EP0998561A1 - Trail receptor - Google Patents
Trail receptorInfo
- Publication number
- EP0998561A1 EP0998561A1 EP98934441A EP98934441A EP0998561A1 EP 0998561 A1 EP0998561 A1 EP 0998561A1 EP 98934441 A EP98934441 A EP 98934441A EP 98934441 A EP98934441 A EP 98934441A EP 0998561 A1 EP0998561 A1 EP 0998561A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- trail
- polypeptide
- trall
- residues
- dna
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
- C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
- C07K14/70578—NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P7/00—Drugs for disorders of the blood or the extracellular fluid
- A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K2319/00—Fusion polypeptide
- C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
- C07K2319/02—Fusion polypeptide containing a localisation/targetting motif containing a signal sequence
Definitions
- TRAIL TNF-related apoptosis-inducing ligand
- TRAIL-R TNF-related apoptosis-inducing ligand
- DNA encoding TRAIL-R, and expression vectors comprising such DNA are provided.
- a method for producing TRAIL-R polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding TRAIL-R, under conditions that promote expression of TRAIL-R, then recovering the expressed TRAIL-R polypeptides from the culture.
- Antibodies that are immunoreactive with TRAIL-R are also provided.
- FIGURES Figures 1A and IB present the nucleotide sequence of a human TRAIL receptor cDNA, as well as the amino acid sequence encoded thereby.
- Figures 2A and 2B present the nucleotide and encoded amino acid sequences for a second human TRAIL receptor cDNA clone.
- the amino acid sequence of Figures 2A-2B differs at two positions from the sequence presented in Figures 1 A- IB.
- TRAIL-R TRAIL receptor
- TRAIL-R binds to the cytokine designated TNF-related apoptosis-inducing ligand
- TRAIL TRAIL
- Certain uses of TRAIL-R flow from this ability to bind TRALL, as discussed further below.
- TRAIL-R finds use in inhibiting biological activities of TRAIL, or in purifying TRAIL by affinity chromatography, for example.
- TRAIL-R protein or immunogenic fragments thereof may be employed as immunogens to generate antibodies that are immunoreactive therewith.
- the antibodies are monoclonal antibodies.
- FIG. 1A to IB The nucleotide sequence of a human TRAIL receptor cDNA is presented in Figures 1A to IB, along with the amino acid sequence encoded by the cDNA (SEQ ID NO: l and SEQ ID NO:2).
- Figures 2A to 2B (SEQ ID NO:3 and SEQ ID NO:4) present the nucleotide sequence of a second human TRAIL receptor cDNA clone, and the amino acid sequence encoded thereby.
- the nucleotide sequences of Figures 1 and 2 differ at two positions.
- the nucleotide at position 145 is a C in the sequence of Figure 1 (SEQ ID NO: l), whereas nucleotide 145 is a T in Figure 2 (SEQ ID NO:3); the nucleotide at position 971 is a C in Figure 1 (SEQ ID NO:l), but is a T in Figure 2 (SEQ ID NO:3).
- the amino acid sequences likewise differ at two positions.
- Residue 35 is Pro in Figure 1 (SEQ ID NO:2) and Ser in Figure 2 (SEQ ID NO:4); residue 310 is Ser in Figure 1 (SEQ ID NO:2) and Leu in Figure 2 (SEQ ID NO:4).
- the TRAIL receptors of Figures 1 and 2 are allelic variants.
- TRAIL receptor protein as a member of the tumor necrosis factor receptor (TNF-R) family of receptors (reviewed in Smith et al., Cell 76:959-962, 1994).
- the TRAIL-R proteins include certain features of other proteins of this family, including cysteine rich repeats in the extracellular domain, as discussed below.
- TRAIL-R lacks a so-called "death domain", which is found in the cytoplasmic region of certain other receptor proteins. Such domains have been reported to be associated with transduction of apoptotic signals, i.e., to play a role in initiating intracellular apoptotic signaling cascades.
- Cytoplasmic death domains have been identified in Fas antigen (Itoh and Nagata, J. Biol. Chem. 268: 10932, 1993), TNF receptor type I (Tartaglia et al. Cell 74:845, 1993), DR3 (Chinnaiyan et al., Science 274:990-992, 1996), and CAR-1 (Brojatsch et al., Cell 87:845- 855, 1996).
- the TRAIL-R proteins of Figure 1 (SEQ ID NO:2) and Figure 2 (SEQ ID NO:4) include an N-terminal hydrophobic region that functions as a signal peptide, followed by an extracellular domain, a transmembrane region comprising amino acids 212 through 232, and a C-terminal cytoplasmic domain comprising amino acids 233 through 386.
- Computer analysis predicts that the signal peptide is likely to be cleaved after residue 55. Cleavage of the signal peptide thus would yield a mature protein comprising amino acids 56 to 386.
- the calculated molecular weight for a mature protein having the amino acid sequence of residues 56 to 386 of Figure 1 is about 36 kilodaltons.
- the isoelectric point (pi) is predicted to be about 5.27.
- the skilled artisan will recognize that the molecular weight of particular preparations of TRAIL-R protein may differ, according to such factors as the degree of glycosylation.
- the glycosylation pattern of a particular preparation of TRAIL-R may vary according to the type of cells in which the protein is expressed, for example, and a given preparation may include multiple differentially glycosylated species of the protein.
- TRAIL-R proteins with or without associated native- pattern glycosylation are provided herein. Expression of TRAIL-R in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. Further, N-glycosylation sites in the native protein may be inactivated, as discussed below.
- the present invention encompasses TRAIL-R in various forms, including those that are naturally occurring or produced through various techniques such as procedures involving recombinant DNA technology.
- forms of TRAIL-R include, but are not limited to, fragments, derivatives, variants, and oligomers of TRAIL-R, as well as fusion proteins containing TRAIL-R or fragments thereof.
- TRAIL-R may be modified to create derivatives thereof by forming covalent or aggregative conjugates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like.
- Covalent derivatives of TRAIL-R may be prepared by linking the chemical moieties to functional groups on TRAIL-R amino acid side chains or at the N-terminus or C-terminus of a TRAIL-R polypeptide.
- Conjugates comprising diagnostic (detectable) or therapeutic agents attached to TRAIL-R are contemplated herein, as discussed in more detail below.
- TRAIL-R derivatives of TRAIL-R within the scope of this invention include covalent or aggregative conjugates of TRAIL-R polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion proteins are discussed below in connection with TRAIL-R oligomers. Further, TRAIL-R-containing fusion proteins can comprise peptides added to facilitate purification and identification of TRAIL-R. Such peptides include, for example, poly-His or the antigenic identification peptides described in U.S. Patent No. 5,011,912 and in Hopp et al., Bio/Technology 6: 1204, 1988.
- Flag® peptide Asp- Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:5), which is highly antigenic and provides an epitope reversibly bound by a specific monoclonal antibody, enabling rapid assay and facile purification of expressed recombinant protein.
- a murine hybridoma designated 4E11 produces a monoclonal antibody that binds the Flag® peptide in the presence of certain divalent metal cations, as described in U.S. Patent 5,011,912, hereby incorporated by reference.
- the 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession no. HB 9259. Monoclonal antibodies that bind the Flag® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut.
- Soluble TRAIL-R may be identified (and distinguished from non-soluble membrane-bound counterparts) by separating intact cells expressing a TRAIL-R polypeptide from the culture medium, e.g., by centrifugation, and assaying the medium (supernatant) for the presence of the desired protein. The presence of TRAIL-R in the medium indicates that the protein was secreted from the cells and thus is a soluble form of the desired protein.
- Soluble forms of receptor proteins typically lack the transmembrane region that would cause retention of the protein on the cell surface.
- a soluble TRAIL-R polypeptide comprises the extracellular domain of the protein.
- soluble TRAIL-R polypeptides are fragments of the extracellular domain.
- a soluble TRAIL-R polypeptide may include the cytoplasmic domain, or a portion thereof, as long as the polypeptide is secreted from the cell in which it is produced.
- soluble TRAIL-R examples include, but are not limited to, polypeptides comprising amino acids 56 through 211 of Figure 1 or 2 (the extracellular domain) or amino acids 56 to 208 of Figure 1 or 2 (a fragment of the extracellular domain).
- An expression vector encoding a soluble TRAIL-R polypeptide comprising amino acids 1 through 208 of Figures 1 or 2, fused to an antibody-derived Fc polypeptide, is described in example 3 below.
- Further examples include, but are not limited to, fragments of the extracellular domain that include the cysteine-rich repeats of TRAIL-R, as described below.
- Soluble forms of TRAIL-R possess certain advantages over the membrane-bound form of the protein. Purification of the protein from recombinant host cells is facilitated, since the soluble proteins are secreted from the cells. Further, soluble proteins are generally more suitable for certain applications, e.g., for intravenous administration.
- TRAIL-R fragments are provided herein. Such fragments may be prepared by any of a number of conventional techniques. Desired peptide fragments may be chemically synthesized. An alternative involves generating TRAIL-R fragments by enzymatic digestion, e.g., by treating the protein with an enzyme known to cleave proteins at sites defined by particular amino acid residues.
- a TRAIL-R DNA may be digested with suitable restriction enzymes, to derive a DNA fragment encoding a desired polypeptide fragment.
- Another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment, by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the DNA fragment are employed as the 5' and 3' primers in the PCR.
- TRAIL-R polypeptide fragments may be employed as immunogens, in generating antibodies. Particular embodiments are directed to TRAIL-R polypeptide fragments that retain the ability to bind TRAIL. Such a fragment may be a soluble TRAIL-R polypeptide, as described above.
- TRAIL-R fragments include the cysteine-rich repeat motifs found in the extracellular domain.
- the human TRAIL-R proteins of Figures 1 and 2 contain two such cysteine rich repeats, the first including residues 98 through 139, and the second including residues 140 through 181.
- Receptors of the TNF-R family contain such cysteine-rich repeats in their extracellular domains (Marsters et al., J. Biol. Chem. 261 :51 l -5150, 1992; Smith et al., Cell 76:959-962, 1994). These repeats are believed to be important for ligand binding.
- Residues 182 through 211 of Figures 1 and 2 constitute a spacer region.
- This spacer is the C-terminal portion of the extracellular domain, positioned between the cysteine rich repeats and the transmembrane region.
- Such spacer regions have been identified in certain other proteins of the TNF-R family, and reportedly are not critical for ligand binding.
- TRAIL-R fragments lacking the spacer region are provided herein.
- Naturally occurring variants of the TRAIL-R protein of Figures 1 and 2 are provided herein. Such variants include, for example, proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the TRAIL-R protein. Alternate splicing of mRNA may, for example, yield a truncated but biologically active TRAIL-R protein, such as a naturally occurring soluble form of the protein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the TRAIL-R protein (generally from 1-5 terminal amino acids). TRAIL-R proteins in which differences in amino acid sequence are attributable to genetic polymorphism (allelic variation among individuals producing the protein) are also contemplated herein.
- a protein preparation may include a mixture of protein molecules having different N-terminal amino acids, resulting from cleavage of the signal peptide at more than one site.
- TRAIL-R DNAs and polypeptides include those derived from non-human species. Homologs of the human TRAIL-R of Figures 1 or 2, from other mammalian species, are contemplated herein, for example. Probes based on the human DNA sequence of Figure 1 or 2 may be used to screen cDNA libraries derived from other mammalian species, using cross-species hybridization techniques.
- TRAIL-R DNA sequences may vary from the native sequences disclosed herein. Due to the known degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in Figure 1 (SEQ ID NO:l) or Figure 2 (SEQ ID NO:3) and still encode a TRAIL-R protein having the amino acid sequence presented in those Figures (SEQ ID NO:2 OR 4, respectively). Such variant DNA sequences may result from silent mutations (e.g., occurring during PCR amplification), or may be the product of deliberate mutagenesis of a native sequence.
- native TRAIL-R sequences e.g., cDNA comprising the nucleotide sequence presented in Figures 1 or 2
- DNA that is degenerate as a result of the genetic code to a native TRAIL-R DNA sequence e.g., cDNA comprising the nucleotide sequence presented in Figures 1 or 2
- TRAIL-R polypeptides that retain a biological activity of a native TRAIL-R.
- Such variants include polypeptides that are substantially homologous to native TRAIL-R, but which have an amino acid sequence different from that of a native TRAIL-R because of one or more deletions, insertions or substitutions.
- Particular embodiments include, but are not limited to, TRALL-R polypeptides that comprise from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native TRAEL-R sequence.
- the TRAIL-R-encoding DNAs of the present invention include variants that differ from a native TRAIL-R DNA sequence because of one or more deletions, insertions or substitutions, but that encode a biologically active TRAIL-R polypeptide.
- One biological activity of TRAIL-R is the ability to bind TRAIL.
- Nucleic acid molecules capable of hybridizing to the DNA of Figures 1 or 2 under moderately stringent or highly stringent conditions, and which encode a biologically active TRAIL-R, are provided herein.
- Such hybridizing nucleic acids include, but are not limited to, variant DNA sequences and DNA derived from non-human species, e.g., non- human mammals.
- Moderately stringent conditions include conditions described in, for example, Sambrook et al, Molecular Cloning: A Laboratory Manual, 2nd ed., Vol. 1, pp 1.101-
- Conditions of moderate stringency include use of a prewashing solution of 5X SSC, 0.5% SDS,
- One embodiment of the invention is directed to DNA sequences that will hybridize to the DNA of Figures 1 or 2 under highly stringent conditions, wherein said conditions include hybridization at 68°C followed by washing in 0.1X SSC/0.1% SDS at 63-68°C.
- Certain DNAs and polypeptides provided herein comprise nucleotide or amino acid sequences, respectively, that are at least 80% identical to a native TRAIL-R sequence.
- a TRAIL-R DNA or polypeptide comprises a sequence that is at least 90% identical, at least 95% identical, or at least 98% identical to a native TRAIL-R sequence.
- the percent identity may be determined, for example, by comparing sequence information using the GAP computer program, version 6.0 described by Devereux et al. (Nucl. Acids Res. 12:387, 1984) and available from the University of Wisconsin Genetics Computer Group (UWGCG).
- the preferred default parameters for the GAP program include: (1) a unary comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides, and the weighted comparison matrix of Gribskov and Burgess, Nucl. Acids Res.
- the percent identity is calculated by comparing the sequence of the fragment with the corresponding portion of a native TRAIL-R.
- a variant TRAIL-R polypeptide differs in amino acid sequence from a native TRAIL-R, but is substantially equivalent to a native TRAIL-R in a biological activity.
- a variant TRAIL-R that binds TRAIL with essentially the same binding affinity as does a native TRAIL-R. Binding affinity can be measured by conventional procedures, e.g., as described in U.S. Patent no. 5,512,457.
- Variant amino acid sequences may comprise conservative substitution(s), meaning that one or more amino acid residues of a native TRAIL-R is replaced by a different residue, but that the conservatively substituted TRALL-R polypeptide retains a desired biological activity of the native protein (e.g., the ability to bind TRAIL).
- a given amino acid may be replaced by a residue having similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as He, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gin and Asn. Other conservative substitutions, e.g., involving substitutions of entire regions having similar hydrophobicity characteristics, are well known.
- sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be deleted or replaced with other amino acids, preventing formation of incorrect intramolecular disulfide bridges upon renaturation.
- Cysteine residues within the above-described cysteine rich repeat domains advantageously remain unaltered in TRAIL-R variants, when retention of TRAIL-binding activity is desired.
- Other variants are prepared by modification of adjacent dibasic amino acid residues, to enhance expression in yeast systems in which KEX2 protease activity is present.
- EP 212,914 discloses the use of site-specific mutagenesis to inactivate KEX2 protease processing sites in a protein.
- KEX2 protease processing sites are inactivated by deleting, adding or substituting residues to alter Arg-Arg, Arg-Lys, and Lys-Arg pairs to eliminate the occurrence of these adjacent basic residues.
- Human TRAIL-R contains such adjacent basic residue pairs at amino acids 75-76, 233-234, 260-261, 261-262, 328-329, and 329-330 of Figure 1 and 2. Lys-Lys pairings are considerably less susceptible to KEX2 cleavage, and conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivating KEX2 sites.
- N-glycosylation sites in a native TRAIL-R are inactivated.
- N-glycosylation sites can be modified to preclude glycosylation, allowing expression of a more homogeneous, reduced carbohydrate analog in mammalian and yeast expression systems.
- N-glycosylation sites in eukaryotic polypeptides are characterized by an amino acid triplet Asn-X-Y, wherein X is any amino acid except Pro and Y is Ser or Thr.
- the TRAIL-R protein of Figure 1 and 2 comprises three such triplets, at amino acids 127- 129, 171-173, and 182-184. Appropriate substitutions, additions or deletions to the nucleotide sequence encoding these triplets will result in prevention of attachment of carbohydrate residues to the Asn side chain.
- TRAIL-R polypeptides can be tested for biological activity in any suitable assay.
- the ability of a TRALL-R polypeptide to bind TRAIL can be confirmed in conventional binding assays, examples of which are described below.
- the present invention also provides recombinant cloning and expression vectors containing TRAIL-R DNA, as well as host cell containing the recombinant vectors.
- Expression vectors comprising TRAIL-R DNA may be used to prepare TRAIL-R polypeptides encoded by the DNA.
- a method for producing TRAIL-R polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding TRAIL-R, under conditions that promote expression of TRAIL-R, then recovering the expressed TRAIL-R polypeptides from the culture.
- the skilled artisan will recognize that the procedure for purifying the expressed TRAIL-R will vary according to such factors as the type of host cells employed, and whether the TRAIL-R is membrane-bound or a soluble form that is secreted from the host cell.
- the vectors include a DNA encoding a TRAIL-R polypeptide, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such as those derived from a mammalian, microbial, viral, or insect gene.
- suitable transcriptional or translational regulatory nucleotide sequences such as those derived from a mammalian, microbial, viral, or insect gene.
- regulatory sequences include transcriptional promoters, operators, or enhancers, an mRNA ribosomal binding site, and appropriate sequences which control transcription and translation initiation and termination.
- Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the TRAIL-R DNA sequence.
- a promoter nucleotide sequence is operably linked to an TRAIL-R DNA sequence if the promoter nucleotide sequence controls the transcription of the TRAIL-R DNA sequence.
- An origin of replication that confers the ability to replicate in the desired host cells, and a selection gene by which transformants are identified, are generally incorporated into the expression vector.
- a sequence encoding an appropriate signal peptide can be incorporated into expression vectors.
- a DNA sequence for a signal peptide secretory leader
- a signal peptide that is functional in the intended host cells promotes extracellular secretion of the TRAIL-R polypeptide. The signal peptide is cleaved from the TRALL-R polypeptide upon secretion of TRAIL-R from the cell.
- Suitable host cells for expression of TRAIL-R polypeptides include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, and mammalian cellular hosts are described, for example, in Pouwels et al. Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems could also be employed to produce TRAIL-R polypeptides using RNAs derived from DNA constructs disclosed herein.
- Prokaryotes include gram negative or gram positive organisms, for example, E. coli or Bacilli. Suitable prokaryotic host cells for transformation include, for example, E. coli, Bacillus subtilis, Salmonella typhimurium, and various other species within the genera Pseudomonas, Streptomyces, and Staphylococcus.
- a TRAIL-R polypeptide may include an N-terminal methionine residue to facilitate expression of the recombinant polypeptide in the prokaryotic host cell. The N- terminal Met may be cleaved from the expressed recombinant TRAIL-R polypeptide.
- Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes.
- a phenotypic selectable marker gene is, for example, a gene encoding a protein that confers antibiotic resistance or that supplies an autotrophic requirement.
- useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017).
- pBR322 contains genes for ampicillin and tetracycline resistance and thus provides simple means for identifying transformed cells.
- An appropriate promoter and a TRAIL-R DNA sequence are inserted into the pBR322 vector.
- Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMl (Promega Biotec, Madison, WI, USA).
- Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include ⁇ -lactamase (penicillinase), lactose promoter system (Chang et al., Nature 275:615, 1978; and Goeddel et al., Nature 281:544, 1979), tryptophan (tip) promoter system (Goeddel et al., Nucl. Acids Res. 8:4051, 1980; and EP-A-36776) and tac promoter (Maniatis, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, p. 412, 1982).
- a particularly useful prokaryotic host cell expression system employs a phage ⁇ PL promoter and a cI857ts thermolabile repressor sequence.
- Plasmid vectors available from the American Type Culture Collection which incorporate derivatives of the ⁇ P promoter include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR1 , ATCC 53082).
- TRAIL-R alternatively may be expressed in yeast host cells, preferably from the Saccharomyces genus (e.g., S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, may also be employed.
- yeast vectors will often contain an origin of replication sequence from a 2 ⁇ yeast plasmid, an autonomously replicating sequence (ARS), a promoter region, sequences for polyadenylation, sequences for transcription termination, and a selectable marker gene.
- Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3 -phosphogly cerate kinase (Hitzeman et al., J. Biol. Chem.
- glycolytic enzymes Hess et al., J. Adv. Enzyme Reg. 7: 149, 1968; and Holland et al., Biochem. 17:4900, 1978
- enolase glyceraldehyde-3-phosphate dehydrogenase
- hexokinase hexokinase
- pyruvate decarboxylase phosphofructokinase
- glucose-6-phosphate isomerase 3 -phosphogly cerate mutase
- pyruvate kinase triosephosphate isomerase
- phospho-glucose isomerase phospho-glucose isomerase
- glucokinase glucokinase
- yeast vectors and promoters for use in yeast expression are further described in Hitzeman, EPA-73,657.
- Another alternative is the glucose-repressible ADH2 promoter described by Russell et al. (J. Biol. Chem. 258:2614, 1982) and Beier et al. (Nature 300:124, 1982).
- Shuttle vectors replicable in both yeast and E. coli may be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Amp r gene and origin of replication) into the above-described yeast vectors.
- the yeast ⁇ -factor leader sequence may be employed to direct secretion of the TRAIL polypeptide.
- the ⁇ -factor leader sequence is often inserted between the promoter sequence and the structural gene sequence. See, e.g., Kurjan et al., Cell 30:933, 1982 and Bitter et al., Proc. Natl. Acad. Sci. USA 81:5330, 1984.
- Other leader sequences suitable for facilitating secretion of recombinant polypeptides from yeast hosts are known to those of skill in the art.
- a leader sequence may be modified near its 3' end to contain one or more restriction sites. This will facilitate fusion of the leader sequence to the structural gene.
- Yeast transformation protocols are known to those of skill in the art.
- One such protocol is described by Hinnen et al., Proc. Natl. Acad. Sci. USA 75:1929, 1978.
- Hinnen et al. Proc. Natl. Acad. Sci. USA 75:1929, 1978.
- Hinnen et al. protocol selects for Trp + transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 ⁇ g/ml adenine and 20 ⁇ g/ml uracil.
- Yeast host cells transformed by vectors containing an ADH2 promoter sequence may be grown for inducing expression in a "rich" medium.
- a rich medium is one consisting of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 ⁇ g/ml adenine and 80 ⁇ g/ml uracil. Derepression of the ADH2 promoter occurs when glucose is exhausted from the medium.
- Mammalian or insect host cell culture systems also may be employed to express recombinant TRALL-R polypeptides.
- Baculovirus systems for production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio/Technology 6:47 (1988). Established cell lines of mammalian origin also may be employed.
- suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23: 115, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (CHO) cells, HeLa cells, and BHK (ATCC CRL 10) cell lines, and the CV1/EBNA cell line (ATCC CRL 10478) derived from the African green monkey kidney cell line CV1 (ATCC CCL 70) as described by McMahan et al. (EMBO J. 10: 2821, 1991).
- Transcriptional and translational control sequences for mammalian host cell expression vectors may be excised from viral genomes.
- Commonly used promoter sequences and enhancer sequences are derived from Polyoma virus, Adenovirus 2, Simian Virus 40 (SV40), and human cytomegalovirus.
- DNA sequences derived from the SV40 viral genome for example, SV40 origin, early and late promoter, enhancer, splice, and polyadenylation sites may be used to provide other genetic elements for expression of a structural gene sequence in a mammalian host cell.
- Viral early and late promoters are particularly useful because both are easily obtained from a viral genome as a fragment which may also contain a viral origin of replication (Fiers et al., Nature 275: 113, 1978). Smaller or larger SV40 fragments may also be used, provided the approximately 250 bp sequence extending from the Hind III site toward the Bgl I site located in the SV40 viral origin of replication site is included.
- Expression vectors for use in mammalian host cells can be constructed as disclosed by Okayama and Berg (Mol. Cell. Biol. 3:280, 1983), for example.
- a useful system for stable high level expression of mammalian cDNAs in C 127 murine mammary epithelial cells can be constructed substantially as described by Cosman et al. (Mol. Immunol. 23:935, 1986).
- a high expression vector, PMLSV N1/N4 described by Cosman et al., Nature 312:16%, 1984 has been deposited as ATCC 39890. Additional mammalian expression vectors are described in EP-A-0367566, and in WO 91/18982.
- the vector may be derived from a retrovirus.
- the native signal peptide of TRALL-R may be replaced by a heterologous signal peptide or leader sequence, if desired.
- the choice of signal peptide or leader may depend on factors such as the type of host cells in which the recombinant TRAIL-R is to be produced.
- heterologous signal peptides that are functional in mammalian host cells include the signal sequence for interleukin-7 (IL-7) described in United States Patent 4,965,195, the signal sequence for interleukin-2 receptor described in Cosman et al., Nature 312:768 (1984); the interleukin-4 receptor signal peptide described in EP 367,566; the type 1 interleukin- 1 receptor signal peptide described in U.S. Patent 4,968,607; and the type II interleukin- 1 receptor signal peptide described in EP 460,846.
- Another example is a leader peptide derived from cytomegalovirus, as described in WO 97/01633, hereby incorporated by reference.
- TRAIL-R polypeptides of the present invention may be produced by recombinant expression systems as described above, or purified from naturally occurring cells.
- TRAIL-R may be purified by any of a number of suitable methods, which may employ conventional protein purification techniques. As is known to the skilled artisan, procedures for purifying a given protein are chosen according to such factors as the types of contaminants that are to be removed, which may vary according to the particular cells in which the TRAIL-R is expressed. For recombinant proteins, other considerations include the particular expression systems employed and whether or not the desired protein is secreted into the culture medium.
- cells expressing the protein are disrupted by any of the numerous known techniques, including freeze-thaw cycling, sonication, mechanical disruption, or by use of cell lysing agents.
- a soluble TRAIL-R may be expressed and secreted from the cell.
- the subsequent purification process may include affinity chromatography, e.g., employing a chromatography matrix containing TRAIL.
- the chromatography matrix may instead comprise an antibody that binds TRAIL-R.
- the TRAIL-R polypeptides can be recovered from an affinity chromatography column using conventional techniques (e.g., elution in a high salt buffer), then dialyzed into a lower salt buffer for use.
- a suitable affinity chromatography matrix is a Flag®-TRAIL affi- gel column (10 mg of recombinant protein coupled to 1 ml of affi-gel beads).
- the Affi-gel support is an N-hydroxysuccinimide ester of a derivatized, crosslinked agarose gel bead (available from Biorad Laboratories, Richmond, CA).
- the Flag® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys provides an epitope reversibly bound by specific monoclonal antibodies, which allows rapid assay and facile purification of expressed recombinant protein.
- Flag®-TRAIL fusion proteins comprising Flag® fused to a soluble TRAIL polypeptide
- the Flag®-TRAIL fusion protein is attached to the affi-gel beads by conventional techniques.
- the culture medium first may be concentrated using a commercially available protein concentration filter, for example, an Amicon or Millipore Pellicon ultrafiltration unit.
- a purification matrix such as a gel filtration matrix.
- an anion exchange resin can be employed, for example, a matrix or substrate having pendant diethylaminoethyl (DEAE) groups.
- the matrices can be acrylamide, agarose, dextran, cellulose or other support materials commonly employed in protein purification.
- a cation exchange step can be employed.
- Suitable cation exchangers include various insoluble matrices comprising sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred.
- one or more reversed-phase high performance liquid chromatography (RP-HPLC) steps employing hydrophobic RP-HPLC media, (e.g., silica gel having pendant methyl or other aliphatic groups) can be employed. Some or all of the foregoing purification steps, in various combinations, may be employed.
- RP-HPLC reversed-phase high performance liquid chromatography
- Recombinant protein produced in bacterial culture can be isolated by initial disruption of the host cells, centrifugation, extraction from cell pellets if an insoluble polypeptide, or from the supernatant fluid if a soluble polypeptide, followed by one or more concentration, salting-out, ion exchange, affinity purification or size exclusion chromatography steps. Finally, RP-HPLC can be employed for final purification steps. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.
- TRAIL-R is preferably expressed as a secreted polypeptide, to simplify purification.
- Recombinant polypeptides secreted from a yeast host cell fermentation can be purified by methods analogous to those disclosed by Urdal et al. (J. Chromatog. 296: 171, 1984). Urdal et al. describe two sequential, reversed-phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.
- TRAIL-R polypeptides are purified such that no protein bands corresponding to other (non-TRAIL-R) proteins are detectable upon analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).
- SDS-PAGE SDS-polyacrylamide gel electrophoresis
- TRAIL-R most preferably is purified to substantial homogeneity, as indicated by a single protein band upon analysis by SDS-PAGE. The protein band may be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography. Oligomeric Forms of TRAIL-R
- TRAIL-R oligomers that contain TRAIL-R polypeptides.
- TRAIL-R oligomers may be in the form of covalently-linked or non- covalently-linked dimers, trimers, or higher oligomers.
- One embodiment of the invention is directed to oligomers comprising multiple TRAIL-R polypeptides joined via covalent or non-covalent interactions between peptide moieties fused to the TRAIL-R polypeptides.
- Such peptides may be peptide linkers (spacers), or peptides that have the property of promoting oligomerization.
- Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of TRAIL-R polypeptides attached thereto, as described in more detail below.
- the oligomers comprise from two to four TRAIL-R polypeptides.
- the TRAIL-R moieties of the oligomer may be soluble polypeptides, as described above.
- a TRALL-R oligomer is prepared using polypeptides derived from immunoglobulins. Preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of antibody-derived polypeptides (including the Fc domain) has been described, e.g., by Ashkenazi et al. (PNAS USA 88: 10535, 1991); Byrn et al. (Nature 344:677, 1990); Hollenbaugh and Aruffo ("Construction of Immunoglobulin Fusion Proteins", in Current Protocols in Immunology, Suppl. 4, pages 10.19.1 - 10.19.11, 1992); Smith et al. (Cell 73:1349-1360, 1993); and Fanslow et al. (J. Immunol. 149:655-660, 1992).
- One embodiment of the present invention is directed to a TRAIL-R dimer comprising two fusion proteins created by fusing TRAIL-R to the Fc region of an antibody.
- a gene fusion encoding the TRAIL-R/Fc fusion protein is inserted into an appropriate expression vector.
- TRAIL-R/Fc fusion proteins are expressed in host cells transformed with the recombinant expression vector, and allowed to assemble much like antibody molecules, whereupon interchain disulfide bonds form between the Fc moieties to yield divalent TRALL-R.
- fusion proteins comprising a TRAIL-R polypeptide fused to an Fc polypeptide derived from an antibody.
- DNA encoding such fusion proteins, as well as dimers containing two fusion proteins joined via disulfide bonds between the Fc moieties thereof, are also provided.
- the term "Fc polypeptide” as used herein includes native and mutein forms of polypeptides derived from the Fc region of an antibody. Truncated forms of such polypeptides containing the hinge region that promotes dimerization are also included.
- Fc polypeptide is a single chain polypeptide extending from the N-terminal hinge region to the native C-terminus of the Fc region of a human IgGl antibody.
- Another useful Fc polypeptide is the Fc mutein described in U.S. Patent 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994).
- the amino acid sequence of this mutein is identical to that of the native Fc sequence presented in WO 93/10151, except that amino acid 19 has been changed from Leu to Ala, amino acid 20 has been changed from Leu to Glu, and amino acid 22 has been changed from Gly to Ala.
- TRAIL-R may be substituted for the variable portion of an antibody heavy or light chain. If fusion proteins are made with both heavy and light chains of an antibody, it is possible to form a TRAIL-R oligomer with as many as four TRAIL-R extracellular regions.
- the oligomer is a fusion protein comprising multiple TRAIL-R polypeptides, with or without peptide linkers (spacer peptides).
- suitable peptide linkers are those described in U.S. Patents 4,751,180 and 4,935,233, which are hereby incorporated by reference.
- a DNA sequence encoding a desired peptide linker may be inserted between, and in the same reading frame as, the DNA sequences encoding TRAIL-R, using any suitable conventional technique.
- a chemically synthesized oligonucleotide encoding the linker may be ligated between sequences encoding TRAIL-R.
- a fusion protein comprises from two to four soluble TRAIL-R polypeptides, separated by peptide linkers.
- Leucine zipper domains are peptides that promote oligomerization of the proteins in which they are found. Leucine zippers were originally identified in several DNA- binding proteins (Landschulz et al., Science 240:1759, 1988), and have since been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or trimerize. Examples of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308, and the leucine zipper derived from lung surfactant protein D (SPD) described in Hoppe et al.
- SPD lung surfactant protein D
- Oligomeric TRAIL-R has the property of bivalent, trivalent, etc. binding sites for TRAIL.
- the above-described fusion proteins comprising Fc moieties (and oligomers formed therefrom) offer the advantage of facile purification by affinity chromatography over Protein A or Protein G columns.
- DNA sequences encoding oligomeric TRAIL-R, or encoding fusion proteins useful in preparing TRAIL-R oligomers, are provided herein.
- TRALL-R proteins may be tested for the ability to bind TRAIL in any suitable assay.
- TRALL-R may be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like).
- a detectable reagent e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction, and the like.
- the labeled TRALL-R is contacted with cells expressing TRAIL.
- the cells then are washed to remove unbound labeled TRAIL-R, and the presence of cell-bound label is determined by a suitable technique, chosen according to the nature of the label.
- TRAIL comprises an N-terminal cytoplasmic domain, a transmembrane region, and a C- terminal extracellular domain.
- CVl-EBNA-1 cells in 10 cm 2 dishes are transfected with the recombinant expression vector.
- CV-l/EBNA-1 cells (ATCC CRL 10478) constitutively express EBV nuclear antigen- 1 driven from the CMV immediate-early enhancer/promoter.
- CVl-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).
- the transfected cells are cultured for 24 hours, and the cells in each dish then are split into a 24-well plate. After culturing an additional 48 hours, the transfected cells (about 4 x 10 4 cells/well) are washed with BM-NFDM, which is binding medium (RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2) to which 50 mg/ml nonfat dry milk has been added. The cells then are incubated for 1 hour at 37°C with various concentrations of a soluble TRAIL-R/Fc fusion protein.
- BM-NFDM binding medium
- RPMI 1640 containing 25 mg/ml bovine serum albumin, 2 mg/ml sodium azide, 20 mM Hepes pH 7.2
- Cells then are washed and incubated with a constant saturating concentration of a 125 I- mouse anti-human IgG in binding medium, with gentle agitation for 1 hour at 37°C. After extensive washing, cells are released via trypsinization.
- the mouse anti-human IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA.
- the antibody is radioiodinated using the standard chloramine-T method.
- the antibody will bind to the Fc portion of any TRAIL-R/Fc protein that has bound to the cells.
- non-specific binding of 125 1- antibody is assayed in the absence of TRAIL-R/Fc, as well as in the presence of TRAIL-R/Fc and a 200-fold molar excess of unlabeled mouse anti-human IgG antibody.
- Cell-bound 1 5 I-antibody is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N.Y. Acad. Sci. 51 :660, 1949) are generated on RS/1 (BBN Software, Boston, MA) run on a Microvax computer.
- TRAIL-R variant Another type of suitable binding assay is a competitive binding assay.
- biological activity of a TRAIL-R variant may be determined by assaying for the variant's ability to compete with a native TRAIL-R for binding to TRAIL.
- TRAIL-R and intact cells expressing TRAIL (endogenous or recombinant) on the cell surface.
- a radiolabeled soluble TRAIL-R fragment can be used to compete with a soluble TRAIL-R variant for binding to cell surface TRALL.
- a soluble TRAIL/Fc fusion protein bound to a solid phase through the interaction of Protein A or Protein G (on the solid phase) with the Fc moiety.
- Chromatography columns that contain Protein A and Protein G include those available from Pharmacia Biotech, Inc., Piscataway, NJ.
- Another type of competitive binding assay utilizes radiolabeled soluble TRAIL, such as a soluble TRALL/Fc fusion protein, and intact cells expressing TRALL-R.
- Qualitative results can be obtained by competitive autoradiographic plate binding assays, while Scatchard plots (Scatchard, Ann. N.Y. Acad.
- TRAIL-R polypeptide for the ability to block TRAIL-mediated apoptosis of target cells, such as the human leukemic T-cell line known as Jurkat cells, for example.
- target cells such as the human leukemic T-cell line known as Jurkat cells
- TRAIL-mediated apoptosis of the cell line designated Jurkat clone E6-1 is demonstrated in assay procedures described in PCT application WO 97/01633, hereby incorporated by reference.
- Uses of TRAIL-R include, but are not limited to, the following. Certain of these uses of TRAIL-R flow from its ability to bind TRAIL.
- TRAIL-R finds use as a protein purification reagent.
- TRAIL-R polypeptides may be attached to a solid support material and used to purify TRAIL proteins by affinity chromatography.
- a TRAIL-R polypeptide (in any form described herein that is capable of binding TRALL) is attached to a solid support by conventional procedures.
- chromatography columns containing functional groups that will react with functional groups on amino acid side chains of proteins are available (Pharmacia Biotech, Inc., Piscataway, NJ).
- a TRAIL-R/Fc protein is attached to Protein A- or Protein G-containing chromatography columns through interaction with the Fc moiety.
- TRAIL-R proteins also find use in measuring the biological activity of TRAIL proteins in terms of their binding affinity for TRAIL-R.
- TRAIL-R proteins thus may be employed by those conducting "quality assurance" studies, e.g., to monitor shelf life and stability of TRAIL protein under different conditions.
- TRAIL-R may be employed in a binding affinity study to measure the biological activity of a TRAIL protein that has been stored at different temperatures, or produced in different cell types.
- TRAIL-R also may be used to determine whether biological activity is retained after modification of a TRAIL protein (e.g., chemical modification, truncation, mutation, etc.).
- TRAIL-R also finds use in purifying or identifying cells that express TRALL on the cell surface.
- TRALL-R polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable substrate.
- magnetic microspheres can be coated with TRAIL-R and held in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing TRAIL-expressing cells are contacted with the solid phase having TRAIL-R thereon. Cells expressing TRAIL on the cell surface bind to the fixed TRAIL-R, and unbound cells then are washed away.
- TRAIL-R can be conjugated to a detectable moiety, then incubated with cells to be tested for TRAIL expression. After incubation, unbound labeled TRAIL- R is removed and the presence or absence of the detectable moiety on the cells is determined.
- mixtures of cells suspected of containing TRAIL * cells are incubated with biotinylated TRALL-R. Lncubation periods are typically at least one hour in duration to ensure sufficient binding.
- the resulting mixture then is passed through a column packed with avidin-coated beads, whereby the high affinity of biotin for avidin provides binding of the desired cells to the beads.
- Procedures for using avidin-coated beads are known (see Berenson, et al. 7. Cell. Biochem., 10D:239, 1986). Washing to remove unbound material, and the release of the bound cells, are performed using conventional methods.
- TRALL-R polypeptides also find use as carriers for delivering agents attached thereto to cells bearing TRAIL.
- Cells expressing TRALL include those identified in Wiley et al. (Immunity, 3:673-682, 1995).
- TRAIL-R proteins thus can be used to deliver diagnostic or therapeutic agents to such cells (or to other cell types found to express TRAIL on the cell surface) in in vitro or in vivo procedures.
- Detectable (diagnostic) and therapeutic agents that may be attached to a TRAIL-R polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction, and the like, with the particular agent being chosen according to the intended application.
- toxins include ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal inactivating proteins, mycotoxins such as trichothecenes, and derivatives and fragments (e.g., single chains) thereof.
- Radionuclides suitable for diagnostic use include, but are not limited to, 1 3 I, 131 I, 99m Tc, ⁇ ⁇ In, and 76 Br.
- Examples of radionuclides suitable for therapeutic use are 131 I, 21 1 At, 77 Br, 186 Re, 188 Re, 2 12pb, 212 B i, 109 Pd) 64 CUj and 67 Cu .
- TRAIL-R being a protein
- TRAIL-R comprises functional groups on amino acid side chains that can be reacted with functional groups on a desired agent to form covalent bonds, for example.
- the protein or agent may be derivatized to generate or attach a desired reactive functional group.
- the derivatization may involve attachment of one of the bifunctional coupling reagents available for attaching various molecules to proteins (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabeling proteins are known.
- Radionuclide metals may be attached to TRAIL-R by using a suitable bifunctional chelating agent, for example. Conjugates comprising TRALL-R and a suitable diagnostic or therapeutic agent
- conjugates are thus prepared.
- the conjugates are administered or otherwise employed in an amount appropriate for the particular application.
- TRALL-R DNA and polypeptides of the present invention may be used in developing treatments for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of, TRALL-R.
- TRAIL-R polypeptides may be administered to a mammal afflicted with such a disorder. Alternatively, a gene therapy approach may be taken. Disclosure herein of native TRAIL-R nucleotide sequences permits the detection of defective TRAIL-R genes, and the replacement thereof with normal TRAIL-R- encoding genes.
- Defective genes may be detected in in vitro diagnostic assays, and by comparison of a native TRALL-R nucleotide sequence disclosed herein with that of a TRAIL-R gene derived from a person suspected of harboring a defect in this gene.
- TRALL-R polypeptides also may be employed in in vitro assays for detecting TRAIL or TRALL-R or the interactions thereof.
- a purified TRAIL-R polypeptide may be used to bind TRALL, thus inhibiting the binding of TRALL to endogenous cell surface TRALL receptors.
- TRALL receptors include the TRAIL-R disclosed herein, as well as TRAIL-binding proteins that are distinct from the TRAIL-R of the present invention.
- Certain ligands of the TNF family (of which TRAIL is a member) have been reported to bind to more than one distinct cell surface receptor protein.
- TRAIL likewise may bind to multiple cell surface proteins.
- a receptor protein designated DR4 that reportedly binds TRAIL, but is distinct from the TRAIL-R of the present invention, is described in Pan et al. (Science 276: 111-113, 1997; hereby incorporated by reference).
- TRAIL-R may be used to inhibit a biological activity of TRALL, in in vitro or in vivo procedures. By inhibiting binding of TRALL to cell surface receptors, TRALL-R consequently inhibits biological effects that result from the binding of TRALL to endogenous receptors.
- Various forms of TRALL-R may be employed, including, for example, the above-described TRAIL-R fragments, oligomers, derivatives, and variants that are capable of binding TRAIL.
- a soluble TRAIL-R is employed to inhibit a biological activity of TRAIL, e.g., to inhibit TRALL-mediated apoptosis of cells susceptible to such apoptosis.
- TRAIL-R may be administered to a mammal to treat a TRAIL-mediated disorder.
- Such TRAIL-mediated disorders include conditions caused (directly or indirectly) or exacerbated by TRAIL.
- TRAIL-R may be useful for treating thrombotic microangiopathies.
- One such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, H.C., Semin. Hematol, 24:71, 1987; Thompson et al., Blood, 80: 1890, 1992).
- TTP thrombotic thrombocytopenic purpura
- Increasing TTP-associated mortality rates have been reported by the U.S. Centers for Disease Control (Torok et al., Am. J. Hematol. 50:84, 1995).
- Plasma from patients afflicted with TTP induces apoptosis of human endothelial cells of dermal microvascular origin, but not large vessel origin (Laurence et al., Blood, 87:3245, April 15, 1996). Plasma of TTP patients thus is thought to contain one or more factors that directly or indirectly induce apoptosis.
- TRAIL is present in the serum of TTP patients, and may play a role in inducing apoptosis of microvascular endothelial cells.
- Another thrombotic microangiopathy is hemolytic-uremic syndrome (HUS)
- One embodiment of the invention is directed to the use of TRALL-R to treat the condition that is often referred to as "adult HUS" (even though it can strike children as well).
- TRALL-R to treat the condition that is often referred to as "adult HUS” (even though it can strike children as well).
- a disorder known as childhood/diarrhea-associated HUS differs in etiology from adult HUS.
- TRALL-R clotting of small blood vessels
- Such conditions include but are not limited to the following. Cardiac problems seen in about 5-10% of pediatric AIDS patients are believed to involve clotting of small blood vessels. Breakdown of the microvasculature in the heart has been reported in multiple sclerosis patients. As a further example, treatment of systemic lupus erythematosus (SLE) is contemplated.
- SLE systemic lupus erythematosus
- the present invention thus provides a method for treating a thrombotic microangiopathy, involving use of an effective amount of TRAIL-R.
- a TRAIL-R polypeptide may be employed in in vivo or ex vivo procedures, to inhibit TRAIL-mediated damage to (e.g., apoptosis of) microvascular endothelial cells.
- TRAIL-R may be employed in conjunction with other agents useful in treating a particular disorder.
- TRAIL-R may be employed in conjunction with other agents useful in treating a particular disorder.
- Another embodiment of the present invention is directed to the use of TRAIL-R to reduce TRAIL-mediated death of T cells in HIV-infected patients.
- the role of T cell apoptosis in the development of AIDS has been the subject of a number of studies (see, for example, Meyaard et al., Science 257:217-219, 1992; Groux et al., J Exp. Med., 175:331, 1992; and Oyaizu et al., in Cell Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-114).
- At least some of the T cell death seen in HIV + patients is believed to be mediated by TRAIL. While not wishing to be bound by theory, such TRAIL-mediated T cell death is believed to occur through the mechanism known as activation-induced cell death (AICD).
- AICD activation-induced cell death
- AICD activated-induced cell death
- the present invention provides a method of inhibiting TRAIL-mediated T cell death in HIV + patients, comprising administering TRAIL-R (preferably, a soluble TRAIL- R polypeptide) to the patients.
- TRAIL-R preferably, a soluble TRAIL- R polypeptide
- the patient is asymptomatic when treatment with TRAIL-R commences.
- peripheral blood T cells may be extracted from an HIV + patient, and tested for susceptibility to TRAIL- mediated cell death by conventional procedures.
- a patient's blood or plasma is contacted with TRAIL-R ex vivo.
- the TRAIL-R may be bound to a suitable chromatography matrix by conventional procedures.
- the patient's blood or plasma flows through a chromatography column containing TRAIL-R bound to the matrix, before being returned to the patient.
- the immobilized TRALL-R binds TRALL, thus removing TRALL protein from the patient's blood.
- TRALL-R may be employed in combination with other inhibitors of T cell apoptosis. Fas-mediated apoptosis also has been implicated in loss of T cells in H1V + individuals (Katsikis et al., J. Exp. Med. 181:2029-2036, 1995).
- a patient susceptible to both Fas ligand (Fas-L)-mediated and TRAIL-mediated T cell death may be treated with both an agent that blocks TRALL/TRALL receptor interactions and an agent that blocks Fas-L/Fas interactions.
- Suitable agents for blocking binding of Fas-L to Fas include, but are not limited to, soluble Fas polypeptides; oligomeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis; an ti -Fas-L antibodies that block binding of Fas-L to Fas; and muteins of Fas-L that bind Fas but don't transduce the biological signal that results in apoptosis.
- TRAIL-R can be combined in admixture, either as the sole active material or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (e.g., saline, Tris-HCl, acetate, and phosphate buffered solutions), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and/or carriers.
- Suitable formulations for pharmaceutical compositions include those described in Remington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, East on, PA.
- compositions can contain TRAIL-R complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
- PEG polyethylene glycol
- metal ions or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts.
- PEG polyethylene glycol
- metal ions or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc., or incorporated into lip
- compositions of the present invention may contain a TRAIL-R polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers, and biologically active fragments.
- the composition comprises a soluble TRAIL-R polypeptide or an oligomer comprising soluble TRAIL-R polypeptides.
- TRALL-R can be administered in any suitable manner, e.g., topically, parenterally, or by inhalation.
- parenteral includes injection, e.g., by subcutaneous, intravenous, or intramuscular routes, also including localized administration. Sustained release from implants is also contemplated.
- suitable dosages will vary, depending upon such factors as the nature of the disorder to be treated, the patient's body weight, age, and general condition, and the route of administration. Preliminary doses can be determined according to animal tests, and the scaling of dosages for human administration are performed according to art-accepted practices.
- compositions comprising TRALL-R nucleic acids in physiologically acceptable formulations are also contemplated.
- TRALL-R DNA may be formulated for injection, for example.
- TRAIL-R protein of Figure 1 or 2 may be employed as an immunogen in producing antibodies immunoreactive therewith.
- another form of TRAIL- R such as a fragment or fusion protein, is employed as the immunogen.
- the present invention thus provides antibodies obtained by immunizing an animal with the TRALL-R of Figure 1 or 2, or an immunogenic fragment thereof.
- a method for producing antibodies comprises immunizing an animal with a TRALL-R polypeptide, whereby antibodies directed against the TRALL-R are generated in said animal.
- the desired antibodies may be purified, e.g., from the animal's serum, by conventional techniques.
- the antibodies are specific for the TRALL-R of the present invention, and do not cross-react with other (non-TRAIL-R) proteins. Screening procedures by which such antibodies may be identified are well known, and may involve immunoaffinity chromatography, for example. Hybridoma cell lines that produce monoclonal antibodies specific for TRAIL-R are also contemplated herein. Such hybridomas may be produced and identified by conventional techniques.
- the antibodies are use in assays to detect the presence of TRALL-R polypeptides, either in vitro or in vivo.
- the antibodies also may be employed in purifying TRAIL-R proteins by immunoaffinity chromatography.
- the antibodies additionally can block binding of TRAIL to TRAIL-R.
- Such antibodies may be employed to inhibit binding of TRALL to cell surface TRALL-R, for example.
- Blocking antibodies may be identified using conventional assay procedures.
- Such an antibody may be employed in an in vitro procedure, or administered in vivo to inhibit a TRALL-R-mediated biological activity. Disorders caused or exacerbated (directly or indirectly) by the interaction of TRAIL with cell surface TRALL-R thus may be treated.
- a therapeutic method involves in vivo administration of a blocking antibody to a mammal in an amount effective in inhibiting a TRAIL-mediated biological activity. Disorders caused or exacerbated by TRAIL, directly or indirectly, are thus treated.
- Monoclonal antibodies are generally preferred for use in such therapeutic methods.
- an antigen-binding antibody fragment is employed.
- compositions comprising an antibody that is directed against TRALL-R, and a physiologically acceptable diluent, excipient, or carrier, are provided herein. Suitable components of such compositions are as described above for compositions containing TRALL-R proteins.
- conjugates comprising a detectable (e.g., diagnostic) or therapeutic agent, attached to an antibody directed against TRALL-R. Examples of such agents are presented above.
- the conjugates find use in in vitro or in vivo procedures.
- TRAIL-R nucleotide sequences comprise at least about 30, or at least 60, contiguous nucleotides of a TRALL-R DNA sequence.
- Nucleic acids provided herein include DNA and RNA complements of said fragments, along with both single-stranded and double- stranded forms of the TRALL-R DNA.
- Other useful fragments of the TRALL-R nucleic acids include antisense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding to target TRALL-R mRNA (sense) or TRALL-R DNA (antisense) sequences.
- binding of antisense or sense oligonucleotides to target nucleic acid sequences results in the formation of duplexes that block transcription or translation of the target sequence by one of several means, including enhanced degradation of the duplexes, premature termination of transcription or translation, or by other means.
- the antisense oligonucleotides thus may be used to block expression of TRALL-R proteins.
- Antisense or sense oligonucleotides further comprise oligonucleotides having modified sugar- phosphodiester backbones (or other sugar linkages, such as those described in WO91/06629) and wherein such sugar linkages are resistant to endogenous nucleases.
- Such oligonucleotides with resistant sugar linkages are stable in vivo (i.e., capable of resisting enzymatic degradation) but retain sequence specificity to be able to bind to target nucleotide sequences.
- sense or antisense oligonucleotides include those oligonucleotides which are covalently linked to organic moieties, such as those described in WO 90/10448, and other moieties that increases affinity of the oligonucleotide for a target nucleic acid sequence, such as poly-(L-lysine).
- intercalating agents such as ellipticine, and alkylating agents or metal complexes may be attached to sense or antisense oligonucleotides to modify binding specificities of the antisense or sense oligonucleotide for the target nucleotide sequence.
- Antisense or sense oligonucleotides may be introduced into a cell containing the target nucleic acid sequence by any gene transfer method, including, for example, CaPO-t- mediated DNA transfection, electroporation, or by using gene transfer vectors such as Epstein-Barr virus.
- an antisense or sense oligonucleotide is inserted into a suitable retroviral vector.
- a cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo.
- Suitable retroviral vectors include, but are not limited to, those derived from the murine retrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the double copy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).
- Sense or antisense oligonucleotides also may be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.
- Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors.
- conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block entry of the sense or antisense oligonucleotide or its conjugated version into the cell.
- a sense or an antisense oligonucleotide may be introduced into a cell containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex, as described in WO 90/10448.
- the sense or antisense oligonucleotide-lipid complex is preferably dissociated within the cell by an endogenous lipase.
- the two amino acids that differ in the proteins of Figures 1 and 2 are found in the signal peptide (at position 35) and in the cytoplasmic domain (at position 310).
- the extracellular domains of the proteins of Figures 1 and 2 thus are identical.
- TRAIL-R was tested for the ability to bind TRALL, in a slide binding assay.
- DNA encoding the full length TRALL-R of Figure 1 was inserted into a mammalian expression vector designated pDC409.
- pDC409 was derived from the pDC406 vector described in McMahan et al. (EMBO J. 10:2821-2832, 1991; hereby incorporated by reference).
- pDC409 features added to pDC409 (compared to pDC406) include additional unique restriction sites in the multiple cloning site (mcs); three stop codons (one in each reading frame) positioned downstream of the mcs; and a T7 polymerase promoter, downstream of the mcs, that faciliates sequencing of DNA inserted into the mcs.
- CV-1/EBNA cells were transfected with the recombinant expression vector, and cultured on glass slides to allow expression of TRALL-R.
- a slide binding assay was conducted generally as described in Gearing et al. (EMBO J. 8:3667, 1989); McMahan et al. (EMBO J. 10:2821, 1991) and Goodwin et al. (Eur. J. Immunol. 23:2631, 1993), hereby incorporated by reference. Briefly, the transfected cells were incubated with an LZ-TRA1L fusion protein (described below) in binding media. Slides were then washed, and an 125 I-labeled antibody specific for the leucine zipper (LZ) moiety of the fusion protein was added. After incubation with the antibody, the slides were washed, fixed, and dipped in photographic emulsion. The assay demonstrated that the TRAIL-R protein binds TRAIL.
- the LZ-TRAIL employed in the assay is a fusion protein comprising a leucine zipper peptide fused to the N-terminus of a soluble TRAIL polypeptide (LZ-TRAIL) was employed in the assay.
- An expression construct was prepared, essentially as described for preparation of the Flag®-TRAIL expression construct in Wiley et al. (Immunity, 3:673- 682, 1995; hereby incorporated by reference), except that DNA encoding the Flag® peptide was replaced with a sequence encoding a modified leucine zipper that allows for trimerization.
- the construct in expression vector pDC409, encoded a leader sequence derived from human cytomegalovirus, followed by the leucine zipper moiety fused to the N-terminus of a soluble TRALL polypeptide.
- the TRAIL polypeptide comprised amino acids 95-281 of human TRALL (a fragment of the extracellular domain), as described in Wiley et al. (supra).
- the LZ-TRAIL was expressed in CHO cells, and purified from the culture supernatant.
- a DNA fragment encoding amino acids 1 to 208 of Figure 1 was isolated by polymerase chain reaction (PCR). Oligonucleotides that defined the desired termini of the DNA fragment were employed as the 3' and 5' primers in the PCR, and the cDNA clone represented in Figure 1 (SEQ ID NO:l) was employed as the template.
- the Fc moiety of the fusion protein was a mutein of a human IgGl Fc region polypeptide. DNA and amino acid sequence information for this Fc mutein are described in U.S. Patent 5,457,035 and in Baum et al., (EMBO J. 13:3992-4001, 1994), hereby incorporated by reference.
- Suitable immunogens that may be employed in generating such antibodies include, but are not limited to, purified TRAIL-R protein or an immunogenic fragment thereof such as the extracellular domain, or fusion proteins containing TRAIL-R (e.g., a soluble TRAIL-R/Fc fusion protein).
- TRAIL-R can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in U.S. Patent 4,411,993. Briefly, mice are immunized with TRAIL-R immunogen emulsified in complete Freund's adjuvant, and injected in amounts ranging from 10-100 ⁇ g subcutaneously or intraperitoneally. Ten to twelve days later, the immunized animals are boosted with additional TRAIL-R emulsified in incomplete Freund's adjuvant. Mice are periodically boosted thereafter on a weekly to bi-weekly immunization schedule. Serum samples are periodically taken by retro-orbital bleeding or tail-tip excision to test for TRALL-R antibodies by dot blot assay, EL1SA (Enzyme-Linked Immunosorbent Assay) or inhibition of TRAIL binding.
- EL1SA Enzyme-Linked Immunosorbent Assay
- TRAIL-R TRAIL-R
- saline a murine myeloma cell line
- NS1 or preferably P3x63Ag8.653 ATCC CRL 1580. Fusions generate hybridoma cells, which are plated in multiple microtiter plates in a HAT (hypoxanthine, aminopterin and thymidine) selective medium to inhibit proliferation of non-fused cells, myeloma hybrids, and spleen cell hybrids.
- HAT hypoxanthine, aminopterin and thymidine
- hybridoma cells are screened by ELISA for reactivity against purified TRALL-R by adaptations of the techniques disclosed in Engvall et al., Immunochem. 8:871, 1971 and in U.S. Patent 4,703,004.
- a preferred screening technique is the antibody capture technique described in Beckmann 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.
- hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques.
- Monoclonal antibodies produced in mouse ascites can be purified by ammonium sulfate precipitation, followed by gel exclusion chromatography.
- affinity chromatography based upon binding of antibody to Protein A or Protein G can also be used, as can affinity chromatography based upon binding to TRAIL-R.
- NAME Anderson, Kathryn A.
- GGA GCC AGG ACA GCG TCG
- GGA ACC AGA CCA TGG CTC CTG
- GAC CCC
- AAG 150 Gly Ala Arg Thr Ala Ser Gly Thr Arg Pro Trp Leu Leu Asp Pro Lys 25 30 35
- Lys Gly lie Cys Ser Gly Gly Gly Gly Gly Pro Glu Arg Val His Arg 245 250 255
- Gin Val Ser Glu Gin Glu lie Gin Gly Gin Glu Leu Ala Glu Leu Thr 290 295 300
- CAG GAA ATC CAA GGT CAG GAG CTG GCA GAG CTA ACA GGT GTG ACT GTA 966 Gin Glu He Gin Gly Gin Glu Leu Ala Glu Leu Thr Gly Val Thr Val 295 300 305
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US89211997A | 1997-07-15 | 1997-07-15 | |
US892119 | 1997-07-15 | ||
PCT/US1998/014410 WO1999003992A1 (en) | 1997-07-15 | 1998-07-10 | Trail receptor |
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JP (1) | JP2001510042A (en) |
AU (1) | AU8395798A (en) |
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US6506569B1 (en) | 1997-05-30 | 2003-01-14 | Human Genome Sciences, Inc. | Antibodies to human tumor necrosis factor receptor TR10 |
WO1998054202A1 (en) | 1997-05-30 | 1998-12-03 | Human Genome Sciences, Inc. | Human tumor necrosis factor receptor tr10 |
US6252050B1 (en) | 1998-06-12 | 2001-06-26 | Genentech, Inc. | Method for making monoclonal antibodies and cross-reactive antibodies obtainable by the method |
ATE324377T1 (en) * | 1999-05-28 | 2006-05-15 | Human Genome Sciences Inc | HUMAN TUMOR NECROSIS FACTOR RECEPTOR FC FUSION PROTEINS TR10 |
US6627199B1 (en) | 1999-07-09 | 2003-09-30 | Amgen Inc | Isolation, identification and characterization of tmst2, a novel member of the TNF-receptor supergene family |
AU783682B2 (en) | 1999-08-04 | 2005-11-24 | Amgen, Inc. | Fhm, a novel member of the TNF ligand supergene family |
AU6517800A (en) | 1999-08-04 | 2001-03-05 | Amgen, Inc. | Ntr3, a member of the tnf-receptor supergene family |
US7476383B2 (en) | 2000-05-02 | 2009-01-13 | The Uab Research Foundation | Antibody selective for DR4 and uses thereof |
US7279160B2 (en) | 2000-05-02 | 2007-10-09 | The Uab Research Foundation | Combinations of DR5 antibodies and other therapeutic agents |
TWI318983B (en) | 2000-05-02 | 2010-01-01 | Uab Research Foundation | An antibody selective for a tumor necrosis factor-related apoptosis-inducing ligand receptor and uses thereof |
KR100436089B1 (en) * | 2000-07-06 | 2004-06-14 | 설대우 | DNA cassette for the production of secretable recombinant trimeric TRAIL protein, Tetracycline/Doxycycline-inducible Adeno-associated virus(AAV), and combination of both and the gene therapy method thereof |
KR20020056565A (en) * | 2000-12-29 | 2002-07-10 | 황규언 | Three-dimensional structure of human derived apoptotic factor and receptor thereof by X-ray chrystallography and TRAIL deletion mutant protein |
CA2446723C (en) | 2001-05-25 | 2014-01-21 | Human Genome Sciences, Inc. | Antibodies that immunospecifically bind to trail receptors |
JP5562521B2 (en) | 2005-02-02 | 2014-07-30 | ザ ユーエービー リサーチ ファンデーション | Agents and methods relating to reducing resistance to apoptosis-inducing death receptor agonists |
BR112012010698A2 (en) | 2009-11-05 | 2016-11-29 | Uab Research Foundation | method for treating a subject with cancer, method for screening a breast cancer cell, and antibody |
WO2011116344A2 (en) | 2010-03-18 | 2011-09-22 | The Uab Research Foundation | Targeting cancer stem cells |
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WO1998054202A1 (en) * | 1997-05-30 | 1998-12-03 | Human Genome Sciences, Inc. | Human tumor necrosis factor receptor tr10 |
WO1999010484A1 (en) * | 1997-08-26 | 1999-03-04 | Genentech, Inc. | Rtd receptor |
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1998
- 1998-07-10 WO PCT/US1998/014410 patent/WO1999003992A1/en not_active Application Discontinuation
- 1998-07-10 CA CA002295719A patent/CA2295719A1/en not_active Abandoned
- 1998-07-10 EP EP98934441A patent/EP0998561A4/en not_active Withdrawn
- 1998-07-10 IL IL13392098A patent/IL133920A0/en unknown
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WO1998054202A1 (en) * | 1997-05-30 | 1998-12-03 | Human Genome Sciences, Inc. | Human tumor necrosis factor receptor tr10 |
WO1999010484A1 (en) * | 1997-08-26 | 1999-03-04 | Genentech, Inc. | Rtd receptor |
Non-Patent Citations (2)
Title |
---|
DEGLI ESPOSITI M A ET AL: "The novel receptor TRAIL-R4 induces NF-kappaB and protects against TRAIL-mediated apoptosis, yet retains an incomplete death domain" IMMUNITY, CELL PRESS, US, vol. 7, no. 6, 1 December 1997 (1997-12-01), pages 813-820, XP002087442 ISSN: 1074-7613 * |
See also references of WO9903992A1 * |
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