among the uses of the antibodies is used in analyzes to detect the presence of TRAIL-R polypeptides, both in vitro or in vivo. The antibodies can also be used in the purification of TRAIL-R proteins by immunoaffinity chromatography. In one embodiment, the antibodies can additionally block the binding of TRAIL to TRAIL-R. Such antibodies can be used to inhibit the binding of TRAIL to the cell surface TRAIL-R, for example. Blocking antibodies can be identified using conventional analysis methods. Such an antibody can be used in an in vitro procedure, or administered in vivo to inhibit a biological activity mediated by TRAIL-R. The disorders caused or exacerbated (directly or indirectly) by the interaction of TRAIL with the TRAIL-R cell surface can therefore be treated. A therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting a biological activity mediated by TRAIL. The disorders caused or exacerbated by TRAIL, directly or indirectly, are therefore treated. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In one embodiment, a fragment of antigen binding antibody employs. The compositions comprising an antibody that is directed against TRAIL-R, and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described above by compositions containing TRAIL-R proteins. The conjugates are also provided herein which comprise a detectable (eg, diagnostic) or therapeutic agent, linked to an antibody directed against TRAIL-R. Examples of such agents are presented above. The conjugates find use in in vitro or in vivo procedures. Nucleic acids. The present invention provides TRAIL-R nucleic acids. Such nucleic acids include, but are not limited to, the human TRAIL-R DNA of Figures 1 and 2 (SEQ ID NOS: 1 and 3). The nucleic acid molecules of the present invention include TRAIL-R DNA in both single filament and double filament forms, as well as the RNA complement thereof. The TRAIL-R DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR amplified DNA, and combinations thereof. Genomic DNA can be isolated by conventional techniques, for example, using cDNA of Figures 1 and 2 or a suitable fragment thereof, as a test. Particular embodiments, nucleic acids are useful in the production of TRAIL-R polypeptides, for example, in the recombinant expression systems discussed above. DNAs encoding TRAIL-R in any of the forms contemplated herein (e.g., full-length TRAIL-R or fragments thereof) are provided. Particular embodiments of TRAIL-R encoding the DNAs include DNAs encoding the full-length human TRAIL-R of Figures 1 or 2 (including the N-terminal signal peptide), and DNA encoding mature human TRAIL-R from full length Other embodiments include DNA encoding soluble TRAIL-R (e.g., the extracellular domain of the protein, either with or without the signal peptide). The TRAIL-R nucleic acid fragments provided herein have uses including, but not limited to, use as tests or primers. Oligonucleotides derived from the nucleic acid sequences described herein can be used as 3 'and 5' primers in polymerase chain reactions (PCR), for example, where the TRAIL-R DNA fragments are isolated and amplified. Particular fragments of TRAIL-R nucleotide sequences comprise at least about 30, or at least 60, contiguous nucleotides of a TRAIL-R DNA sequence. The nucleic acids provided herein include DNA and RNA supplements of said fragments, along with both single strand and double strand forms of the TRAIL-R DNA. Other useful fragments of the TRAIL-R nucleotide acids include sense and sense oligonucleotides comprising a single strand nucleic acid sequence (either RNA or DNA) capable of binding to the white TRAIL-R mRNA (sense) or DNA sequences TRAIL-R (sense). The antisense or sense oligonucleotides, according to the present invention, may comprise a fragment of the coding region of the TRAIL-R DNA of Figure 1 or 2. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to 30 nucleotides. The ability to derive an antisense or sense oligonucleotide, based on the cDNA sequence encoding a given protein, is described in, for example, Atein and Cohen (Cancer Res. 48: 2659, 1998) and van der Krol et al. (BioTechniques 6: 958, 1988). The binding of sense and sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block the transcription or translation of the target sequence by one of several means, including increased degradation of duplexes, premature termination of the transcription or translation, by other means. The antisense oligonucleotides can therefore be used to block the expression of TRAIL-R proteins. Sense or sense oligonucleotides further comprise oligonucleotides having modified phosphodiester sugar base structures (or other sugar linkages, such as those described in WO 91/06629) and wherein such sugar linkages are resistant to endogenous nucleosides . Such oligonucleotides with resistant sugar linkages are stable in vivo (ie, capable of resisting enzymatic degradation) but retain the sequence specificity to be capable of binding to target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other portions that increase the affinity of the oligonucleotides for a target nucleic acid sequence, such as poly- (L-lysine). In addition, intercalating agents, such as, ellipticine, and alkylating agents or metal complexes can be attached to sense or antisense oligonucleotides to modify the binding specificities of the sense or sense oligonucleotide for the target nucleotide sequence. The sense or sense oligonucleotides can be introduced into a cell containing the target nucleic acid sequence by any method of gene transfer, including, for example, transfection of DNA mediated by CaPO4, electrophoresis, or using gene transfer vectors such as Epstein-Barr virus. In a preferred method, an antisense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, both in vivo and ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retroviruses M-MuLV, N2 (a retrovirus derived from M-MuLV), or from the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90). / 13641).

The sense and antisense oligonucleotides can also be introduced into a cell containing the target nucleotide sequence by the formation of a conjugate with a ligand that binds the molecule, as described in WO 91/04753. Ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or blocking entry of the sense or antisense oligonucleotide or its conjugated version in the cell . Alternatively, a sense or antisense oligonucleotide can be introduced into a cell containing a target nucleic acid sequence by the formation of a complex oligonucleotide-lipid, as described in WO 90/10448. The oligonucleotide-lipid complex is preferably disassociated within the cell by an endogenous lipase. The following examples are provided to further illustrate particular embodiments of the invention, and are not construed as limiting the scope of the present invention. EXAMPLE 1: TRAIL-R cDNA clones TRAIL-R clones were isolated from two cDNA libraries. The first cDNA library was derived from human skin anterior fibroblasts; the second was derived from human peripheral blood lymphocytes (PBL). A TRAIL-R cDNA clone isolated from the PBL library was found to comprise the nucleotide sequence shown in Figure 2 (SEQ ID NO: 3). Both forms of TRAIL-R (the sequence presented in Figure 1 or Figure 2) were represented in clones isolated from the human skin anterior fibroblast library. The nucleotide proteins of Figure 1 (SEQ ID NO: 1) and Figure 2 (SEQ ID NO: 3) differ in two positions. In Figure 1, the nucleotide at position 145 is a C, since nucleotide 145 is a T in Figure 2; the nucleotide at position 971 is a C in Figure 1, but it is a T in Figure 2. The amino acid sequences likewise differ in two positions. Residue 35 is Pro in Figure 1 (SEQ ID NO: 2) and Ser in Figure 2 (SEQ ID NO: 4); residue 310 is Ser in Figure 1 and Leu in Figure 2. One possible explanation is that the TRAIL receptors of Figures 1 and 2 are allelic variants. The TRAIL-R proteins of Figure 1 (SEQ ID NO: 2) and Figure 2 (SEQ ID NO: 4) include an N-terminal hydrophobic region that functions as a signal peptide. A signal peptide separation site provided by computer analysis followed by amino acids 55 of Figures 1 and 2. Separation of the signal peptide (amino acids 1 to 55) would therefore produce a mature protein comprising amino acids 56 through 386. The protein also comprises an extracellular domain (amino acids 56 to 211), a transmembrane region (amino acids 212 through 232), and a cytoplasmic C-terminal domain (amino acids 233 through 386). The two amino acids that differ in the proteins of Figures 1 and 2 are found in the signal peptide (in the position ) and in the cytoplasmic domain (at position 310). The extracellular domains of the proteins of Figures 1 and 2 are therefore identical. EXAMPLE 2: Analysis of union. The TRAIL-R was tested for the ability to join TRAIL, in a slide binding analysis. The DNA encoding the full-length TRAIL-R of Figure 1 was inserted into a mammalian expression vector designated pDC409. PDC409 was derived from the pDC406 vector described in McMahan et al. (EMBO J. 10: 2821-2832, 1991, hereby incorporated by reference). Aspects added to pDC409 (compared to pDC406) include additional single restriction sites at the multiple cloning site (scm); three stop triplets (one in each reading structure) positioned below the scm; and a T7 polymerase promoter, downstream of the scm, which facilitates the sequencing of the DNA inserted into the scm. The CV-1 / EBNA cells were transfected with the recombinant expression vector, and cultured on glass plates to allow the expression of TRAIL-R. A slip binding analysis was generally conducted as described in Gearing et al. (EMBO J. 8: 3667, 1989); McMahan et al. (EMBO J. 10: 2821, 1991) and Goodwin et al. (Eur. J. Immunol., 23: 2631, 1993), incorporated herein by reference. Briefly, the transfected cells are incubated with a CL-TRAIL fusion protein (described below) in binding medium. The plates are then washed and a 125I-labeled antibody specific for the leucine (CL) closing portion of the fusion protein is added. After incubation with the antibody, the plates were washed, fixed and immersed in photographic emulsion. The analysis showed that the TRAIL-R protein binds to TRAIL. The CL-TRAIL used in the analysis is a fusion protein comprising a leucine-binding peptide fused to the N-terminus of a soluble TRAIL polypeptide (CL-TRAIL) was used in the analysis. An expression construct was prepared, essentially as described by the preparation of the Flag®-TRAIL expression construct in Wiley et al. (Immunity, 3: 673-682,1995, incorporated herein by reference), except that DNA encodes the Flag® peptide was replaced with a sequence encoding a modified leucine lock that is allowed for trimerization. The construct, in expression vector pDC409, encoded a leader sequence derived from human cytomegalovirus, followed by the closing portion of leucine fused at the N-terminus of a soluble TRAIL polypeptide. The TRAIL polypeptide comprised amino acids 95-281 of human TRAIL (a fragment of the extracellular domain), as described in Wiley et al. (Supra), CL-TRAIL was expressed in CHO cells, and purified from culture supernatant.

EXAMPLE 3: Preparation of a soluble TRAIL-R An expression vector encoding a soluble TRAIL-R / Fc fusion protein was constructed, as follows. The fusion protein comprised a soluble TRAIL-R polypeptide fused at the N-terminus of a lgG1 Fc region polypeptide mutein. A DNA fragment encoding amino acids 1 through 208 of Figure 1 (SEQ D NO: 2) was isolated by polymerase chain reaction (PCR). Oligonucleotides that defined the desired term of the DNA fragment were used as the 3 'and 5' primers in the PCR, and the cDNA clone depicted in Figure 1 (SEQ ID NO: 1) was used as the standard. The Fc portion of the fusion protein was a mutein of a human IgG1 Fc region polypeptide. The DNA and amino acid sequence information for this Fc mutein is described in the U.S. Patent. 5,457,035 and in Baum et al. (EMBO J. 13: 3992-4001, 1994), incorporated herein by reference. The procedures for preparing an expression vector containing a fusion of TRAIL-R / Fc genes were generally as described in Smith et al (Cell 73: 1349-1360, 1993) and Fanslow et al (J. Immunol., 149: 655 -660, 1992), which are incorporated herein by reference. The expression vector pDC409, described in example 2, was used. The CV1 / EBNA cells were transfected with the resulting recombinant expression vector, and cultured to allow the expression and secretion of the TRAIL-R / Fc from the cells. EXAMPLE 4: Monoclonal antibodies binding TRAIL-R. This example illustrates a method for preparing monoclonal antibodies that bind TRAIL-R. Suitable immunogens that can be employed in the generation of such antibodies include, but are not limited to, purified TRAIL-R protein or an immunogenic fragment thereof such as the extracellular domain, or fusion proteins containing TRAIL-R (e.g., a soluble TRAIL-R / Fc fusion protein). The purified TRAIL-R can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in the U.S. Patent. 4,411,993. Briefly, mice are immunized with TRAIL-R immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10 to 100 μg subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals were raised with additional TRAIL-R emulsified in incomplete Freund's adjuvant. The mice are periodically raised after the same on a weekly or bi-weekly schedule. Serum samples are taken periodically or by retro-orbital purge or end-to-end excision to test TRAIL-R antibodies by spot analysis, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of TRAIL binding.

After detection of an appropriate antibody titer, positive animals are provided with a final intravenous injection of TRAIL-R in saline. Three to four days later, the animals are sacrificed, the spleen cells are recovered, and the spleen cells are fused with a murine myeloma cell line, for example, NSI or preferably P3x63Ag8.653 (ATCC CRL 1580). The fusions generate the hybridoma cells, which are seeded in multiple microtiter plates in a selective HAT (hypoxanthine, aminopterin and thymidine) medium to inhibit the proliferation of unfused cells, myeloma hybrids, and spleen cell hybrids. Hybridoma cells are screened by ELISA for reactivity against TRAIL-R purified by adaptations of the techniques described in Engvall et al., Immunochem. 8: 871, 1971 and in the U.S. Patent. 4,703.004. A preferred screening technique is the technique of capturing antibodies described in Beckmann et al. (J. Immunol., 144: 4212, 1990). The positive hybridoma cells can be injected intraperitoneally into syngeneic BALB / c mice to produce ascites containing high concentrations of anti-TRAIL-R monoclonal antibodies. Alternatively, the hybridoma cells can grow in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by precipitation of ammonium sulfate, followed by gel exclusion chromatography. Alternatively, chromatography affinity can also be used based on the binding of the antibody to Protein A or Protein G, as well as affinity chromatography based on binding to TRAIL-R.

LIST OF SEQUENCES (1) GENERAL INFORMATION: (i) APPLICANTS: IMMUNEX CORPORATION, (ii) TITLE OF THE INVENTION: LIGANDO INDUCTOR RECEIVER OF APOPTOSIS RELATED WITH TNF. (iíi) SEQUENCE NUMBER: 5 (v) CORRESPONDENCE ADDRESS: (A) ADDRESS: Kathryn A. Anderson, Immunex Corporation (B) STREET: 51 University Street (C) CITY: Seattie (D) STATE: WA (E) ) COUNTRY: USA (F) POSTAL CODE: 98101 (v) COMPUTER LEADABLE FORM (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM compatible PC (C) OPERATING SYSTEM: MS-DOS / Windows 95 (D) SOFTWARE: Word for Windows 95, 7.0a (vi) CURRENT APPLICATION DATA (A) APPLICATION NUMBER: -by being assigned- (B) DATE OF SUBMISSION: 10-Jul-1998 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) NUMBER OF APPLICATION: USA 08 / 892,119 (B) DATE OF SUBMISSION: 15-Jul-1998 (C) CLASSIFICATION: (viii) INFORMATION OF POWDER / AGENT (A) NAME: Anderson, Kathryn A. (B) REGISTRATION NUMBER: 32,172 (C) NUMBER OF REFERENCE / CASE: 2630-WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE: 206-587-0430 (B) TELEFAX: 206-233-0644 (2) INFORMATION FOR SEQ ID NO: 1: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 1552 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (vii) ORIGINAL SOURCE (B) CLON: TRAILR4A (ix) CHARACTERISTICS: ( A) NAME / KEY: CDS (B) LOCATION: 43..1203 (xi) DESCRIPTION OF THE SEQUENCE: SEQ ID NO: 1: TGATTGATTT TTGGCGCTTT CGATCCACCC TCCTCCCTTC TC ATG GGA CTT TGG 54 Met Gly Leu Trp 1 GGA CAA AGC GTC CCG ACC GCC TCG AGC GCT CGA GCA GGG CGC TAT CCA 10.

Gly Gln Ser Val Pro Thr Wing Ser Wing Wing Arg Wing Gly Arg Tyr Pro 5 10 15 20 GGA GCC AGG ACA GCG TCG GGA ACC AGA CCA TGG CTC CTG GAC CCC AAG 150 Gly Wing Arg Thr Wing Ser Gly Thr Arg Pro Trp Leu Leu Asp Pro Lys 25 30 35 ATC CTT AAG TTC GTC GTC TTC ATC GTC GCG GTT CTG CTG CCG GTC CGG 198 He Leu Lys Phe Val Val Phe He Val Wing Val Leu Leu Pro Val Arg l 40 45 50 GTT GAC TCT GCC ACC ATC CCC CGG CAG GAC GAA GTT CCC CAG CAG AC 246 Val Asp Ser Wing Thr He Pro Arg Gln Asp Glu Val Pro Gln Gln Thr 55 60 65 GTG GCC CCA CAG CAA CAG AGG CGC AGC CTC AAG GAG GAG GAG TGT CCA 294 Val Ala Pro Gln Gln Gln Arg Arg Ser Leu Lys Glu Glu Glu Cys Pro 70 75 80 10 GCA GGA TCT CAT AGA TCA GAA TAT ACT GGA GCC TGT AAC CCG TGC ACA 342 Wing Gly Ser His Arg Ser Glu Tyr Thr Gly Wing Cys Asn Pro Cys Thr 85 90 95 100 GAG GGT GTG GAT TAC ACC ATT GCT TCC AAC AAT TTG CCT TCT TGC CTG 390 Glu Gly Val Asp Tyr Thr He Ala Ser Asn Asn Leu Pro Ser Cys Leu 105 110 115 CTA TGT ACÁ GTT TGT AAA TCA GGT CAA ACA AAT AAA AGT TCC TGT ACC 438 Leu Cys Thr Val Cys Lys Ser Gly Gln Thr Asn Lys Ser Ser Cys Thr 120 125 130 ACG ACC AGA GAC ACC GTG TGT CAG TGT GAA AAA GGA AGC TTC CAG GAT 486 -te Thr Thr Arg Asp Thr Val Cys Gln Cys Glu Lys Gly Ser Phe Gln Asp 135 140 145 AAA AAC TCC CCT GAG ATG TGC CGG ACG TGT AGA ACA GGG TGT CCC AGA 534 Lys Asn Ser Pro Glu Met Cys Arg Thr Cys Arg Thr Gly Cys Pro Arg 150 155 160 GGG ATG GTC AAG GTC AGT AAT TGT ACG CCC CGG AGT GAC ATC AAG TGC 582 Gly Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser Asp He Lys Cys 165 170 175 180 AAA AAT GAA TCA GCT GCC AGT TCC ACT GGG AAA ACC CCA GCA GCG GAG 630 Lys Asn Glu Ser Ala Ala Ser Ser Thr Gly Lys Thr Pro Ala Ala Glu 185 190 195 20 GAG ACÁ GTG ACC ACC ATC CTG GGG ATG CTT GCC TCT CCC TAT CAC TAC 678 Glu Thr Val Thr Thr He Leu Gly Met Leu Wing Pro Pro Tyr His Tyr 200 205 210 CTT ATC ATC ATA GTG GTT TTA GTC ATC ATT TTA GCT GTG GTT GTG GTT 726 Leu He He He Val Val Leu Val He He Leu Ala Val Val Val Val 215 220 225 GGC TTT TCA TGT CGG AAG AAA TTC ATT TCT TAC CTC AAA GGC ATC TGC 774 Gly Phe Ser Cys Arg Lys Lys Phe He Ser Tyr Leu Lys Gly He Cys 230 235 240 25 TCA GGT GGT GGA GGA GGT CCC GAA CGT GTG CAC AGA GTC CTT TTC CGG 822 Ser Gly Gly Gly Gly Pro Glu Arg Val His Arg Val Leu Phe Arg 245 250 255 260 CGG CGT TCA TGT CCT TCA CGA GTT CCT GGG GCG GAG GAC AAT GCC CGC 870 Arg Arg Ser Cys Pro Ser Arg Val Pro Gly Ala Glu Asp Asn Ala Arg 265 270 275 t. AAC GAG ACC CTG AGT AAC AGA TAC TTG CAG CCC ACC CAG GTC TCT GAG 918 Asn Glu Thr Leu Ser Asn Arg Tyr Leu Gln Pro Thr Gln Val Ser Glu 280 285 290 CAG GAA ATC CAA GGT CAG GAG CTG GCA GAG CTA AC GGT GTG ACT GTA 966 Gln Glu He Gln Gly Gln Glu Leu Wing Glu Leu Thr Gly Val Thr Val 295 300 305 GAG TCG CCA GAG GAG CCA CAG CTG CTG GAA CAG GCA GAA GCT GAA 1014 Glu Ser Pro Glu Glu Pro Gln Arg Leu Leu Glu Gln Ala Glu Wing Glu 310 315 320 0 GGG TGT CAG AGG AGG CTG CTG GTT CCA GTG AAT GAC GCT TAC 1062 Gly Cys Gln Arg Arg Arg Leu Leu Val Pro Val Asn Asp Wing Asp Ser 325 330 335 340 GCT GAC ATC AGC ACC TTG CTG GAT GCC TCG GCA AC CTG GAA GAA GGA 1110 Wing Asp He Ser Thr Leu Leu Asp Wing Ser Wing Thr Leu Glu Glu Gly 345 350 355 CAT GCA AAG GAA ACÁ ATT CAG GAC CAA CTG GTG GGC TCC GAA AAG CTC 1158 His Wing Lys Glu Glu Thr He Gln Asp Gln Leu Val Gly Ser Glu Lys Leu 1 360 365 370 TTT TAT GAA GAA GAT GAC GGC TCT GCT ACG TCC TGC CTG TGA 1203 Phe Tyr Glu Glu Asp Glu Wing Gly Ser Wing Ser Cys Leu * 375 380 385 AAGAATCTCT TCAGGAAACC AGAGCTTCCC TCATTTACCT TTTCTCCTAC AAAGGGAAGC 1263 AGCCTGGAAG AAACAGTCCA GTACTTGACC CATGCCCCAA CAAACTCTAC TATCCAATAT 1323 GGGGCAGCTT ACCAATGGTC CTAGAACTTT GTTAACGCAC TTGGAGTAAT TTTTATGAAA 1383 TACTGCGTGT GATAAGCAAA CGGGAGAAAT TTATATCAGA TTCTTGGCTG CATAGTTATA 1443 CGATTGTGTA TTAAGGGTCG TTTTAGGCCA CATGCGGTGG CTCATGCCTG TAATCCCAGC 1503 0 ACTTTGATAG GCTGAGGCAG GTGGATTGCT TGAGCTCGGG AGTTTGAGA 1552 (2) IN FORMAC TION FOR S EQ ID NO: 2: (i) CHARACTERISTICS OF THE EU NCIA SECTION (A) LO NG ITU D: 386 base pairs (B) TI PO: ami no acids (D) TO PO LOGA: linear? 0 (¡i) TI PO DE MOLÉCU LA: protein (xi) DESCRI PTION OF THE SEQUENCE: SEQ ID NO: 2: Met Gly Leu Trp Gly Gln Ser Val Pro Thr Ala Ser Be Ala Arg Ala 1 5 10 15 Gly Arg Tyr Pro Gly Wing Arg Thr Wing Be Gly Thr Arg Pro Trp Leu 20 25 30 Leu Asp Pro Lys He Leu Lys Phe Val Val Phe He Val Wing Val Leu 35 40 45 Leu Pro Val Arg Val Asp Ser Wing Thr He Pro Arg Gln Asp Glu Val 50 55 60 Pro Gln Gln Thr Val Wing Pro Gln Gln Gln Arg Arg Ser Leu Lys Glu 65 70 75 80 Glu Glu Cys Pro Wing Gly Ser His Arg Ser Glu Tyr Thr Gly Wing Cys 85 90 95 Asn Pro Cys Thr Glu Gly Val Asp Tyr Thr He Wing Ser Asn Asn Leu 100 105 110 Pro Ser Cys Leu Leu Cys Thr Val Cys Lys Ser Gly Gln Thr Asn Lys 115 120 125 Ser Ser Cys Thr Thr Thr Arg Asp Thr Val Cys Gln Cys Glu Lys Gly 130 135 140 Ser Phe Gln Asp Lys Asn Ser Pro Glu Met Cys Arg Thr Cys Arg Thr 145 150 155 160 Gly Cys Pro Arg Gly Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser 165 170 175 Asp Lie Lys Cys Lys Asn Glu Be Ala Wing Ser Thr Gly Lys Thr 180 185 190 Pro Ala Wing Glu Thr Val Thr Thr He Leu Gly Met Leu Wing Ser 195 200 205 Pro Tyr His Tyr Leu He He He Val Val Leu Val He He Leu Ala 210 215 220 Val Val Val Val Gly Phe Ser Cys Arg Lys Lys Phe He Ser Tyr Leu 225 230 235 240 Lys Gly He Cys Ser Gly Gly Gly Gly Pro Glu Arg Val His Arg 245 250 255 Val Leu Phe Arg Arg Arg Ser Cys Pro Ser Arg Val Pro Gly Wing Glu 260 265 270 Asp Asn Wing Arg Asn Glu Thr Leu Ser Asn Arg Tyr Leu Gln Pro Thr 275 280 285 Gln Val Ser Glu Gln Glu He Gln Gly Gln Glu Leu Wing Glu Leu Thr 290 295 300 Gly Val Thr Val Glu Pro Pro Glu Pro Gln Arg Leu Leu Glu Gln 305 310 315 320 Wing Glu Wing Glu Gly Cys Gln Arg Arg Arg Leu Leu Val Pro Val Asn 325 330 335 Asp Wing Asp Being Wing Asp He Being Thr Leu Leu Asp Wing Being Wing Thr 340 345 350 Leu Glu Glu Gly His Wing Lys Glu Thr He Gln Asp Gln Leu Val Gly 355 360 365 Ser Glu Lys Leu Phe Tyr Glu Glu Asp Glu Wing Gly Ser Wing Thr Ser 370 375 380 Cys Leu * 385 (2) INFORMATION FOR SEQ ID NO: 3: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 1296 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (¡) i) TYPE OF MOLECULE: cDNA (vii) ORIGINAL SOURCE (B) CLON: TRAILR4A 12 (ix) CHARACTERISTICS: (A) NAME / KEY: CDS (B) LOCATION: 43..1203 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: TGATTGATTT TTGGCGCTTT CGATCCACCC TCCTCCCTTC TC ATG GGA CTT TGG 54 Met Gly Leu Trp 1 GGA CAA AGC GTC CCG ACC GCC TCG AGC GCT CGA GCA GGG CGC TAT CCA 102 Gly Gln Ser Val Pro Thr Ala Ser Ser Ala Arg Ala Gly Arg Tyr Pro 5 10 15 20 GGA GCC AGG AC GCG TCG GGA ACC AGA CCA TGG CTC CTG GAC TCC AAG 150 Gly Wing Arg Thr Wing Ser Gly Thr Arg Pro Trp Leu Leu Asp Ser Lys 25 30 35 ATC CTT AAG TTC GTC GTC TTC ATC GTC GCG GTT CTG CTG CCG GTC CGG 1 8 He Leu Lys Phe Val Val Phe He Val Wing Val Leu Leu Pro Val Arg 40 45 50 GTT GAC TCT GCC ACC ATC CCC CGG CAG GAC GAA GTT CCC CAG CAG AC 246 Val Asp Ser Ala Thr He Pro Arg Gln Asp Glu Val Pro Gln Gln Thr 55 60 65 GTG GCC CCA CAG CAA CAG AGG CGC AGC CTC AAG GAG GAG GAG TGT CCA 294 Val Wing Pro Gln Gln Gln Arg Arg Ser Leu Lys Glu Glu Glu Cys Pro 70 75 80 GCA GGA TCT CAT AGA TCA GAA TAT ACT GGA GCC TGT AAC CCG TGC ACA 3 2 Wing Gly Ser His Arg Ser Glu Tyr Thr Gly Wing Cys Asn Pro Cys Thr 85 90 95 100 GAG GGT GTG GAT TAC ACC ATT GCT TCC AAC AAT TTG CCT TCT TGC CTG 390 Glu Gly Val Asp Tyr Thr He Wing Ser Asn Asn Leu Pro Ser Cys Leu 105 110 115 CTA TGT ACÁ GTT TGT AAA TCA GGT CAA ACA AAT AAA AGT TCC TGT ACC 438 Leu Cys Thr Val Cys Lys Ser Gly Gln Thr Asn Lys Ser Ser Cys Thr 120 125 130 ACG ACC AGA GAC ACC GTG TGT CAG TGT GAA AAA GGA AGC TTC CAG GAT 486 Thr Thr Arg Asp Thr Val Cys Gln Cys Glu Lys Gly Ser Phe Gln Asp 135 140 145 AAA AAC TCC CCT GAG ATG TGC CGG ACG TGT AGA ACA GGG TGT CCC AGA 534 Lys Asn Ser Pro Glu Met Cys Arg Thr Cys Arg Thr Gly Cys Pro Arg 150 155 160 GGG ATG GTC AAG GTC AGT AAT TGT ACG CCC CGG AGT GAC ATC AAG TGC 582 Gly Met Val Lys Val Ser Asn Cys Thr Pro Arg Be Asp He Lys Cys 165 170 175 180 AAA AAT GAA TCA GCT GCC AGT TCC ACT GGG AAA ACC CCA GCA GCG GAG 630 Lys Asn Glu Ser Ala Ala Ser Ser Thr Gly Lys Thr Pro Ala Ala Glu 185 190 195 GAG AC GTG ACC ACC ATC CTG GGG ATG CTT GCC TCT CCC TAT CAC TAC 678 Glu Thr Val Thr Thr He Leu Gly Met Leu Wing Pro Pro Tyr His Tyr 200 205 210 CTT ATC ATC ATA GTG GTT TTA GTC ATC ATT TTA GCT GTG GTT GTG GTT 726 Leu He He He Val Val Leu Val Heu Leu Val Val Val Val 215 215 225 GGC TTT TCA TGT CGG AAG AAA TTC ATT TCT CTC AAA GGC ATC TGC 774 Gly Phe Ser Cys Arg Lys Lys Phe He Ser Tyr Leu Lys Gly lie C ys 230 235 240 TCA GGT GGT GGA GGA GGT CCC GAA CGT GTG CAC AGA GTC CTT TTC CGG 822 Ser Gly Gly Gly Gly Pro Glu Arg Val His Arg Val Leu Phe Arg 245 250 255 260 CGG CGT TCA TGT CCT TCA CGA GTT CCT GGG GCG GAG GAC AAT GCC CGC 870 Arg Arg Ser Cys Pro Ser Arg Val Pro Gly Wing Glu Asp Asn Wing Arg 265 270 275 AAC GAG ACC CTG AGT AAC AGA TAC TTG CAG CCC ACC CAG GTC TCT GAG 918 Asn Glu Thr Leu Ser Asn Arg Tyr Leu Gln Pro Thr Gln Val Ser Glu 280 285 290 CAG GAA ATC CAA GGT CAG GAG CTG GCA GAG CTA GGT GTG ACT GTA 966 Gln Glu He Gln Gly Gln Glu Leu Glu Glu Leu Thr Gly Val Thr Val 295 300 305 GAG TTG CCA GAG GAG CCA CAG CGT CTG CTG GAA CAG GCA GAA GCT GAA 1014 Glu Leu Pro Glu Glu Pro Gln Arg Leu Leu Glu Gln Wing Glu Wing Glu 310 315 320 GGG TGT CAG AGG AGG AGG CTG CTG GTT CCA GTG AAT GAC GCT GAC TCC 1062 Gly Cys Gln Arg Arg Arg Leu Leu Val Pro Val Asn Asp Wing Asp Ser 325 330 335 340 GCT GAC ATC AGC ACC TTG CTG GAT GCC TCG GCA ACA CTG GAA GAA GGA 1110 Wing Asp He Ser Thr Leu Leu As p Wing Being Wing Thr Leu Glu Glu Gly 345 350 355 CAT GCA AAG GAA ACA ATT CAG GAC CAA CTG GTG GGC TCC GAA AAG CTC 1158 His Wing Lys Glu Thr He Gln Asp Gln Leu Val Gly Ser Glu Lys Leu 360 365 370 TTT TAT GAA GAA GAT GAG GCA GGC TCT GCT ACG TCC TGC CTG TGA 1203 Phe Tyr Glu Glu Asp Glu Wing Gly Ser Wing Ser Cys Leu * 375 380 385 AAGAATCTCT TCAGGAAACC AGAGCTTCCC TCATTTACCT TTTCTCCTAC AAAGGGAAGC 1263 AGCCTGGAAG AAACAGTCCA GTACTTGACC CAT 1296 (2) INFORMATION FOR SEQ ID NO: 4: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 386 base pairs (B) TYPE: amino acids (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 4: Met Gly Leu Trp Gly Gln Ser Val Pro Thr Wing Being Wing Arg Wing 1 5 10 15 Gly Arg Tyr Pro Gly Wing Arg Thr Wing Ser Gly Thr Arg Pro Trp Leu 20 25 30 Leu Asp Ser Lys He Leu Lys Phe Val Val Phe He Val Wing Val Leu 35 40 45 Leu Pro Val Arg Val Asp Ser Wing Thr He Pro Arg Gln Asp Glu Val 50 55 60 Pro Gln Gln Thr Val Wing Pro Gln Gln Gln Arg Arg Ser Leu Lys Glu 65 70 75 80 Glu Glu Cys Pro Wing Gly Ser His Arg Ser Glu Tyr Thr Gly Wing Cys 85 90 95 Asn Pro Cys Thr Glu Gly Val Asp Tyr Thr He Wing Ser Asn Asn Leu 100 105 110 Pro Ser Cys Leu Leu Cys Thr Val Cys Lys Ser Gly Gln Thr Asn Lys 115 120 125 Being Ser Cys Thr Thr Arg Asp Thr Val Cys Gln Cys Glu Lys Gly 130 135 140 Be Phe Gln Asp Lys Asn Ser Pro Glu Met Cys Arg Thr Cys Arg Thr 145 150 155 160 Gly Cys Pro Arg Gly Met Val Lys Val Ser Asn Cys Thr Pro Arg Ser 165 170 175 Asp He Lys Cys Lys Asn Glu Be Wing Wing Ser Thr Gly Lys Thr 180 185 190 Pro Wing Wing Glu Glu Thr Val Thr Th Leu Gly Met Leu Wing Ser 195 200 205 Pro Tyr His Tyr Leu He He He Val Val Leu Val He He Leu Ala 210 215 220 Val Val Val Val Gly Phe Ser Cys Arg Lys Lys Phe He Ser Tyr Leu 225 230 235 240 Lys Gly He Cys Ser Gly Gly Gly Gly Pro Glu Arg Val His Arg 245 250 255 Val Leu Phe Arg Arg Arg Ser Cys Pro Ser Arg Val Pro Gly Wing Glu 260 265 270 Asp Asn Wing Arg Asn Glu Thr Leu Ser Asn Arg Tyr Leu Gln Pro Thr 275 280 285 Gln Val Ser Glu Gln Glu He Gln Gly Gln Glu Leu Wing Glu Leu Thr 290 295 300 Gly Val Thr Val Glu Leu Pro Glu Pro Gln Arg Leu Leu Glu Gln 305 310 315 320 Wing Glu Wing Glu Gly Cys Gln Arg Arg Arg Leu Leu Val Pro Val Asn 325 330 335 Asp Wing Asp Being Wing Asp He Being Thr Leu Leu Asp Wing Being Wing Thr 340 345 350 Leu Glu Glu Gly His Wing Lys Glu Thr He Gln Asp Gln Leu Val Gly 355 360 365 Ser Glu Lys Leu Phe Tyr Glu Glu Asp Glu Wing Gly Ser Ala Thr Ser 370 375 380 Cys Leu * 385 (2) INFORMATION FOR SEQ ID NO: 5: (i) CHARACTERISTICS OF THE SEQUENCE (A) LENGTH: 8 amino acids (B) TYPE: amino acids (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: peptide (iii) HYPOTHETIC: No (iv) ANTI-SENSE: No. (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 5: yr Lys Asp Asp Asp Asp Lys 5

Claims (27)

1. CLAIMS 1. An isolated DNA comprising a nucleotide sequence encoding a TRAIL receptor polypeptide (TRAIL-R), wherein said TRAIL-R is selected from the group consisting of: a) the TRAIL-R polypeptide of the Figure 1; b) the TRAIL-R polypeptide of Figure 2; and c) a fragment of the polypeptide of (a) or (b), wherein said fragment is capable of binding TRAIL.
2. 2. A DNA of claim 1, wherein said fragment is a soluble TRAIL-R polypeptide.
3. 3. A DNA of claim 2, wherein said polypeptide Soluble TRAIL-R comprises the extracellular domain of the TRAIL-R of the Figure 1 or 2.
4. 4. A DNA of claim 1, wherein said TRAIL-R lacks a transmembrane region.
5. 5. A DNA of claim 1, wherein said TRAIL-R polypeptide comprises the amino acid sequence of residues x and y of Figure 2, wherein x represents an integer from 1 to 98, and Y represents an integer of 181 to 386.
6. A DNA of claim 5, wherein x is an integer selected from the group consisting of 1 and 56, and Y is an integer selected from the group consisting of 181, 208, 211, and 386.
7. 7. An isolated DNA encoding a TRAIL-R polypeptide, wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a) residues 1 to 386 of the Figure 1; b) residues 1 to 386 of Figure 2; c) residues 56 to 386 of Figure 1; d) residues 56 to 386 of Figure 2; e) residues 1 to 211 of Figure 1; f) residues 1 to 211 of Figure 2; and g) residues 56 to 211 of Figure 1.
8. 8. A DNA of claim 7, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: a) residues 1 to 386 of Figure 1; b) residues 1 to 386 of Figure 2; c) residues 56 to 386 of Figure 1; d) residues 56 to 386 of Figure 2; e) residues 1 to 211 of Figure 1; f) residues 1 to 211 of Figure 2; and g) residues 56 to 211 of Figure 1.
9. 9. An isolated DNA comprising at least 60 contiguous nucleotides of the nucleotide sequence of Figure 1 or Figure 2.
10. 10. An expression vector comprising a DNA according to claim 1, 2, 3, 5, 7, or 8.
11. 11. A process for preparing a TRAIL-R polypeptide, comprising culturing a host cell containing a vector according to claim 10, under conditions that promote the expression of TRAIL-R, and recovery of the TRAIL-R polypeptide.
12. 12. A purified TRAIL-R polypeptide selected from the group consisting of: a) the TRAIL-R polypeptide of Figure 1 in mature form; b) the TRAIL-R polypeptide of Figure 2 in mature form; and c) a fragment of the polypeptide of (a) or (b), wherein said fragment is capable of binding TRAIL.
13. 13. A TRAIL-R of claim 13, wherein said fragment is a soluble TRAIL-R polypeptide.
14. 14. A TRAIL-R of claim 13, wherein said soluble TRAIL-R polypeptide comprises the extracellular domain of TRAIL-R of Figure 1 or 2.
15. 15. A TRAIL-R of claim 12, wherein said TRAIL -R lacks a transmembrane region.
16. 16. A TRAIL-R of claim 12, wherein said TRAIL-R polypeptide comprises the amino acid sequence of residues x up to and of Figure 1 or Figure 2, wherein X represents an integer from 1 to 98, and Y represents an integer from 181 to 386.
17. 17. A TRAIL-R of claim 16, wherein x is an integer selected from the group consisting of 1 and 56, and Y is an integer selected from the group consisting of 181, 208, 211, and 386.
18. 18. A purified TRAIL-R polypeptide comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of: a) residues of 56 to 386 of Figure 1; b) residues 56 to 386 of Figure 2; and c) residues 56 to 211 of Figure 1.
19. 19. A TRAIL-R of claim 18, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of: a) residues 56 to 386 of Figure 1; b) residues 56 to 386 of Figure 2; and c) residues 56 to 211 of Figure 1.
20. 20. An oligomer comprising at least two polypeptides TRAIL-R of claim 12, 13, 18, or 19.
21. 21. An oligomer of claim 20, wherein said oligomer comprises two to four soluble TRAIL-R polypeptides.
22. 22. A fusion protein comprising a TRAIL-R polypeptide and an antibody derived from Fc polypeptide, wherein said TRAIL-R polypeptide is a soluble fragment of the TRAIL-R protein of Figure 1 or Figure 2, wherein said fragment is able to join TRAIL.
23. 23. A dimer comprising two fusion proteins of claim 22.
24. 24. A composition comprising a polypeptide of claim 12, and a physiologically acceptable diluent, excipient or carrier.
25. 25. A composition comprising an oligomer of claim 20, and a physiologically acceptable diluent, excipient, or carrier.
26. 26. An antibody that is directed against a TRAIL-R polypeptide of claim 12, or an antigen-binding fragment of said antibody.
27. 27. An antibody of claim 26, wherein said antibody is a monoclonal antibody.