JP5334773B2 - Receptor binding to TRAIL - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide antibodies that bind TNF-Related Apoptosis-Inducing Ligand (TRAIL) or antigen-binding fragments thereof. <P>SOLUTION: The antibodies or antigen-binding fragments thereof directed to a TRAIL receptor (TRAIL-R) polypeptides are polypeptides in which the TRAIL receptor polypeptides include the amino acid sequence VPANEGD and are capable of binding TRAIL. Also provided are expression vectors containing DNA sequences encoding TRAIL-R polypeptides, as well as host cells genetically transformed with such recombinant expression vectors. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  A protein known as TNF-related apoptosis-inducing ligand (TRAIL) is a member of the tumor necrosis factor family of ligands (Wiley et al., Immunity, 3: 673-682, 1995). TRAIL has shown the ability to induce apoptosis in certain transformed cells, including not only virus-infected cells but also many different types of cancer cells (PCT applications WO 97/01633 and Wiley et al., Ibid.).

  The identification of receptor proteins that bind to TRAIL would be beneficial for further study of TRAIL biological activity. However, no TRAIL receptor has been reported prior to the present invention.

WO97 / 01633

Wiley et al., Immunity, 3: 673-682, 1995.

  The present invention is directed to a novel protein called TRAIL receptor (TRAIL-R) that binds to a protein known as TNF-related apoptosis-inducing ligand (TRAIL). DNA encoding TRAIL-R and expression vectors containing such DNA are provided. A method of producing a TRAIL-R polypeptide comprises culturing a host cell transformed with a recombinant expression vector encoding TRAIL-R under conditions that promote expression of TRAIL-R, and then expressing the expressed TRAIL-R. Recovering the R polypeptide from the culture. Antibodies that immunoreact with TRAIL-R are also provided.

FIG. 1 shows the nucleotide sequence of the human TRAIL receptor DNA fragment and the amino acid sequence encoded thereby. This DNA fragment is described in Example 3. FIG. 2 shows the results of the assay described in Example 7. In this assay, soluble TRAIL-R / Fc fusion protein blocked TRAIL-induced apoptosis of Jurkat cells. FIG. 3 shows the results of the experiment described in Example 8. The compounds shown have been shown to inhibit apoptosis of cells expressing the TRAIL receptor.

  Provided herein is a novel protein called the TRAIL receptor (TRAIL-R). TRAIL-R binds to a cytokine called TNF-related apoptosis-inducing ligand (TRAIL). As discussed below, certain uses of TRAIL-R stem from this ability to bind TRAIL. For example, TRAIL-R finds use in inhibiting TRAIL biological activity or purifying TRAIL by affinity chromatography.

  The nucleotide sequence of the coding region of human TRAIL receptor DNA is displayed as SEQ ID NO: 1. The amino acid sequence encoded by the DNA sequence of SEQ ID NO: 1 is shown in SEQ ID NO: 2. This sequence information identifies the TRAIL receptor protein as a member of the tumor necrosis factor receptor (TNF-R) family of receptors (Smith et al., Cell 76: 959-962, 1994). The extracellular domain contains a cysteine-rich repeat, but this motif has been reported to be important for ligand binding at other receptors in this family. TRAIL-R contains so-called “death domains” in the cytoplasmic region, but such domains of certain other receptors are associated with the transmission of apoptotic signals. These and other features of this protein are discussed in more detail below.

  TRAIL-R proteins or immunogenic fragments thereof can be utilized as immunogens to produce antibodies that immunoreact with them. In one aspect of the invention, the antibody is a monoclonal antibody.

  Human TRAIL-R protein was purified as described in Example 1. In Example 2, amino acid sequence information derived from the TRAIL-R fragment is shown. One aspect of the present invention is directed to a purified human TRAIL-R protein capable of binding to TRAIL, wherein TRAIL-R is amino acid sequence VPANEGD (SEQ ID NO: 2 at positions 327-333). Amino acid). In another aspect, TRAIL-R additionally comprises the sequence ETLRQCFDDDFADLVVPFDSWEPLMRMKLGLMDNEIKVAKAEAAGHRDTTLXTML (amino acids at positions 336 to 386 of SEQ ID NO: 2, one unknown amino acid is denoted as X). Also provided are TRAIL-R fragments comprising only one of these characteristic amino acid sequences.

  The nucleotide sequence of the TRAIL-R DNA fragment and the amino acid sequence encoded thereby are shown in FIG. 1 (SEQ ID NO: 3 and SEQ ID NO: 4): see Example 3. The amino acid sequence shown in FIG. 1 has the so-called “death domain” characteristics found in the cytoplasmic region of certain other receptor proteins. Such domains have been reported to be involved in the transmission of apoptotic signals. The cytoplasmic death domain is Fas antigen (Itoh and Nagata, J. Biol. Chem. 268: 10932, 1993), TNF receptor type I (Tartaglia et al., Cell 74: 845, 1993), DR3 (Chinyanian et al., Science 274). : 990-992, 1996) and CAR-1 (Broyasch et al., Cell 87: 845-855, 1996). The role of these death domains in the initiation of the intracellular apoptosis signal cascade is discussed further below.

  SEQ ID NO: 1 shows the nucleotide sequence of the coding region of human TRAIL receptor DNA including the start codon (ATG) and stop codon (TAA). The amino acid sequence encoded by the DNA of SEQ ID NO: 1 is shown in SEQ ID NO: 2. The fragment displayed in FIG. 1 corresponds to the region of TRAIL-R shown as amino acids at positions 336 to 386 of SEQ ID NO: 2.

  The TRAIL-R protein of SEQ ID NO: 2 contains an N-terminal hydrophobic region that functions as a signal peptide, followed by an extracellular domain, a transmembrane region containing amino acids 211 to 231 and a C-terminal cytoplasmic domain . Computer analysis predicts that the signal peptide corresponds to residues 1 to 51 of SEQ ID NO: 2. Therefore, when the signal peptide is cleaved, a mature protein containing amino acids 52 to 440 of SEQ ID NO: 2 is produced. The calculated molecular weight of the mature protein comprising residues 52-440 of SEQ ID NO: 2 is about 43 kilodaltons. The next possible signal peptidase cleavage sites predicted by the computer are after amino acids 50 and 58 (in descending order) of SEQ ID NO: 2.

  In another aspect of the invention, the N-terminal residue of the mature TRAIL-R protein is an isoleucine residue at position 56 of SEQ ID NO: 2. The sequence of several trypsin digested peptide fragments of TRAIL-R was determined by a combination of N-terminal sequencing and nano ES MS / MS (nanoelectrospray tandem mass spectrometry). One N-terminal amino acid of this peptide fragment was isoleucine at position 56 of SEQ ID NO: 2. Since this fragment was not preceded by a trypsin cleavage site, this (Ile) 56 residue may correspond to the N-terminal residue resulting from cleavage of the signal peptide.

  A further aspect of the invention is directed to mature TRAIL-R having amino acid 54 as the N-terminal residue. In one preparation of TRAIL-R (soluble TRAIL-R / Fc fusion protein expressed in CV-1 / EBNA cells), the signal peptide was cleaved after the residue at position 53 of SEQ ID NO: 2. .

  One skilled in the art will recognize that the molecular weight of a specially prepared TRAIL-R protein may vary depending on factors such as the degree of glycosylation. The specially prepared TRAIL-R glycosylation pattern may differ, for example, depending on the type of cell in which the protein is expressed. Furthermore, certain preparations may contain many types of proteins that are variously glycosylated. TRAIL-R polypeptides with or without an associated native glycosylation pattern are provided herein. Expression of a TRAIL-R polypeptide in a bacterial expression system such as E. coli provides an unglycosylated molecule.

  In one aspect, the protein is characterized by a molecular weight in the range of about 50-55 kilodaltons, which is the molecular weight determined for the preparation of natural full length human TRAIL-R. . Molecular weight can be determined by SDS polyacrylamide gel electrophoresis (SDS-PAGE).

  Example 1 shows one method for purifying TRAIL-R protein. Jurkat cells are disrupted and subsequent purification methods include affinity chromatography (utilizing a chromatography matrix containing TRAIL) and reverse phase HPLC.

  The TRAIL-R polypeptides of the invention can also be purified by other suitable methods using known protein purification techniques. In one alternative approach, the chromatography matrix contains antibodies that bind to TRAIL-R instead. Other cell types that express TRAIL-R (eg, PS-1 cells described in Example 2) can be substituted for Jurkat cells. The cells can be disrupted by any of a number of existing techniques including freeze-thaw cycling, sonication, mechanical disruption or the use of cytolytic agents.

  The degree of purification desired will depend on the intended use of the protein. A relatively high purity is desired, for example, when the protein is administered in vivo. Advantageously, the TRAIL-R polypeptide is purified such that no protein bands corresponding to other (non-TRAIL-R) proteins are detected by SDS polyacrylamide electrophoresis (SDS-PAGE). One skilled in the art will recognize that multiple bands corresponding to TRAIL-R protein may be visualized by SDS-PAGE due to differences in glycosylation, post-translational processing, and the like. . TRAIL-R is most preferably purified to be substantially homogeneous, as indicated by a single protein band on analysis by SDS-PAGE. Protein bands may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography.

  The present invention encompasses various forms of TRAIL-R, including those that are naturally occurring or produced through various techniques such as methods involving recombinant DNA technology. Such forms of TRAIL-R include, but are not limited to, TRAIL-R fragments, derivatives, mutants and oligomers, as well as fusion proteins comprising TRAIL-R or fragments thereof.

  TRAIL-R can be modified to create derivatives thereof by forming covalent or aggregated conjugates with other chemical components such as glycosyl groups, lipids, phosphates, acetyl groups. A covalent derivative of TRAIL-R can be produced by attaching a chemical moiety to a functional group on the TRAIL-R amino acid side chain or at the N-terminus or C-terminus of the TRAIL-R polypeptide. Conjugates with diagnostic (detection) or therapeutic agents attached to TRAIL-R are contemplated herein as discussed in more detail below.

  Other TRAIL-R derivatives within the scope of the present invention are covalent or aggregated conjugates of TRAIL-R polypeptides with other proteins or polypeptides, such as synthesis in recombinant cultures such as N-terminal or C-terminal fusions. including. Examples of fusion proteins are discussed below in connection with TRAIL-R oligomers. Further, the TRAIL-R containing fusion protein may comprise a peptide added to facilitate the purification and identification of TRAIL-R. Such peptides include, for example, poly-His or the antigen identification peptides described in US Pat. No. 5,011,912 and Hopp et al., Bio / Technology 6: 1204, 1988. One such peptide is the Flag® peptide Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which is highly antigenic and reversibly bound to a specific monoclonal antibody. Provide epitopes and allow rapid assay and convenient purification of expressed recombinant protein. The mouse hybridoma designated 4E11 is conjugated to Flag® peptide in the presence of certain divalent metal cations as described in US Pat. No. 5,011,912, incorporated herein by reference. Produces monoclonal antibodies that bind. The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under the deposit number HB9259. Monoclonal antibodies that bind to the Flag® peptide are available from Eastman Kodak Company, Scientific Imaging Systems Division, New Haven, Connecticut.

  Both cell membrane bound and soluble (secreted) forms of TRAIL-R are provided herein. Soluble TRAIL-R is the separation of living cells expressing TRAIL-R polypeptide from the culture medium, for example by centrifugation, and assaying the culture medium (supernatant) for the presence of the desired protein. (Simultaneously distinguished from the insoluble membrane-bound form). The presence of TRAIL-R in the culture medium indicates that this protein is secreted from the cells and becomes a soluble form of the desired protein.

  Soluble forms of receptor proteins usually lack a transmembrane region that allows the protein to be retained on the cell surface. In one aspect of the invention, the soluble TRAIL-R polypeptide comprises the extracellular domain of this protein. A soluble TRAIL-R polypeptide may comprise a cytoplasmic domain or a portion thereof as long as it is secreted from the cell in which it is produced. One example of soluble TRAIL-R is soluble human TRAIL-R comprising amino acids 52-210 of SEQ ID NO: 2. Other soluble TRAIL-R polypeptides include, but are not limited to, polypeptides comprising amino acids X to 210 of SEQ ID NO: 2, wherein X is an integer from 51 to 59.

  The soluble form of TRAIL-R has several advantages over the membrane-bound form of this protein. Soluble protein is secreted from the cell, which facilitates purification of the protein from the recombinant host cell. Furthermore, soluble proteins are generally more suitable for certain applications, such as intravenous administration.

  TRAIL-R fragments are provided herein. Such fragments can be prepared by any of a number of conventional techniques. The desired peptide fragment may be chemically synthesized. One alternative is to produce a TRAIL-R fragment by enzymatic digestion, for example by treating the protein with an enzyme known to cleave the protein at a site defined by a particular amino acid residue. Accompany. Another suitable technique involves isolating a DNA fragment encoding the desired polypeptide fragment and amplifying it by polymerase chain reaction (PCR). Oligonucleotides that define the desired ends of the DNA fragments are used as 5 'and 3' primers in PCR.

  Examples of fragments are those comprising at least 20 or at least 30 consecutive amino acids in the sequence of SEQ ID NO: 2. Fragments from the cytoplasmic domain find use in the study of TRAIL-R mediated signaling and in regulating cellular processes associated with biological signaling. Fragments of TRAIL-R polypeptides can also be used as immunogens in antibody production. Certain embodiments are directed to polypeptide fragments of TRAIL-R that retain the ability to bind TRAIL. Such a fragment may be a soluble TRAIL-R polypeptide as described above.

  Provided herein are naturally occurring variants of the TRAIL-R protein of SEQ ID NO: 2. Such variants include, for example, proteins resulting from alternative mRNA splicing or from proteolytic cleavage of the TRAIL-R protein. Alternative mRNA splicing may result in a truncated biologically active TRAIL-R protein, such as the naturally occurring soluble form of the protein. Mutations resulting from proteolysis include expression in various types of host cells, for example by proteolytic removal of one or more terminal amino acids (usually 1 to 5 terminal amino acids) from the TRAIL-R protein. There is a difference in the N or C terminus at times. Also contemplated herein are TRAIL-R proteins whose differences in amino acid sequence are due to genetic polymorphisms (allelic variation between individuals producing the protein).

  Those skilled in the art will also recognize that the cleavage site of the signal peptide may differ from that predicted by the computer program and that the type of host cell utilized to express the recombinant TRAIL-R polypeptide. It will be appreciated that it may vary depending on various factors. Certain protein preparations may include a mixture of protein molecules having various N-terminal amino acids, as the signal peptide is cleaved at one or more sites. As already discussed, certain aspects of the mature TRAIL-R protein provided herein have a residue at position 51, 52, 54, 56 or 59 of SEQ ID NO: 2 as the N-terminal amino acid. Although it is protein, it is not limited to it.

  With respect to the discussion regarding the various domains of TRAIL-R, one skilled in the art will appreciate that the above boundaries of such regions of the protein are approximate. Illustratively, the transmembrane region boundaries may differ from those described above (although they can be predicted using a computer program provided for that purpose). Thus, soluble TRAIL-R polypeptides in which the C-terminus of the extracellular domain is different from the residues identified above are contemplated herein.

  Other naturally occurring TRAIL-R DNAs and polypeptides include those derived from non-human species. For example, homologues from other mammalian species of human TRAIL-R, SEQ ID NO: 2, are contemplated herein. Probes based on the human DNA sequence of SEQ ID NO: 3 or SEQ ID NO: 1 can be utilized to screen cDNA libraries from other mammalian species using conventional cross-species hybridization techniques.

  The TRAIL-R DNA sequence may be different from the native sequence disclosed herein. Due to the degeneracy of the known genetic code in which one or more codons encode the same amino acid, the DNA sequence has the amino acid sequence of SEQ ID NO: 2 even if it differs from that shown in SEQ ID NO: 1 It is possible to encode a TRAIL-R protein. Such mutated DNA sequences may arise from silent mutations (eg, occurring during PCR amplification) or may be the product of planned mutagenesis against the native sequence. Thus, among the DNA sequences provided herein, the results of the genetic code for the native TRAIL-R sequence (eg, a cDNA comprising the nucleotide sequence shown in SEQ ID NO: 1) and the native TRAIL-R DNA sequence There is a degenerate DNA.

  Among the TRAIL-R polypeptides provided herein are variants of natural TRAIL polypeptides that retain the biological activity of native TRAIL-R. Such a variant is a polypeptide that is substantially homologous to native TRAIL-R but has an amino acid sequence that differs from native TRAIL-R due to one or more deletions, insertions or substitutions. including. Particular embodiments include, but are not limited to, TRAIL-R polypeptides in which 1 to 10 amino acid residues have been deleted, inserted or substituted relative to the native TRAIL-R sequence. TRAIL-R encoding DNA of the present invention differs from native TRAIL-R DNA due to one or more deletions, insertions or substitutions, but mutants that encode biologically active TRAIL-R polypeptides including. One of the biological activities of TRAIL-R is the ability to bind to TRAIL.

  A nucleic acid encoding a biologically active TRAIL-R that can hybridize to DNA of SEQ ID NO: 1 or SEQ ID NO: 3 under moderately stringent or highly stringent conditions is provided herein. Provided. Such hybridizing nucleic acids include, but are not limited to, mutated DNA sequences and DNA from non-human species such as non-human mammals.

  Moderate stringent conditions include, for example, those described in Sambrook et al., “Molecular Cloning: Experimental Manual” 2nd Edition, Volume 1, pages 1.101-104, Cold Spring Harbor Laboratory Press, 1989. Moderate stringency conditions as defined by Sambrook et al. Are about 5 ° SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) pre-wash solution and about 55 ° C., 5XSSC, overnight. Includes hybridization conditions. High stringency conditions include hybridization and washing operations at higher temperatures. One aspect of the present invention is directed to DNA sequences that hybridize to DNA with SEQ ID NO: 1 or 3 under highly stringent conditions, wherein the conditions are 63 to 63 after hybridization at 68 ° C. Washing with 0.1XSSC / 0.1% SDS at 68 ° C.

  Certain DNAs and polypeptides provided herein each contain a nucleotide or amino acid that is at least 80% identical to the native TRAIL-R sequence. Also contemplated are embodiments in which the TRAIL-R DNA or polypeptide comprises a sequence that is at least 90% identical, at least 95% identical, or at least 98% identical to a native TRAIL-R sequence. This identity ratio is described, for example, by Devereux et al. (Nucl. Acids Res. 12: 387, 1984) using the GAP computer program, version 6.0, available from the University of Wisconsin Genetics Computer Group (UWGCG). And can be determined by comparing the sequence information. Preferred default parameters for the GAP program are (1) a single comparison matrix for nucleotides (including 1 for the identification symbol and 0 for the non-identification symbol), and Schwartz and Dayhoff, “Protein Sequence and Structure Atlas”, Gribskov and Burgess, Nucl., As described in National Biomedical Research Foundation, pages 353-358, 1979. Acids Res. 14: 6745, 1986, (2) a penalty of 3.0 for each gap and an additional 0.10 penalty for each symbol of each gap, and (3) for the end gap Including no penalty.

  In certain aspects of the invention, the mutant TRAIL-R polypeptide differs in amino acid sequence from native TRAIL-R, but is substantially equivalent to native TRAIL-R in biological activity. One example is a mutant TRAIL-R that binds to TRAIL with substantially the same binding affinity as native TRAIL-R. Binding affinity can be measured by conventional methods such as those described, for example, in US Pat. No. 5,512,457.

  The mutated amino acid sequence has one or more amino acid residues of native TRAIL-R replaced with another, but this conservatively substituted TRAIL-R polypeptide has a native protein. Conservative substitutions that retain the desired biological activity (eg, the ability to bind TRAIL) may be included. Certain amino acids can be substituted with residues having similar physicochemical properties. Examples of conservative substitutions are the substitution of one lipophilic residue for another with another liposoluble residue such as Ile, Val, Leu or Ala, or one polar residue with another polarity. Substituent substitution with each other, for example between Lys and Arg, Glu and Asp, or Gln and Asn. It is also well known to involve other conservative substitutions, such as substitution of entire regions with similar hydrophobic properties.

  In another variant example, a sequence encoding a Cys residue that is not essential for biological activity is altered such that the Cys residue is deleted or replaced with another amino acid, resulting in an incorrect intramolecular Disulfide bond formation is prevented. One receptor of the TNF-R family contains a cysteine-rich repeat motif in its extracellular domain (Marsters et al., J. Biol. Chem. 267: 5747-5750, 1992). These repeating moieties are believed to be important for ligand binding. Illustratively, Marsters et al., Supra, shows that the binding affinity for TNFα and TNFβ of a soluble TNF-R1 type polypeptide lacking one of these repeats is reduced by a factor of 10 and that further repeats are deleted. Showed that the detectable binding of this ligand was completely lost. The human TRAIL-R of SEQ ID NO: 2 contains two such cysteine-rich repeats, the first containing residues 94-137 and the second containing residues 138-178 . Cysteine residues within the cysteine-rich domain remain beneficially unchanged in TRAIL-R variants when retention of TRAIL binding activity is desired.

  Other variants can be produced by modification of adjacent dibasic amino acid residues to enhance expression in yeast systems where KEX2 protease activity is present. EP 212,914 discloses the use of site-directed mutagenesis to inactivate the KEX2 protease processing site of a protein. The KEX2 protease processing site is inactivated by deletion, addition or substitution of residues that alter the Arg-Arg, Arg-Lys and Lys-Arg pairs to eliminate the appearance of these adjacent basic residues. Mature human TRAIL-R contains such adjacent basic residue pairs at amino acids 72-73, 154-155, 322-323, 323-324 and 359-360 of SEQ ID NO: 2. Since Lys-Lys pairing is almost insensitive to cleavage by KEX2, converting Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivate the KEX2 site. ing.

  TRAIL-R polypeptides can be tested for biological activity in any suitable assay, including variants and fragments thereof. The ability of a TRAIL-R polypeptide to bind to TRAIL is confirmed by conventional binding assays, examples of which are described below.

Expression System The present invention also provides a recombinant cloning and expression vector comprising TRAIL-R DNA and a host cell comprising the recombinant vector. Expression vectors containing TRAIL-R DNA can be used to produce the TRAIL-R polypeptide encoded by the DNA. TRAIL-R polypeptide is produced by culturing host cells transformed with a recombinant expression vector encoding TRAIL-R under conditions that promote TRAIL-R expression, and then expressing the expressed TRAIL-R polypeptide. Recovering from the culture. Those skilled in the art will know how to purify the expressed TRAIL-R depending on the type of host used and whether it is a membrane-bound or a soluble form secreted from the host cell. Would admit.

  Any suitable expression system can be utilized. Vectors include DNA encoding a TRAIL-R polypeptide operably linked to appropriate transcriptional or translational regulatory nucleotide sequences such as those derived from mammalian, microbial, viral or insect genes. Examples of regulatory sequences include transcriptional promoters, operators or enhancers, mRNA ribosome binding sites, and appropriate sequences that control the initiation and termination of transcription and translation. A nucleotide sequence is operably linked when the regulatory sequence is functionally related to the TRAIL-R DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a TRAIL-R DNA sequence if the promoter nucleotide sequence controls transcription of the TRAIL-R DNA sequence. An origin of replication that confers replication capability in the desired host cell and a selection gene that identifies the transformant are generally introduced into the expression vector.

  In addition, a coding sequence for a suitable signal peptide (natural or heterologous) can be introduced into the expression vector. The DNA sequence of the signal peptide (secretory leader) can be fused in frame with the TRAIL-R sequence so that TRAIL-R is first translated as a fusion protein containing the signal peptide. A signal peptide that is functional in the desired host cell promotes extracellular secretion of the TRAIL-R polypeptide. This signal peptide is cleaved from the TRAIL-R polypeptide when TRAIL-R is secreted from the cell.

  Suitable host cells for expression of a TRAIL-R polypeptide include prokaryotic, yeast, or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Suitable cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cell hosts are described, for example, in Pouwels et al., “Cloning Vectors: Experimental Manual”, Elsieber, New York (1985). Cell-free translation systems can also be utilized to produce TRAIL-R polypeptides using RNA derived from the DNA constructs disclosed herein.

  Prokaryotes include gram negative or gram positive bacteria, such as E. coli and Bacillus. Prokaryotic host cells suitable for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species included in the genera Pseudomonas, Streptomyces, Staphylococcus. In prokaryotic host cells such as E. coli, the TRAIL-R polypeptide can include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. This N-terminal Met can be cleaved from the expressed recombinant TRAIL-R polypeptide.

  Expression vectors used in prokaryotic host cells usually contain a marker gene that can be selected by one or more phenotypes. A phenotypic selectable marker gene is, for example, a coding gene for a protein that confers antibacterial resistance or confers autotrophic requirements. Examples of expression vectors useful for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). Since pBR322 contains ampicillin and tetracycline resistance genes, it provides a convenient way to identify transformed cells. Appropriate promoter and TRAIL-R DNA sequences are inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEM1 (Promega Biotech, Madison, WI, USA).

Promoter sequences commonly used as expression vectors for recombinant prokaryotic host cells include β-lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275: 615, 1978 and Goeddel et al., Nature 281: 544, 1979. ), Tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4057, 1980; and EP-A-36776) and the tac promoter (Maniatis, “Molecular Cloning: Experimental Manual” Cold Spring Harbor Laboratory, page 412 1982). Particularly useful prokaryotic host expression systems utilizes λ phage _{P L} promoter and cI857ts thermolabile repressor sequence. Plasmid vectors that incorporate derivatives of the λP _L promoter available from the American Type Culture Collection include plasmids pHUB2 (present in E. coli strain JMB9, ATCC 37092) and pPLc28 (present in E. coli PR1, ATCC 53082).

TRAIL-R may alternatively be expressed in yeast host cells, preferably in the genus Saccharomyces (eg S. cerevisiae). Other genera of yeast such as Pichia or Criveromyces can also be utilized. Yeast vectors often contain an origin of replication sequence derived from a 2μ yeast plasmid, an automatic replication sequence (ARS), a promoter region, a polyadenylation sequence, a transcription termination sequence and a selectable marker gene. Suitable promoter sequences for yeast vectors include metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073, 1980), or enolase, glyceraldehyde-3-phosphate dehydrogenase, Other glycolytic systems such as hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triose phosphate isomerase, phospho-glucose isomerase and glucokinase Promoters for the enzymes (Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968; and Holland et al., Biochem. 17: 4900, 1978) are included. Other suitable vectors and promoters used for expression in yeast are further described in Hitzeman, EPA-73,657. Another alternative is the glucose repressible ADH2 promoter described by Russel et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al. (Nature 300: 724, 1982). Shuttle vector to be replicated either yeast and E. coli, the DNA sequences from pBR322 for selection and replication in E. coli (Amp ^r gene and origin of replication) may be constructed by inserting the yeast vector.

  The yeast alpha factor leader sequence can be utilized to direct secretion of the TRAIL polypeptide. This alpha factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, for example, Kurjan et al., Cell 30: 933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81: 5330, 1984. Other leader sequences suitable for promoting secretion of recombinant polypeptides from yeast hosts are also known to those skilled in the art. The leader sequence can be modified near its 3 'end to include one or more restriction sites. This facilitates fusion of the leader sequence to the structural gene.

Yeast transformation protocols are known to those skilled in the art. Such a protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75: 1929, 1978. The Hinnen et al protocol selects Trp ⁺ transformants in a selective medium, where the selective medium is 0.67% yeast nitrogen base, 0.5% casamino acid, 2% glucose, 10 μg / mL. Contains adenine and 20 μg / mL uracil.

  Yeast host cells transformed with a vector containing the ADH2 promoter sequence can be grown for expression induction in "rich" media. An example of a rich medium is 1% yeast extract, 2% peptone and 1% glucose supplemented with 80 μg / mL adenine and 80 μg / mL uracil. When glucose is consumed from the medium, derepression of the ADH2 promoter occurs.

  Mammalian or insect host culture systems can also be utilized to express recombinant TRAIL-R polypeptides. Baculovirus strains for the production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988). Established cell lines of mammalian origin can also be utilized. Examples of suitable mammalian host cell lines include COS-7 strain 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 line, and African green monkey kidney cell line described by McMahan et al. (EMBO J. 10: 2821, 1991), CV1 (ATCC CCL 70) CV1 / EBNA cell line derived from

  Transcriptional and translational control sequences for mammalian host cell expression vectors can be extracted from the viral genome. Commonly used promoter and enhancer sequences are derived from polyoma virus, adenovirus 2, simian virus 40 (SV40) and human cytomegalovirus. For example, DNA sequences from the SV40 viral genome, SV40 origins of replication, early and late promoters, enhancers, splices and polyadenylation sites are used to provide other genetic elements for expression of structural gene sequences in mammalian host cells. Can be done. The early and late promoters of the virus are particularly useful because both are readily obtained from the viral genome as fragments that can also contain the viral replication initiation site (Fiers et al., Nature 273: 113, 1978). Shorter or longer SV40 fragments can be used as long as they contain an approximately 250 bp sequence extending from the HindIII site located at the SV40 origin of replication to the BglI site.

  Expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3: 280, 1983). A useful system for expressing mammalian cDNAs at stable high levels in C127 mouse mammary epithelial cells can be constructed substantially by the method described by Cosman et al. (Mol. Immunol. 23: 935, 1986). The high expression vector PMLSV N1 / N4 described in Cosman et al., Nature 312: 768, 1984 has been deposited as ATCC 39890. Further mammalian expression vectors are described in EP-A-0367566 and WO 91/18982. As one alternative, the vector may be derived from a retrovirus. Overexpression of full-length TRAIL-R resulted in membrane blebbing and nuclear enrichment in transformed CV-1 / EBNA cells, indicating that cell death mechanisms are apoptotic. It shows that there is. For host cells in which such TRAIL-R-mediated apoptosis occurs, an appropriate apoptosis inhibitor may be included in the expression system.

  In order to inhibit TRAIL-R induced apoptosis of a host cell expressing recombinant TRAIL-R, the cell may be co-transformed with an expression vector encoding a polypeptide that functions as an apoptosis inhibitor. Expression vectors encoding such polypeptides can be produced by conventional methods. Another approach involves adding an apoptosis inhibitor to the culture medium. Examples 6 and 8 show the reduction of host cell death using poxvirus CrmA, baculovirus P35, C-terminal fragment of FADD and the tripeptide derivative zVAD-fmk.

  zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) is a tripeptide-based compound provided by Enzyme Systems Products, Dublin, California. As shown in Example 8, zVAD-fmk may be added to the medium in which cells expressing TRAIL-R are cultured.

  A 38 kilodalton cowpox-derived protein, later referred to as CrmA, is described in Pickup et al. (Proc. Natl. Acad. Sci. USA 83: 7698-7702, 1986; incorporated herein). The sequence information of CrmA is shown in FIG. One approach to producing and purifying the CrmA protein is described in Ray et al. (Cell, 69: 597-604, 1992; incorporated herein).

  A 35 kilodalton protein encoded by the baculovirus nuclear polyhedrosis virus Autographa californica is described in Friesen and Miller (J. Virol. 61: 2264-2272, 1987; incorporated herein). The sequence information of this protein, denoted here as baculovirus p35, is shown in Friesen and Miller, FIG. 5 above.

  A death domain-containing cytoplasmic protein, FADD (also known as MORT1), is described in Boldin et al. (J. Biol. Chem. 270: 7795-7798, 1995; incorporated herein). FADD has been reported to bind directly or indirectly to the cytoplasmic death domain of certain receptors that mediate apoptosis (Boldin et al., Cell 85: 803-815, June 1996; Hsu et al., Cell 84: 299-308, January 1996 issue).

  In one aspect of the invention, a truncated FADD polypeptide that contains a death domain (located in the C-terminal portion of the protein) but lacks an N-terminal region attributed to apoptotic effector function reduces apoptosis. Is used for. Inhibiting the death of cells expressing other apoptosis-inducing receptors using a polypeptide of a particular FADD deletion mutant, truncated at the N-terminus, is described in Hsu et al. (Cell 84: 299 308, January 1996; incorporated herein by reference).

  This approach is demonstrated in Example 8, which corresponds to amino acids 117-245 in the amino acid sequence of MORT1 shown in Boldin et al. (J. Biol. Chem. 270: 7795-7798, 1995). One suitable FADD dominant negative (FADD-DN) polypeptide having an amino acid sequence is used. In Example 8, cells were co-transformed with an expression vector encoding TRAIL-R and an expression vector encoding the Flag® peptide fused to the N-terminus of the FADD-DN polypeptide.

  While not wishing to be bound by theory, one possible interpretation is that the C-terminal fragment of FADD binds to the intracellular death domain of the receptor but lacks the N-terminal portion of the protein necessary to cause apoptosis. (Hsu et al., Cell 84: 299-308, January 1996 issue; Boldin et al., Cell 85: 803-815, June 1996 issue). Truncated FADD may block endogenous full-length FADD from binding to the receptor's death domain; thus inhibiting apoptosis that would be initiated by such endogenous FADD. It is.

  Other apoptosis inhibitors useful in the expression system of the present invention can be identified by conventional assays. Such an assay for testing compounds for their ability to reduce apoptosis of TRAIL-R expressing cells is described in Example 8.

  Poxvirus CrmA, baculovirus P35 and zVAD-fmk are viral caspase inhibitors. Other caspase inhibitors can be tested for their ability to reduce TRAIL-R mediated cell death.

  The use of CrmA, baculovirus p35 and certain peptide derivatives (including zVAD-fmk) as apoptosis inhibitors in certain cells / systems is described by Sarin et al. (J. Exp. Med. 184: 2445-2450, 1996). December issue; incorporated herein). The role of interleukin-1β converting enzyme (ICE) family proteases in signal transduction cascades leading to programmed cell death, and the use of inhibitors of such proteases in the prevention of apoptosis is Sarin et al., Ibid. And Muzio et al. ( Cell 85: 817-827, 1996).

  In general, it is not necessary to utilize an apoptosis inhibitor for the expression of a TRAIL-R polypeptide lacking a cytoplasmic domain (ie lacking a protein region involved in signal transduction). Thus, an expression system that produces a soluble TRAIL-R polypeptide that includes only the extracellular domain (or fragment thereof) need not include one of the apoptosis inhibitors.

  For signal peptides that can be utilized to produce TRAIL-R, the native signal peptide of TRAIL-R may be replaced with a heterologous signal peptide or leader sequence, if desired. The choice of signal peptide or leader can depend on factors such as the type of host cell in which recombinant TRAIL-R is produced. Illustratively, an example of a heterologous signal peptide that is functional in mammalian host cells is the signal sequence of interleukin-7 (IL-7) described in US Pat. No. 4,965,195, Cosman et al., Nature 312. : Signal sequence of interleukin-2 receptor described in 768 (1984); signal peptide of interleukin-4 receptor described in EP367,566; I described in US Pat. No. 4,968,607 Type interleukin-1 receptor signal peptide; and type II interleukin-1 receptor signal peptide described in EP460,846.

TRAIL-R oligomeric forms The invention also encompasses oligomers comprising TRAIL-R polypeptide (s). TRAIL-R oligomers can exist in the form of covalently or non-covalently linked dimers, trimers or higher order oligomers.

  One aspect of the invention is directed to an oligomer comprising a number of TRAIL-R polypeptides linked via covalent or non-covalent interactions between peptide components fused to the TRAIL-R polypeptide. Such a peptide can be a peptide linker (spacer) or a peptide having the property of promoting oligomerization. Certain polypeptides derived from leucine zippers and antibodies are among the peptides that can attach to TRAIL-R polypeptides and promote their oligomerization, as described in more detail below.

In a particular embodiment, the oligomer consists of 2 to 4 TRAIL-R polypeptides. The oligomeric TRAIL-R component may be a soluble polypeptide, as described above.
As one alternative, TRAIL-R oligomers are prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including Fc domains) is described, for example, by Ashkenazi et al. (PNAS USA 88: 10535, 1991); Byrn et al. (Nature 344). : 677, 1990) and Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Protein", Latest Protocol of Immunology, Supplement 4, 10, 10.9.11-10.111, 1992). .

  One aspect of the present invention is directed to a TRAIL-R dimer comprising two fusion proteins made by fusing TRAIL-R to an antibody Fc region. A fusion gene encoding a TRAIL-R / Fc fusion protein is inserted into an appropriate expression vector. TRAIL-R / Fc fusion proteins are expressed in host cells transformed with a recombinant expression vector and associate in much the same way as antibody molecules, but interchain disulfide bonds are formed between the Fc components, Bivalent TRAIL-R is produced.

  Provided herein are fusion proteins comprising a TRAIL-R polypeptide fused to an antibody-derived Fc polypeptide. Not only are dimers comprising two fusion proteins linked via a disulfide bond between Fc components, but also DNA encoding such fusion proteins. The term “Fc polypeptide” as used herein includes natural and variant forms of polypeptides derived from antibody Fc regions. Also included are truncated forms of such polypeptides that include a hinge portion that promotes dimerization.

  One suitable Fc polypeptide described in PCT application WO 93/10151 (incorporated herein) is a short polypeptide that extends from the N-terminal hinge of the Fc region of a human IgG1 antibody to the native C-terminus. . Another useful Fc polypeptide is the Fc variant described in US Pat. No. 5,457,035 and Baum et al. (EMBO J. 13: 3992-4001, 1994). The amino acid sequence of this mutant is that of the natural Fc sequence shown in WO93 / 10151, the amino acid at position 19 is changed from Leu to Ala, the amino acid at position 20 is changed from Leu to Glu, And the amino acid at position 22 is identical except that the amino acid at position 22 is changed from Gly to Ala. This variant shows a reduced affinity for the Fc receptor.

  In another embodiment, TRAIL-R may be substituted with the variable region of an antibody heavy or light chain. If the fusion protein is made with both the heavy and light chains of an antibody, it is possible to use as many as four TRAIL-R extracellular regions to form a TRAIL-R oligomer.

  An oligomer is also a fusion protein comprising various TRAIL-R polypeptides with or without a peptide linker (spacer peptide). Among the suitable peptide linkers are those described in US Pat. Nos. 4,751,180 and 4,935,233, incorporated herein by reference. The DNA sequence encoding the desired peptide linker can be inserted in the same reading frame into the DNA sequence encoding TRAIL-R using any suitable conventional technique. For example, a chemically synthesized oligonucleotide encoding a linker can be linked within the TRAIL-R coding sequence. In certain aspects, the fusion protein comprises 2-4 soluble TRAIL-R polypeptides separated by a peptide linker.

  Another method for producing oligomeric TRAIL-R involves the use of leucine zippers. A leucine zipper domain is a peptide that promotes oligomerization of the protein in which it is present. Originally leucine zippers were identified among several DNA binding proteins (Landschulz et al., Science 240: 1759, 1988) and have since been found in a wide variety of proteins. Known leucine zippers include naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and leucine zippers from lung surfactant protein D (SPD) are described in Hoppe et al. (FEBS Letters 344: 191, 1994). ) (Incorporated herein). The use of a modified leucine zipper to allow stable trimerization of heterologous proteins fused thereto has been described by Fanslow et al. (Semin. Immunol. 6: 267-278, 1994). A recombinant fusion protein comprising a soluble TRAIL-R polypeptide fused to a leucine zipper peptide is expressed in a suitable host cell and the soluble oligomeric TRAIL-R formed is recovered from the culture supernatant.

  The oligomeric TRAIL-R has a characteristic of, for example, a bivalent or trivalent binding site for TRAIL. The fusion protein (and the oligomer formed therefrom) containing the Fc component provides the advantage that it can be easily purified by affinity chromatography on a protein A or protein G column. Provided herein are DNAs that encode oligomeric TRAIL-R or that encode fusion proteins useful for the production of TRAIL-R oligomers.

Assays TRAIL-R proteins (including fragments, variants, oligomers and other forms of TRAIL-R) can be tested for their ability to bind TRAIL in any suitable assay, such as conventional binding assays. Illustratively, TRAIL-R can be labeled with a detectable reagent (eg, a radionuclide, a chromophore, a colorimetric or fluorescent catalytic enzyme, etc.). Labeled TRAIL-R is contacted with cells expressing TRAIL. The cells are then washed to remove unbound labeled TRAIL-R and the presence of the label bound to the cells is determined by an appropriate technique selected depending on the nature of the label.

One example of a binding assay is as follows. A recombinant expression vector containing the TRAIL-R cDNA is constructed, for example, as described in PCT application WO 97/01633 (incorporated herein). DNA and amino acid sequence information of human and mouse TRAIL is shown in WO97 / 01633. TRAIL contains an N-terminal cytoplasmic domain, a transmembrane region and a C-terminal extracellular domain. CV1-EBNA-1 cells in 10 cm ² dishes are transformed with the recombinant expression vector. CV-1 / EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1 driven from the CMV early-early enhancer / promoter. CV1-EBNA-1 was derived from the African green monkey kidney cell line, CV-1 (ATCC CCL 70), as described by McMahan et al. (EMBO J. 10: 2821, 1991).

Transformed cells are cultured for 24 hours and the cells in each dish are divided into 24-well plates. After further incubation for 48 hours, the transformed cells (approximately ⁴ × 10 ⁴ cells / well) are washed with BM-NFDM. BM-NFDM is a binding medium supplemented with 50 mg / mL non-fat dry milk (RPMI 1640 containing 25 mg / mL bovine serum albumin, 2 mg / mL sodium azide, 20 mM Hepes pH 7.2) It is. The cells are then incubated with various concentrations of soluble TRAIL / Fc fusion protein for 1 hour at 37 ° C. Cells are then washed and incubated with a constant saturating concentration of ¹²⁵ I-mouse anti-human IgG in binding medium for 1 hour at 37 ° C. with gentle agitation. After extensive washing, the cells are released by trypsinization.

The mouse anti-human IgG utilized above is directed against the Fc region of human IgG and is available from Jackson ImmunoResearch Laboratories, West Grove, PA. This antibody is radioiodinated using the standard chloramine-T method. The antibody binds to the Fc portion of any TRAIL-R / Fc protein bound to the cell. In all assays, ¹²⁵ I-antibody non-specific binding was assayed in the absence of TRAIL-R / Fc and in the presence of TRAIL-R / Fc and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody. The

¹²⁵ I-antibody bound to the cells is quantified with a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. NY Acad. Sci. 51: 660, 1949) are made on the basis of RS / 1 (BBN software, Boston, Mass.) Processed on a microvax computer.

  Another type of suitable binding assay is a competitive binding assay. Illustratively, the biological activity of a TRAIL-R variant can be determined by assaying the variant's ability to compete with native TRAIL-R for binding to TRAIL.

  Competitive binding assays can be performed by conventional methods. Reagents that can be utilized in competitive binding assays include radiolabeled TRAIL-R and intact cells expressing TRAIL (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble TRAIL-R fragment can be used to compete with a soluble TRAIL-R variant for binding to cell surface TRAIL. Instead of intact cells, soluble TRAIL / Fc fusion protein bound to the solid phase can be replaced via interaction (on the solid phase) with the Fc component of protein A or protein G. Chromatographic columns containing protein A and protein G include those available from Pharmacia Biotech, Piscataway, NJ. Another type of competitive binding assay utilizes intact cells expressing radiolabeled soluble TRAIL and TRAIL-R, such as soluble TRAIL / Fc fusion proteins. Qualitative results are obtained by competitive autoradiography plate binding assays, while Scatchard plots (Scatchard, Ann. NY Acad. Sci. 51: 660, 1949) show quantitative results. Can be used to produce.

  Another type of assay for biological activity involves testing TRAIL-R polypeptides for their ability to block TRAIL-mediated apoptosis of target cells, such as the human leukemia T cell line known as Jurkat cells. . TRAIL-mediated apoptosis of a cell line designated Jurkat clone E6-1 (ATCC TIB 152) is shown in the assay method described in PCT application WO 97/01633 (incorporated herein).

Uses of TRAIL-R Uses of TRAIL-R include, but are not limited to: Some of these TRAIL-R uses stem from their ability to bind to TRAIL.

  TRAIL-R finds use as a protein purification reagent. TRAIL-R polypeptides can be used to attach to a solid support material and purify TRAIL by affinity chromatography. In certain aspects, the TRAIL-R polypeptide (any form capable of binding to TRAIL described herein) is attached to a solid support by conventional methods. As one example, a chromatographic column containing functional groups that react with functional groups on the amino acid side chains of proteins is obtained (Pharmacia Biotech, Piscataway, NJ). Alternatively, the TRAIL / Fc protein is attached to a protein A or protein G containing chromatography column via interaction with the Fc component.

  TRAIL-R protein also finds use in measuring the biological activity of TRAIL protein with respect to binding affinity to TRAIL-R. Thus, TRAIL-R protein can be utilized, for example, by people who perform “quality assurance” tests that monitor the shelf life and stability of TRAIL protein under various conditions. Illustratively, TRAIL-R can be stored at various temperatures or utilized in binding affinity studies that measure the biological activity of TRAIL protein produced in various cell types. TRAIL-R can also be utilized to determine whether it retains biological activity after modification (eg, chemical modification, truncation, mutation, etc.) of TRAIL protein. The binding affinity of the modified TRAIL protein to TRAIL-R is compared to that of the unmodified TRAIL protein to detect the adverse effects of that modification on the biological activity of TRAIL. Then, the biological activity of a TRAIL protein can be confirmed, for example, before using it in research studies.

  TRAIL-R also finds use in purifying or identifying cells that express TRAIL on the cell surface. TRAIL-R polypeptides bind to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, magnetic microspheres can be coated with TRAIL-R and kept in a heat-insulated container with a magnetic field. A suspension of the cell mixture containing TRAIL-expressing cells is contacted with a solid phase to which TRAIL-R is attached. Cells expressing TRAIL on the cell surface bind to the immobilized TRAIL-R, and cells that do not bind are washed away.

  TRAIL-R can also bind to a detectable moiety and incubate with the cell being tested for expression of TRAIL. After incubation, unbound labeled TRAIL-R is removed, confirming the presence or absence of detectable components on the cell surface.

In yet another, a cell mixture suspected of containing TRAIL ^- cells is incubated with biotinylated TRAIL-R. Incubation time is usually at least one hour to ensure sufficient binding. The resulting mixture is passed through a column packed with avidin-coated beads, thus the high affinity of biotin for avidin allows the desired cells to bind to the beads. Methods using avidin-coated beads are known (see Berenson et al., J. Cell. Biochem. 10D: 239, 1986). Washing to remove unbound material and release of the bound cells is performed using conventional methods.

  TRAIL-R polypeptides also find use as carriers that carry drugs attached to them to the cells responsible for TRAIL. TRAIL expressing cells include those identified by Wiley et al. (Immunity, 3: 673-682, 1995). Thus, TRAIL-R protein can be used to deliver diagnostic or therapeutic agents to such cells (or other cell types found to express TRAIL on the cell surface) in an in vitro or in vivo manner.

Diagnostic (detection) or therapeutic agents that can attach to TRAIL-R polypeptides include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, colorimetric or fluorescent reaction catalytic enzymes, and the like, A particular drug is selected depending on its intended application. Toxins include ricin, abrin, diphtheria toxin, Pseudomonas exotoxin A, ribosome inactivating protein, fungal toxins such as trichothecenes, and derivatives or fragments thereof (eg, short chains). Radionuclides suitable for diagnostic use include, but are not limited to, ¹²³ I, ¹³¹ I, ^99m Tc, ¹¹¹ In, and ⁷⁶ Br. Examples of radionuclides suitable for therapy are ¹³¹ I, ²¹¹ At, ⁷⁷ Br, ¹⁸⁶ Re, ¹⁸⁸ Re, ²¹² Pb, ²¹² Bi, ¹⁰⁹ Pd, ⁶⁴ Cu, and ⁶⁷ Cu.

  Such agents can be attached to TRAIL-R by any suitable conventional method. TRAIL-R, a protein, contains a functional group in the side chain of an amino acid, which reacts with the functional group of the desired drug to form, for example, a covalent bond. Alternatively, the protein or agent may be derivatized to produce or attach the desired reactive functional group. Derivatization may involve attaching one of the bifunctional linking reagents (Pierce Chemical Company, Rockford, Ill.) That serves to attach various molecules to the protein. Many techniques for radiolabeling proteins are known. The radionuclide metal can be attached to TRAIL-R, for example, by using a suitable bifunctional chelator.

  A conjugate comprising TRAIL-R and a suitable diagnostic or therapeutic agent (preferably covalently linked) is thus produced. The conjugate is administered or applied in an amount appropriate for the particular application.

  The TRAIL-R DNA and polypeptides of the present invention can be used in the development of methods for treating disorders caused by deletions or insufficient amounts of TRAIL-R (directly or indirectly). The TRAIL-R polypeptide may be administered to a mammal suffering from such a disorder. A gene therapy approach may also be employed. The disclosure of the native TRAIL-R nucleotide sequence allows detection of the deleted TRAIL-R gene and its replacement with the normal TRAIL-R encoding gene. Deletion genes are those of the TRAIL-R gene derived from humans suspected of having a deletion of this gene in in vitro diagnostic assays and with the native TRAIL-R nucleotide sequence disclosed herein. By comparing it, it can be detected.

  Another use of the proteins of the invention is as a research tool to study the biological effects resulting from inhibiting TRAIL / TRAIL-R interactions in various cell types. TRAIL-R polypeptides may also be used in in vitro assays to detect TRAIL or TRAIL-R or their interactions.

  TRAIL-R may be utilized to inhibit TRAIL biological activity in an in vitro or in vivo manner. Purified TRAIL-R polypeptide can be used to inhibit TRAIL from binding to endogenous cell surface TRAIL-R. In this way, the biological effects resulting from binding of TRAIL to endogenous receptors are inhibited. For example, various forms of TRAIL-R can be utilized, including the TRAIL-R fragments, oligomers, derivatives, and variants described above that can bind to TRAIL. In one aspect, soluble TRAIL-R is utilized to inhibit TRAIL biological activity, eg, to inhibit TRAIL-mediated apoptosis of specific cells.

  TRAIL-R may be administered to mammals to treat TRAIL-mediated disorders. Such TRAIL-mediated disorders include conditions caused or exacerbated (directly or indirectly) by TRAIL.

  TRAIL-R appears to be useful for the treatment of thrombotic microangiopathy. One such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, HC, Semin. Hematol. 24:71, 1987; Thompson et al., Blood 80: 1890, 1992). An increase in mortality due to TTP has been reported by the American Center for Disease Control (Torok et al., Am. J. Hematol. 50:84, 1995).

Plasma from individuals with TTP (including HIV ⁺ and HIV ⁻ patients) induces apoptosis of human epithelial cells of skin microvascular origin but not macrovascular origin (Laurence et al., Blood, 87: 3245, April 15, 1996). Thus, it is believed that the plasma of TTP patients contains one or more factors that induce apoptosis directly or indirectly. As described in PCT application WO 97/01633 (incorporated herein), TRAIL is present in the plasma of TTP patients and may play a role in inducing apoptosis of microvascular epithelial cells. is there.

  Another thrombotic microangiopathy is the hemolytic uremic syndrome (HUS) (Moake, JL Lancet, 343: 393, 1994; Melnyk et al., Arch. Inter. Med., 155: 2077, 1995; Thompson et al., Ibid.). One aspect of the present invention is directed to the use of TRAIL-R to treat a condition often referred to as “adult HUS” (which also affects children). The disorder known as pediatric / diarrhea-related HUS has a different etiology from adult HUS.

  Other pathologies characterized by small blood vessel clot formation can be treated using TRAIL-R. Such pathological conditions are as follows, but are not limited thereto. It is believed that small vessel clot formation is involved in heart problems seen in about 5-10% of pediatric AIDS patients. Intracardiac microvascular damage has been reported in patients with multiple sclerosis. As a further example, the treatment of systemic lupus erythematosus (SLE) is being considered.

  In one embodiment, the patient's blood or plasma is contacted with TRAIL-R ex vivo. TRAIL-R can be coupled to appropriate chromatography by conventional methods. The patient's blood or plasma is flowed through a chromatography column containing TRAIL-R bound to a matrix and then returned to the patient. The immobilized receptor binds to TRAIL, thereby removing TRAIL protein from the patient's blood.

TRAIL-R can also be administered in vivo to patients suffering from thrombotic microangiopathy. In one aspect, a soluble form of TRAIL-R is administered to the patient.
Accordingly, the present invention provides a method for treating thrombotic microvascular disorders through the use of an effective amount of TRAIL-R. TRAIL-R polypeptides can be utilized in in vivo or in vitro methods to inhibit TRAIL-mediated damage to microvascular epithelial cells (eg, apoptosis).

  TRAIL-R can be utilized in conjunction with other agents useful for treating certain disorders. In an in vitro study reported by Laurence et al. (Blood 87: 3245, 1996), TTP plasma-mediated microvascular epithelial cells were used by using normal plasma depleted of anti-Fas blocking antibodies, aurintricarboxylic acid or cold precipitate. Reduction of apoptosis was achieved.

  Thus, an agent that inhibits Fas ligand-mediated apoptosis of epithelial cells can be combined with an agent that inhibits TRAIL-mediated apoptosis of epithelial cells to treat the patient. In one aspect, TRAIL-R and anti-Fas blocking antibody are administered together to a subject suffering from a disorder characterized by thrombotic microvascular disorders such as TTP and HUS. An example of a monoclonal blocking antibody directed against the Fas antigen (CD95) is described in PCT Application Publication No. WO95 / 10540 (incorporated herein).

  Another aspect of the invention is directed to the use of TRAIL-R to reduce TRAIL-mediated T cell death in HIV infected patients. The role of T cell apoptosis in the development of AIDS has been the subject of much research (eg, Meyaard et al., Science 257: 217-219, 1992; Groux et al., J. Exp. Med., 175: 331, 1992; Oyaizu et al., "Cell activation and apoptosis in HIV infection", edited by Andrieu and Lu, Plenum Press, New York, 1995, pages 101-114). Although certain researchers have studied the role of Fas-mediated apoptosis, the involvement of interleukin-1β converting enzyme (ICE) has also been explored (Estaquier et al., Blood 87: 4959-4966, 1996; Mitra). Et al., Immunology 87: 581-585, 1996; Katsikis et al., J. Exp. Med. 181: 2029-2036, 1995). T cell apoptosis appears to be expressed through a number of mechanisms.

It is believed that TRAIL is mediated by at least some of the T cell death seen in HIV ⁺ patients. Without wishing to be bound by theory, it is believed that such TRAIL-mediated T cell death is expressed through a mechanism known as activation-induced cell death (AICD).

Activated human T cells trigger programmed cell death (apoptosis) when triggered by a process termed activation-induced cell death (AICD) via the CD3 / T cell receptor complex. Be guided to perform. AICD of CD4 ⁺ T cells isolated from asymptomatic individuals with HIV infection has been reported (Groux et al., Ibid). Thus, AICD may play a role in CD4 ⁺ T cell depletion and progression to AIDS in HIV-infected individuals.

The present invention provides a method of inhibiting TRAIL-mediated T cell death in an HIV ⁺ patient comprising administering to the patient TRAIL-R (preferably a soluble TRAIL-R polypeptide). In one aspect, the patient is asymptomatic when treatment with TRAIL-R is initiated. If desired, prior to treatment, peripheral blood T cells may be extracted from HIV ⁺ patients and tested for sensitivity to TRAIL-mediated cell death by conventional methods.

  In one embodiment, the patient's blood or plasma is contacted with TRAIL-R ex vivo. TRAIL-R can be coupled to appropriate chromatography by conventional methods. The patient's blood or plasma is flowed through a chromatography column containing TRAIL-R bound to a matrix and then returned to the patient. The immobilized receptor binds to TRAIL, thereby removing TRAIL protein from the patient's blood.

In the treatment of HIV ⁺ patients, TRAIL-R can be utilized in combination with other T cell apoptosis inhibitors. Fas-mediated apoptosis has also been implicated in T cell loss in HIV ⁺ individuals (Katikis et al., J. Exp. Med. 181: 2029-2036, 1995). Thus, patients susceptible to Fas ligand (Fas-L) -mediated and TRAIL-mediated T cell death may be treated with agents that block the TRAIL / TRAIL-R interaction and drugs that block the Fas-L / Fas interaction. Both can be treated. Agents suitable for blocking the binding of Fas-L to Fas include, but are not limited to, soluble Fas polypeptides; oligomeric forms of soluble Fas polypeptides (eg, dimers of sFas / Fc); An anti-Fas antibody that binds to Fas without transmitting; an anti-Fas-L antibody that blocks binding of Fas-L to Fas; and a variant of Fas-L that binds Fas but does not transmit a biological signal that results in apoptosis; It is. Preferably, the antibody utilized in this method is a monoclonal antibody. Examples of agents suitable for blocking Fas-L / Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540 and are incorporated herein.

  Provided herein are compositions comprising an effective amount of a TRAIL-R polypeptide of the invention in combination with other ingredients such as a physiologically acceptable diluent, carrier or excipient. TRAIL-R can be formulated according to known methods used to produce pharmaceutically useful compositions. TRAIL-R is a pharmaceutically acceptable diluent (eg, saline, Tris-HCl, acetate and phosphate buffer as a single active substance or with other known active substances suitable for certain indications). ), Preservatives (eg, thimerosal, benzyl alcohol, paraoxybenzoates [parabens]), emulsifiers, stabilizers, adjuvants and / or carriers. Suitable formulations for pharmaceutical compositions include those described in "Remington's Pharmaceutical Sciences" 16th edition, 1980, Mac Publishing Company, Easton, PA.

  Further, such compositions can be polyethylene glycol (PEG), complexed with metal ions or incorporated into polymer compounds such as polyacetic acid, polyglycolic acid, hydrogel, dextran, or liposomes, microemulsions, It may include TRAIL-R incorporated into micelles, monolayer or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such compositions affect the physical state, solubility, stability, in vivo release rate and in vivo clearance rate of TRAIL-R and are selected according to the intended application. TRAIL-R expressed on the cell surface may find similar use.

  The compositions of the invention may comprise any form of TRAIL-R polypeptide described herein, such as natural proteins, variants, derivatives, oligomers and biologically active fragments. In certain embodiments, the composition comprises a soluble TRAIL-R polypeptide or an oligomer comprising a soluble TRAIL-R polypeptide.

  TRAIL-R can be administered in any suitable manner, such as topical, parenteral, or inhalation. The term “parenteral” includes, for example, subcutaneous, intravenous, intramuscular injection, and topical administration, eg, at the site of disease or injury. Sustained release from the implant is also considered. Those skilled in the art will recognize that the appropriate dosage will vary depending on such factors as the nature of the disorder being treated, the weight, age and general condition of the patient, and the route of administration. The initial dose is determined by animal studies, and human dose assessment is performed by industry accepted methods.

Also contemplated are compositions comprising TRAIL-R nucleic acids in a physiologically acceptable formulation. TRAIL-R DNA can be formulated, for example, for injection.
Provided herein are antibodies that immunoreact with the antibody TRAIL-R polypeptide. Such an antibody specifically binds to TRAIL-R in that the antibody binds to TRAIL-R via the antibody's antigen binding site (as opposed to non-specific binding).

  TRAIL-R protein produced as described in Example 1 can be used as an immunogen in producing antibodies that immunoreact with it. Other forms of TRAIL-R such as fragments or fusion proteins are also utilized as immunogens.

  Polyclonal and monoclonal antibodies can be produced by conventional techniques. For example, “monoclonal antibodies, hybridomas: a new dimension in biological analysis” Kennet et al. (Eds.), Plenum Press, New York (1980); See Harbor, NY (1988). Production of monoclonal antibodies directed against TRAIL-R is further illustrated in Example 4.

Such antigen-binding fragments of antibodies can be produced by conventional techniques, but are encompassed by the present invention. 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.

  Monoclonal antibodies of the invention include chimeric antibodies, such as humanized versions of mouse monoclonal antibodies. Such humanized antibodies can be produced by known techniques and provide the advantage of reduced immunogenicity when the antibody is administered to a human. In one embodiment, the humanized antibody comprises a murine antibody variable region (or only its antigen binding site) and a human antibody-derived constant region. Alternatively, the humanized antibody fragment comprises an antigen binding site of a mouse monoclonal antibody and a variable fragment derived from a human antibody (devoid of the antigen binding site). Methods for producing chimerized and further engineered monoclonal antibodies are described by Riechmann et al. (Nature 332: 323, 1988), Liu et al. (PNAS 84: 3439, 1987), Larick et al. (Bio / Technology 7: 934, 1989) and Includes those described in Winter and Harris (TIPS 14: 139, May 1993).

  Among the uses of this antibody are in assays that detect the presence of TRAIL-R polypeptide, either in vitro or in vivo. This antibody can also be utilized when purifying TRAIL-R protein by immunoaffinity chromatography.

  The above-described antibodies that can further block the binding of TRAIL-R to TRAIL can be used to inhibit biological activity resulting from such binding. Such blocking antibodies may be identified using any suitable assay, such as testing antibodies for the ability to inhibit TRAIL binding to TRAIL-R expressing cells. Examples of such cells are Jurkat cells and PS1 cells described in Example 2 below. Alternatively, blocking antibodies may be identified in an assay for the ability to inhibit biological activity resulting from binding of TRAIL to target cells. For example, antibodies can be assayed for the ability to inhibit TRAIL-mediated lysis of Jurkat cells.

  Such antibodies can be utilized in in vitro methods, or administered in vivo to inhibit TRAIL-R mediated biological activity. Disorders caused or exacerbated (directly or indirectly) by cell surface TRAIL receptors and TRAIL interactions may be treated in this way. One therapeutic method involves administering to a mammal in vivo an amount of a blocking antibody effective to inhibit a TRAIL-mediated biological response. Disorders caused or exacerbated directly or indirectly by TRAIL are thus treated. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In one embodiment, antigen-binding antibody fragments are utilized.

  Blocking antibodies directed against TRAIL-R can substitute for TRAIL-R in the above-described methods of treating thrombotic microvascular disorders such as treating TTP or HUS. This antibody can be administered in vivo to inhibit TRAIL-mediated damage (such as apoptosis) to microvascular epithelial cells.

  Antibodies produced against TRAIL-R can be screened by their agonistic (ie, mimicking ligand) properties. Such an antibody, when bound to cell surface TRAIL-R, has a biological effect similar to that produced when TRAIL binds to cell surface TRAIL-R (eg, transmission of biological signals). cause. Agonist antibodies can be used to cause apoptosis of certain cancer cells or virus-infected cells, as reported for TRAIL. The ability of TRAIL to kill cancer cells (including but not limited to leukocytes, lymphomas, melanoma cells) and virus-infected cells is described in Wiley et al. (Immunity 3: 673-682, 1995) and PCT application WO 97/01633. .

  Provided herein are compositions comprising an antibody directed against TRAIL-R and a physiologically acceptable diluent, excipient or carrier. Suitable components for such compositions are those described above for compositions comprising TRAIL-R protein.

  Also provided herein is a conjugate comprising a detectable (eg, diagnostic) or therapeutic agent attached to an antibody directed against TRAIL-R. Examples of such agents are shown above. This conjugate finds use in in vitro or in vivo methods.

Nucleic acids The present invention provides TRAIL-R nucleic acids. Such nucleic acids include, but are not limited to, DNA encoding the peptide described in Example 2. Such DNA can be identified from knowledge of the genetic code. Other nucleic acids of the invention include isolated DNA consisting of the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 3.

  The present invention provides isolated nucleic acids useful for the production of TRAIL-R polypeptides, such as the recombinant expression systems discussed above. Such nucleic acids include, but are not limited to, human TRAIL-R DNA with SEQ ID NO: 1. The nucleic acid molecules of the present invention include not only TRAIL-R DNA in either single-stranded or double-stranded form, but also its RNA complement. TRAIL-R DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, DNA amplified by PCR, and combinations thereof. Genomic DNA can be isolated by conventional techniques using, for example, SEQ ID NO: 1 or 3 cDNA or appropriate fragments thereof as probes.

  DNA encoding any form of TRAIL-R (full-length TRAIL-R or a fragment thereof) contemplated herein is provided. Specific embodiments of TRAIL-R encoding DNA encode DNA encoding SEQ ID NO: 2 (including N-terminal signal peptide) full-length human TRAIL-R, and full-length mature human TRAIL-R Contains DNA. Other embodiments include DNA that encodes soluble TRAIL-R (eg, encodes the extracellular domain of the protein of SEQ ID NO: 2 with or without a signal peptide).

  One aspect of the invention is directed to a fragment of a TRAIL-R nucleotide sequence comprising at least about 17 contiguous nucleotides of the TRAIL-R DNA sequence. In other embodiments, the DNA fragment comprises at least 30 or at least 60 contiguous nucleotides of the TRAIL-R DNA sequence. The nucleic acids provided herein include the DNA and RNA complements of the fragments along with TRAIL-R DNA in single and double stranded form.

  Among the uses of TRAIL-R nucleic acid fragments are use as probes or primers. Using the genetic code and knowledge of the amino acid sequence shown in Example 2, a number of degenerate oligonucleotides can be produced. Such oligonucleotides find use as primers, for example in the polymerase chain reaction (PCR) in which TRAIL-R DNA fragments are isolated and amplified.

  Other useful fragments of TRAIL-R nucleic acids include either target TRAIL-R mRNA (sense) or TRAIL-R DNA (antisense) sequences that can bind to single-stranded nucleic acid sequences (either RNA or DNA). An antisense or sense oligonucleotide. An antisense or sense oligonucleotide according to the present invention comprises a fragment of the coding region of TRAIL-R DNA. Such fragments generally comprise at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive antisense or sense oligonucleotides based on a cDNA sequence encoding a protein is described, for example, by Stein and Cohen (Cancer Res. 48: 2659, 1988) and van der Krol et al. (BioTechniques 6: 958, 1988). )It is described in.

  When the antisense or sense oligonucleotide binds to the target nucleic acid sequence, a double stranded molecule is formed, several means including enhanced degradation of the double stranded, immature termination of transcription or translation, or other means One of them prevents transcription or translation of the target sequence. Antisense oligonucleotides can thus be used to block the expression of TRAIL-R protein. In addition, antisense or sense oligonucleotides include oligonucleotides having modified sugar phosphodiester backbones (or other sugar linkages as described in WO 91/06629), where such sugar linkages are endogenous nucleases. Resistant to Such oligonucleotides with resistant sugar linkages are stable in vivo (ie, can resist enzymatic degradation), but retain sequence specificity that can bind to target nucleotide sequences.

  Other examples of sense or antisense oligonucleotides are shared by organic components as described in WO 90/10448 and other components that increase affinity for the target nucleic acid sequence, such as poly- (L-lysine). Includes bound oligonucleotides. Furthermore, intercalating agents such as ellipticine, and alkylating agents or metal complexes can also bind to the sense or antisense oligonucleotide to modify the binding specificity of the antisense or sense oligonucleotide to the target nucleotide sequence.

Antisense or sense oligonucleotides, for example, CaPO ₄ mediated DNA transfection, by any gene transfer methods including electroporation, or by using gene transfer vectors such as Epstein-Barr virus, the target nucleic acid sequence Can be introduced into the containing cells. In a preferred method, the antisense or sense oligonucleotide can be inserted into a suitable retroviral vector. The cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, the murine retroviruses M-MuLV, N2 (M-MuLV-derived retrovirus) or DCT5A, DCT5B and DCT5C (see WO90 / 13641). Includes heavy copy vectors.

  Sense or antisense oligonucleotides can also be introduced into cells 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, the binding of the ligand binding molecule is due to the ability of the ligand binding molecule to bind to its corresponding molecule or receptor or to prevent the sense or antisense oligonucleotide or its bound transformant from entering the cell. Virtually no interference.

  Sense or antisense oligonucleotides can also be introduced into cells containing the target nucleotide sequence by formation of oligonucleotide-lipid complexes, as described in WO 90/10448. The sense or antisense oligonucleotide-lipid complex is preferably separated by endogenous lipase inside the cell.

The following examples are provided to further illustrate certain embodiments of the present invention and should not be construed as limiting the scope of the invention.
Example 1: Purification of TRAIL-R protein Human TRAIL receptor (TRAIL-R) protein was prepared by the following method. TRAIL-R was isolated from the cell membrane of Jurkat cells, a human acute T leukemia cell line. Jurkat cells were selected from Jurkat cells that were biotinylated on the surface using Flag® peptides covalently linked to Affi-gel beads (BioRad Laboratories, Richmond, Calif.). This is because these bands can be affinity precipitated. This precipitation band has a molecular weight of about 52 kD. A minor specific band of approximately 42 kD was also present, presumably interpreted as a proteolytic product or less glycosylated form of TRAIL-R.

Approximately 50 billion Jurkat cells were harvested by centrifugation (80 mL cell pellet), washed once with PBS, and then shock frozen in liquid nitrogen. The plasma membrane was isolated with the following five modifications to the third method described by Maeda et al. (Biochim. Et Biophys. Acta, 731: 115, 1983; incorporated herein).
1. The following protease inhibitors were included in all solutions at the specified concentrations: aprotinin, 150 nM; EDTA, 5 mM; leupeptin, 1 μM; pAPMSF, 20 μM; pephroblock, 500 μM; pepstatin A, 1 μM; PMSF, 500 μM.
2. Dithiothreitol was not used.
3. DNAase was not used in the homogenization solution.
For 4.1 mL of cell pellet, 1.25 mL of homogenization buffer was used.
5. Homogenization was achieved by passing 5 times through a ground glass dance homogenizer.

  After isolation of the cell membrane, the isolated cell membrane was resuspended in 50 mL PBS containing 1% octyl glucoside and all of the above protease inhibitors at the above concentrations. The resulting solution was then vortexed repeatedly during a 30 minute incubation at 4 ° C. This solution was centrifuged at 20,000 rpm with a LE-80 Beckman ultracentrifuge (Beckman Instruments, Palo Alto, Calif.) And a SW28 rotor to obtain a supernatant (membrane extract).

Chromatography The first step of purifying TRAIL-R from the membrane extract produced as described above was affinity chromatography. First, the membrane extract was passed through an anti-Flag (registered trademark) M2 Affigel column (10 mL of monoclonal antibody M2 bound to 2 mL of Affigel beads) to adsorb nonspecifically bound substances. Next, the flow-through fraction was applied to a Flag (registered trademark) TRAIL Affigel column (10 mL of recombinant protein bound to 1 mL of Affigel beads).

  The Affigel support is an N-hydroxysuccinimide ester of derivatized and cross-linked agarose gel beads (available from Biorad Laboratories, Richmond, CA). As discussed above, the Flag® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, provides an epitope reversibly bound by a specific monoclonal antibody and is expressed Allows rapid assay and convenient purification of recombinant proteins. M2 is a monoclonal that binds to Flag (registered trademark). Monoclonal antibodies that bind to Flag® peptides, and other reagents for making and using Flag® fusion proteins, are available from Eastman Kodak, Scientific Imaging Systems Division, New Haven, Connecticut. Is done. The manufacture of Flag® TRAIL fusion proteins (including Flag® fused to soluble TRAIL polypeptides) is described in further detail in PCT application WO 97/01633, incorporated herein by reference.

The column was washed in the following order with 25 mL each of the following buffers:
PBS containing 1.1% octyl glucoside
2. PBS
3. PBS with additional 200 mM NaCl
4). PBS
The bound material was eluted several times with 1 mL fractions of 50 mM sodium citrate (pH 3) and each fraction was immediately neutralized with 300 μL of 1 M Tris-HCl (pH 8.5). The TRAIL binding activity of each fraction was determined by the TRAIL-R specific ELISA described below. Fractions with high TRAIL binding activity were pooled, made into a 0.1% trifluoroacetic acid (TFA) solution, and then packed with a capillary reverse phase HPLC column [ID 500 μm × 25 cm, 5 μm Vydac C ₄ material (Vydac, Hesperia, Calif.). In a fused silicon capillary column] using a linear gradient solution (2% per minute) of 0% to 100% acetonitrile in water containing 0.1% TFA. Fractions with high TRAIL binding activity were then determined as described above, pooled and lyophilized if desired.

TRAIL-R specific ELISA
Serial dilutions of TRAIL-R containing samples (50 mM NaHCO ₃ solution adjusted to pH 9 with NaOH) were added to a 96-well flat bottom E. coli of Linbro / Titrek. I. A. A microtiter plate (ICN Biomedical, Aurora, OH) was loaded at 100 μL / well. After incubation at 4 ° C. for 16 hours, the wells were washed 6 times with 200 μL of PBS containing 0.05% Tween 20 (PBS-Tween). The wells were incubated for 90 minutes (100 μL of each well) with 1 μg / mL Flag® TRAIL in PBS-Tween containing 5% fetal calf serum (FCS) and then washed as above. Each well was then incubated with 1 μg / mL anti-Flag® monoclonal antibody M2 in PBS-Tween containing 5% FCS for 90 minutes (100 μL per well) and then washed as above. Subsequently, the wells were incubated for 90 minutes (100 μL of each well) with a polyclonal goat anti-mIgG1-specific horseradish peroxidase-binding antibody (commercial stock diluted to 1/5000 in PBS-Tween solution containing 5% FCS). HRP-binding antibodies were obtained from Southern Biotechnology Associates, Birmingham, Alabama. The wells were then washed 6 times as described above.

  For ELISA development, the substrate mixture [100 μL of each well, a 1: 1 premix of TMB peroxidase substrate and peroxidase solution B (Kilkegaard Perry Laboratories, Gaithersburg, MD)] was added to the wells. After sufficient color reaction, the enzyme reaction was terminated by adding 2N sulfuric acid (50 μL per well). Color intensity (indicating TRAIL binding activity) was determined by measuring absorbance at 450 nm with a VMax plate reader (Molecular Device, Sanivale, CA).

Example 2: Amino acid sequence (a) TRAIL-R purified from Jurkat cells
TRAIL-R protein isolated from Jurkat cells was digested with trypsin using conventional methods. Amino acid sequence analysis was performed on one of the peptide fragments produced by this trypsin digestion. This fragment was found to contain the following sequence, which corresponds to amino acids 327-333 of the sequence shown in SEQ ID NO: 2, ie, VPANEGD.

(B) TRAIL-R purified from PS-1 cells
TRAIL-R protein was also isolated from PS-1 cells. PS-1 is a human B cell line that appeared spontaneously after lethal irradiation of human peripheral blood lymphocytes (PBLs). The TRAIL-R protein was digested with trypsin using conventional methods. Amino acid sequence analysis was performed on several peptide fragments resulting from this trypsin digestion. One of the fragments was found to contain the following sequence, which, like the fragment shown in (a), is the amino acid at positions 327 to 333 of the sequence shown in SEQ ID NO: 2, ie VPANEGD Corresponding to

Example 3: DNA and amino acid sequences TRAIL-R was further digested with trypsin to determine the amino acid sequence of the peptide fragment. Based on the two amino acid sequences of this peptide, degenerate oligonucleotides were produced. TRAIL-R DNA fragment was isolated and amplified by polymerase chain reaction (PCR) using degenerate oligonucleotides as 5 ′ and 3 ′ primers. PCR was performed according to a conventional method using the cDNA derived from PS-1 cell line described in Example 2 as a template. The nucleotide sequence of the isolated TRAIL-R DNA fragment (excluding the portion corresponding to the portion of the primer) and the amino acid sequence encoded thereby are shown in FIG. 1 (SEQ ID NO: 3 and 4). The sequence of the entire TRAIL-R DNA fragment isolated by PCR corresponds to nucleotides 988-1164 of SEQ ID NO: 1, which encodes amino acids 330-388 of SEQ ID NO: 2.

  The amino acid sequence of SEQ ID NO: 4 has significant homology with the so-called death domain found in certain other receptors. The cytoplasmic region of Fas and TNF receptor type I each contain a death domain that is associated with the transmission of apoptotic signals (Tartaglia et al., Cell 74: 845, 1993; Itoh and Nagata, J. Biol. Chem. 268: 10392, 1993). Thus, the sequence shown in SEQ ID NO: 4 is believed to be found within the cytoplasmic domain of TRAIL-R.

  The probe from the above isolated fragment was used to screen a cDNA library (cDNA from human foreskin fibroblasts in λgt10 vector), and human TRAIL-R cDNA was isolated. The nucleotide sequence of the coding region of this cDNA is shown in SEQ ID NO: 1, and the amino acid sequence encoded thereby is shown in SEQ ID NO: 2.

Example 4: Monoclonal antibody binding to TRAIL-R This example demonstrates how to produce a monoclonal antibody that binds to TRAIL-R. Suitable immunogens that can be utilized to produce such antibodies include, but are not limited to, purified TRAIL-R protein or immunogenic fragments thereof such as the extracellular domain, or fusion protein comprising TRAIL-R (E.g., soluble TRAIL / Fc fusion protein).

  Purified TRAIL-R can be used to produce monoclonal antibodies that immunoreact with it using conventional techniques such as those described in US Pat. No. 4,411,993. Briefly, mice are immunized with TRAIL-R immunogen emulsified with complete Freund's adjuvant and injected in an amount of 10-100 μg subcutaneously or intraperitoneally. After 10-12 days, the immunized animals are further boosted with TRAIL-R emulsified with incomplete Freund's adjuvant. Mice are then boosted regularly on a 1 to 2 week immunization schedule. Serum samples are collected periodically by retroorbital bleeding or tail tip excision and tested for TRAIL-R antibodies by dot blotting, ELISA (enzyme-linked immunosorbent assay) or inhibition of TRAIL binding.

  After detecting a suitable antibody titer, TRAIL-R physiological saline solution is finally injected into a positive animal once intravenously. Three to four days later, the animals are sacrificed, spleen cells are harvested and the spleen cells are fused to a mouse myeloma cell line such as NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580). Fusion produces hybridoma cells that are plated in multi-microtiter plates containing HAT (hypoxanthine, aminopterin and thymidine) selective media that inhibit the growth of non-fused cells, myeloma hybrids and spleen cell hybrids.

  Engvall et al., Immunochem. 8: 871, 1971 and US Pat. No. 4,703,004 by screening the hybridoma cells in an ELISA for reactivity to purified TRAIL-R. A preferred screening technique is the antibody capture technique described in Beckman et al. (J. Immunol. 144: 4212, 1990). Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB / c mice to produce ascites containing high concentrations of anti-TRAIL-R monoclonal antibodies. Alternatively, 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 gel exclusion chromatography after ammonium sulfate precipitation. Affinity chromatography based on binding of antibodies to protein A or protein G can also be used, as can affinity chromatography based on binding to TRAIL-R.

Example 5: Northern blot analysis The tissue distribution of TRAIL-R mRNA was examined by Northern blot analysis as follows. Two different human multi-tissue northern blots (Clontech, Palo Alto, CA; Biochain, Palo Alto, CA) were applied to a partial sample of the radiolabeled probe (the same radiolabeled probe used for the cDNA library screening of Example 3). Added. Hybridization was performed in 50% formamide at 63 ° C. overnight, as previously described (March et al., Nature 315: 641-647, 1985). The blot was then washed with 68 ° C. for 30 minutes with 2 × SSC, 0.1% SDS. To normalize RNA loadings, glycerol-aldehyde-phosphate dehydrogenase (GAPDH) was used.

  Numerous tissues along the spleen, thymus, peripheral blood lymphocytes (PBLs), prostate, testis, ovary, uterus, placenta and gastrointestinal tract (including esophagus, stomach, duodenum, jejunum / ileum, colon, rectum and small intestine) A single 4.4 kilobase (kb) transcript was present in all tissues examined. Cells and tissues with the highest levels of TRAIL-R mRNA are PBLs, spleen and ovary, as shown by comparison to control hybridization with GAPDH specific probes.

Example 6: Binding assay Full-length human TRAIL-R was expressed and tested for its ability to bind TRAIL. This binding assay was performed as follows.

  A fusion protein comprising a leucine zipper peptide (LZ-TRAIL) fused to the N-terminus of the soluble TRAIL polypeptide was utilized in this assay. Essentially, Wiley et al. (Immunity, 3: 673-682, 1995; except that the DNA encoding the Flag® peptide was replaced with a sequence encoding a modified leucine zipper that allows trimerization. The expression construct was prepared as described for the manufacture of the Flag® TRAIL expression construct in the specification. This construct, the expression vector pDC409, encoded a leader sequence from human cytomegalovirus followed by a leucine zipper component fused to the N-terminus of the soluble TRAIL polypeptide. The TRAIL polypeptide contained amino acids 95-281 of human TRAIL (a fragment of the extracellular domain) as described by Wiley et al. LZ-TRAIL was expressed in CHO cells and purified from the culture supernatant.

  The expression vector designated pDC409 is a mammalian expression vector derived from the pDC406 vector described in McMahan et al. (EMBO J. 10: 2821-2832, 1991; incorporated herein). Features added to pDC409 (compared to pDC406) include the addition of unique restriction sites in the multiple cloning site (mcs), three stop codons located downstream of mcs (one for each reading frame), and mcs. A T7 polymerase promoter that facilitates sequencing of the DNA inserted downstream of the mcs.

  For expression of the full-length human TRAIL-R protein, the complete coding region (ie the DNA sequence shown in SEQ ID NO: 1) was amplified by polymerase chain reaction (PCR). The template utilized for PCR was a cDNA clone isolated from the human foreskin fibroblast cDNA library described in Example 3. The isolated and amplified DNA was inserted into the expression vector pDC409, producing a construct designated pDC409-TRAIL-R.

  As discussed above and in Example 8, the CrmA protein was utilized to inhibit apoptosis of host cells expressing recombinant TRAIL-R. An expression vector called pDC409-CrmA contained DNA encoding the poxvirus CrmA in pDC409. A 38 kilodalton cowpox-derived protein labeled CrmA is described in Pickup et al. (Proc. Natl. Acad. Sci. USA 83: 7698-7702, 1986; incorporated herein).

  CV-1 / EBNA cells were either co-transformed with pDC409-TRAIL-R and pDC409-CrmA or transformed with pDC409-CrmA alone. The cells were cultured in DMEM supplemented with 10% fetal calf serum, penicillin, streptomycin and glutamine. 48 hours after transformation, cells were non-enzymatically isolated and incubated with LZ-TRAIL (5 μg / mL), biotinylated anti-LZ monoclonal antibody (5 μg / mL) and phycoerythrin-binding streptavidin (1: 400). And analyzed by fluorescence activated cell scanning (FACS). Hemocytometric analysis was performed by FACscan (Becton Dickinson, San Jose, CA).

  CV-1 / EBNA cells co-transformed with vectors encoding TRAIL-R and CrmA significantly enhanced LZ-TRAIL binding compared to cells transformed with the vector encoding CrmA alone. Showed that.

Example 7: TRAIL-R blocks TRAIL-induced apoptosis of target cells.
TRAIL-R was tested for its ability to block TRAIL-induced apoptosis of Jurkat cells. TRAIL-R used in this assay is a form of a fusion protein called sTRAIL-R / Fc, which contains the extracellular domain of human TRAIL-R fused to the N-terminus of an Fc polypeptide derived from human IgG1. there were.

  CV-1 / EBNA cells can be transformed with a recombinant expression vector containing a fusion gene encoding sTRAIL-R / Fc protein in the pDC409 vector described in Example 6 to allow expression of the fusion protein Was cultured as follows. The sTRAIL-R / Fc fusion protein was recovered from the culture supernatant. A method for fusing a DNA encoding an IgG1 Fc polypeptide to a DNA encoding a heterologous protein is described in Smith et al. (Cell 73: 1349-1360, 1993). used.

  The fusion protein called TNF-R2-Fc used as a control is known as p75 or p80TNF-R (Smith et al., Science 248: 1019-1023, 1990; Smith et al., Cell 76: 959-996, 1994). It contained the extracellular domain of the TNF receptor protein and was fused to an Fc polypeptide. A mouse monoclonal antibody, a blocking antibody directed against human TRAIL, was also utilized in this assay.

  Jurkat cells are mixed with various or constant concentrations of LZ-TRAIL (described in Example 6 for LZ-TRAIL fusion protein), as well as various concentrations of sTRAIL-R-Fc, TNF-R2-Fc or TRAIL specific. Incubated in the absence or presence of monoclonal antibody. MTT cell activity assay described previously (Mosmann, J. Immunol. Methods 65: 55-63, 1983) (MTT is 3- [4,5-dimethylthiazol-2-yl] -2,5-diphenyltetrazolium Cell death was quantified using bromide). The results are shown in FIG. 2, which is variable concentration with no treatment (Δ) or variable concentration (▲) or constant concentration (○, ●, □, ■) LZ-TRAIL (13 ng / mL). Represents the cell death rate of Jurkat cells treated in the presence or absence (●) of TRAIL-R2-Fc (■), TNF-R2-Fc (□), or anti-TRAIL antibody (◯) . Variable concentrations for all materials were serially diluted starting from 10 μg / mL.

  Anti-TRAIL monoclonal antibody and sTRAIL-R / Fc each blocked TRAIL-induced apoptosis in a dose-dependent manner, whereas TNF-R2-Fc did not. Thus, the extracellular domain of TRAIL-R can bind to TRAIL and inhibit TRAIL-mediated apoptosis of target cells.

Example 8: TRAIL-induced apoptosis is blocked by caspase inhibitors and FADD-DN.
CV-1 / EBNA cells were prepared by DEAE-dextran method using TRAIL-R expression plasmid (pDC409-TRAIL-R) in the presence or absence of z-VAD-fmk (10 μM in medium). Transformation was performed with a fold excess of empty expression vector (pDC409) or with a 3-fold excess of expression vector pDC409-CrmA, pDC409-p35 or pDC409-FADD-DN. In addition, the expression vector for E. coli lacz gene (400 ng / slide) was cotransformed with all DNA mixtures. The transformed cells were cultured in a box fixed on a slide table.

  The mammalian expression vector pDC409 and the pDC409-TRAIL-R vector encoding full-length human TRAIL-R are described in Example 6. The tripeptide derivative zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone) is supplied by Enzyme Systems Products, Dublin, California.

  A 38 kilodalton protein derived from cowpox labeled CrmA is described in Pickup et al. (Proc. Natl. Acad. Sci. USA 83: 7698-7702, 1986; incorporated herein). The sequence information of CrmA is shown in FIG.

  A 35 kilodalton protein encoded by the baculovirus nuclear polyhedrosis virus, Autographa californica, is described in Friesen and Miller (J. Virol. 61: 2264-2272, 1987; incorporated herein). . The sequence information of this protein, denoted here as baculovirus p35, is shown in Friesen and Miller, FIG. 5 above.

  FADD (also referred to as MORT1) is described in Boldin et al. (J. Biol. Chem. 270: 7795-7798, 1995; incorporated herein). This protein, called FADD-DN (FADD dominant negative), is a C-terminal fragment of FADD containing the death domain. DNA encoding FADD-DN fused to an N-terminal Flag® epitope tag (described above) was inserted into the pDC409 expression vector described in Example 6 to form pDC409-FADD-DN. This FADD-DN polypeptide corresponds to amino acids at positions 117 to 245 of the MORT1 amino acid sequence shown above, such as Boldin et al.

  48 hours after transformation, cells were washed with PBS, fixed with glutaraldehyde, and then incubated with X-gal (5-bromo-4-chloro-3-indoxyl-β-D-galactopyranoside). Cells expressing β-galactosidase are stained blue. A decrease in the proportion of stained cells indicates a loss of β-galactosidase expression and is associated with the death of cells expressing this protein cotransformed with the lacz gene.

  The results are shown in FIG. 3, where the numbers plotted here represent the mean and standard deviation of at least 3 individual experiments. Poxvirus CrmA, baculovirus p35, FADD-DN and z-VAD-fmk all effectively reduced the death of transformed cells expressing TRAIL-R.

The present invention also provides the following aspects 1-39.
Aspect 1. A purified TRAIL receptor (TRAIL-R) polypeptide capable of binding to TRAIL, characterized in that it comprises the amino acid sequence VPANEGD.
Aspect 2. The TRAIL-R polypeptide of embodiment 1, further characterized in that the polypeptide has a molecular weight of about 50-55 kilodaltons.
Aspect 3. The TRAIL-R polypeptide according to aspect 1, further characterized in that the polypeptide comprises the amino acid sequence ETLRQCFFDDFADLVVPFDSWEPLMRKLGLMNEIKVAKAEAAGHRDTTLXTML.
Aspect 4. The TRAIL-R polypeptide of embodiment 3, further characterized in that the polypeptide has a molecular weight of about 50-55 kilodaltons.
Aspect 5 A purified TRAIL-R polypeptide selected from the group comprising: a) a TRAIL-R polypeptide of SEQ ID NO: 2, and b) a fragment of the polypeptide of (a) that can bind to TRAIL.
Aspect 6 The TRAIL-R polypeptide according to aspect 5, wherein the polypeptide includes amino acids at positions x to 440 of SEQ ID NO: 2, and x is an integer of 51 to 59.
Aspect 7. The TRAIL-R polypeptide according to aspect 6, wherein the polypeptide comprises amino acids 54 to 440 of SEQ ID NO: 2.
Aspect 8 6. A TRAIL-R polypeptide according to aspect 5, wherein the fragment is a soluble TRAIL-R comprising the extracellular domain of the TRAIL-R protein of SEQ ID NO: 2.
Aspect 9. A purified TRAIL-R polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2.
Aspect 10 The TRAIL-R polypeptide of embodiment 9, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2.
Aspect 11 The TRAIL-R polypeptide of embodiment 10, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 2.
Aspect 12 The TRAIL-R polypeptide according to aspect 9, wherein the polypeptide is obtained in nature.
Aspect 13 An oligomer comprising 2 to 4 TRAIL-R polypeptides according to aspect 5.
Aspect 14. An oligomer comprising 2 to 4 TRAIL-R polypeptides according to aspect 8.
Aspect 15 A composition comprising the TRAIL-R polypeptide according to aspect 5 and a physiologically acceptable diluent, excipient or carrier.
Aspect 16. A composition comprising a TRAIL-R polypeptide according to aspect 8 and a physiologically acceptable diluent, excipient or carrier.
Aspect 17 An isolated TRAIL-R DNA comprising the nucleotide sequence shown in FIG.
Aspect 18. An isolated TRAIL- encoding a polypeptide selected from the group comprising: a) a TRAIL-R polypeptide of SEQ ID NO: 2, and b) a fragment of the polypeptide of (a) that can bind to TRAIL. R DNA.
Aspect 19 The TRAIL-R DNA according to aspect 18, wherein the DNA encodes amino acids 1 to 440 of SEQ ID NO: 2.
Aspect 20 The TRAIL-R DNA according to embodiment 18, wherein the polypeptide comprises amino acids at positions x to 440 of SEQ ID NO: 2, and x is an integer from 51 to 59.
Aspect 21. 21. The TRAIL-R DNA according to aspect 20, wherein the polypeptide comprises amino acids 54 to 440 of SEQ ID NO: 2.
Aspect 22 The TRAIL-R DNA according to aspect 18, wherein the fragment is a soluble TRAIL-R comprising the extracellular domain of the TRAIL-R protein of SEQ ID NO: 2.
Aspect 23. An isolated TRAIL-R DNA encoding a polypeptide wherein the DNA comprises an amino acid sequence that is at least 80% identical to the amino acid sequence shown in SEQ ID NO: 2.
Aspect 24. 24. The TRAIL-R DNA according to embodiment 23, wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence shown in SEQ ID NO: 2.
Aspect 25 25. TRAIL-R DNA according to embodiment 24, wherein said polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence shown in SEQ ID NO: 2.
Aspect 26. 24. TRAIL-R DNA according to embodiment 23, wherein the polypeptide is obtained in nature.
Aspect 27. An expression vector comprising DNA according to embodiment 18.
Aspect 28. An expression vector comprising DNA according to embodiment 19.
Aspect 29 An expression vector comprising the DNA according to aspect 20.
Aspect 30 An expression vector comprising DNA according to embodiment 22.
Aspect 31 An expression vector comprising DNA according to aspect 23.
Aspect 32. A host cell transformed with the expression vector according to embodiment 27.
Aspect 33. A host cell transformed with the expression vector according to aspect 28.
Aspect 34. A host cell transformed with the expression vector according to aspect 29.
Aspect 35. A host cell transformed with the expression vector according to aspect 30.
Aspect 36. A host cell transformed with the expression vector according to embodiment 31.
Aspect 37 An isolated TRAIL-R DNA comprising at least 60 nucleotides of the sequence of SEQ ID NO: 1 or its DNA or RNA complement.
Aspect 38. An antibody directed against the TRAIL-R polypeptide or the antigen-binding fragment thereof according to aspect 5.
Aspect 39 The antibody is a monoclonal antibody. 39. The antibody according to aspect 38.

Claims (9)

An antibody or antigen-binding fragment thereof directed against a TRAIL-R polypeptide, wherein the TRAIL-R polypeptide comprises:
a) TRAIL-R polypeptide with SEQ ID NO: 2;
b) a polypeptide comprising an amino acid at positions x to 440 of SEQ ID NO: 2, wherein x is an integer from 51 to 59; and c) an amino acid at positions 54 to 440 of SEQ ID NO: 2. A polypeptide;
The antibody or antigen-binding fragment thereof selected from the group consisting of:   An antibody or antigen-binding fragment thereof directed against a TRAIL-R polypeptide, wherein the TRAIL-R polypeptide is an amino acid at positions x to 210 of SEQ ID NO: 2, wherein x is from 51 to 59 The antibody or antigen-binding fragment thereof, which is a soluble TRAIL-R, including an integer.   The antibody or antigen-binding fragment thereof according to claim 2, wherein the TRAIL-R polypeptide comprises amino acids 52 to 210 of SEQ ID NO: 2.   An antibody or antigen-binding fragment thereof directed against a TRAIL-R polypeptide, wherein the TRAIL-R polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence set forth in SEQ ID NO: 2; The antibody or antigen-binding fragment thereof.   The antibody or antigen-binding fragment thereof according to claim 4, wherein the TRAIL-R polypeptide is naturally occurring.   The antibody or antigen-binding fragment thereof according to any one of claims 1 to 5, wherein the antibody is a monoclonal antibody. A composition comprising the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 , and a physiologically acceptable diluent, excipient or carrier. A conjugate comprising a detectable agent or therapeutic agent attached to the antibody or antigen-binding fragment thereof according to any one of claims 1 to 6 . A hybridoma cell producing the isolated monoclonal antibody according to claim 6 .

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