AU724826B2 - Cytokine that includes apoptosis - Google Patents

Description

AUSTRALIA

Patents Act COMPLETE SPECIFICATION

(ORIGINAL)

Class Int. Class Application Number: Lodged: Complete Specification Lodged: Accepted: Published: Priority Related Art: Name of Applicant: S Immunex Corporation Actual Inventor(s): Steven R. Wiley Raymond G. Goodwin Address for Service: PHILLIPS ORMONDE FITZPATRICK Patent and Trade Mark Attorneys 367 Collins Street Melbourne 3000 AUSTRALIA Invention Title: CYTOKINE THAT INDUCES APOPTOSIS Our Ref 604450 POF Code: 44735/44735 The following statement is a full description of this invention, including the best method of performing it known to applicant(s): IP Australia 9

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Documents received on: 0 2 0 OCT 1999 Batch No:

TITLE

CYTOKINE THAT INDUCES APOPTOSIS The present invention is a divisional application from Australian patent application number AU 708239, the entire disclosure of which is incorporated herein by reference.

BACKGROUND OF THE INVENTION The programmed cell death known as apoptosis is distinct from cell death due to necrosis. Apoptosis occurs in embryogenesis, metamorphosis, endocrine-dependent tissue atrophy, normal tissue turnover, and death of immune thymocytes (induced 10 through their antigen-receptor complex or by glucocorticoids) (Itoh et al., Cell 66:233, 1991). During maturation of T-cells in the thymus, T-cells that recognize self-antigens are destroyed through the apoptotic process, whereas others are positively selected.

The possibility that some T-cells recognizing certain self epitopes inefficiently 1 processed and presented antigenic determinants of a given self protein) escape this elimination process and subsequently play a role in autoimmune diseases has been suggested (Gammon et al., Immunology Today 12:193, 1991).

A cell surface antigen known as Fas has been reported to mediate apoptosis and is believed to play a role in clonal deletion of self-reactive T-cells (Itoh et al., Cell 66:233, 1991; Watanabe-Fukunage et al., Nature 356:314, 1992). Cross-linking a 20 specific monoclonal antibody to Fas has been reported to induce various cell lines to undergo apoptosis (Yonehara et al., J. Exp. Med., 169:1747, 1989; Trauth et al., Science, 245:301, 1989). However, under certain conditions, binding of a specific monoclonal antibody to Fas can have a costimulatory effect on freshly isolated T cells (Alderson et al., J. Exp. Med. 178:2231, 1993).

26 DNAs encoding a rat Fas ligand (Suda et al., Cell, 75:1169, 1993) and a human Fas ligand (Takahashi et al., International Immunology 6:1567, 1994) have been isolated. Binding of the Fas ligand to cells expressing Fas antigen has been demonstrated to induce apoptosis (Suda et al., supra, and Takahashi et al., supra).

Investigation into the existence and identity of other molecule(s) that play a role in apoptosis is desirable. Identifying such molecules would provide an additional means of regulating apoptosis, as well as providing further insight into the development of self-tolerance by the immune system and the etiology of autoimmune diseases.

MR C:\WINWORD\MARY\NODELETE\708239.DOC SUMMARY OF THE INVENTION The present invention provides a novel cytokine protein, as well as isolated DNA encoding the cytokine and expression vectors comprising the isolated DNA. Properties of the novel cytokine, which is a member of the tumor necrosis factor (TNF) family of ligands, include the ability to induce apoptosis of certain types of target cells. This protein thus is designated TNF Related Apoptosis Inducing Ligand (TRAIL). Among the types of cells that are killed by contact with TRAIL are cancer cells such as leukemia, lymphoma, and melanoma cells, and cells infected with a virus.

A method for producing TRAIL polypeptides involves culturing host cells transformed with a recombinant expression vector that contains TRAIL- S* encoding DNA under conditions appropriate for expression of TRAIL, then recovering the expressed TRAIL polypeptide from the culture. Antibodies directed against TRAIL polypeptides are also provided.

15 An aspect of the present invention is an E. coli cell transformed with an expression vector encoding a soluble TRAIL polypeptide, wherein said polypeptide comprises an amino acid sequence that is at least 80% identical to a sequence selected from: a) the extracellular domain of the human TRAIL protein of SEQ ID 20 NO:2; b) the extracellular domain of the mouse TRAIL protein of SEQ ID SNO:6; or c) a fragment of the extracellular domain of or wherein said soluble TRAIL polypeptide is capable of killing cancer cells.

Another aspect of the present invention is an E. coli cell transformed with a DNA comprising a nucleotide sequence selected from: a) nucleotides 88 to 933 of the sequence presented as SEQ ID NO:1; b) nucleotides 47 to 922 of the sequence presented as SEQ ID c) a fragment of the nucleotide sequence of or d) a fragment of the nucleotide sequence of wherein said DNA encodes a polypeptide capable of inducing apoptosis of a TRAIL-sensitive target cell.

2 MR C:\WINWORD\MARY\NODELETE\708239.DOC The present invention also provides a pharmaceutical composition comprising a TRAIL polypeptide as hereinbefore described. Another aspect of the present invention is a method of treating cancer, comprising administering an effective amount of said polypeptide.

Throughout the description and claims of this specification, the word "comprise" and variations of the word, such as "comprising" and "comprises", is not intended to exclude other additives, components, integers or steps.

BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 presents the results of an assay described in example 8. The assay demonstrated that a soluble human TRAIL polypeptide induced death of Jurkat cells, which are a leukemia cell line.

Figure 2 presents the results of an assay described in example 11.

Contact with a soluble human TRAIL polypeptide induced death of cytomegalovirus-infected human fibroblasts, whereas non-virally infected 15 fibroblasts were not killed.

DETAILED DESCRIPTION OF THE INVENTION A novel protein designated TRAIL is provided herein, along with DNA encoding TRAIL and recombinant expression vectors comprising TRAIL DNA.

A method for producing recombinant TRAIL polypeptides involves cultivating 20 host cells transformed with the recombinant expression vectors under conditions appropriate for expression of TRAIL, and recovering the expressed

TRAIL.

The present invention also provides antibodies that specifically bind TRAIL proteins. In one embodiment, the antibodies are monoclonal antibodies.

The TRAIL protein induces apoptosis of certain types of target cells, such as transformed cells that include but are not limited to cancer cells and virally-infected cells. As demonstrated in examples 5, 8, 9 and 10 below, TRAIL induced apoptosis of human leukemia, lymphoma, and melanoma cell lines. Among the uses of TRAIL is use in killing cancer cells. TRAIL finds further use in treatment of viral infections. Infection with cytomegalovirus (CMV) rendered human fibroblasts susceptible to 2a MR C:\WINWORD\MARY\NODELETE\708239.DOC Ah.

apoptosis when contacted with TRAIL, whereas uninfected fibroblasts were not killed through contact with TRAIL (see example 11).

Isolation of a DNA encoding human TRAIL is described in example 1 below.

The nucleotide sequence of the human TRAIL DNA isolated in example 1 is presented in SEQ ID NO: 1, and the amino acid sequence encoded thereby is presented in SEQ ID NO:2. This human TRAIL protein comprises an N-terminal cytoplasmic domain (amino acids 1-18), a transmembrane region (amino acids 19-38), and an extracellular domain (amino acids 39-281). The extracellular domain contains a receptor-binding region.

E. coli strain DH1OB cells transformed with a recombinant vector containing this human TRAIL DNA were deposited with the American Type Culture Collection on June 14, 1995, and assigned accession no. 69849. The deposit was made under the terms of the Budapest Treaty. The recombinant vector in the deposited strain is the expression vector pDC409 (described in example The vector was digested with 15 SalI and NotI, and human TRAIL DNA that includes the entire coding region shown in SEQ ID NO:1 was ligated into the vector.

DNA ncoding a second human TRAIL protein was isolated as described in example 2. The nucleotide sequence of this DNA is presented in SEQ ID NO:3, and the amino acid sequence encoded thereby is presented in SEQ ID NO:4. The encoded 20 protein comprises an N-terminal cytoplasmic domain (amino acids 1-18), a transmembrane region (amino acids 19-38), and an extracellular domain (amino acids 39-101).

The DNA of SEQ ID NO:3 lacks a portion of the DNA of SEQ ID NO: 1, and is thus designated the human TRAIL deletion variant (huTRAILdv) clone. Nucleotides 18 through 358 of SEQ ID NO: 1 are identical to nucleotides 8 through 348 of the huTRAILdv DNA of SEQ ID NO:3. Nucleotides 359 through 506 of SEQ ID NO: 1 are missing from the cloned DNA of SEQ ID NO:3. The deletion causes a shift in the reading frame, which results in an in-frame stop codon after amino acid 101 of SEQ ID NO:4. The DNA of SEQ ID NO:3 thus encodes a truncated protein. Amino acids 1 through 90 of SEQ ID NO:2 are identical to amino acids 1 through 90 of SEQ ID NO:4.

However, due to the deletion, the C-terminal portion of the huTRAILdv protein (amino acids 91 through 101 of SEQ ID NO:4) differs from the residues in the corresponding positions in SEQ ID NO:2. In contrast to the full length huTRAIL protein, the truncated huTRAILdv protein does not exhibit the ability to induce apoptosis of the T cell leukemia cells of the Jurkat cell line.

DNA encoding a mouse TRAIL protein has also been isolated, as described in example 3. The nucleotide sequence of this DNA is presented in SEQ ID NO:5 and the amino acid sequence encoded thereby is presented in SEQ ID NO:6. The encoded protein comprises an N-terminal cytoplasmic domain (amino acids 1-17), a transmembrane region (amino acids 18-38), and an extracellular domain (amino acids 39-291). This mouse TRAIL is 64% identical to the human TRAIL of SEQ ID NO:2 at the amino acid level. The coding region of the mouse TRAIL nucleotide sequence is identical to the coding region of the human nucleotide sequence of SEQ ID NO: 1.

One embodiment of the present invention is directed to human TRAIL protein characterized by the N-terminal amino acid sequence MetAlaMetMetGluValGlnGly GlyProSerLeuGlyGlnThr (amino acids 1-15 of SEQ ID NOS:2 and Mouse TRAIL proteins characterized by the N-terminal amino acid sequence MetProSerSerGlyAla LeuLysAspLeuSerPheSerGlnHis (amino acids 1-15 of SEQ ID NO:6) are also provided herein.

The TRAIL of the present invention is distinct from the protein known as Fas ligand (Suda et al., Cell, 75:1169, 1993; Takahashi et al., International Immunology 15 6:1567, 1994). Fas ligand induces apoptosis of certain cell types, via the receptor known as Fas. As demonstrated in example 5, TRAIL-induced apoptosis of target cells is not mediated through Fas. The human TRAIL amino acid sequence of SEQ ID NO:2 is about 20% identical to the human Fas ligand amino acid sequence that is presented in Takahashi et al., supra. The extracellular domain of human TRAIL is about 28.4% 20 identical to the extracellular domain of human Fas ligand.

The amino acid sequences disclosed herein reveal that TRAIL is a member of the TNF family of ligands (Smith et al. Cell, 73:1349, 1993; Suda et al., Cell, 75:1169, 1993; Smith et al., Cell, 76:959, 1994). The percent identities between the human TRAIL extracellular domain amino acid sequence and the amino acid sequence of the extracellular domain of other proteins of this family are as follows: 28.4% with Fas ligand, 22.4% with lymphotoxin-p, 22.9% with TNF-a, 23.-1% with TNF-P, 22.1% with CD30 ligand, and 23.4% with CD40 ligand.

TRAIL was tested for ability to bind receptors of the TNF-R family of receptors. The binding analysis was conducted using the slide autoradiography procedure of Gearing et al. (EMBO J. 8:3667, 1989). The analysis revealed no detectable binding of human TRAIL to human CD30, CD40, 4-1BB, OX40, TNF-R form), CD27, or LTR (also known as TNFR-RP). The results in example indicate that human TRAIL does not bind human Fas.

The TRAIL polypeptides of the present invention include polypeptides having amino acid sequences that differ from, but are highly homologous to, those presented in SEQ ID NOS:2 and 6. Examples include, but are not limited to, homologs derived from otheriammalian sneeies. variants (both inaturally occurring variants and those generated by recombinant DNA technology), and TRAIL fragments that retain a desired biological activity. Such polypeptides exhibit a biological activity of the TRAIL proteins of SEQ ID NOS:2 and 6, and preferably comprise an amino acid sequence that is at least 80% identical (most preferably at least 90% identical) to the amino acid sequence presented in SEQ ID NO:2 or SEQ ID NO:6. These embodiments of the present invention are described in more detail below.

Conserved sequences located in the C-terminal portion of proteins in the TNF family are identified in Smith et al. (Cell, 73:1349, 1993, see page 1353 and Figure 6); Suda et al. (Cell, 75:1169, 1993, see figure Smith et al. (Cell, 76:959, 1994, see figure and Goodwin et al. (Eur. J. Immunol., 23:2631, 1993, see figure 7 and pages 2638-39), hereby incorporated by reference. Among the amino acids in the human TRAIL protein that are conserved (in at least a majority of TNF family members) are those in positions 124-125 136 154 169 174 180 182 187 190 193 and 275-276 (FG) of SEQ ID NO:2.

15 Another structural feature of TRAIL is a spacer region between the C-terminus of the trans-membrane region and the portion of the extracellular domain that is believed to be most important for biological activity. This spacer region, located at the N-terminus of the extracellular domain, consists of amino acids 39 through 94 of SEQ ID NO:2.

Analogous spacers are found in other family members, CD40 ligand. Amino S 20 acids 138 through 153 correspond to a loop between the B sheets of the folded (three dimensional) human TRAIL protein.

Provided herein are membrane-bound TRAIL proteins (comprising a cytoplasmic domain, a transmembrane region, and an extracellular domain) as well as TRAIL fragments that retain a desired biological property of the full length TRAIL protein. In one embodiment, TRAIL fragments are soluble TRAIL polypeptides comprising all or part of the extracellular domain, but lacking the transmembrane region that would cause retention of the polypeptide on a cell membrane. Soluble TRAIL proteins are capable of being secreted from the cells in which they are expressed.

Advantageously, a heterologous signal peptide is fused to the N-terminus such that the soluble TRAIL is secreted upon expression.

Soluble TRAIL may be identified (and distinguished from its non-soluble membrane-bound counterparts) by separating intact cells which express the desired protein from the culture medium, by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein. The presence of TRAIL in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the TRAIL protein. Naturally-occurring soluble forms of TRAIL are encompassed by the present invention.

(r The use of soluble forms of TRAIL is advantageous for certain applications.

Purification of the proteins from recombinant host cells is facilitated, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for intravenous administration.

Examples of soluble TRAIL polypeptides are those containing the entire extracellular domain amino acids 39 to 281 of SEQ ID NO:2 or amino acids 39 to 291 of SEQ ID NO:6). Fragments of the extracellular domain that retain a desired biological activity are also provided. Such fragments advantageously include regions of TRAIL that are conserved in proteins of the TNF family of ligands, as described above.

Additional examples of soluble TRAIL polypeptides are those lacking not only the cytoplasmic domain and transmembrane region, but also all or part of the abovedescribed spacer region. Soluble human TRAIL polypeptides thus include, but are not .limited to, polypeptides comprising amino acids x to 281, wherein x represents any of 15 the amino acids in positions 39 through 95 of SEQ ID NO:2. In the embodiment in which residue 95 is the N-terminal amino acid, the entire spacer region has been deleted.

TRAIL fragments, including soluble polypeptides, may be prepared by any of a number of conventional techniques. A DNA sequence encoding a desired TRAIL S. 20 fragment may be subcloned into an expression vector for production of the TRAIL fragment. The TRAIL-encoding DNA sequence advantageously is fused to a sequence encoding a suitable leader or signal peptide. The desired TRAIL-encoding DNA fragment may be chemically synthesized using known techniques. DNA fragments also may be produced by restriction endonuclease digestion of a full length cloned DNA sequence, and isolated by electrophoresis on agarose gels. If necessary, oligonucleotides that reconstruct the 5' or 3' terminus to a desired-point may be ligated to a DNA fragment generated by restriction enzyme digestion. Such oligonucleotides may additionally contain a restriction endonuclease cleavage site upstream of the desired coding sequence, and position an initiation codon (ATG) at the N-terminus of the coding sequence.

The well known polymerase chain reaction (PCR) procedure also may be employed to isolate and amplify a DNA sequence encoding a desired protein fragment.

Oligonucleotides that define the desired termini of the DNA fragment are employed as and 3' primers. The oligonucleotides may additionally contain recognition sites for restriction endonucleases, to faciliate insertion of the amplified DNA fragment into an expression vector. PCR techniques are described in Saiki et al., Science 239:487 (1988); Recombinant DNA Methodology, Wu et al., eds., Academic Press, Inc., San

J

Diego (1989), pp. 189-196; and PCR Protocols: A Guide to Methods and Applications, Innis et al., eds., Academic Press, Inc. (1990).

As will be understood by the skilled artisan, the transmembrane region of each TRAIL protein discussed above is identified in accordance with conventional criteria for identifying that type of hydrophobic domain. The exact boundaries of a transmembrane region may vary slightly (most likely by no more than five amino acids on either end) from those presented above. Computer programs useful for identifying such hydrophobic regions in proteins are available.

The TRAIL DNA of the present invention includes cDNA, chemically synthesized DNA, DNA isolated by PCR, genomic DNA, and combinations thereof.

Genomic TRAIL DNA may be isolated by hybridization to the TRAIL cDNA disclosed herein using standard techniques. RNA transcribed from the TRAIL DNA is also encompassed by the present invention.

A search of the NCBI databank identified five expressed sequence tags (ESTs) 15 having regions of identity with TRAIL DNA. These ESTs (NCBI accession numbers T90422, T82085, T10524, R31020, and Z36726) are all human cDNA fragments.

The NCBI records do not disclose any polypeptide encoded by the ESTs, and do not indicate what the reading frame, if any, might be. However, even if the knowledge of the reading frame revealed herein by disclosure of complete TRAIL coding regions is S: 20 used to express the ESTs, none of the encoded polypeptides would have the apoptosisinducing property of the presently-claimed TRAIL polypeptides. In other words, if each of the five ESTs were inserted into expression vectors downstream from an initiator methionine codon, in the reading frame elucidated herein, none of the resulting •expressed polypeptides would contain a sufficient portion of the extracellular domain of TRAIL to induce apoptosis of Jurkat cells.

Certain embodiments of the present invention provide isolated DNA comprising a nucleotide sequence selected from the group consisting of nucleotides 88 to 933 of SEQ ID NO: 1 (human TRAIL coding region); nucleotides 202 to 933 of SEQ ID NO: 1 (encoding the human TRAIL extracellular domain); nucleotides 47 to 922 of SEQ ID NO:5 (mouse TRAIL coding region); and nucleotides 261 to 922 of SEQ ID (encoding the mouse TRAIL extracellular domain). DNAs encoding biologically active fragments of the proteins of SEQ ID NOS:2 and 6 are also provided. Further embodiments include sequences comprising nucleotides 370 to 930 of SEQ ID NO: 1 and nucleotides 341 to 919 of SEQ ID NO:5, which encode the particular human and murine soluble TRAIL polypeptides, respectively, described in example 7.

Due to degeneracy of the genetic code, two DNA sequences may differ, yet encode the same amino acid sequence. The present invention thus provides isolated DNA sequences encoding biologically active TRAIL, selected from DNA comprising the coding region of a native human or murine TRAIL cDNA, or fragments thereof, and DNA which is degenerate as a result of the genetic code to the native TRAIL DNA sequence.

Also provided herein are purified TRAIL polypeptides, both recombinant and non-recombinant. Variants and derivatives of native TRAIL proteins that retain a desired biological activity are also within the scope of the present invention. In one embodiment, the biological activity of an TRAIL variant is essentially equivalent to the biological activity of a native TRAIL protein. One desired biological activity of TRAIL is the ability to induce death of Jurkat cells. Assay procedures for detecting apoptosis of target cells are well known. DNA laddering is among the characteristics of cell death via apoptosis, and is recognized as one of the observable phenomena that distinguish apoptotic cell death from necrotic cell death. Examples of assay techniques suitable for detecting death or apoptosis of target cells include those described in examples 5 and 8 15 to 11. Another property of TRAIL is the ability to bind to Jurkat cells.

TRAIL variants may be obtained by mutations of native TRAIL nucleotide 0 sequences, for example. A TRAIL variant, as referred to herein, is a polypeptide substantially homologous to a native TRAIL, but which has an amino acid sequence different from that of native TRAIL because of one or a plurality of deletions, insertions or substitutions. TRAIL-encoding DNA sequences of the present invention encompass sequences that comprise one or more additions, deletions, or substitutions of nucleotides when compared to a native TRAIL DNA sequence, but that encode an TRAIL protein that is essentially biologically equivalent to a native TRAIL protein.

The variant amino acid or DNA sequence preferably is at least 80% identical to a native TRAIL sequence, most preferably at least 90% identical. The degree of homology (percent identity) between a native and a mutant sequence may be determined, for example, by comparing the two sequences using computer programs commonly employed for this purpose. One suitable program is the GAP computer program, version 6.0, described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).

The GAP program utilizes the alignment method of Needleman and Wunsch Mol.

Biol. 48:443, 1970), as revised by Smith and Waterman (Adv. Appl. Math 2:482, 1981). Briefly, the GAP program defines identity as the number of aligned symbols nucleotides or amino acids) which are identical, divided by the total number of symbols in the shorter of the two sequences. The preferred default parameters for the GAP program include: a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. 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; a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol in each gap; and no penalty for end gaps.

Alterations of the native amino acid sequence may be accomplished by any of a number of known techniques. Mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion.

Alternatively, oligonucleotide-directed site-specific inutagenesis-procedures can be employed to provide an altered gene having particular codons altered according to the substitution, deletion, or insertion required. Techniques for making such alterations include those disclosed by Walder et al. (Gene 42:133, 1986); Bauer et al. (Gene 37:73, 1985); Craik (BioTechniques, January 1985, 12-19); Smith et al. (Genetic Engineering: Principles and Methods, Plenum Press, 1981); and U.S. Patent Nos.

4,518,584 and 4,737,462, which are incorporated by reference herein.

Variants may comprise conservatively substituted sequences, meaning that one or more amino acid residues of a native TRAIL polypeptide are replaced by different 20 residues, but that the conservatively substituted TRAIL polypeptide retains a desired biological activity that is essentially equivalent to that of a native TRAIL polypeptide.

Examples of conservative substitutions include substitution of amino acids that do not alter the secondary and/or tertiary structure of TRAIL. Other examples involve substitution of amino acids outside of the receptor-binding domain, when the desired S 25 biological activity is the ability to bind to a receptor on target cells and induce apoptosis of the target cells. A given amino acid may be replaced by a residue having similar physiochemical characteristics, substituting one aliphatic residue for another (such as Ile, Val, Leu, or Ala for one another), or substitution of one polar residue for another (such as between Lys and Arg; Glu and Asp; or Gin and Asn). Other such conservative substitutions, substitutions of entire regions having similar hydrophobicity characteristics, are well known. TRAIL polypeptides comprising conservative amino acid substitutions may be tested in one of the assays described herein to confirm that a desired biological activity of a native TRAIL is retained. DNA sequences encoding TRAIL polypeptides that contain such conservative amino acid substitutions are encompassed by the present invention.

Conserved amino acids located in the C-terminal portion of proteins in the TNF family, and believed to be important for biological activity, have been identified. These conserved sequences are discussed in Smith et al. (Cell, 73:1349, 1993, see page 1353 and Figure Suda et al. (Cell, 75:1169, 1993, see figure Smith et al. (Cell, 76:959, 1994, see figure and Goodwin et al. (Eur. J. Immunol., 23:2631, 1993, see figure 7 and pages 2638-39). Advantageously, the conserved amino acids are not altered when generating conservatively substituted sequences. If altered, amino acids found at equivalent positions in other members of the TNF family are substituted.

TRAIL also may be modified to create TRAIL derivatives by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate; acetyl groups and the like. Covalent derivatives of TRAIL may be prepared by linking the chemical moieties to functional groups on TRAIL amino acid side chains or at the N-terminus or C-terminus of a TRAIL polypeptide or the extracellular domain thereof. Other derivatives of TRAIL within the scope of this invention include covalent or aggregative conjugates of TRAIL or its fragments with other proteins or polypeptides, such as by synthesis in recombinant culture as N- 15 terminal or C-terminal fusions. For example, the conjugate may comprise a signal or leader polypeptide sequence the a-factor leader of Saccharomyces) at the Nterminus of a TRAIL polypeptide. The signal or leader peptide co-translationally or *post-translationally directs transfer of the conjugate from its site of synthesis to a site inside or outside of the cell membrane or cell wall.

20 TRAIL polypeptide fusions can comprise peptides added to facilitate purification and identification of TRAIL. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., Bio/Technology 6:1204, 1988. One such peptide is the FLAG® peptide, Asp-Tyr-Lys-Asp-Asp-Aspp-A-Lys (DYKDDDDK) (SEQ ID NO:7), which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, thus enabling rapid assay and facile purification of expressed recombinant protein.

This sequence is also specifically cleaved by bovine mucosal enterokinase at the residue immediately following the Asp-Lys pairing. Fusion proteins capped with this peptide may also be resistant to intracellular degradation in E. coli.

A murine hybridoma designated 4E11 produces a monoclonal antibody that binds the peptide DYKDDDDK (SEQ ID NO:7) in the presence of certain divalent metal cations (as described in U.S. Patent 5,011,912), and has been deposited with the American Type Culture Collection under accession no HB 9259. Expression systems useful for producing recombinant proteins fused to the FLAG® peptide, as well as monoclonal antibodies that bind the peptide and are useful in purifying the recombinant proteins, are available from Eastman Kodak Company, Scientific Imaging Systems, New Haven, Connecticut.

The present invention further includes TRAIL polypeptides with or without associated native-pattern glycosylation. TRAIL expressed in yeast or mammalian expression systems may be similar to or significantly different from a native TRAIL polypeptide in molecular weight and glycosylation pattern, depending upon the choice of expression system. Expression of TRAIL polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules.

Glycosylation sites in the TRAIL extracellular domain can be modified to preclude glycosylation while allowing expression of a homogeneous, reduced carbohydrate analog using yeast or mammalian expression systems. N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr. Appropriate modifications to the nucleotide sequence encoding this triplet will result in substitutions, additions or deletions that prevent attachment of carbohydrate residues at the Asn side chain. Known procedures for inactivating N-glycosylation sites in 15 proteins include those described in U.S. Patent 5,071,972 and EP 276,846. A potential N-glycosylation site is found at positions 109-111 in the human protein of •SEQ ID NO:2 and at positions 52-54 in the murine protein of SEQ ID NO:6.

In another example, sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation. Other variants are prepared by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems in which KEX2 protease activity is present. EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein. KEX2 protease processing 25 sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites. Potential KEX2 protease processing sites are found at positions 89-90 and 149-150 in the protein of SEQ ID NO:2, and at positions 85-86, 135-136, and 162-163 in the protein of SEQ ID NO:6.

Naturally occurring TRAIL variants are also encompassed by the present invention. Examples of such variants are proteins that result from alternative mRNA splicing events (since TRAIL is encoded by a multi-exon gene) or from proteolytic cleavage of the TRAIL protein, wherein a desired biological activity is retained.

Alternative splicing of mRNA may yield a truncated but biologically active TRAIL protein, such as a naturally occurring soluble form of the protein, for example.

Variations attributable to proteolysis include, for example, differences in the N- or Ctermini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the TRAIL protein. In addition, proteolytic cleavage may release a soluble form of TRAIL from a membrane-bound form of the protein. Allelic variants are also encompassed by the present invention.

Oligomers The present invention encompasses TRAIL polypeptides in the form of oligomers, such as dimers, trimers, or higher oligomers. Oligomers may be formed by disulfide bonds between cysteine residues on different TRAIL polypeptides, or by noncovalent interactions between TRAIL polypeptide chains, for example. In other embodiments, oligomers comprise from two to four TRAIL polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the TRAIL polypeptides. Such peptides may be peptide linkers (spacers), or peptides that have the 15 property of promoting oligomerization. Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of TRAIL polypeptides attached thereto, as described in more detail below. The TRAIL polypeptides preferably are soluble.

Preparation of fusion proteins comprising heterologous polypeptides fused to 20 various portions of antibody-derived polypeptides (including the Fc domain) has been described, by Ashkenazi et al. (PNAS USA 88:10535, 1991); Byrn et al. (Nature 344:667, 1990); and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Supplement 4, pages 10.19.1 10.19.11, 1992), hereby incorporated by reference. In one embodiment of the invention, an TRAIL dimer is created by fusing TRAIL to an Fc region polypeptide derived from an antibody. The term "Fc polypeptide" includes native and mutein forms, as well as truncated Fc polypeptides containing the hinge region that promotes dimerization. The Fc polypeptide preferably is fused to a soluble TRAIL comprising only the extracellular domain).

A gene fusion encoding the TRAIL/Fc fusion protein is inserted into an appropriate expression vector. The TRAIL/Fc fusion proteins are allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc polypeptides, yielding divalent TRAIL. In other embodiments, TRAIL may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form an TRAIL oligomer with as many as four TRAIL extracellular regions.

One suitable Fc polypeptide is the native Fc region polypeptide derived from a human IgG I, which is described in PCT application WO 93/10151, hereby incorporated by reference. Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035. The amino acid sequence of the mutein is identical to that of 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 acid 22 has been changed from Gly to Ala. This mutein Fc exhibits reduced affinity for immunoglobulin receptors.

Alternatively, oligomeric TRAIL may comprise two or more soluble TRAIL polypeptides joined through peptide linkers. Examples include those peptide linkers described in United States Patent 5,073,627 (hereby incorporated by reference).

Fusion proteins comprising multiple TRAIL polypeptides separated by peptide linkers may be produced using conventional recombinant DNA technology.

Another method for preparing oligomeric TRAIL polypeptides involves use of a 15 leucine zipper. Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers 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 zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize.

20 Examples of leucine zipper domains suitable for producing soluble oligomeric TRAIL proteins are those described PCT application WO 94/10308, hereby incorporated by reference. Recombinant fusion proteins comprising a soluble TRAIL polypeptide fused to a peptide that dimerizes or trimerizes in solution are expressed in suitable host cells, and the resulting soluble oligomeric TRAIL is recovered from the culture supernatant.

25 Certain members of the TNF family of proteins are believed to exist in trimeric form (Beutler and Huffel, Science 264:667, 1994; Banner et al., Cell 73:431, 1993).

Thus, trimeric TRAIL may offer the advantage of enhanced biological activity.

Preferred leucine zipper moieties are those that preferentially form trimers. One example is a leucine zipper derived from lung surfactant protein D (SPD), as described in Hoppe etal. (FEBS Letters 344:191, 1994)andin U.S. Patent 5,716,805 hereby incorporated by reference. Other peptides derived from naturally occurring trimeric proteins may be employed in preparing trimeric TRAIL.

As described in example 7, a soluble Flag®-TRAIL polypeptide expressed in CV-1/EBNA cells spontaneously formed oligomers believed to be a mixture of dimers and trimers. The cytotoxic effect of this soluble Flag®-TRAIL in the assay of example 8 was enhanced by including an anti-Flag® antibody, possibly because the antibody facilitated cross-linking of TRAIL/receptor complexes. In one embodiment of the invention, biological activity of TRAIL is enhanced by employing TRAIL in conjunction with an antibody that is capable of cross-linking TRAIL. Cells that are to be killed may be contacted with both a soluble TRAIL polypeptide and such an antibody.

As one example, cancer or virally infected cells are contacted with an anti-Flag® antibody and a soluble Flag®-TRAIL polypeptide. Preferably, an antibody fragment lacking the Fc region is employed. Bivalent forms of the antibody may bind the Flag® moieties of two soluble Flag®-TRAIL polypeptides that are found in separate dimers or trimers. The antibody may be mixed or incubated with a Flag®-TRAIL polypeptide prior to administration in vivo.

Expression Systems The present invention provides recombinant expression vectors for expression S. of TRAIL, and host cells transformed with the expression vectors. Any suitable 15 expression system may be employed. The vectors include a DNA encoding a TRAIL polypeptide, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene. Examples of regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences 20 which control transcription and translation initiation and termination. Nucleotide o• sequences are operably linked when the regulatory sequence functionally relates to the TRAIL DNA sequence. Thus, a promoter nucleotide sequence is operably linked to an TRAIL DNA sequence if the promoter nucleotide sequence controls the transcription of I the TRAIL DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are S* generally incorporated into the expression vector.

In addition, a sequence encoding an appropriate signal peptide can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) may be fused in frame to the TRAIL sequence so that the TRAIL is initially translated as a fusion protein comprising the signal peptide. A signal peptide that is functional in the intended host cells promotes extracellular secretion of the TRAIL polypeptide. The signal peptide is cleaved from the TRAIL polypeptide upon secretion of TRAIL from the cell.

Suitable host cells for expression of TRAIL polypeptides include prokaryotes, yeast or higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems could also be employed to produce TRAIL polypeptides using RNAs derived from DNA constructs disclosed herein.

Prokaryotes include gram negative or gram positive organisms, for example, E.

coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus. In a prokaryotic host cell, such as E. coli, a TRAIL polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The Nterminal Met may be cleaved from the expressed recombinant TRAIL polypeptide.

Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning 15 vector pBR322 (ATCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells. An appropriate promoter and a TRAIL DNA sequence are inserted into the pBR322 vector.

SOther commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotec, Madison, WI, USA).

20 Promoter sequences commonly used for recombinant prokaryotic host cell Sexpression vectors include p-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8:4057, 1980; and EP-A- 36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982). A particularly useful prokaryotic host cell .expression system employs a phage X PL promoter and a ci857ts thermolabile repressor sequence. Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the X PL promoter include plasmid pHUB2 (resident in E.

coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1, ATCC 53082).

TRAIL alternatively may be expressed in yeast host cells, preferably from the Saccharomyces genus S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors will often contain an origin of replication sequence from a 2g. yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255:2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzyme Reg. 7:149, 1968; and Holland et al., Biochem. 17:4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase. Other suitable vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657. Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. Biol. Chem.

258:2674, 1982) and Beier et al. (Nature 300:724, 1982). Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) into the above-described yeast vectors.

The yeast a-factor leader sequence may be employed to direct secretion of the TRAIL polypeptide. The a-factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, Kurjan et al., Cell 15 30:933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984. Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art. A leader sequence may be modified S near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.

20 Yeast transformation protocols are known to those of skill in the art. One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978.

The Hinnen et al. protocol selects for Trp+ transformants in a selective medium, D o. wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 gg/ml adenine and 20 gg/ml uracil.

Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 gg/ml adenine and 80 gg/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.

Mammalian or insect host cell culture systems could also be employed to express recombinant TRAIL polypeptides. Bacculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be employed. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23:175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CVI/EBNA cell line derived from the African green monkey kidney cell line CVI (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).

Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes. Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell. 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 larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind II site toward the Bgl I site S• located in the SV40 viral origin of replication site is included.

15 Expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983), for example. A useful system for stable high level expression of mammalian cDNAs in C127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al.

(Mol. Immunol. 23:935, 1986). A high expression vector, PMLSV N1/N4, described 20 by Cosman et al., Nature 312:768, 1984 has been deposited as ATCC 39890.

Additional mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982. As one alternative, the vector may be derived from a retrovirus. Additional suitable expression systems are described in the examples below.

One preferred expression system employs Chinese hamster ovary (CHO) cells and an expression vector designated PG5.7. This expression vector is described in :PCT application W097/25420, which is hereby incorporated by reference. PG5.7 components include a fragment of CHO cell genomic DNA, followed by a CMV-derived promoter, which is followed by a sequence encoding an adenovirus tripartite leader, which in turn is followed by a sequence encoding dihydrofolate reductase (DHFR). These components were inserted into the plasmid vector pGEM1 (Promega, Madison, WI). DNA encoding a TRAIL polypeptide (or fuion protein containing TRAIL) may be inserted between the sequences encoding the tripartite leader and DHFR. Methotrexate may be added to the culture medium to increase expression levels, as is recognized in the field.

The fragment of CHO cell genomic DNA in vector PG5.7 enhances expression of TRAIL. A phage lysate containing a fragment of genomic DNA isolated from CHO cells was deposited with the American Type Culture Collection on January 4, 1996, and assigned accession number ATCC 97411. Vector PG5.7 contains nucleotides 8671 through 14507 of the CHO genomic DNA insert in strain deposit ATCC 97411.

For expression of TRAIL, a type II protein lacking a native signal sequence, a heterologous signal sequence or leader functional in mammalian host cells may be added. Examples include the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195, the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type I interleukin-1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin-1 receptor signal peptide described in EP 460,846.

A preferred expression system employs a leader sequence derived from cytomegalovirus (CMV). Example 7 illustrates the use of one such leader. In example 7, mammalian host cells were transformed with an expression vector encoding the peptide Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr Val Ala Leu S. 15 Ala Ala Pro Ser Gin Lys Ser Lys Arg Arg Thr Ser Ser (SEQ ID NO:9) fused to the Nterminus of an octapeptide designated FLAG® (SEQ ID NO:7, described above), .which in turn is fused to the N-terminus of a soluble TRAIL polypeptide. Residues 1 through 29 of SEQ ID NO:9 constitute a CMV-derived leader sequence, whereas residues 30 through 32 are encoded by oligonucleotides employed in constructing the 20 expression vector described in example 7. In one embodiment, DNA encoding a poly- His peptide a peptide containing six histidine residues) is positioned between the sequences encoding the CMV leader and the FLAG® peptide.

-Expression systems that employ such CMV-derived leader peptides are useful for expressing proteins other than TRAIL. Expression vectors comprising a DNA sequence that encodes amino acids 1 through 29 of SEQ ID NO:9 are provided herein.

In another embodiment, the vector comprises a sequence that encodes amino acids 1 through 28 of SEQ ID NO:9. DNA encoding a desired heterologous protein is positioned downstream of, and in the same reading frame as, DNA encoding the leader.

Additional residues those encoded by linkers or primers) may be encoded by DNA positioned between the sequences encoding the leader and the desired heterologous protein, as illustrated by the vector described in example 7. As is understood in the pertinent field, the expression vectors comprise promoters and any other desired regulatory sequences, operably linked to the sequences encoding the leader and heterologous protein.

The leader peptide presented in SEQ ID NO:9 may be cleaved after the arginine residue at position 29 to yield the mature secreted form of a protein fused thereto.

Alternatively or additionally, cleavage may occur between amino acids 20 and 21, or between amino acids 28 and 29, of SEQ ID NO:9.

The skilled artisan will recognize that the position(s) at which the signal peptide is cleaved may vary according to such factors as the type of host cells employed, whether murine or human TRAIL is expressed by the vector, and the like. Analysis by computer program reveals that the primary cleavage site may be between residues and 21 of SEQ ID NO:9. Cleavage between residues 22 and 23, and between residues 27 and 28, is predicted to be possible, as well. To illustrate, expression and secretion of a soluble murine TRAIL polypeptide resulted in cleavage of a CMV-derived signal peptide at multiple positions. The three most prominent species of secreted protein (in descending order) resulted from cleavage between amino acids 20 and 21 of SEQ ID NO:9, cleavage between amino acids 22 and 23, and cleavage between amino acids 27 and 28.

A method for producing a heterologous recombinant protein involves culturing 15 mammalian host cells transformed with such an expression vector under conditions that promote expression and secretion of the heterologous protein, and recovering the protein from the culture medium. Expression systems employing CMV leaders may be used to produce any desired protein, examples of which include, but are not limited to, colony stimulating factors, interferons, interleukins, other cytokines, and cytokine 20 receptors.

Purified TRAIL Protein The present invention provides purified TRAIL proteins, which may be produced by recombinant expression systems as described above or purified from 25 naturally occurring cells. The desired degree of purity may depend 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, TRAIL polypeptides are purified such that no protein bands corresponding to other proteins are detectable by SDSpolyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by one skilled in the pertinent field that multiple bands corresponding to TRAIL protein may be detected by SDS-PAGE, due to differential glycosylation, variations in posttranslational processing, and the like, as discussed above. A preparation of TRAIL protein is considered to be purified as long as no bands corresponding to different (non-TRAIL) proteins are visualized. TRAIL most preferably is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-PAGE. The protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography.

One process for producing the TRAIL protein comprises culturing a host cell transformed with an expression vector comprising a DNA sequence that encodes TRAIL under conditions such that TRAIL is expressed. The TRAIL protein is then recovered from the culture (from the culture medium or cell extracts). As the skilled artisan will recognize, procedures for purifying the recombinant TRAIL will vary according to such factors as the type of host cells employed and whether or not the TRAIL is secreted into the culture medium.

For example, when expression systems that secrete the recombinant protein are employed, the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit. Following the concentration step, the concentrate can be applied to a purification matrix such as a gel filtration medium. Alternatively, an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups. The matrices can be acrylamide, agarose, dextran, cellulose or other 15 types commonly employed in protein purification. Alternatively, a cation exchange step can be employed. Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.

Finally, one or more reversed-phase high performance liquid chromatography (RP- HPLC) steps employing hydrophobic RP-HPLC media, silica gel having 20 pendant methyl or other aliphatic groups) can be employed to further purify TRAIL.

Some or all of the foregoing purification steps, in various combinations, can be employed to provide a purified TRAIL protein.

b* Recombinant protein produced in bacterial culture may be isolated by initial *i disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freezethaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

Transformed yeast host cells are preferably employed to express TRAIL as a secreted polypeptide. This simplifies purification. Secreted recombinant polypeptide from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. Chromatog. 296:171, 1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.

Alternatively, TRAIL polypeptides can be purified by immunoaffinity chromatography. An affinity column containing an antibody that binds TRAIL may be prepared by conventional procedures and employed in purifying TRAIL. Example 4 describes a procedure for generating monoclonal antibodies directed against TRAIL.

Properties and Uses of TRAIL Programmed cell death (apoptosis) occurs during embryogenesis, metamorphosis, endocrine-dependent tissue atrophy, normal tissue turnover, and death of immune thymocytes. Regulation of programmed cell death is vital for normal functioning of the immune system. To illustrate, T cells that recognize self-antigens are destroyed through the apoptotic process during maturation of T-cells in the thymus, whereas other T cells are positively selected. The possibility that some T-cells recognizing certain self epitopes inefficiently processed and presented antigenic determinants of a given self protein) escape this elimination process and subsequently play a role in autoimmune diseases has been proposed (Gammon et al., Immunology Today 12:193, 1991).

Insufficient apoptosis has been implicated in certain conditions, while elevated levels of apoptotic cell death have been associated with other diseases. The desirability of identifying and using agents that regulate apoptosis in treating such disorders is recognized (Kromer, Advances in Immunology, 58:211, 1995; Groux et al., J. Exp.

Med. 175:331, 1992; Sachs and Lotem, Blood 82:15, 1993).

20 Abnormal resistance of T cells toward undergoing apoptosis has been linked to lymphocytosis, lymphadenopathy, splenomegaly, accumulation of self-reactive T cells, autoimmune disease, development of leukemia, and development of lymphoma :(Kromer, supra; see especially pages 214-215). Conversely, excessive apoptosis of T cells has been suggested to play a role in lymphopenia, systemic immunodeficiency, and specific immunodeficiency, with specific examples being virus-induced *immunodeficient states associated with infectious mononucleosis and cytomegalovirus infection, and tumor-mediated immunosuppression (Kromer, supra; see especially page 214). Depletion of CD4+ T cells in HIV-infected individuals may be attributable to inappropriate activation-induced cell death (AICD) by apoptosis (Groux et al., J. Exp.

Med. 175:331, 1992).

As demonstrated in examples 5 and 8, TRAIL induces apoptosis of the acute T cell leukemia cell line designated Jurkat clone E6-1. TRAIL thus is a research reagent useful in studies of apoptosis, including the regulation of programmed cell death.

Since Jurkat cells are a leukemia cell line arising from T cells, the TRAIL of the present invention finds use in studies of the role TRAIL may play in apoptosis of other transformed T cells, such as other malignant cell types arising from T cells.

TRAIL binds Jurkat cells, as well as inducing apoptosis thereof. TRAIL did not cause death of freshly isolated murine thymocytes, or peripheral blood T cells (PBTs) freshly extracted from a healthy human donor. A number of uses flow from these properties of TRAIL.

TRAIL polypeptides may be used to purify leukemia cells, or any other cell type to which TRAIL binds. Leukemia cells may be isolated from a patient's blood, for example. In one embodiment, the cells are purified by affinity chromatography, using a chromatography matrix having TRAIL bound thereto. The TRAIL attached to the chromatography matrix may be a full length protein, an TRAIL fragment comprising the extracellular domain, an TRAIL-containing fusion protein, or other suitable TRAIL polypeptide described herein. In one embodiment, a soluble TRAIL/Fc fusion protein is bound to a Protein A or Protein G column through interaction of the Fc moiety with the Protein A or Protein G. Alternatively, TRAIL may be used in isolating leukemia cells by flow cytometry.

15 The thus-purified leukemia cells are expected to die following binding of TRAIL, but the dead cells will still bear cell surface antigens, and may be employed as immunogens in deriving anti-leukemia antibodies. The leukemia cells, or a desired cell .:surface antigen isolated therefrom, find further use in vaccine development.

Since TRAIL binds and kills leukemia cells (the Jurkat cell line), TRAIL also 20 may be useful in treating leukemia. A therapeutic method involves contacting leukemia cells with an effective amount of TRAIL. In one embodiment, a leukemia patient's blood is contacted ex vivo with an TRAIL polypeptide. The TRAIL may be immobilized on a suitable matrix. TRAIL binds the leukemia cells, thus removing them °from the patient's blood before the blood is returned into the patient.

Alternatively or additionally, bone marrow extracted from a leukemia patient may be contacted with an amount of TRAIL effective in inducing death of leukemia cells in the bone marrow. Bone marrow may be aspirated from the stemum or iliac crests, for example, and contacted with TRAIL to purge leukemia cells. The thustreated marrow is returned to the patient.

TRAIL also binds to, and induces apoptosis of, lymphoma and melanoma cells (see examples 5, 9, and 10). Thus, uses of TRAIL that are analogous to those described above for leukemia cells are applicable to lymphoma and melanoma cells.

TRAIL polypeptides may be employed in treating cancer, including, but not limited to, leukemia, lymphoma, and melanoma. In one embodiment, the lymphoma is Burkitt's lymphoma. Table I in example 9 shows that TRAIL had a cytotoxic effect on several Burkitt's lymphoma cell lines. Epstein-Barr virus is an etiologic agent of Burkitt's lymphoma.

TRAIL polypeptides also find use in treating viral infections. Contact with TRAIL caused death of cells infected with cytomegalovirus, but not of the same cell type when uninfected, as described in example 11. The ability of TRAIL to kill cells infected with other viruses can be confirmed using the assay described in example 11.

Such viruses include, but are not limited to, encephalomyocarditis virus, Newcastle disease virus, vesicular stomatitis virus, herpes simplex virus, adenovirus-2, bovine viral diarrhea virus, HIV, and Epstein-Barr virus.

An effective amount of TRAIL is administered to a mammal, including a human, afflicted with a viral infection. In one embodiment, TRAIL is employed in conjunction with interferon to treat a viral infection. In the experiment described in example 11, pretreatment of CMV-infected cells with y-interferon enhanced the level of killing of the infected cells that was mediated by TRAIL. TRAIL may be administered in conjunction with other agents that exert a cytotoxic effect on cancer cells or virusinfected cells.

In another embodiment, TRAIL is used to kill virally infected cells in cell preparations, tissues, or organs that are to be transplanted. To illustrate, bone marrow may be contacted with TRAIL to kill virus infected cells that may be present therein, before the bone marrow is transplanted into the recipient.

The TRAIL of the present invention may be used in developing treatments for 20 any disorder mediated (directly or indirectly) by defective or insufficient amounts of TRAIL. A therapeutically effective amount of purified TRAIL protein is administered to a patient afflicted with such a disorder. Alternatively, TRAIL DNA sequences may be employed in developing a gene therapy approach to treating such disorders.

Disclosure herein of native TRAIL nucleotide sequences permits the detection of 25 defective TRAIL genes, and the replacement thereof with normal TRAIL-encoding o* genes. Defective genes may be detected in in vitro diagnostic assays, and by comparision of the native TRAIL nucleotide sequence disclosed herein with that of a TRAIL gene derived from a person suspected of harboring a defect in this gene.

The present invention provides pharmaceutical compositions comprising purified TRAIL and a physiologically acceptable carrier, diluent, or excipient. Suitable carriers, diluents, and excipients are nontoxic to recipients at the dosages and concentrations employed. Such compositions may comprise buffers, antioxidants such as ascorbic acid, low molecular weight (less than about 10 residues) polypeptides, proteins, amino acids, carbohydrates including glucose, sucrose or dextrins, chelating agents such as EDTA, glutathione and other stabilizers and excipients commonly employed in pharmaceutical compositions. Neutral buffered saline or saline mixed with conspecific serum albumin are among the appropriate diluents. The composition may be formulated as a lyophilizate using appropriate excipient solutions sucrose) as diluents.

For therapeutic use, purified proteins of the present invention are administered to a patient, preferably a human, for treatment in a manner appropriate to the indication.

Thus, for example, the pharmaceutical compositions can be administered locally, by intravenous injection, continuous infusion, sustained release from implants, or other suitable technique. Appropriate dosages and the frequency of administration will depend, of course, on such factors as the nature and severity of the indication being.

treated, the desired response, the condition of the patient and so forth.

The TRAIL protein employed in the pharmaceutical compositions preferably is purified such that the TRAIL protein is substantially free of other proteins of natural or endogenous origin, desirably containing less than about 1% by mass of protein contaminants residual of production processes. Such compositions, however, can contain other proteins added as stabilizers, carriers, excipients or co-therapeutics.

The TRAIL-encoding DNAs disclosed herein find use in the production of o. TRAIL polypeptides, as discussed above. Fragments of the TRAIL nucleotide S. sequences are also useful. In one embodiment, such fragments comprise at least about 17 consecutive nucleotides, more preferably at least 30 consecutive nucleotides, of the human or murine TRAIL DNA disclosed herein. DNA and RNA complements of said 20 fragments are provided herein, along with both single-stranded and double-stranded forms of the TRAIL DNA of SEQ ID NOS:1, 3 and Among the uses of such TRAIL nucleic acid fragments are use as a probe or as primers in a polymerase chain reaction (PCR). As one example, a probe corresponding to the extracellular domain of TRAIL may be employed. The probes find use in 25 detecting the presence of TRAIL nucleic acids in in vitro assays and in such procedures as Northern and Southern blots. Cell types expressing TRAIL can be identified as well. Such procedures are well known, and the skilled artisan can choose a probe of suitable length, depending on the particular intended application. For PCR, 5' and 3' primers corresponding to the termini of a desired TRAIL DNA sequence are employed to isolate and amplify that sequence, using conventional techniques.

Other useful fragments of the TRAIL nucleic acids are antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target TRAIL mRNA (sense) or TRAIL DNA (antisense) sequences. Such a fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to create an antisense or a sense oligonucleotide, based upon a cDNA sequence for a given protein is described in, 1 for example, Stein and Cohen, Cancer Res. 48:2659, 1988 and van der Krol et al., BioTechniques 6:958, 1988.

Binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block translation (RNA) or transcription (DNA) by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides thus may be used to block expression of TRAIL proteins.

Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar-phosphodiester backbones (or other sugar linkages, such as those described in W091/06629) and wherein such sugar linkages are resistant to endogenous nucleases. Such oligonucleotides with resistant sugar linkages are stable in vivo capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences. Other examples of sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(Llysine). Further still, intercalating agents, such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oliginucleotide for the target nucleotide 20 sequence.

Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, i CaPO4-mediated DNA transfection, electroporation, or other gene transfer vectors such as Epstein-Barr virus. Antisense or sense oligonucleotides are preferably introduced into a cell containing the target nucleic acid sequence by insertion of the antisense or sense oligonucleotide into a suitable retroviral vector, then contacting the cell with the retrovirus vector containing the inserted sequence, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or or the double copy vectors designated DCT5B and DCT5C (see PCT Application WO 90/13641). Alternatively, other promotor sequences may be used to express the oligonucleotide.

Sense or antisense oligonucleotides may also be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable 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, 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 block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.

Alternatively, a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.

Antibodies Immunoreactive with TRAIL The TRAIL proteins of the present invention, or immunogenic fragments thereof, may be employed in generating antibodies. The present invention thus provides antibodies that specifically bind TRAIL, the antibodies bind to TRAIL via the antigen-binding sites of the antibody (as opposed to non-specific binding).

S, Polyclonal and monoclonal antibodies may be prepared by conventional .techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New 15 Dimension in Biological Analyses, Kennet et al. Plenum Press, New York (1980); and Antibodies: A Laboratory Manual Harlow and Land Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). Production of monoclonal antibodies that are immunoreactive with TRAIL is further illustrated in example 4 below.

20 Antigen-binding fragments of such antibodies, which may be produced by conventional techniques, are also encompassed by 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.

25 The monoclonal antibodies of the present invention include chimeric antibodies, humanized versions of murine monoclonal antibodies. Such humanized antibodies may 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 fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment (lacking the antigen-binding site) derived from a human antibody. Procedures for the production of chimeric and further 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).

Among the uses of the antibodies is use in assays to detect the presence of TRAIL polypeptides, either in vitro or in vivo. The antibodies find further use in purifying TRAIL by affinity chromatography.

Those antibodies that additionally can block binding of TRAIL to target cells may be used to inhibit a biological activity of TRAIL. A therapeutic method involves in vivo administration of such an antibody in an amount effective in inhibiting a TRAILmediated biological activity. Disorders mediated or exacerbated by TRAIL, directly or indirectly, are thus treated. Monoclonal antibodies are generally preferred for use in such therapeutic methods.

Antibodies directed against TRAIL may be useful for treating thrombotic microangiopathies. One such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, Semin. Hematol., 24:71, 1987; Thompson et al., Blood, 80:1890, 1992). Increasing TTP-associated mortality rates have been reported by the U.S.

Centers for Disease Control (Torok et al., Am. J. Hematol. 50:84, 1995).

15 Plasma from patients afflicted with TTP (including HIV' and HIV patients) induces apoptosis of human endothelial cells of dermal microvascular origin, but not large vessel origin (Laurence et al., Blood, 87:3245, April 15, 1996). Plasma of TTP patients thus is thought to contain one or more factors that directly or indirectly induce apoptosis. In the assay described in example 13 below, polyclonal antibodies raised 20 against TRAIL inhibited TTP plasma-induced apoptosis of dermal microvascular endothelial cells. The data presented in example 13 suggest that TRAIL is present in the serum of TTP patients, and may play a role in inducing apoptosis of microvascular endothelial cells.

Another thrombotic microangiopathy is hemolytic-uremic syndrome (HUS) (Moake, Lancet, 343:393, 1994; Melnyk et al., (Arch. Intern. Med., 155:2077, 1995; Thompson et al., supra). One embodiment of the invention is directed to use of an anti-TRAIL antibody to treat the condition that is often referred to as "adult HUS" (even though it can strike children as well). A disorder known as childhood/diarrheaassociated HUS differs in etiology from adult HUS.

Other conditions characterized by clotting of small blood vessels may be treated using anti-TRAIL antibodies. Such conditions include but are not limited to the following. Cardiac problems seen in about 5-10% of pediatric AIDS patients are believed to involve clotting of small blood vessels. Breakdown of the microvasculature in the heart has been reported in multiple sclerosis patients. As a further example, treatment of systemic lupus erythematosus (SLE) is contemplated.

In one embodiment, a patient's blood or plasma is contacted with an anti- TRAIL antibody ex vivo. The antibody (preferably a monoclonal antibody) may be bound to a suitable chromatography matrix by conventional procedures. The patient's blood or plasma flows through a chromatography column containing the antibody bound to the matrix, before being returned to the patient. The immobilized antibody binds TRAIL, thus removing TRAIL protein from the patient's blood.

In an alternative embodiment, the antibodies are administered in vivo, in which case blocking antibodies are desirably employed. Such antibodies may be identified using any suitable assay procedure, such as by testing antibodies for the ability to inhibit binding of TRAIL to target cells. Alternatively, blocking antibodies may be identified in assays for the ability to inhibit a biological effect of the binding of TRAIL to target cells. Example 12 illustrates one suitable method of identifying blocking antibodies, wherein antibodies are assayed for the ability to inhibit TRAIL-mediated lysis of Jurkat cells.

The present invention thus provides a method for treating a thrombotic microangiopathy, involving use of an effective amount of an antibody directed against 15 TRAIL. Antibodies of the present invention may be employed in in vivo or ex vivo procedures, to inhibit TRAIL-mediated damage to apoptosis of) microvascular S" endothelial cells.

Anti-TRAIL antibodies may be employed in conjunction with other agents useful in treating a particular disorder. In an in vitro study reported by Laurence et al.

20 (Blood 87:3245, 1996), some reduction of TTP plasma-mediated apoptosis of microvascular endothelial cells was achieved by using an anti-Fas blocking antibody, aurintricarboxylic acid, or normal plasma depleted of cryoprecipitate.

Thus, a patient may be treated with an agent that inhibits Fas-ligand-mediated apoptosis of endothelial cells, in combination with an agent that inhibits TRAIL- 25 mediated apoptosis of endothelial cells. In one embodiment, an anti-TRAIL blocking :antibody and an anti-FAS blocking antibody are both administered to a patient afflicted with a disorder characterized by thrombotic microangiopathy, such as TTP or HUS.

Examples of blocking monoclonal antibodies directed against Fas antigen (CD95) are described in PCT application publication number WO 95/10540, hereby incorporated by reference.

Pharmaceutical compositions comprising an antibody that is immunoreactive with TRAIL, and a suitable, diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described above for the compositions containing TRAIL proteins.

The following examples are provided to illustrate particular embodiments of the present invention, and are not to be construed as limiting the scope of the invention.

EXAMPLE 1: Isolation of a Human TRAIL DNA DNA encoding a human TRAIL protein of the present invention was isolated by the following procedure. A TBLASTN search of the dbEST data base at the National Center for Biological Information (NCBI) was performed, using the query sequence LVVXXXGLYYVYXQVXF (SEQ ID NO:8). This sequence is based upon the most conserved region of the TNF ligand family (Smith et al., Cell, 73:1349, 1993). An expressed sequence tag (EST) file, GenBank accession number Z36726, was identified using these search parameters. The GenBank file indicated that this EST was obtained from a human heart atrium cDNA library.

Two 30-bp oligonucleotides based upon sequences from the 3' and 5' ends of this EST file were synthesized. The oligonucleotide from the 3' end had the sequence TGAAATCGAAAGTATGTITGGGAATAGATG (complement of nucleotides 636 to *"665 of SEQ ID NO: 1) and the 5' oligonucleotide was TGACGAAGAGAGTATGAA 15 CAGCCCCTGCTG (nucleotides 291 to 320 of SEQ ID NO: The oligonucleotides -were 5' end labeled with 32 P y-ATP and polynucleotide kinase. Two Xgtl0 cDNA libraries were screened by conventional methods with an equimolar mixture of these labeled oligonucleotides as probe. One library was a human heart 5' stretch cDNA library (Stratagene Cloning Systems, La Jolla, CA). The other was a peripheral blood 20 lymphocyte (PBL) library prepared as follows: PBLs were obtained from normal human volunteers and treated with 10 ng/ml of OKT3 (an anti-CD3 antibody) and ng/ml of human IL-2 for six days. The PBL cells were washed and stimulated with 500 ng/ml of ionomycin (Calbiochem) and 10 ng/ml PMA for 4 hours. Messenger RNA was isolated from the stimulated PBL cells. cDNA synthesized on the mRNA template was packaged into Xgtl0 phage vectors (Gigapak®, Stratagene Cloning Systems, La Jolla, CA).

Recombinant phages were plated onto E. coli strain C600-HFL and screened using standard plaque hybridization techniques. Nitrocellulose filters were lifted from these plates in duplicate, and hybridized with the 32 P-labeled oligonucleotides overnight at 67 0 C in a solution of 60 mM Tris pH 8.0, 2 mM EDTA, 5x Denhardt's Solution, 6x SSC, 1 mg/ml n-lauroyl sarcosine, 0.5% NP40, and 4 gg/ml SS salmon sperm DNA. The filters were then washed in 3x SSC at 67 0 C for thirty minutes.

From the heart 5' stretch cDNA library, one positive plaque was obtained out of approximately one million plaques. This clone did not include the 3' end of the gene.

Using the PBL library, approximately 50 positive plaques were obtained out of 500,000 plaques. Fifteen of these first round positive plaques were picked, and the inserts from the enriched pools were amplified using oligonucleotide primers designed to amplify phage inserts. The resulting products were resolved by 1.5% agarose gel electrophoresis, blotted onto nitrocellulose, and analyzed by standard Southern blot technique using the 32 P-labeled 30-mer oligonucleotides as probes. The two plaque picks that produced the largest bands by Southern analysis were purified by secondary screening, and isolated phage plaques were obtained using the same procedures described above.

DNA from the isolated phages was prepared by the plate lysis method, and the cDNA inserts were excised with EcoRI, purified by electrophoresis using agarose in Tris-Borate-EDTA buffer, and ligated into the pBluescript® plasmid.

These inserts were then sequenced by conventional methods, and the resulting sequences were aligned.

The nucleotide sequence of a human TRAIL DNA is presented in SEQ ID NO: 1 and the amino acid sequence encoded thereby is presented in SEQ ID NO:2. This human TRAIL protein comprises an N-terminal cytoplasmic domain (amino acids 1- 15 18), a transmembrane region (amino acids 19-38), and an extracellular domain (amino acids 39-281). The calculated molecular weight of this protein is 32,508 daltons.

E. coli strain DH IOB cells transformed with a recombinant vector containing this TRAIL DNA were deposited with the American Type Culture Collection on June 14, 1995, and assigned accession no. 69849. The deposit was made under the terms 20 of the Budapest Treaty. The recombinant vector in the deposited strain is the expression vector pDC409 (described in example The vector was digested with Sall and NotI, and human TRAIL DNA that includes the entire coding region shown in SEQ ID NO: 1 was ligated into the digested vector.

25 EXAMPLE 2: Isolation of DNA Encoding a Truncated TRAIL DNA encoding a second human TRAIL protein was isolated as follows. This truncated TRAIL does not exhibit the ability to induce apoptosis of Jurkat cells.

PCR analysis, using the 30-mers described in example 1 as the 5' and 3' primers, indicated that 3 out of 14 of the first round plaque picks in example 1 contained shorter forms of the TRAIL DNA. One of the shortened forms of the gene was isolated, ligated into the pBluescript® cloning vector (Stratagene Cloning Systems, La Jolla, CA) and sequenced.

The nucleotide sequence of this DNA is presented in SEQ ID NO:3. The amino acid sequence encoded thereby is presented in SEQ ID NO:4. The encoded protein comprises an N-terminal cytoplasmic domain (amino acids 1-18), a transmembrane region (amino acids 19-38), and an extracellular domain (amino acids 39-101).

The DNA of SEQ ID NO:3 lacks nucleotides 359 through 506 of the DNA of SEQ ID NO: 1, and is thus designated the human TRAIL deletion variant (huTRAILdv) clone. The deletion causes a shift in the reading frame, which results in an in-frame stop codon after amino acid 101 of SEQ ID NO:4. The DNA of SEQ ID NO:3 thus encodes a truncated protein. Amino acids 1 through 90 of SEQ ID NO:2 are identical to amino acids 1 through 90 of SEQ ID NO:4. However, due to the deletion, the Cterminal portion of the huTRAILdv protein (amino acids 91 through 101 of SEQ ID NO:4) differs from the residues in the corresponding positions in SEQ ID NO:2.

The huTRAILdv protein lacks the above-described conserved regions found at the C-terminus of members of the TNF family of proteins. The inability of this huTRAILdv protein to cause apoptotic death of Jurkat cells further confirms the importance of these conserved regions for biological activity.

EXAMPLE 3: DNA encoding a murine TRAIL 15 DNA encoding a murine TRAIL was isolated by the following procedure. A cDNA library comprising cDNA derived from the mouse T cell line 7B9 in the vector XZAP was prepared as described in Mosley et al. (Cell 59:335, 1989). DNA from the library was transferred onto nitrocellulose filters by conventional techniques.

Human TRAIL DNA probes were used to identify hybridizing mouse cDNAs on the filters. Two separate probes were used, in two rounds of screening. PCR reaction products about 400bp in length, isolated and amplified using the human TRAIL DNA as template, were employed as the probe in the first round of screening.

These PCR products consisted of a fragment of the human TRAIL coding region. The probe used in the second round of screening consisted of the entire coding region of the 25 human TRAIL DNA of SEQ ID NO:1. A random primed DNA labeling kit (Stratagene, La Jolla, CA) was used to radiolabel the probes.

Hybridization was conducted at 37 0 C in 50% formamide, followed by washing with 1 x SSC, 0.1% SDS at 50 0 C. A mouse cDNA that was positive in both rounds of screening was isolated.

The nucleotide sequence of this DNA is presented in SEQ ID NO:5 and the amino acid sequence encoded thereby is presented in SEQ ID NO:6. The encoded protein comprises an N-terminal cytoplasmic domain (amino acids 1-17), a transmembrane region (amino acids 18-38), and an extracellular domain (amino acids 39-291). This mouse TRAIL is 64% identical to the human TRAIL of SEQ ID NO:2, at the amino acid level. The coding region of the mouse TRAIL nucleotide sequence is identical to the coding region of the human nucleotide sequence of SEQ ID NO: 1.

EXAMPLE 4: Antibodies that bind TRAIL This example illustrates the preparation of monoclonal antibodies that specifically bind TRAIL. Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified TRAIL protein or an immunogenic fragment thereof the extracellular domain), fusion proteins containing TRAIL polypeptides soluble TRAIL/Fc fusion proteins), and cells expressing recombinant TRAIL on the cell surface.

Known techniques for producing monoclonal antibodies include those described in U.S. Patent 4,411,993. Briefly, mice are immunized with TRAIL as an immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 gg subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional TRAIL emulsified in incomplete Freund's adjuvant.

15 Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision for testing by dot blot assay or ELISA (Enzyme-Linked Immuno-sorbent Assay) for TRAIL antibodies.

Following detection of an appropriate antibody titer, positive animals are 20 provided one last intravenous injection of TRAIL in saline. Three to four days later, the animals are sacrificed, spleen cells harvested, and spleen cells are fused to a murine S.myeloma cell line such as NS1 or, preferably, P3x63Ag 8.653 (ATCC CRL 1580).

Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit 25 proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.

The hybridoma cells are screened by ELISA for reactivity against purified TRAIL by adaptations of the techniques disclosed in Engvall et al. (Immunochem.

8:871, 1971) and in U.S. Patent 4,703,004. Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB/c mice to produce ascites containing high concentrations of anti-TRAIL monoclonal antibodies. Alternatively, hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography. Alternatively, affinity chromatography based upon binding of antibody to protein A or protein G can be used, as can affinity chromatography based upon binding to TRAIL.

EXAMPLE 5: DNA Laddering Anontosis Assay Human TRAIL was expressed and tested for the ability to induce apoptosis.

Oligonucleotides were synthesized that corresponded to the 3' and 5' ends of the coding region of the human TRAIL gene, with Sall and NotI restriction sites incorporated at the ends of the oligonucleotides. The coding region of the human TRAIL gene was amplified by standard PCR techniques, using these oligonucleotides as primers. The PCR reaction products were digested with the restriction endonucleases Sall and NotI, then inserted into Sall/NotI-digested vector pDC409. pDC409 is an expression vector for use in mammalian cells, but is also replicable in E. coli cells.

pDC409 is derived from an expression vector designated pDC406 (described in McMahan et al., EMBO J. 10:2821, 1991, and in PCT application WO 91/18982, hereby incorporated by reference). pDC406 contains origins of replication derived from SV40, Epstein-Barr virus and pBR322 and is a derivative of HAV-EO described 15 by Dower et al., J. Immunol. 142:4314 (1989). pDC406 differs from HAV-EO by the deletion of an intron present in the adenovirus 2 tripartite leader sequence in HAV-EO.

DNA inserted into a multiple cloning site (containing a number of restriction endonuclease cleavage sites) is transcribed and translated using regulatory elements derived from HIV and adenovirus. The vector also contains a gene that confers 20 ampicillin resistance.

pDC409 differs from pDC406 in that a Bgl II site outside the mcs has been deleted so that the mcs Bgl II site is unique. Two Pme 1 sites and one Srf 1 site have been added to the mcs, and three stop codons (TAG) have been positioned downstream of the mcs to function in all three reading frames. A T7 primer/promoter has been 25 added to aid in the DNA sequencing process.

The monkey kidney cell line CV- /EBNA-1 (ATCC CRL 10478) was derived by transfection of the CV-1 cell line (ATCC CCL 70) with a gene encoding Epstein- Barr virus nuclear antigen-1 (EBNA-1) that constitutively expresses EBNA-1 driven from the human CMV intermediate-early enhancer/promoter, as described by McMahan et al., supra. The EBNA-1 gene allows for episomal replication of expression vectors, such as pDC409, that contain the EBV origin of replication.

CV1/EBNA cells grown in Falcon T175 flasks were transfected with 15 ptg of either "empty" pDC409 or pDC409 containing the human TRAIL coding region. The transformed cells were cultured for three days at 37 0 C and 10% CO 2 The cells then were washed with PBS, incubated for 20 minutes at 37 0 C in 50 mM EDTA, scraped off of the flask with a cells scraper, and washed once in PBS. Next, the cells were fixed in 1% paraformaldehyde PBS for 10 minutes at 4 0 C, and washed 3x in PBS.

Jurkat cells were used as the target cells in this assay, to determine whether the TRAIL-expressing cells could induce apoptosis thereof. The Jurkat cell line, clone E6- 1, is a human acute T cell leukemia cell line available from the American Type Culture Collection under accession no. ATCC TIB 152, and described in Weiss et al. (J.

Immunol. 133:123-128, 1984). The Jurkat cells were cultured in RPMI media supplemented with 10% fetal bovine serum and 10 L.g/ml streptomycin and penicillin to a density of 200,000 to 500,000 cells per ml. Four million of these cells per well were co-cultured in a 6 well plate with 2.5 mis of media with various combinations of fixed cells, supematants from cells transfected with Fas ligand, and various antibodies, as indicated below.

After four hours the cells were washed once in PBS and pelleted at 1200 RPM for 5 minutes in a desktop centrifuge. The pellets were resuspended and incubated for ten minutes at 4 0 C in 500 pl of buffer consisting of 10 mM Tris-HC1, 10 mM EDTA, pH 7.5, and 0.2% Triton X-100, which lyses the cells but leaves the nuclei intact. The 15 lysate was then spun at 4 0 C for ten minutes in a micro-centrifuge at 14,000 RPM. The supernatants were removed and extracted three times with 1 ml of 25:24:1 phenolchloroform-isoamyl alcohol, followed by precipitation with NaOAC and ethanol in the presence of 1 tg of glycogen carrier (Sigma).

The resulting pellets were resuspended in 10 mM Tris-HC1, 10 mM EDTA, pH 20 7.5. and incubated with 10 gig/ml RNase A at 37 0 C for 20 minutes. The DNA solutions were then resolved by 1.5% agarose gel electrophoresis in Tris-Borate EDTA buffer, Sstained with ethidium bromide and photographed while trans-illuminated with UV light.

The results were as follows. Fixed CV1/EBNA cells transfected with either pDC409 or pDC409-TRAIL produced no detectable DNA laddering. pDC409-TRAIL S 25 fixed cells co-cultured with Jurkat cells produced DNA laddering, but pDC409 fixed cells co-cultured with Jurkat cells did not.

DNA laddering was also seen when Jurkat cells were co-cultured with concentrated supernatants from COS cells transfected with DNA encoding human Fas ligand in pDC409. The supematants are believed to contain soluble Fas ligand that is proteolytically released from the cell surface. The Fas ligand-induced DNA laddering could be blocked by adding 10 tg/ml of a soluble blocking monoclonal antibody directed against Fas. This same antibody could not inhibit laddering of Jurkat DNA by the pDC409-TRAIL cells, which indicates that TRAIL does not induce apoptosis through Fas.

In the same assay procedure, fixed CV1/EBNA cells transfected with pDC409- TRAIL induced DNA laddering in U937 cells. U937 (ATCC CRL 1593) is a human histiocytic lymphoma cell line. The ratio of effector to target cells was 1:4 (the same as in the assay on Jurkat target cells).

The fragmentation of cellular DNA into a pattern known as DNA laddering is a hallmark of apoptosis. In the foregoing assay, TRAIL induced apoptosis of a leukemia cell line and a lymphoma cell line.

EXAMPLE 6: Northern Blot Analysis Expression of TRAIL in a number of different tissue types was analysed in a conventional northern blot procedure. Northern blots containing poly A RNA from a variety of adult human tissues (multiple tissue northern blots I and II) were obtained from Clonetech (Palo Alto, CA). Other blots were prepared by resolving RNA samples on a 1.1% agarose-formaldehyde gel, blotting onto Hybond-N as recommended by the manufacturer (Amersham Corporation), and staining with methylene blue to monitor RNA concentrations. The blots were probed with an antisense RNA riboprobe 15 corresponding to the entire coding region of human TRAIL.

Human TRAIL mRNA was detected in peripheral blood lymphocytes, colon, small intestine, ovary, prostate, thymus, spleen, placenta, lung, kidney, heart, pancreas, and skeletal muscle. TRAIL transcripts were found to be abundant in the large cell anaplastic lymphoma cell line Karpas 299 (Fischer et al., Blood, 72:234, 1988) and in tonsilar T cells. TRAIL message was present to a lesser degree in the Burkitt lymphoma cell line designated Raji.

STRAIL mRNA was not detected in testis, brain, or liver, or in several T cell lines. Little or no TRAIL transcripts were detected in freshly isolated peripheral blood T cells (PBT), either unstimulated or stimulated with PMA and calcium ionophore for 25 20 hours.

EXAMPLE 7: Production of a Soluble TRAIL Polvnentide A soluble human TRAIL polypeptide comprising amino acids 95 to 281 of SEQ ID NO:2 was prepared as follows. This polypeptide is a fragment of the extracellular domain, lacking the spacer region discussed above.

An expression vector encoding soluble human TRAIL was constructed by fusing in-frame DNA encoding the following amino acid sequences (listed from N- to C-terminus): a leader sequence derived from human cytomegalovirus (CMV), a synthetic epitope designated Flag®, and amino acids 95-281 of human TRAIL. The Flag® octapeptide (SEQ ID NO:7) facilitates purification of proteins fused thereto, as described above and in Hopp et al. (Biotechnology 6:1204-1210, 1988).

The TRAIL-encoding DNA fragment was isolated and amplified by polymerase chain reaction (PCR), using oligonucleotide primers that defined the termini of a DNA fragment encoding amino acids 95-281 of SEQ ID NO:2. The 3' primer was a 31-mer that additionally added a NotI site downstream of the TRAIL-encoding sequence. The 5' primer added an SpeI site and a Flag® epitope encoding sequence upstream of the TRAIL-encoding sequence. PCR was conducted by conventional procedures, using the above-described human TRAIL cDNA as the template.

The reaction products were digested with SpeI and NotI, and inserted into the expression vector pDC409 (described in example which had been cleaved with Sail and NotI. Annealed oligonucleotides that form a SalI-Spel fragment encoding a CMV open reading frame leader were also ligated into the vector. The amino acid sequence of the CMV-derived leader is presented as SEQ ID NO:9. Amino acids 1 to 29 of SEQ ID NO:9 are encoded by CMV DNA, whereas amino acids 30 to 32 are encoded by oligonucleotides employed in constructing the vector. E. coli cells were transfected 15 with the ligation mixture, and the desired recombinant expression vector was isolated therefrom.

CV1-EBNA cells (ATCC CRL 10478; described in example 5) were transfected with the recombinant vector, which is designated pDC409-Flag-shTRAIL, and cultured to allow expression and secretion of the soluble Flag®-TRAIL polypeptide. Culture 20 supernatants were harvested 3 days after transfection and applied to a column containing an anti-Flag® antibody designated M2 immobilized on a solid support. The column then was washed with PBS. The monoclonal antibody M2 is described in Hopp et al., supra, and available from Kodak Scientific Imaging Systems, New Haven, Connecticut. 8 00pl fractions were eluted from the column with 50 mM citrate, and 25 immediately neutralized in 0.45 ml 1M Tris (pH Fractions were adjusted to glycerol and stored at -20 0 C until needed.

This soluble recombinant Flag®/human TRAIL expressed in CV1/EBNA cells has an apparent molecular weight of 28 kD when analyzed by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). The Flag® moiety contributes an estimated 880 daltons to the total molecular weight. Gel filtration analysis of purified soluble Flag®/TRAIL suggests that the molecule is multimeric in solution with a size of -80 kD. While not wishing to be bound by theory, the gel filtration analysis suggests that the soluble recombinant Flag®/human TRAIL naturally formed a combination of dimers and trimers, with trimers predominating.

An expression vector designated pDC409-Flag-smTRAIL, which encodes a CMV leader-Flag®-soluble murine TRAIL protein, was constructed by analogous procedures. A DNA fragment encoding a soluble murine TRAIL polypeptide was isolated and amplified by PCR. Oligonucleotides that defined the termini of DNA encoding amino acids 99 to 291 of the murine TRAIL sequence of SEQ ID NO:6 were employed as the 5' and 3' primers in the PCR.

EXAMPLE 8: Lvsis of Leukemia Cells by Soluble TRAIL In example 5, cells expressing human TRAIL induced apoptosis of Jurkat cells (a leukemia cell line). In the following study, a soluble human TRAIL polypeptide killed Jurkat cells.

Jurkat cells were cultured to a density of 200,000 to 500,000 cells per ml in RPMI medium supplemented with 10% fetal bovine serum, 100 uig/ml streptomycin, and 100 Wg/ml penicillin. The cells (in 96-well plates at 50,000 cells per well in a volume of 100 l) were incubated for twenty hours with the reagents indicated in Figure 1. "TRAIL supe." refers to conditioned supernatant (10 Pl per well) from CV1/EBNA cells transfected with pDC409-Flag-shTRAIL (see example "Control supe." refers 15 to supernatant from CV1/EBNA cells transfected with empty vector. Where indicated, immobilized anti-Flag® antibody M2 ("Imm. M2") was added at a concentration of jig/ml in a v6lume of 100 pl per well and allowed to adhere either overnight at 4oC or for 2 hours at 370C, after which wells were aspirated and washed twice with PBS to remove unbound antibody. Jurkat cells treated with Fas ligand or M3, a blocking 20 monoclonal antibody directed against Fas, (Alderson et al., J. Exp. Med. 181:71, 1995; and PCT application WO 95/10540) were included in the assay as indicated.

.Metabolic activity of the thus-treated cells was assayed by metabolic conversion of alamar Blue dye, in the following procedure. Alamar Blue conversion was measured by adding 10 pl of alamar Blue dye (Biosource International, Camarillo, CA) per well, 25 and subtracting the optical density (OD) at 550-600 nm at the time the dye was added i: from the OD 550-600 nm after four hours. No conversion of dye is plotted as 0 percent viability, and the level of dye conversion in the absence of TRAIL is plotted as 100 percent viability. Percent viability was calculated by multiplying the ratio of staining of experimental versus control cultures by 100.

The results are presented in Figure 1. Error bars represent the standard deviation of measurements from four independent wells, and the values are the average of these measurements.

The TRAIL-containing supernatant caused a significant reduction in viability of Jurkat cells. A greater reduction of cell viability resulted from contact with a combination of TRAIL-containing supematant and immobilized anti-Flag® antibody M2. One possible explanation is that M2 facilitates cross-linking of the Flag®/TRAILreceptor complexes, thereby increasing the strength of signaling.

Fas ligand demonstrated the ability to kill Jurkat cells. The anti-Fas antibody M3 inhibited the activity of Fas ligand, but not the activity of TRAIL.

In order to confirm that the changes in dye conversion in the alamar Blue assay were due to cell death, the decrease in cell viability induced by TRAIL was confirmed by staining the cells with trypan blue.

EXAMPLE 9: Lvsis of Leukemia and Lvmohoma Cells In examples 5 and 8, TRAIL induced apoptosis of a leukemia cell line (Jurkat) and a lymphoma cell line (U937). The following study further demonstrates the ability of TRAIL to kill leukemia and lymphoma cells.

The human cell lines indicated in Table I were cultured to a density of 200,000 to 500,000 cells per ml in RPMI medium supplemented with 10% fetal bovine serum, 100 jlg/ml streptomycin, and 100 pg/ml penicillin. The cells (in 96-well plates at 50,000 cells per well in a volume of 100 pl) were incubated for twenty hours with 15 conditioned supernatants (10 pl per well) from pDC409-Flag-shTRAIL transfected CV1/EBNA cells.

Metabolic activity was assayed by conversion of alamar Blue dye, in the assay procedure described in example 8. The results are presented in Table I.

In order to confirm that the changes in dye conversion in the alamar Blue assay 20 were due to cell death, the decrease in cell viability induced by TRAIL was confirmed by staining the cells with trypan blue. Crystal violet staining, performed as described by Flick and Gifford Immunol. Methods 68:167-175, 1984), also confirmed the results seen in the alamar Blue assay. The apoptotic nature of the cell death was confirmed by trypan blue staining and visualization of apoptotic fragmentation by microscopy.

As shown in Table I, many cancer cell lines were sensitive to TRAIL mediated killing. The susceptibility of additional cell types to TRAIL mediated apoptosis can be determined using the assay procedures described in this examples section.

TRAIL exhibited no significant cytotoxic effect on the cell lines THP-1 K562, Karpas 299, and MP-1. K299, also known as Karpas 299, (DSM-ACC31) was established from peripheral blood of a male diagnosed with high grade large cell anaplastic lymphoma (Fischer et al., Blood, 72:234, 1988). MP-1 is a spontaneously derived EBV-transformed B cell line (Goodwin et al., Cell 73:447, 1993). While not wishing to be bound by theory, it is possible that these four cell lines do not express a receptor for TRAIL, or are characterized by upregulation of a gene that inhibits apoptosis.

Table 1. Effect of soluble TRAIL on cell line viability Cell Line Description Percent Viabilitya Bjab Burkitt lymphoma 0.5 3.8 Ramos Burkitt lymphoma 12.1 2.1 U937 histiocytic lymphoma 25.2 8.2 promyelocytic leukemia 59.5 3.2 Raji Burkitt lymphoma 64.9 Daudi Burkitt lymphoma 70.2 4.2 THP-1 monocytic cell line 92.3 6.8 K562 chronic myelogenous leukemia 97.1 4.8 K 299 large cell anaplastic lymphoma 99.0 4.3 MP-1 spontaneous B cell line 104.9+ 11.7 a Results are means SEMs of 4 wells for each data point EXAMPLE 10: Cross-Snecies Activity of TRAIL Interspecies cross-reactivity of human and murine TRAIL was tested as follows. Murine and human TRAIL were incubated with the human melanoma cell line A375 (ATCC CRL 1619). Since this is an adherent cell line, a crystal violet assay, rather than alamar Blue, was used to determine cell viability. A375 cells were cultured in DMEM supplemented with 10% fetal bovine serum, 100 gtg/ml streptomycin, and 100 itg/ml penicillin. The cells (in 96-well plates at 10,000 cells per well in a volume of 100 pl) were incubated for 72 hours with the soluble murine TRAIL described in example 7. Crystal violet staining was performed as described by (Flick and Gifford 15 Immunol. Methods 68:167-175, 1984). The results demonstrated that both human and murine TRAIL are active on these human cells, in that murine and human TRAIL killed A375 cells.

The ability of human TRAIL to act on murine cells was tested, using the immortalized murine fibroblast cell line L929. Incubation of L929 cells with either human or murine TRAIL resulted in a decrease in crystal violet staining, thus demonstrating that human and murine TRAIL are active on (induced apoptosis of) murine cells. In addition to crystal violet, cell death was confirmed by trypan-blue staining.

EXAMPLE 11: Lvsis of CMV-Infected Cells The following experiment demonstrates that the soluble Flag®-human TRAIL protein prepared in example 7 has a cytotoxic effect on virally infected cells.

Normal human gingival fibroblasts were grown to confluency on 24 well plates in 10% C02 and DMEM medium supplemented with 10% fetal bovine serum, 100 gg/ml streptomycin, and 100 uig/ml penicillin. Samples of the fibroblasts were treated as indicated in Figure 2. Concentrations of cytokines were 10 ng/ml for y-interferon and 30 ng/ml of soluble Flag®-human TRAIL. All samples receiving TRAIL also received a two-fold excess by weight of anti-Flag® antibody M2 (described above), which enhances TRAIL activity (presumably by crosslinking).

Pretreatment of cells with the indicated cytokines was for 20 hours. To infect cells with cytomegalovirus (CMV), culture media were aspirated and the cells were infected with CMV in DMEM with an approximate MOI (multiplicity of infection) of After two hours the virus containing media was replaced with DMEM and cytokines added as indicated. After 24 hours the cells were stained with crystal violet dye as described (Flick and Gifford, 1984, supra). Stained cells were washed twice with water, disrupted in 200 l of 2% sodium deoxycholate, diluted 5 fold in water, and the OD taken at 570 nm. Percent maximal staining was calculated by normalizing ODs to the sample that showed the greatest staining. Similar results were obtained from several 15 independent experiments.

The results presented in Figure 2 demonstrate that TRAIL specifically killed CMV infected fibroblasts. This cell death was enhanced by pretreatment of the cells with y-interferon. No significant death of non-virally infected fibroblasts resulted from contact with TRAIL.

EXAMPLE 12: Assay to Identify Blockine Antibodies Blocking antibodies directed against TRAIL may be identified by testing antibodies for the ability to inhibit a particular biological activity of TRAIL. In the following assay, a monoclonal antibody was tested for the ability to inhibit TRAIL- 25 mediated apoptosis of Jurkat cells. The Jurkat cell line is described in example A hybridoma cell line producing a monoclonal antibody raised against a Flag®/soluble human TRAIL fusion protein was employed in the assay. Supernatants from the hybridoma cultures were incubated with 20 ng/ml Flag®/soluble human TRAIL crosslinked with 40 ng/ml anti-Flag® monoclonal antibody M2, in RPMI complete media in a 96 well microtiter plate. An equivalent amount of fresh hybridoma culture medium was added to control cultures. The Flag®/soluble human TRAIL fusion protein and the monoclonal antibody designated M2 are described in example 7.

The hybridoma supernatant was employed at a 1:50 dilution (starting concentration), and at two fold serial dilutions thereof. After incubation at 37 0 C,

CO

2 for 30 minutes, 50,000 Jurkat cells were added per well, and incubation was continued for 20 hours.

Cell viability was then assessed measuring metabolic conversion of alamar blue dye. An alamar blue conversion assay procedure is described in example 8. The monoclonal antibody was found to inhibit the apoptosis of Jurkat cells induced by Flag®/soluble human TRAIL.

EXAMPLE 13: TRAIL Blocking Study Human microvascular endothelial cells of dermal origin were treated for 16-18 hours with plasma from patients with thrombotic thrombocytopenic purpura (TTP) or with control plasma, either alone or in the presence of anti-TRAIL polyclonal antiserum. A 1:2000 dilution of the antiserum was employed. The plasma was from two 'TP patients, designated #1 and #2 below, The cells employed in the assays were MVEC-1 (HMVEC 2753, purchased from Clonetics, San Diego, CA) and MVEC-2 (DHMVEC 30282, purchased from Cell Systems, Kirkland, WA). Cultures of these cells can be maintained as described in Laurence et al. (Blood, 87:3245, 1996).

15 The results were as follows. The data shown are from DNA histograms of cells stained with propidium iodide, and "Ao peak" represents the apoptotic peak (see Oyaizu et al., Blood, 82:3392, 1993; Nicoletti et al., J. Immunol. Methods, 139:271, 1991; and Laurence et al., Blood, 75:696, 1990).

20 Microvascular EC Plasma Antibody A peak Experiment 1 Dermal MVEC-1 control 0 DermalMVEC-1 TTP -19.5 DermalMVEC-1 TTP 0.3 S* 25 Experiment 2 Dermal MVEC-2 control 0 Dermal MVEC-2 TTP 20.0 Dermal MVEC-2 TTP control Ab 13.1 .i Dermal MVEC-2 TTP 0.2 Experiment 3 DermalMVEC-1 TTP -50.1 DermalMVEC-1 TTP 10.6 Experiment 4 Dermal MVEC-2 control 0 Dermal MVEC-2 TTP -13.9 Dermal MVEC-2 TTP control Ab 14.1 Dermal MVEC-2 TTP 0.6 The data reveal that plasma derived from TTP patients induces apoptosis of microvascular endothelial cells of dermal origin. This apoptosis was inhibited by polyclonal antibodies directed against TRAIL.

The discussion of documents, acts, materials, devices, articles and the like is included in this specification solely for the purpose of providing a context for the present invention. It is not suggested or represented that any or all of these matters formed part of the prior art base or were common general knowledge in the field relevant to the present invention as it existed in Australia before the priority date of each claim of this application.

e C 41a MR C:\WINWORD\MARY\NODELETE\708239.DOC SEQUENCE LISTING GENERAL INFORMATION: APPLICANT: Immunex Corporation.

(ii) TITLE OF INVENTION: Cytokine That Induces Apoptosis (iii) NUMBER OF SEQUENCES: 9 (iv) CORRESPONDENCE ADDRESS: ADDRESSEE: Kathryn A. Anderson, Immunex Corporation STREET: 51 University Street CITY: Seattle STATE: WA COUNTRY: USA ZIP: 98101 COMPUTER READABLE FORM: MEDIUM TYPE: Floppy disk COMPUTER: Apple Macintosh OPERATING SYSTEM: Apple 7.5.2 SOFTWARE: Microsoft Word, Version 6.0.1 (vi) CURRENT APPLICATION DATA: APPLICATION NUMBER: to be assigned-- FILING DATE: 25-JUN-1996

CLASSIFICATION:

(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/496,632 FILING DATE: 29-JUN-1995

CLASSIFICATION:

*(vii) PRIOR APPLICATION DATA: APPLICATION NUMBER: US 08/548,368 FILING DATE: 01-NOV-1995

CLASSIFICATION:

(viii) ATTORNEY/AGENT

INFORMATION:

NAME: Anderson, Kathryn A.

REGISTRATION NUMBER: 32,172 REFERENCE/DOCKET NUMBER: 2835-WO (ix) TELECOMMUNICATION

INFORMATION:

TELEPHONE: (206) 587-0430 TELEFAX: (206) 233-0644 TELEX: 756822 INFORMATION FOR SEQ ID NO:1: SEQUENCE CHARACTERISTICS: LENGTH: 1751 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear MOLECULE TYPE: CDNA to rrRNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: huAIC (ix) FEATURE: NAME/KEY: CDS LOCATION: 88. .933 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: CCTCACTGAC TATAAAAGAA TAGAGAAGGA AGGGCTTCAG TGACCGGOTG CCTGGCTGAC TTACAGCAGT CAGACTCTGA CAGGATC ATG GCT ATG ATG GAG GTC CAG GGG Met Ala Met Met Giu Val Gin Gly 9

S

GGA

Gly

CTC

Leu

GAG

Giu

TTC

Phe

ATG

Met

AGA

Arg

GAA

Giu 105

AGA

Arg

TCT

Ser

TCC

CCC

Pro 10

CTG

Leu

CTG

Leu

TTA

Leu

AAC

Asn

AAG,

Lys

AAG

Lys

GTA

Va 1

TCT

Ser

TGG

AGO

Ser

CAG

Gin

AAG

Lys

AAA

Lys

AGC

Ser 75

ATG

met

CAA

Gin

GCA

Ala

CCA

Pro

GAA

CTG

Leu

TCT

Ser

CAG

Gin

GAA

Giu 60

CCC

Pro

ATT

Ile

CAA

Gin

GCT

Ala

AAC

Asn 140

TCA

GGA

Giy

CTC

Leu

ATG

Met

GAT

Asp

TGC

Cys

TTG

Leu

AAT

As n

CAC

His 125

TCC

Ser

TCA

CAG

Gin

TGT

Cys 30

CAG

Gin

GAO

Asp

TGG

Trp

AGA

Arg

ATT

Ile 110

ATA

Ile

AAG

Lys

AGG

ACC

Thr 15

GTG

Vai

GAO

Asp

AGT

Ser

CAA

Gin

ACC

Thr 95

TCT

Ser

ACT

Thr

AAT

Asn TGC GTG CTG ATO GTG ATC TTO ACA GTG Ile

TAC

Tyr

AAA

Lys

CCC

Pro

CAA

Gin

ACC

Th r

AGA

Arg 115

GGA

Giy

CTG

Leu Val

GTG

Val

AGT

Ser

AAT

Asn

CTC

Leu

ATT

Ile 100

GAA

Giu

AGA

Arg

GGC

Gly Ile

TAC

Tyr

GGC

Giy

GAC

Asp

CGT

Arg

TCT

Ser

AGA

Arg

AGC

Ser

CGC

Arg Phe

TTT

Phe

ATT

Ile

GAA

Giu

CAG

Gin

ACA

Thr

GGT

Giy

AAC

Asn

AAA

Lys 150 Thr

ACC

Thr

GOT

Ala

GAG

Giu

CTC

Leu

GTT

Val1

CCT

Pro

ACA

Thr 135

ATA

Ile ill 159 207 255 303 351 399 447 495 543 591 AGT GGG CAT TCA TTC CTG AGC AAC TTG CAC Ser Trp Glu 155 TTG AGG AAT Leu Argj Asn 170 Ser Ser Arg Ser GGT GAA CTG GTC Gly Giu Leu Val 175 ACA TAC TTT CGA Thr Tyr Phe Arg His Ser Phe Leu Asn Leu His TAC TAO ATC Tyr Tyr Ile CAT GAA AAA His Giu Lys

GGG

Gly 180

ATA

Ile

TAT

Tyr 185

AAG

Lys TCC CAA Ser Gin TTT CAG GAG Phe Gin Giu

GAA

Giu 195

TAC

Tyr AAA GAA AAC Lys Giu Asn

ACA

Thr 200 AAC GAC AAA Asn Asp Lys

CAA

Gin 205

TTG

Leu GTC CAA TAT Vai Gin Tyr AAA TAO ACA Lys Tyr Thr AGT TAT Ser Tyr 215 0* 9.

CCT GAO OCT Pro Asp Pro AAA GAT GCA Lys Asp Ala 235 GAG CTT AAG Giu Leu Lys 250 TTG ATA GAO Leu Ile Asp 265

ATA

Ile 220

GAA

Glu TTG ATG AAA Leu Met Lys

AGT

Ser 225

TCC

Ser TAT GGA CTO Tyr Giy Leu ATC TAT CAA Ile Tyr Gin

GGG

Giy 245

ACA

Thr AGA A.AT AGT Arg Asn Ser TGT TGG TOT Cys Trp Ser 230 GGA ATA TTT Giy Ile Phe AAT GAG CAC Asn Giu His 639 687 735 783 831 879 927 983 GAA AAT GAC Giu Asn Asp ATG GAO OAT Met Asp His 270

AGA

Arg 255

GAA

Glu TTT GTT TOT Phe Vai Ser

GTA

Vali 2.60

GGG

Giy GOC AGT TTT TTO Ala Ser Phe Phe GOO TTT TTA GTT Aia Phe Leu Vai 280 :TAT TCAGTTTTOA GGO TAA CTGAOOTGGA AAGAAAAAGO AATAAOOTOA AAGTGAC 9*e*

GGATGATAOA

AAAACAAAAA

AOACACTGTT

OAGGAOTOTA

ATTAATGOAA

GAAAGOGOAA

AAGGGAOAAC

OTGGTGCAGT

OGAGATOAAG

TGOAA-AAATT

GGOAGGAGAA

AOAOTCOAGC

CAGTAAGTAC

CTATGAAGAT

AACCTCTATG

CTGAAAGTGA

OCTOATATOA

TGGTTAACAT

CAATCCATCT

ATOOTTAAGT

GGOTCAOAOC

AGATOAAGAC

AGOTGGGTGT

TCGTTTGAAC

OTGGOGAOAG

GTGTTATTTT

GTTTOAAAAA

CAATCTGAGT

CTCAOTTATC

GTTTGOTAGO

OTTOTGTCTT

CTCAAGTAGT

CAAAAGAGAG

TGTAATCCCA

OATAGTGACO

GTTGGOAOAT

OOGGGAGGCA

AGCGAGACTT

TTTCAATAAA

ATOTGAOOA.A

AGAGOAGOCA

CCAAGAAAAT

AGAAATOTAG,

TATAATOTAO

GTATOAOAGT

AAGAGGCACO

AOATTTTGcGG

AAOATAGTGA

GOOTGTAGTo

GAGGTTGOAG-

AAOAAACAAA

OAAOOAAAAA

GAAATTGCTG

AAGACTGTCA

TCCTTGTAAA

AGTAGOCTCO

ACTAAAAGAT

AACOAAGGT

AACOOCATCT

OOAGOTAOTT

TGTGGTGAGA

CAGAAAACAG

ATTOTACAAC

AAAGATCTTT

GCTTOCAAAC

GACTGTAGAA

AGGTTTCOTT

OGOAGTTTGO

GGGTAGATOA

CTACTGAAAG

GAGAGGOTGA

TCATGCCACT

AAAAAAACTT

1043 1103 1163 1223 1283 1343 1403 1463 1523 1583 1643 1703 1751 GGTTTOAAAA AAAAAAAAAA ATTOTATTAC AGTATGTO INFORMATION FOR SEQ ID NO:2: SEQUENCE CHARACTERISTICS: LENGTH: 281 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:2: Met Ala Met 1 Met Glu Val Gin Giy Gly Pro Ser Leu Gly Gin Thr Cys a a a a .a a a.

a Val1 Val1 Tyr Trp 65 Lys Giu Leu Thr Lys 145 His His Gin Tyr Ser 225 Ile Tyr Lys Pro Gin Th r Arg 115 Giy Leu Phe Lys Giu 195 Tyr Arg Val Val Ser Asn Leu Ile 100 Giu Arg Giy Leu Gly 180 Ile Lys Asn Ile Tyr Gly Asp Arg Ser Arg Ser Arg Ser 165 Phe Lys Tyr Ser Gly 245 Phe Phe Ile Giu 70 Gin Thr Giy As n Lys 150 Asn Tyr Giu Thr Cys 230 Thr Thr Ala 55 Giu Leu Val1 Pro Thr 135 Ile Leu Tyr As n Ser 215 T rp Val1 Asn 40 Cys Ser Val Gin Gin 120 Leu Asn His Ile Thr 200 Tyr Ser Leu 25 Giu Phe Met Arg Giu 105 Arg Se r Se r Leu Tyr 185 Lys Pro Lys Leu Leu Leu Asn Lys 90 Lys Val Ser Trp Arg 170 Ser Asn Asp Asp Leu 250 Gin Ser Lys Gin Lys Glu Ser Pro 75 Met Ile Gin Gin Ala Ala Pro Asn 140 Giu Ser 155 Asn Gly Gin Thr Asp Lys Pro Ile 220 Aia'Giu 235 Lys Giu Leu Met Asp Cys Leu Asn His 125 Ser Ser Giu Tyr Gin 205 Leu Tyr Asn Cys Gin Asp Trp Arg Ile 110 Ile Lys Arg Leu Phe 190 Met Leu Gly Asp Val1 Asp Ser Gin Thr Ser Thr Asn Ser Val1 175 Arg Vali Met Leu Arg 255 Ala Lys Tyr Val1 Se r Pro Giy Giu Gly 160 Ile P he Gin Lys Tyr 240 Ile Ser Ile Tyr Gin Gly Ile Phe Glu Phe Val Ser Val Thr Asn Giu His Leu Ile Asp Met Asp His Giu Ala 260 265 270 Ser Phe Phe Gly Ala Phe Leu Val Gly* 275 280 INFORMATION FOR SEQ ID NO:3: SEQUENCE CHARACTERISTICS: LENGTH: 1521 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to mRNA (iii) HYPOTHETICAL:

NO

(iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: HuAIC-dv (ix) FEATURE: NAME/KEY: CDS LOCATION: 78. .383 (xi) SEQUENCE DESCRIPTION: SEQ ID NO:3: AATTCCGGAA TAGAGAAGGA AGGGCTTCAG TGACCGGCTG CCTGGCTGAC TTACAGCAGT CAGACTCTGA CAGGATO ATG GCT ATG ATG GAG GTC CAG GGG GGA CCC AGC Met Ala Met Met Giu Val Gin Gly Gly Pro Ser a a. a a.

a. 4 110 158 206 CTG GGA CAG Leu Gly Gin TCT CTC TGT Ser Leu Cys

ACC

Thr TGC GTG CTG ATC Cys Val Leu Ile ATC TTC ACA GTG Ile Phe Thr Val CTC CTG CAG Leu Leu Gin GAG CTG AAG Glu Leu Lys GTG OCT GTA ACT Val Ala Val Thr

TAC

Tyr 35 GTG TAC TTT ACC Val Tyr Phe Thr

AAC

Asn CAG ATG Gin Met CAG GAC AAG TAC Gin Asp Lys Tyr

TCC

Ser AAA AGT GOC ATT Lys Ser Gly Ile TOT TTC TTA AAA Cys Phe Leu Lys

GAA

Giu GAT GAC AGT TAT Asp Asp Ser Tyr GAG CCC AAT GAC Asp Pro Asn Asp

GAA

Giu GAG AGT ATO AAC Giu Ser Met Asn CCC TGO TGO Pro Cys Trp CAA GTC Gin Val AAG TOG CAA CTC Lys Trp Gin Leu

COT

Arg 85 CAG CTC OTT AGA Gin Leu Vai Arg AAG ACT Lys Thr CCA AGA ATG AAA AGO CTC TOG GCC OCA AAA TAA Pro Arg~ Met Lys Arg Leu Trp, Ala Aia Lys* ACTCCTGGGA ATCATCAAGG 100 AGTGGGCATT CATTCCTGAG CAACT TGCAC TTGAGGAATG GTGAACTGGT 9 9 9 *99 9 9 4 .9 9.

AAAGGGTTTT

AACACAAAGA

CCTATATTGT

CTCTATTCCA

TCTGTAACAA

TTAGTTGGCT

CAGGATGATA

AGAAAACAAA

ACACACACTG

TTCAGGACTC

ACATTAATGC

AAGAAAGCGC

TTAAGGGACA

GCCTGGTGCA

CACGAGATCA

AGTGCAAAAA

GAGGCAGGAG

CTACACTCCA

ACTACATCTA

ACGACAAACA

TGATGAAAAG

TCTATCAAGG

ATGAGCACTT

AACTGACCTG

CACTATGAAG

AAAACCTCTA

TTCTGAAAGT

TACCTCATAT

AGTGGTTAAC

AACAATCCAT

ACATCCTTAA

GTGGCTCACA

AGAGATCAAG

TTAGCTGGGT

AATCGTTTGA

GCCTGGCGAC

TTCCCAAACA

AATGGTCCAA

TGCTAGAAAT

GGGAATATTT

GATAGACATG

GAA.AGAAAAA

ATGTTTCAAA

TGCAATCTGA

GACTCACTTA

CAGTTTGCTA

ATCTTCTGTC

CTCTCAAGTA

GTCAAAAGAG

CCTGTAATCC

ACCATAGTGA

GTGTTGGCAC

ACCCGGGAGG

AGAGCGAGAC

TACTTTCGAT

TATATTTACA

AGTTGTTGGT

GAGCTTAAGG

GACCATGAAG

GCAATAACCT

AAATCTGACC

GTAGAGCAGC

TCCCAAGAGA

GCAGAAATCT

TTTATAATCT

GTGTATCACA

AGAAGAGGCA

CAACATTTTG

CCAACATAGT

ATGCCTGTAG

CAGAGGTTGC

TTGGTTTC

TTCAGGAGGA

AATACACAAG

CTAAAGATGC

AAAATGACAG

CCAGTTTTTT

CAAAGTGACT

AAAACAAACA

CACAACCAAA

ATGAAATTGC

AGAAGACTGT

ACTCCTTGTA

GTAGTAGCCT

CCACTAAAAG

GGAACCCAAG

GAAACCCCAT

TCCCAGCTAC

AGTGTGGTGA

CATCCATGAA

AATAAAAGAA

TTATCCTGAC

AGAATATGGA

AATTTTTGTT

CGGGGCCTTT

ATTCAGTTTT

AACAGAAAAC

AAATTCTACA

TGAAAGATCT

CAGCTTCCAA

AAGACTGTAG

CCAGGTTTCC

ATCGCAGTTT

GTGGGTAGAT

CTCTACTGAA

TTGAGAGGCT

GATCATGCCA

463 523 583 643 703 763 823 883 943 1003 1063 1123 1183 1243 1303 136.3 1423 1483 1521 .9 INFORMATION FOR SEQ ID NO:4: SEQUENCE CHARACTERISTICS: LENGTH: 101 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:4: Met Ala Met Met Glu Val Gin Gly Gly Pro Ser Leu Gly Gin Thr Cys 1 5 10 Val Leu Ile Val Ile Phe Thr Val Leu Leu Gin Ser Leu Cys Val Ala 25 Vai Thr Tyr Val Tyr Phe Thr Asn Glu Leu Lys Gin Met Gin.Asp Lys 40 Tyr Ser Lys Ser Gly Ile Ala Cys Phe Leu Lys Giu 55 Trp Asp Pro Asn Asp Giu Giu Ser Met Asn Ser Pro 70 Lys Trp Gin Leu Arg Gin Leu Val Arg Lys Thr Pro 90 Leu Trp Ala Ala Lys* 100 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS: LENGTH: 1366 base pairs TYPE: nucleic acid STRANDEDNESS: single TOPOLOGY: linear (ii) MOLECULE TYPE: cDNA to rnRNA (iii) HYPOTHETICAL: No (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: MuAIC (ix) FEATURE: NAME/KEY: CDS LOCATION: 47. .919 (xi) SEQUENCE DESCRIPTION: SEQ ID Asp Asp Ser Tyr Cys Trp Gin Arg Met Lys Arg

S

S

TGCTGGGCTG CAAGTCTGCA TTGGGAAGTC AGACCTGGAC AGCAGT ATG CCT TCC Met Pro Ser 1 TCA GGG Ser Gly GCC CTG AAG GAC Ala Leu Lys Asp

CTC

Leu AGC TTC AGT CAG Ser Phe Ser Gin TTC AGG ATG ATG Phe Arg Met Met

GTG

Val1 ATT TGC ATA GTG CTC CTG CAG GTG CTC Ile Cys Ile Val Leu Leu Gin Val Leu 25

CTG

Leu 30 CAG GCT GTG TCT Gin Ala Val Ser

GTG

Val1 GCT GTG ACT TAC Ala Val Thr Tyr TAC TTC ACC AAC Tyr Phe Thr Asn ATG AAG CAG CTG Met Lys Gin Leu GAG GAC Gin Asp AAT TAC TCC Asn Tyr Ser

AAA

Lys ATT GGA CTA GCT Ile Gly Leu Ala

TGC

Cys 60 TTC TCA AAG ACG GAT GAG GAT Phe Ser Lys Thr Asp Giu Asp TTC TGG GAC TCC ACT GAT GGA GAG ATC TTG AAC AGA CCC TGC TTG GAG Phe Trp, Asp Ser Thr Asp Gly Giu Ile Leu Asn Arg Pro Cys, Leu Gin .9 a *999 a. a a.

GTT

Val1

TTT

Phe 100

CCC

Pro

GGG

Gly

GGA

Gly

GGG

Gly

ATC

Ile 180

TTT

Phe

AGA

Arg

GAT

Asp

GAT

Asp

CTA

Leu 260

ATG

AAG

Lys

GAG

Gin

TTG

Leu

ATC

Ile

AAG

Lys

CAT

His 165

GAG

Giu

GAG

Gin

ACC

Thr

CCC

Pro

GCG

Ala 245

AAA

Lys

GAG

AGG

Arg

GAC

Asp

CCC

Pro

ACT

Thr

ACC

Thr 150

TCA

Ser

CAG

Gin

GAA

Giu

AAA

Lys

ATA

Ile 230

GAG

Giu

AAA

Lys

CTG

CAA

Gin

ACC

Thr

AGA

Arg

CGG

Arg 135

TTA

Leu

TTT

Phe

GAG

Giu

GCT

Ala

CAG

Gin 215

GTG

Val1

TAG

Tyr

AAT

Asn

GAT

CTG

Le u

ATT

Ile

GGT

Gly 120

AGA

Arg

GGG

Gly

CTC

Leu

GGC

Gly

GAA

Giu 200

CTG

Leu

CTC

Leu

GGA

Giy

GAC

Asp

CAA

GAG

Gin 90

AGA

Thr

AGA

Arg

AAC

Asn

AAG,

Lys

GAG

His 170

TAT

Tyr

GGT

Ala

GAG

Gin

AAG

Lys

TAG

Tyr 250

ATT

Ile

GCC

ATT

Ile

CCA

Pro

GAG

Gin

GOT

Ala 140

GAA

Giu

CTC

Leu

ATC

Ile

AAG

Lys

ATC

Ile 220

GCC

Ala

ATC

Ile

GTT

Val1

TTC

GAA

Giu

GAA

Giu

AAA

Lys 125

TTA

Leu

TGG

Ser

TTT

Phe

TAT

Tyr

ATG

Met 205

TAG

Tyr

AGA

Arg

TAT

Tyr

TGT

Ser

TTT

GTG

Val1

GAG

Gin

GGA

Ala

GGA

Pro

GAG

Giu

AAT

Asn 175

CAA

Gin

TCA

Ser

TAG

Tyr

AGG

Se r

GGA

Gly 255

AGA

Thr

ACT

Thr

GTA

Leu

GCT

Ala

ATC

Ile

TC

Ser 160

GGA

Gly

ACA

Thr

AAG

Lys

AGG

Thr

TGT

Gys 240

GGA

Gly

AAT

Asn AGA ACC Arg Thr ACT CCT Thr Pro 115 ATT ACT Ile Thr 130 AAG GAT Lys Asp CGG AAA Arg Lys GTG GTG Leu Val TTG CGA Phe Arg 195 AAG GTG Lys Val 210 TAT CG Tyr Pro TCC AGA Ser Arg TTG GAG Phe Giu CAT TTG His Leu 275 343 391 439 487 535 265 583 631 679 727 775 823 871 919 Met Asp Leu Asp Gin Giu Ala Ser Phe Phe Gly Ala Phe Leu Ile Asn 280 285 290 TAAATGACGA GTAAAGATCA AACAGAGCCC TAAAGTACCC GGATGCGTGG AAAGCGACTG AAGTGGTTAG GATATGGCCT AGATGTGAGA GAAAGGCAGG TGGAACTCAG CAGCGAGAGG

AGTAATCTTC

GGCTGTAGAA

CCAACAGTCC

TAGGTTGAAG

ACGTGAGGAG

AGCGACAGAC

979 1039 1099 ACTTTCGGTG TTTCATCGAG AGACTTGCTT TCTTTCCGCA AAATGAGATC ACTGTAGCCT 1159 TTCAATGATC TACCTGGTAT CAGTTTGCAG AGATCTAGAA GACGTCCAGT TTCTAAATAT 1219 TTATGCAACA ATTGACAATT TTCACCTTTG TTATCTGGTC CAGGGGTGTA AAGCCAAGTG 1279 CTCACAAGCT GTGTGCAGAC CAGGATAGCT ATGAATGCAG GTCAGCATAA AAATCACAGA 1339 ATATCTCACC TACTAAAAAA AAAAA 1366 INFORMATION FOR SEQ ID NO:6: SEQUENCE CHARACTERISTICS: LENGTH: 291 amino acids TYPE: amino acid TOPOLOGY: linear (ii) MOLECULE TYPE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:6: 9Met Pro Ser Ser Gly Ala Leu Lys Asp Leu Ser Phe Ser Gin His Phe 9.*1 5 10 **Arg Met Met Val Ile Cys Ile Val Leu Leu Gin Val Leu Leu Gin Ala 25 Val Ser Val Ala Val Thr Tyr Met Tyr Phe Thr Asn Giu Met Lys Gin 40 Leu Gin Asp Asn Tyr Ser Lys Ile Gly Leu Ala Cys Phe Ser Lys Thr 55 Asp Giu Asp Phe Trp Asp Ser Thr Asp Giy Glu Ile Leu Asn Arg Pro 70 75 Cys Leu Gin Vai Lys Arg Gin Leu Tyr Gin Leu Ile Giu Giu Val Thr *985 90 Leu Arg Thr Phe Gin Asp Thr Ile Ser Thr Vai Pro Giu Lys Gin Leu 100 105 110 Ser Thr Pro Pro Leu Pro Arg Giy Giy Arg Pro Gin Lys Val Ala Ala 115 120 125 His Ile Thr Gly Ile Thr Arg Arg Ser Asn Ser Ala Leu Ile Pro Ile 130 135 140 Ser Lys Asp Gly Lys Thr Leu Giy Gin Lys Ile Giu Ser Trp Giu Ser 145 150 155 160 Ser Arg Lys Gly His Ser Phe Leu Asn His Vai Leu Phe Arg Asn Gly 165 170 175 Giu Leu Val Ile Giu Gin Giu Giy Leu Tyr Tyr. Ile Tyr Ser Gin Thr 180 185 190 Tyr Phe Arg Phe Gin Giu Ala Giu Asp Ala Ser Lys Met Val Ser Lys 195 200 205 Asp Ser 225 Trp Leu Glu Leu (2) Lys Val Arg Thr Lys Gin Leu Val Gin 210 215 STyr Pro Asp Pro Ile Val Leu Met Lys 230 Ser Arg Asp Ala Glu Tyr Gly Leu Tyr 245 250 Phe Glu Leu Lys Lys Asn Asp Arg Ile 260 265 His Leu Met Asp Leu Asp Gin Glu Ala 275 280 Ile Asn 290 INFORMATION FOR SEQ ID NO:7: SEQUENCE CHARACTERISTICS: LENGTH: 8 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: FLAG peptide Tyr Ile 220 Ser Ala 235 Ser Ile Phe Val Ser Phe Tyr Lys Tyr Thr Arg Asn Ser Cys 240 Tyr Gln Gly Gly 255 Ser Val Thr Asn 270 Phe Gly Ala Phe 285 a *9* a a a a *aa.

*Waa a (xi) SEQUENCE DESCRIPTION: SEQ ID NO:7: Asp Tyr Lys Asp Asp Asp Asp Lys 1 INFORMATION FOR SEQ ID NO:8: SEQUENCE CHARACTERISTICS: LENGTH: 17 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: conserved peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO:8: Leu Val Val Xaa Xaa Xaa Gly Leu Tyr Tyr Val Tyr Xaa Gin Val Xaa 1 5 10 Phe INFORMATION FOR SEQ ID NO:9: SEQUENCE CHARACTERISTICS: LENGTH: 32 amino acids TYPE: amino acid STRANDEDNESS: not relevant TOPOLOGY: not relevant (ii) MOLECULE TYPE: peptide (iii) HYPOTHETICAL: NO (iv) ANTI-SENSE: NO (vii) IMMEDIATE SOURCE: CLONE: CMV leader (xi) SEQUENCE DESCRIPTION: SEQ ID NO:9: Met Ala Arg Arg Leu Trp Ile Leu Ser Leu Leu Ala Val Thr Leu Thr 1 5 10 Val Ala Leu Ala Ala Pro Ser Gln Lys Ser Lys Arg Arg Thr Ser Ser 20 25

Claims (27)

1. An E. coli cell transformed with an expression vector encoding a soluble TRAIL polypeptide, wherein said polypeptide comprises an amino acid sequence that is at least 80% identical to a sequence selected from: a) the extracellular domain of the human TRAIL protein of SEQ ID NO:2; b) the extracellular domain of the mouse TRAIL protein of SEQ ID NO:6; or c) a fragment of the extracellular domain of or wherein said soluble TRAIL polypeptide is capable of killing cancer cells. 2 A transformed cell according to claim 1, wherein said TRAIL polypeptide comprises an amino acid sequence selected from: 15 a) the extracellular domain of the human TRAIL protein of SEQ ID NO:2; b) the extracellular domain of the mouse TRAIL protein of SEQ ID NO:6; or c) a fragment of the extracellular domain of or wherein said TRAIL polypeptide is capable of killing cancer cells. i 3. An E. coli cell transformed with an expression vector that encodes a TRAIL polypeptide, wherein said polypeptide is a fragment of the extracellular domain of the protein of SEQ ID NO:2, wherein said polypeptide is capable of inducing apoptosis of a TRAIL-sensitive target cell.

4. A transformed cell according to any one of claims 1 to 3, wherein said expression vector additionally encodes a methionine residue fused to the N- terminus of said TRAIL polypeptide. An E. coli cell transformed with a DNA comprising a nucleotide sequence selected from: a) nucleotides 88 to 933 of the sequence presented as SEQ ID NO:1; MR C:\WINWORD\MARY\NODELETE\708239.DOC 53 b) nucleotides 47 to 922 of the sequence presented as SEQ ID c) a fragment of the nucleotide sequence of or d) a fragment of the nucleotide sequence of wherein said DNA encodes a polypeptide capable of inducing apoptosis of a TRAIL-sensitive target cell.

6. A transformed cell according to claim 5, wherein said DNA additionally encodes a methionine residue at the N-terminus of said polypeptide.

7. An isolated DNA encoding a TRAIL polypeptide capable of inducing apoptosis of a TRAIL-sensitive target cell, wherein said polypeptide comprises deletions in the amino acid sequence presented in SEQ ID NO:2 or SEQ ID NO:6; and wherein said polypeptide lacks a transmembrane region. 15 8. An isolated DNA encoding a TRAIL polypeptide comprising amino acids 124 to 276 of SEQ ID NO:2.

9. An isolated DNA encoding a soluble TRAIL polypeptide, wherein said polypeptide comprises a soluble fragment of the extracellular domain of the 20 protein of SEQ ID NO:2, and wherein the N-terminal amino acid of said fragment is selected from residues 39 to 124 of SEQ ID NO:2, and the C-terminal amino acid of said fragment is selected from residues 276 to 281 of SEQ ID NO:2. A DNA according to any one of claims 7 to 9, wherein said DNA encodes a TRAIL polypeptide that additionally comprises an N-terminal methionine residue.

11. An expression vector comprising a DNA according to any one of claims 7 to

12. A host cell transformed with an expression vector of claim 11.

13. A transformed host cell of claim 12, wherein said host cell is an E. coli cell. MR C:\WINWORD\MARY\NODELETE\708239.DOC 54

14. A process for preparing a TRAIL polypeptide, comprising culturing a transformed cell according to any one of claims 1 to 6, 12 or 13 under conditions that promote expression of TRAIL, and recovering the TRAIL polypeptide.

15. A process of claim 14, wherein said TRAIL polypeptide is in the form of an oligomer.

16. A TRAIL polypeptide produced by culturing a transformed cell according to any one of claims 1 to 6, 12 or 13, under conditions that promote expression of TRAIL.

17. A TRAIL polypeptide of claim 16, wherein said polypeptide is in the form of an oligomer. 15 18. A purified TRAIL polypeptide capable of inducing apoptosis of a TRAIL- sensitive target cell, wherein said polypeptide comprises deletions in the amino acid sequence presented in SEQ ID NO:2 or SEQ ID NO:6, and wherein said polypeptide lacks a transmembrane region.

19. A purified TRAIL polypeptide comprising amino acids 124 to 276 of SEQ ID NO:2. CC A purified soluble TRAIL polypeptide, wherein said polypeptide comprises a soluble fragment of the extracellular domain of the protein of SEQ ID NO:2, wherein the N-terminal amino acid of said fragment is selected from residues 39 to 124 of SEQ ID NO:2, and the C-terminal amino acid of said fragment is selected from residues 276 to 281 of SEQ ID NO:2.

21. An oligomer comprising two or more TRAIL polypeptides according to any one of claims 18 to

22. An oligomer of claim 21, wherein said oligomer contains three TRAIL polypeptides. MR C:\WINWORD\MARY\NODELETE\708239.DOC

23. A pharmaceutical composition comprising a TRAIL polypeptide according to any one of claims 16 to 20, and a physiologically acceptable carrier, diluent, or excipient.

24. A pharmaceutical composition comprising an oligomer according to claim 21 or 22, and a physiologically acceptable carrier, diluent, or excipient. Use of a polypeptide according to any one of claims 16 to 20, for regulating apoptosis in a mammal.

26. Use of an oligomer according to claim 21 or 22, for regulating apoptosis in a mammal. :27. Use of a composition according to claim 23 or 24, for regulating apoptosis 15 in a mammal.

28. A method of killing cancer cells, comprising contacting cancer cells with a polypeptide, according to any one of claims 16 to *e 20 29. A method of killing cancer cells, comprising contacting cancer cells with an oligomer according to claim 21 or 22. t A method of killing cancer cells, comprising contacting cancer cells with a composition according to claim 23 or 24.

31. A method of treating cancer in a human, comprising administering to said human an effective amount of a polypeptide, according to any one of claims 16 to

32. A method of treating cancer in a human, comprising administering to said human an effective amount of an oligomer according to claim 21 or 22. MR C:\WINWORD\MARY\NODELETE\708239.DOC

33. A method of treating cancer in a human, comprising administering to said human an effective amount of a composition according to claim 23 or 24.

34. Use of a polypeptide, according to any one of claims 16 to 20, for the manufacture of a medicament for treatment of cancer. Use of an oligomer according to claim 21 or 22, for the manufacture of a medicament for treatment of cancer.

36. Use of a composition according to claim 23 or 24, for the manufacture of a medicament for treatment of cancer.

37. A transformed cell according to claim 1, substantially as hereinbefore described.

38. An isolated DNA according to claim 7, substantially as hereinbefore described. :0 0 00.

94.4 0e 0 0000 0 00 OS 0 OSS 0 @0*5 S 0 .SS 00 DATED: 19 October 1999 PHILLIPS ORMONDE FITZPATRICK Attorneys for: IMMUNEX CORPORATION MR CAkWINWORDWMARY"NODELETE\708239.DOC 57