JP2001510042A - TRAIL receptor - Google Patents

Abstract

  (57) [Summary] Proteins called TRAIL receptors bind a protein known as TNF-related apoptosis-inducing ligand (TRAIL). The TRAIL receptor is used when purifying TRAIL or inhibiting its activity. An isolated DNA sequence encoding a TRAIL-R polypeptide is provided, along with an expression vector containing the DNA sequence and a host cell transformed with such a recombinant expression vector. Antibodies that are immunoreactive with TRAIL-R are also provided.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001] proteins known as background TNF-related apoptosis-inducing ligand of the invention (TRAIL) is a member of the tumor necrosis factor family ligands (Wiley et al., Im
Munology, 3: 673-682, 1995). TRAIL has been shown to be capable of inducing apoptosis in a number of transformed cells, including many different types of cancer cells as well as cells infected with the virus (PCT Application No. WO9).
7/01633 and Wiley et al., Supra). The identification of one or more receptor proteins that bind TRAIL would prove useful in further studying the biological activity of TRAIL.

SUMMARY OF THE INVENTION The present invention relates to a novel protein called the TRAIL receptor (TRAIL-R) that binds to a protein known as a TNF-related apoptosis-inducing ligand (TRAIL). DNA encoding TRAIL-R and such D
An expression vector comprising NA is provided. A method for producing a TRAIL-R polypeptide comprises culturing a host cell transformed with a recombinant expression vector encoding TRAIL-R under conditions that promote TRAIL-R expression, and then expressing the TRAIL-R. T
Recovering the RAIL-R polypeptide from the culture. TRAIL-
Antibodies that are immunoreactive with R are also provided.

[0003] DETAILED DESCRIPTION herein the invention provides a novel protein called TRAIL receptor (TRAIL-R). TRAIL-R binds to a cytokine called TNF-related apoptosis-inducing ligand (TRAIL). Some uses of TRAIL-R derive from this ability to bind TRAIL, as discussed further below. TRAIL-R inhibits the biological activity of TRAIL or T
Useful when RAIL is purified, for example, by affinity chromatography.

[0004] TRAIL-R proteins or immunogenic fragments thereof can be used as immunogens to generate antibodies that are immunoreactive with them. In one embodiment of the invention, the antibodies are monoclonal antibodies.

[0005] The nucleotide sequence of the human TRAIL receptor cDNA, together with the amino acid sequence encoded by the cDNA (SEQ ID NO: 1 and SEQ ID NO: 2), is shown in FIG.
1B. Figures 2A-2B (SEQ ID NO: 3 and SEQ ID NO: 4) show the nucleotide sequence and amino acid sequence encoded by another human TRAIL receptor cDNA clone. The nucleotide sequences in FIGS. 1 and 2 differ in two places. The nucleotide at position 145 is C in the sequence of FIG. 1 (SEQ ID NO: 1), while nucleotide 145 in FIG. 2 (SEQ ID NO: 3) is a T; The nucleotide is C, but in FIG. 2 (SEQ ID NO: 3) it is T. The amino acid sequence also differs in two places. Residue 35 is Pro in FIG. 1 (SEQ ID NO: 2) and Ser in FIG. 2 (SEQ ID NO: 4); residue 310 is Ser in FIG. 1 (SEQ ID NO: 2) and FIG. : 4) Leu. One possible explanation is that the TRAIL receptors of FIGS. 1 and 2 are allelic variants.

[0006] The sequence information shown in Figures 1 and 2 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 TRAIL-R protein contains several features of other proteins in this family, including cysteine-rich repeats in the extracellular domain as discussed below. However, TRAIL-R is a so-called "death domain" found in the cytoplasmic region of some other receptor proteins.
It has been reported that such domains are involved in apoptotic signaling, ie, play a role in the initiation of the intracellular apoptotic signal cascade. Fas antigen (Itoh and Nagata, J. Biol. Chem. 268: 10932).
, 1993), TNF receptor type I (Tartaglia et al., Cell 74: 8).
45, 1993), DR3 (Chinnaiyan et al., Science 274).
: 990-992, 1996) and CAR-1 (Brojatsch et al., Ce).
11 87: 845-855, 1996).

[0007] The TRAIL-R protein of FIG. 1 (SEQ ID NO: 2) and FIG. 2 (SEQ ID NO: 4) has an extracellular domain,
It includes a transmembrane region that includes amino acids 212-232 and a C-terminal cytoplasmic domain that includes amino acids 233-386. Computer analysis predicts that the signal peptide is likely to be cleaved after residue 55. Thus, cleavage of the signal peptide would result in a mature protein containing amino acids 56-386.

The calculated molecular weight of the mature protein having the amino acid sequence of residues 56-386 of FIG. 1 is about 36 kilodaltons. The isoelectric point (pI) is expected to be around 5.27. One of skill in the art will appreciate that the molecular weight of a particular preparation of TRAIL-R protein may vary depending on factors such as the degree of glycosylation.
The glycosylation pattern of a particular preparation of TRAIL-R may vary, for example, depending on the type of cell in which the protein is expressed, and a given preparation may contain a large number of different glycosylated species. It may contain proteins. TRAIL- with or without relevant native pattern glycosylation
The R protein is provided herein. Expression of TRAIL-R in a bacterial expression system such as E. coli provides a non-glycosylated molecule. Further, N-glycosylation sites in the native protein may be inactivated as discussed below.

[0009] The present invention encompasses various types of TRAIL-R, including those produced by various techniques, such as procedures involving naturally occurring or recombinant DNA technology. Such forms of TRAIL-R include, but are not limited to, TRAIL-R fragments, derivatives, variants and oligomers, as well as fusion proteins containing TRAIL-R or a fragment thereof. Absent.

[0010] TRAIL-R has been modified to form derivatives thereof by forming covalent or aggregate conjugates with other chemical residues such as glycosyl groups, lipids, phosphate groups, acetyl groups, and the like. May be. The covalent derivative of TRAIL-R is TRAIL
N-terminal or C on the IL-R amino acid side chain or on the TRAIL-R polypeptide
It can be produced by binding those chemical residues to a terminal functional group.
Conjugates comprising a diagnostic (detectable) or therapeutic agent linked to TRAIL-R are included herein, as discussed in more detail below.

Other derivatives of TRAIL-R within the scope of the present invention include TRAIL-R polypeptides and other proteins or polypeptides, such as by synthesis in recombinant culture, such as N-terminal or C-terminal fusions. Or covalent or aggregated conjugates of Examples of fusion proteins are discussed below in connection with TRAIL-R oligomers. In addition, TRAIL-R containing fusion proteins can include peptides added to facilitate TRAIL-R purification and identification. Such peptides include, for example, poly-His or US Pat. No. 5,011,912 and Hop.
p. et al., Bio / Technology 6: 1204, 1988. One such peptide is the 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, allowing for rapid assay and easy purification of the expressed recombinant protein To A murine hybridoma, designated 4E11, is a peptide of Flag® peptide in the presence of certain divalent metal cations, as described in US Pat. No. 5,011,912, which is incorporated herein by reference. Yields a monoclonal antibody that binds The 4E11 hybridoma cell line has been deposited with the American Type Culture Collection under accession number HB9259. Monoclonal antibodies that bind the Flag® peptide are available from Eastman Kodak Co. , Sci
Available from entific Imaging Systems Division, New Haven, Connecticut.

[0012] Provided herein are both TRAIL-R in a cell membrane bound form and in a soluble (secreted) form. Soluble TRAIL-R is TRAIL-R
Intact cells expressing the polypeptide are separated from the medium, for example, by centrifugation, and the medium (supernatant) is identified by assaying for the presence of the desired protein (and distinguished from insoluble membrane-bound counterparts). can do. The presence of TRAIL-R in the medium indicates that the protein is a soluble form of the desired protein by secretion from the cell.

[0013] Soluble forms of the receptor protein typically lack transmembrane regions that are believed to retain the protein on the cell surface. In one embodiment of the present invention,
Soluble TRAIL-R polypeptides include the extracellular domain of a protein. In some other embodiments, the soluble TRAIL-R polypeptide is a fragment of its extracellular domain. A soluble TRAIL-R polypeptide can include its cytoplasmic domain or a portion thereof, as long as it is secreted from the cell in which the polypeptide is produced.

Examples of soluble TRAIL-R include a polypeptide comprising up to amino acids 56-211 of FIG. 1 or 2 (extracellular domain) or up to amino acids 56-208 of FIG. 1 or 2 (fragment of extracellular domain). Including, but not limited to. An expression vector encoding a soluble TRAIL-R polypeptide comprising up to amino acids 1-208 of FIG. 1 or 2 fused to an antibody-derived Fc polypeptide is described in Example 3 below. Still other examples include, but are not limited to, fragments of the extracellular domain containing cysteine-rich repeats of TRAIL-R as described below.

[0015] The soluble form of TRAIL-R has several advantages over the membrane-bound form of the protein. Purification of proteins from recombinant host cells is easy because the soluble proteins are secreted from the cells. In addition, soluble proteins are generally
For certain uses, for example, more suitable for intravenous administration.

[0016] Provided herein are TRAIL-R fragments. Such fragments can be produced by any of a number of conventional techniques. The desired peptide fragment can be synthesized chemically. An alternative method involves generating a TRAIL-R fragment by enzymatic digestion, for example, by treating the protein with an enzyme known to cleave the site defined by a particular amino acid residue. . TRAIL-R
The DNA can be digested with appropriate restriction enzymes to derive a DNA fragment encoding the desired polypeptide fragment. Another suitable technique involves isolating a DNA fragment encoding the desired polypeptide fragment and amplifying it by the polymerase chain reaction (PCR).
Oligonucleotides that define the desired termini of the DNA fragment are
'And 3' primers.

[0017] TRAIL-R polypeptide fragments can be used as immunogens in raising antibodies. Particular embodiments relate 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.

In a specific embodiment, the TRAIL-R fragment comprises a cysteine-rich repeat motif found in the extracellular domain. Human T of FIGS. 1 and 2
The RAIL-R protein contains the first and residues 140 through 98-139.
It contains two such cysteine-rich repeats, the second containing up to -181. The TNF-R family of receptors contains such cysteine-rich repeats in their extracellular domain (Marsters et al., J. Biol).
. Chem. 267: 5747-5750, 1992; Smith et al., Cell.
76: 959-962, 1994). These repeats are considered important for ligand binding. To illustrate, Marsters et al., Supra, disclose that soluble TNF-R1 type polypeptides lacking one of these repeats are TNFα and TNFα.
It showed a 10-fold decrease in binding affinity to NFβ, reporting that deletion of the second repeat completely abolished the detectable binding of their ligands.

Residues 182-211 in FIGS. 1 and 2 constitute a spacer region.
This spacer is the C-terminal portion of the extracellular domain located between the cysteine-rich repeat and the transmembrane region. Such spacer regions have been identified in several other proteins of the TNF-R family and are reportedly not important for ligand binding. Provided herein is a TRAIL-R fragment that lacks a spacer region.

Provided herein are naturally occurring variants of the TRAIL-R proteins of FIGS. Such variants include, for example, proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the TRAIL-R protein. Alternative splicing of mRNA can result in a truncated, biologically active TRAIL-R protein, such as, for example, a naturally occurring soluble form of the protein. Mutations due to proteolysis include, for example, proteolytic removal of one or more terminal amino acids (generally 1-5 terminal amino acids) from the TRAIL-R protein in different types of host cells. Includes N- or C-terminal differences in expression. TRAIL-R proteins in which the amino acid sequence differences are due to genetic polymorphism (allelic variation between individuals producing the protein) are also encompassed herein.

One of skill in the art will appreciate that the location or locations where the signal peptide is cleaved may be different from the locations predicted by the computer program and that the recombinant TRA
It will also be appreciated that factors such as the type of host cell used to express the IL-R polypeptide may vary. A protein preparation can include a mixture of protein molecules having different N-terminal amino acids due to cleavage of the signal peptide at more than one site.

For a discussion herein of the various domains of the TRAIL-R protein,
One skilled in the art will appreciate that the boundaries of such regions of the protein are similar. In particular, the boundaries of the transmembrane region (which can be predicted by using available computer programs for that purpose) can be different from those described above. Accordingly, soluble TRAIL-R polypeptides in which the C-terminus of the extracellular domain differs from the residues so defined above are also encompassed herein.

[0023] Other naturally occurring TRAIL-R DNA and polypeptides include those from non-human species. For example, homologs from other mammalian species of human TRAIL-R of FIG. 1 or 2 are encompassed herein. Human D of FIG. 1 or 2
Probes based on NA sequences can be used to screen cDNA libraries from other mammalian species using interspecies hybridization techniques.

[0024] The TRAIL-R DNA sequence may differ from the native sequence disclosed herein. Due to the known degeneracy of the genetic code in which two or more codons encode the same amino acid, the DNA sequence may differ from the sequence shown in FIG. 1 (SEQ ID NO: 1) or FIG. 2 (SEQ ID NO: 3). Although they can vary, they still encode a TRAIL-R protein having the amino acid sequence shown in those figures (SEQ ID NO: 2 or 4, respectively). Such a mutated DNA sequence can be due to a silent mutation (eg, occurring during PCR amplification) or can be the product of intentional mutagenesis of a native sequence. Accordingly, among the DNA sequences provided herein are native TRAIL-R sequences (eg, cDNAs comprising the nucleotide sequence shown in FIG. 1 or 2) and native TRAIL-R as a result of the genetic code.
Some DNA degenerates into the DNA sequence.

Some of the TRAIL-R polypeptides provided herein include native TRA
There are variants of the native TRAIL-R polypeptide that retain the biological activity of the IL-R. Such variants include those that are substantially homologous to the native TRAIL-R, but that have one or more deletions, insertions or substitutions.
Polypeptides having different amino acids than R are included. Specific embodiments include, but are not limited to, TRAIL-R polypeptides that contain 1-10 deletions, insertions or substitutions of amino acid residues when compared to the native TRAIL-R sequence. Not necessarily. TRAIL-R encoding DN of the present invention
A contains the native TRAIL for one or more deletions, insertions or substitutions.
Includes variants that differ from the -R DNA sequence but encode a biologically active TRAIL-R polypeptide. One biological activity of TRAIL-R is its ability to bind TRAIL.

Provided herein is a nucleic acid molecule that encodes a biologically active TRAIL-R that is capable of hybridizing to the DNA of FIG. 1 or 2 under moderately stringent or high stringency conditions. I do. Such hybridizing nucleic acids include mutated DNA sequences and DNA derived from non-human species, eg, non-human mammals.
NA, but is not limited to these.

[0027] Moderate stringent conditions include, for example, Sambrook et al., Mole.
cultural Cloning: A Laboratory Manual, 2nd
Edition, Volume 1, pages 1.101-104, Cold Spring Harbor L
laboratory Press, 1989. Sa
Moderate stringent conditions, as defined by mbrook et al.
Includes the use of a 5 × SSC, 0.5% SDS, 1.0 mM EDTA (pH 8.0) prewash solution and hybridization conditions at about 55 ° C., 5 × SSC, overnight. High stringency conditions include higher hybridization and washing temperatures. One embodiment of the invention is a high stringency condition comprising hybridization at 68 ° C. followed by washing at 63-68 ° C. in 0.1 × SSC / 0.1% SDS. For the DNA sequence that hybridizes to the DNA of FIG.

Some of the DNA and polypeptides provided herein each contain a nucleotide or amino acid sequence that is at least 80% identical to the native TRAIL-R sequence. Further included are embodiments wherein 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 the native TRAIL-R sequence.

The percent identity is determined, for example, by Devereux et al. (Nucl. Acid
s Res. 12: 387, 1984).
y of Wisconsin Genetics Computer Gro
Up (UWGCG) can be determined by comparing sequence information using the GAP computer program, 6th edition. Preferred default parameters for the GAP program include (1) a unary comparison matrix for nucleotides (containing 1 for matches and 0 for mismatches), and Sch
supervised by wartz and Dayhoff, Atlas of Protein S
equipment and Structure, National Biom
medical Research Foundation, pp. 353-358,
Gribskov and Burgess, as described by 1979
Nucl. Acids Res. 14: 6745,1986 weighted comparison matrix; (2) 3.0 penalty for each gap and an additional 0.10 penalty for each symbol in each gap; and (3) no penalty for terminal gaps. included. The percent identity for a fragment is calculated by comparing the sequence of the fragment to the relevant portion of native TRAIL-R.

In a specific embodiment of the invention, the mutant TRAIL-R polypeptide is
Amino acid sequence differs from native TRAIL-R, but biological activity is similar to native TRAIL-R
Substantially equal to IL-R. One example is a mutant TRAIL-R that binds TRAIL with essentially the same binding affinity as native TRAIL-R. Binding affinity can be measured by conventional methods, for example, as described in US Pat. No. 5,512,457.

[0031] The variant amino acid sequence may include one or more conservative substitutions, wherein one or more amino acid residues of the native TRAIL-R have been replaced by another residue. A conservatively substituted TRAIL-R polypeptide is
It means that it retains the desired biological activity of the native protein (eg, the ability to bind TRAIL). A given amino acid can be replaced by a residue having similar physiochemical properties. Examples of conservative substitutions are substitutions of one aliphatic residue for another, such as Ile, Val, Leu or Ala, or substitution of one polar residue for another, for example L
ys and Arg; Glu and Asp; or between Gln and Asn.
It is well known to make other conservative substitutions, for example, substitutions of all regions having similar hydrophobicity.

In another example of a variant, a sequence encoding a Cys residue that is not essential for biological activity is altered so that the Cys residue is deleted or replaced with another amino acid, The formation of an incorrect intramolecular disulfide bridge during regeneration can be prevented. The cysteine residue in the cysteine-rich repeat domain is T
If retention of RAIL binding activity is desired, it advantageously remains unchanged in the TRAIL-R variant.

[0033] Other variants are made by modifying adjacent dibasic amino acid residues to facilitate expression in yeast systems where KEX2 protease activity has been demonstrated. EP2
No. 12,914 discloses the use of site-directed mutagenesis to inactivate KEX2 protease processing sites in proteins. The KEX2 protease processing sites include Arg-Arg pairs, Arg-Lys pairs and Lys-Ar
Inactivate by deleting, adding or substituting residues so that g pairs are not altered to generate these adjacent basic residues. Human TRAIL-R is derived from amino acids 75-76, 233-234, 260-261, 261-
262, 328-329 and 329-330 contain such adjacent basic residue pairs. The Lys-Lys pairing undergoes little KEX2 cleavage and Arg
Conversion of -Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivation of the KEX2 site.

[0034] In other variants, the N-glycosylation site in native TRAIL-R is inactivated. N-glycosylation sites can be modified to prevent glycosylation and to express more homologous, carbohydrate-reduced analogs in mammalian and yeast expression systems. The N-glycosylation site in a eukaryotic polypeptide is characterized by the amino acid triplet Asn-XY, where X is any amino acid except Pro and Y is Ser or Thr. TRAIL- of FIGS. 1 and 2
The R protein converts three such triplets into amino acids 127-129,
171-173 and 182-184. Appropriate substitutions, additions or deletions in the nucleotide sequence encoding these triplets will prevent the attachment of carbohydrate residues to the Asn side chain. For example, a single nucleotide change in which Asn is selected such that it is replaced by another amino acid is sufficient to deactivate the N-glycosylation site. Known procedures for inactivating N-glycosylation sites in proteins include those described in U.S. Pat.
1,972 and those described in EP 276,846.

[0035] TRAIL-R polypeptides, including variants and fragments, can be tested for biological activity in any suitable assay. T that binds TRAIL
The ability of the RAIL-R polypeptide can be confirmed by conventional binding assays,
Examples of those will be described later.

Expression Systems The present invention further provides recombinant cloning and expression vectors containing TRAIL-R DNA, and host cells containing those recombinant vectors. Expression vectors containing TRAIL-R DNA can be used to produce TRAIL-R polypeptides encoded by the DNA. TRAIL
The method for producing the -R polypeptide comprises 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. Recovering the -R polypeptide from the culture. One skilled in the art will recognize that the procedure for purifying the expressed TRAIL-R depends on factors such as the type of host cell used, and on the TRAIL-R in a membrane-bound form or in a soluble form secreted from the host cell. You will understand that it depends on whether it is.

[0037] Any suitable expression system can be used. The vectors include DNA encoding a TRAIL-R polypeptide operably linked to a suitable transcriptional or translational regulatory nucleotide sequence, such as from a mammalian, microbial, viral or insect gene. 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 its regulatory sequence is operatively related to a TRAIL-R DNA sequence. Thus, a promoter nucleotide sequence is operably linked to a TRAIL-R DNA sequence if the promoter nucleotide sequence controls the transcription of the TRAIL-R DNA sequence. The origin of replication that confers the ability to replicate in the desired host cell, and the selection gene that identifies the transformed cell, are generally included in the expression vector.

In addition, sequences encoding appropriate (natural or atypical) signal peptides can be included in the expression vector. DNA sequence of signal peptide (
The secretory leader) can be fused in frame to the TRAIL-R sequence, so that the TRAIL-R is first translated as a fusion protein containing the signal peptide. The signal peptide that is functional in the intended host cell is T
Promotes extracellular secretion of RAIL-R polypeptide. The signal peptide is
Upon secretion of TRAIL-R from the cell, it is cleaved from the TRAIL-R polypeptide.

[0039] Suitable host cells for expression of a TRAIL-R polypeptide include prokaryotes, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Cloning and expression vectors suitable for use with bacterial, fungal, yeast and mammalian cell hosts are described, for example, in Pouwels et al.
Cloning Vectors: A Laboratory Manual,
Elzeville, New York (1985). Cell-free translation systems
TRAIL-R using RNA derived from the DNA constructs disclosed herein
It is believed that it will be used to produce polypeptides.

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

[0041] Expression vectors for use in prokaryotic host cells generally include 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 auxotrophy. Examples of useful expression vectors for prokaryotic host cells include:
Included are those derived from commercially available plasmids such as the cloning vector pBR322 (ATCC 37017). Since pBR322 contains genes for ampicillin and tetracycline resistance, it provides a simple means to identify transformed cells. Suitable promoter and TRAIL-R
The DNA sequence is inserted into the pBR322 vector. Other commercially available vectors include, for example, pKK223-3 (Pharmacia Fine)
Chemicals, Uppsala, Sweden) and pGEM1 (Prome
ga Biotec, Madison, WI, USA).

[0042] Promoter sequences commonly used in recombinant prokaryotic host cell expression vectors include β-lactamase (penicillinase), lactose promoter system (Ch
ang et al., Nature 275: 615, 1978; and Goeddel et al., Nature 281: 544, 1979), the tryptophan (trp) promoter system (Goeddel et al., Nucl. Acids Res. 8: 4057).
And EP-A-36776) and the tac promoter (Ma
niatis, Molecular Cloning: A Laborato
ry Manual, Cold Spring Harbor Laboratory
ory, p. 412, 1982). A particularly useful prokaryotic host cell expression system uses the phage λPL promoter and the cI857ts thermolabile repressor sequence. American including derivatives of its λPL promoter
Plasmid vectors available from Type Culture Collection include plasmid pHUB2 (resident in E. coli strain JMB9, ATCC 370).
92) and pPLc28 (ends in E. coli strain RR1, ATCC 53082).

TRAIL-R can alternatively be expressed in yeast host cells, preferably from the genus Saccharomyces (eg, S. cerevisiae). Other genera of yeast, such as Pichia or Kluyveromyces, can also be used. Yeast vectors will often contain an origin of replication sequence from a 2μ 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, in particular, metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073).
1980) or enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase,
Other glycolytic enzymes such as pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase and glucokinase (Hess et al., J. Adv. E.
nzyme Reg. 7: 149, 1968; and Holland et al., Bio.
chem. 17: 4900, 1978). Other suitable vectors and promoters for use in yeast expression are described in Hitzeman, EPA-73,6.
No. 57 is further described. Another option is Russell et al. (J.B.
iol. Chem. 258: 2674, 1982) and Beier et al. (Na
glucose 300: 724, 1982).
DH2 promoter. A shuttle vector capable of replication in both yeast and E. coli contains a DNA sequence from pBR322 (Amp) for selection and replication in E. coli.
r gene and origin of replication) into the yeast vector.

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

[0045] Yeast transformation protocols are known to those skilled in the art. One such protocol is described in Hinnen et al., Proc. Natl. Acad. Sci. USA
75: 1929, 1978. The protocol of Hinnen et al. Selects for Trp ⁺ transformants in a selective medium, wherein the selective medium comprises 0.67% yeast nitrogen base, 0.5% casamino acid, 2% glucose,
Consists of 10 μg / ml adenine and 20 μg / ml uracil.

[0046] Yeast host cells transformed with a vector containing the ADH2 promoter sequence can be grown for expression in "rich" media. An example of a rich medium consists of 1% yeast extract, 2% peptone, and 1% glucose supplemented with 80 μg / ml adenine and 80 μg / ml uracil.
The derepression of the ADH2 promoter occurs when the medium is depleted of glucose.

[0047] Mammalian or insect host cell culture systems can also be used to express recombinant TRAIL-R polypeptide. Baculovirus systems for the production of atypical proteins in insect cells are described in Luckow and Summers, Bio / T.
technology 6:47 (1988). Established cell lines of mammalian origin can also be used. Examples of suitable mammalian host cell systems include:
Monkey kidney cell COS-7 line (ATCC CRL 1651) (Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells (
ATCC CCL 163), Chinese hamster ovary (CHO) cells, H
eLa and BHK (ATCC CRL 10) cell lines, and McMah
CV1 / EBN from African green monkey kidney cell line CV1 (ATCC CCL 70) as described by An et al. (EMBO J. 10: 2821, 1991).
A cell line (ATCC CRL 10478) is included.

[0048] Transcriptional and translational regulatory sequences of a mammalian host cell expression vector can be generated from the viral genome. Commonly used promoter and enhancer sequences include polyomavirus, adenovirus 2, simian virus 40
(SV40) and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, such as the SV40 origin of replication, early and late promoters, enhancers, splices, and polyadenylation sites, are used to generate other genetic elements for expression of the structural gene sequence in mammalian host cells. Can be given. Viral early and late promoters are particularly useful because both are readily obtained from the viral genome as fragments that can contain the viral origin of replication (Fiers et al., Nature 273: 113, 1978). Smaller or larger SV40 fragments can also be used, provided that they include an approximately 250 bp sequence extending from a HindIII site to a BglI site located at the SV40 viral origin of replication.

[0049] Expression vectors for use in mammalian host cells are described, for example, in Okayama
And Berg (Mol. Cell. Biol. 3: 280, 1983). A useful system for stable, high-level expression of mammalian cDNA in C127 murine mammary epithelial cells is described in Cosman
(Mol. Immunol. 23: 935, 1986). Cosman et al., Nature 312: 768,
1984. The high expression vector PMLSV N1 / N4 described by AT.
Deposited as CC 39890. Yet another mammalian expression vector is E. coli.
It is described in PA-0367566 and WO91 / 18982. In one alternative, the vector may be derived from a retrovirus.

[0050] With respect to signal peptides that can be used to produce TRAIL-R, the native signal peptide of TRAIL-R can be replaced by a variant 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 the recombinant TRAIL-R is produced. To illustrate, examples of atypical signal peptides that are functional in mammalian host cells include the signal sequence of interleukin-7 (IL-7) described in U.S. Pat. No. 4,965,195, Cosman et al., Natu
re 312: 768 (1984); the signal sequence of the interleukin-2 receptor described in EP 367,566; the interleukin-4 receptor signal peptide described in EP 367,566; described in US Pat. No. 4,968,607. A type I interleukin-1 receptor signal peptide; and a type II interleukin-1 receptor signal peptide described in EP 460,846. Another example is a leader peptide derived from cytomegalovirus described in WO 97/01633, which is incorporated herein. Purified Proteins The TRAIL-R polypeptides of the invention can be produced by recombinant expression systems as described above, or can be purified from naturally occurring cells. TRAIL-R can be purified by any of a number of suitable methods that can employ conventional protein purification techniques. As is known to those skilled in the art, the procedure for purifying a given protein will be selected by factors such as the type of contaminant being removed, and can be modified by the particular cell expressing TRAIL-R. . Other requirements for a recombinant protein include the particular expression system used and whether the desired protein is secreted into the medium.

In one method, cells expressing the protein are disrupted by any of a number of known techniques, including freeze-thaw cycling, sonication, mechanical disruption, or the use of cell lysing agents. Alternatively, soluble TRAIL-R can be expressed and secreted from cells. Subsequent purification methods can include, for example, affinity chromatography using a chromatography matrix containing TRAIL. The chromatography matrix may alternatively include an antibody that binds TRAIL-R. The TRAIL-R polypeptide can be recovered from the affinity chromatography column using conventional techniques (e.g., elution into a high salt buffer) and then dialyzed into a low salt buffer for use. .

One example of a suitable affinity chromatography matrix is Flag (
® -TRAIL affi-gel column (10 mg of recombinant protein bound to 1 ml of Affigel beads). Affigel support is N-hydroxysuccinimide ester of derivatized cross-linked agarose gel beads (available from Biorad Laboratories, Richmond, CA). As discussed above, Flag® Peptide A
sp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys (SEQ ID NO:
5) provides an epitope reversibly bound by a particular monoclonal antibody, allowing rapid assay and easy purification of the expressed recombinant protein.
Purification of Flag®-TRAIL fusion proteins (including Flag® fused to a soluble TRAIL polypeptide) is further described in PCT application WO 97/01633, which is incorporated herein. The Flag®-TRAIL fusion protein is attached to Affigel beads by conventional techniques.

In another approach, when using an expression system that secretes recombinant protein, first the medium is filtered from a commercially available concentration filter such as Ami.
Concentration can be achieved using a con or Millipore Pellicon ultrafiltration device. After the concentration step, the concentrate can be added to a purification matrix, such as a gel filtration matrix. Alternatively, an anion exchange resin, such as a matrix or substrate having pendant diethylaminoethyl (DEAE) groups, can be used. The matrices can be acrylamide, agarose, dextran, cellulose, or other support materials commonly used in protein purification. Alternatively, a cation exchange step can be used. Suitable cation exchangers include various insoluble matrices containing sulfopropyl or carboxymethyl groups. Sulfopropyl groups are preferred. In addition, one or more reverse phase high performance liquid chromatography (RP-HPLC) steps can be performed on hydrophobic RP
-Can be performed using HPLC bases (e.g. silica gel with methyl or other aliphatic side groups). Some or all of the foregoing purification steps can be performed in various combinations.

The recombinant protein produced in the bacterial culture can be initially disrupted by host cells, centrifuged, extracted from the cell pellet if insoluble polypeptide, or from the supernatant if soluble polypeptide, followed by It can be isolated by one or more concentration, salting out, ion exchange, affinity purification, or size exclusion chromatography steps. Finally, RP-HPLC can be used for the final purification step. Microbial cells can be disrupted by any convenient method, including freeze-thaw cycling, sonication, mechanical disruption, or use of cell lysing agents.

[0055] In yeast host cells, TRAIL-R is preferably expressed as a secreted polypeptide to facilitate purification. Recombinant polypeptides secreted from yeast host cell fermentations are described in Urdal et al. (J. Chromatog. 296: 171,
1984).
Describe two consecutive reverse phase HPLC steps for purification of recombinant human IL-2 on a preparative HPLC column.

The desired degree of purification depends on the intended use of the protein. Relatively high degrees of purification are desired, for example, when administering the protein in vivo. Conveniently, TR
The AIL-R polypeptide was subjected to SDS-polyacrylamide gel electrophoresis (SDS
-PAGE) so that protein bands corresponding to other (non-TRAIL-R) proteins cannot be detected by analysis by PAGE. One skilled in the art will appreciate that a number of bands corresponding to TRAIL-R proteins can be visualized by SDS-PAGE, such as by differential glycosylation, differential post-translational processing, and the like. TRAIL-R is most preferably purified to substantially homogeneity as indicated by a single protein band on analysis by SDS-PAGE. The protein band can be visualized by silver staining, Coomassie blue staining, or (if the protein is radiolabeled) by autoradiography. Oligomeric Forms of TRAIL-R Included by the present invention are oligomers containing a TRAIL-R polypeptide. TRAIL-R oligomers may be in the form of covalently or non-covalently linked dimers, trimers or higher oligomers.

One embodiment of the present invention is directed to multiple TRAILs linked by covalent or non-covalent interactions between peptide residues fused to a TRAIL-R polypeptide.
-R oligomers comprising the polypeptide. Such a peptide may be a peptide linker (spacer) or a peptide having a property to promote oligomerization. Leucine zippers, and some polypeptides derived from antibodies, have TRAI bound to them, as described in more detail below.
It is in a peptide that can promote oligomerization of the LR polypeptide.

In a specific embodiment, the oligomer comprises 2-4 TRAIL-R
Including polypeptides. The TRAIL-R residue of the oligomer may be a soluble polypeptide as described above.

[0059] In one option, TRAIL-R oligomers are produced using polypeptides derived from immunoglobulins. The production of fusion proteins comprising several heterologous polypeptides fused to various portions of an antibody-derived polypeptide (including the Fc domain) is described, for example, in Ashkenazi et al. (PNAS USA 88: 10535).
(1991); Byrn et al. (Nature 344: 677, 1990); Ho.
llenbaugh and Aruffo (“Construction of
Immunoglobulin Fusion Proteins ", Curr
ent Protocols in Immunology, Addendum 4, 10.1
9.1-10.19.11, p. 1992); Smith et al. (Cell 73:
1349-1360, 1993); and Fanslow et al. (J. Immuno.
l. 149: 655-660, 1992).

One embodiment of the present invention relates to a TRAIL-R dimer comprising two fusion proteins generated by fusing TRAIL-R to the Fc region of an antibody. The gene fusion encoding the TRAIL-R / Fc fusion protein is inserted into a suitable expression vector. TRAIL-R / Fc fusion proteins are expressed in host cells transformed with recombinant expression vectors and assemble like antibody molecules, where interchain disulfide bonds form between the Fc residues. , Bivalent TRA
This produces IL-R.

Provided herein are T-fused to Fc polypeptides derived from antibodies.
A fusion protein containing a RAIL-R polypeptide. DNAs encoding such fusion proteins, as well as dimers containing two fusion proteins linked by disulfide bonds between Fc residues of the fusion proteins are also provided. The term “Fc polypeptide” as used herein refers to the Fc of an antibody.
Includes native and mutein forms of the polypeptide from the region. Also included are truncated forms of such polypeptides that contain a hinge region that promotes dimerization.

One suitable Fc polypeptide described in PCT application WO 93/10151 (incorporated herein) is the Fc polypeptide from the N-terminal hinge region of a human IgG1 antibody.
A single chain polypeptide that extends to the natural C-terminus of the c region. Another useful Fc polypeptide is described in US Pat. No. 5,457,035 and Baum et al.
BOJ. 13: 3992-4001, 1994). The amino acid sequence of this mutein shows 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 to Gl.
It is identical to that of the native Fc sequence shown in WO 93/10151, except that y has been changed to Ala. The mutein shows reduced affinity for the Fc receptor.

In another embodiment, TRAIL-R can be substituted for a variable portion of an antibody heavy or light chain. If the fusion protein is produced together with both the heavy and light chains of the antibody, it is possible to form TRAIL-R oligomers containing as few as four TRAIL-R extracellular regions.

Alternatively, the oligomer is a fusion protein comprising multiple TRAIL-R polypeptides, with or without a peptide linker (spacer peptide). Among suitable peptide linkers are those described in US Pat.
51,180 and 4,935,233. The DNA sequence encoding the desired peptide linker can be inserted between and in the same reading frame as the TRAIL-R encoding DNA sequence using any suitable conventional technique. it can. For example, a chemically synthesized oligonucleotide encoding the linker can be linked between the sequences encoding TRAIL-R. In a specific embodiment, 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. Leucine zipper domains are peptides that promote oligomerization of the protein in which they are found. Leucine zipper contains some DNA
First identified in binding proteins (Landschulz et al., Science
ce 240: 1759, 1988), and has been found in a variety of different proteins. Among the known leucine zippers are naturally occurring peptides and their dimerizing or trimerizing derivatives. Examples of suitable leucine zipper domains for producing soluble oligomeric proteins are described in PCT application WO 94/10308, which is incorporated herein by reference, and leucine zippers derived from lung surfactant protein D (SPD) are Hoppe. (FEBS
Letters 344: 191, 1994). The use of modified leucine zippers to allow stable trimerization of heterologous proteins fused to leucine zippers 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 oligomer TRAIL-R has the property of a bivalent, trivalent equivalent binding site to TRAIL. Such fusion proteins containing Fc residues (and oligomers formed therefrom) offer the advantage of easy purification by affinity chromatography on Protein A or Protein G columns. Provided herein is a DNA sequence encoding an oligomeric TRAIL-R or encoding a fusion protein useful in making TRAIL-R oligomers. Assays TRAIL-R proteins (including fragments, variants, oligomers and other forms of TRAIL-R) can be tested for the ability to bind TRAIL in any suitable assay. TRAIL-R is a detectable reagent (eg,
Radionuclides, chromophores, enzymes that catalyze colorimetric or fluorometric reactions). The 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 confirmed by an appropriate technique selected depending on the nature of the label.

One example of a binding assay is as follows. The recombinant expression vector containing the TRAIL cDNA can be obtained, for example, using the PCT application WO 97/0, incorporated herein by reference.
Construct as described in 1633. DNA and amino acid sequence information for human and mouse TRAIL is provided in WO 97/01633. 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 transfected with the recombinant expression vector. CV1 / EBNA-1 cells (ATCC C
RL 10478) constitutively expresses EBV nuclear antigen-1 driven from the CMV immediate enhancer / promoter. CV1-EBNA-1 is McMahan
(EMBO J. 10: 2821, 1991) and was derived from the African green monkey kidney cell line CV-1 (ATCC CCL 70).

After culturing the transfected cells for 24 hours, the cells in each dish are split into 24-well plates. After further culturing for 48 hours, the transfected cells (about 4 × 10 ⁴ cells / well) were combined with a binding medium (25 mg / ml bovine serum albumin, 2 mg / ml azide) supplemented with 50 mg / ml non-fat dry milk. RPMI 164 containing sodium, 20 mM Hepes pH 7.2
Wash with BM-NFDM which is 0). The cells are then incubated with various concentrations of the soluble TRAIL-R / Fc fusion protein for 1 hour at 37 ° C. The 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 trypsination.

The mouse anti-human IgG used above is directed against the Fc region of human IgG and is described in Jackson Immunoresearch Laboratories.
s, Inc. Available from 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 bound to the cell. In all assays, the non-specific binding of ¹²⁵ I-antibodies was determined 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. Test below.

The ¹²⁵ I-antibody bound to the cells is quantified on a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. NY Aca)
d. Sci. 51: 660, 1949) occurs in RS / 1 (BBN Software, Boston, Mass.) Performed on a Microvax computer.

Another type of suitable binding assay is a competitive binding assay. To explain, T
The biological activity of the RAIL-R variant is similar to that of the native TR for binding to TRAIL.
It can be measured by assaying for the ability of those variants to compete with AIL-R.

[0072] Competitive binding assays can be performed by conventional methods. Reagents that can be used in competitive binding assays include radiolabeled TRAIL-R and intact cells that express 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 TRAI bound to solid phase by interaction of Fc residues with protein A or protein G (on solid phase)
It is believed that the LR / Fc fusion protein can be substituted. Chromatography columns containing Protein A and Protein G include Pharmacia B
iotech, Inc. Inc., Piscataway, NJ. Another type of competitive binding assay utilizes soluble TRAIL-R / Fc fusion proteins and radiolabeled soluble TRAIL, such as intact cells expressing TRAIL-R. Qualitative results can be obtained by competitive autoradiographic plate binding assays, but with Scatchard plots (Scatch
ard, Ann. N. Y. Acad. Sci. 51: 660, 1949) can be used to generate quantitative results.

Another type of assay for biological activity involves translating TRAIL-R polypeptides into T cells of a target cell, such as the human leukemia T cell line known as Jurkat cells.
Investigating the ability to block RAIL-mediated apoptosis. Jur
Kat of the cell line designated clone E6-1 (ATCC TIB 152)
AIL-mediated apoptosis is described in PCT application WO 97/01, incorporated herein by reference.
No. 633. Uses of TRAIL-R Uses of TRAIL-R include, but are not limited to: Some of these uses for TRAIL-R derive from its ability to bind TRAIL.

TRAIL-R is used as a protein purification reagent. TRAIL-R polypeptides can be bound to a solid support material and can be used to purify TRAIL proteins by affinity chromatography. In a specific embodiment, the TRAIL-R polypeptide (all forms described herein that can bind TRAIL) is attached to a solid support by conventional methods. As an example, chromatographic columns are available that contain functional groups that react with functional groups on the amino acid side chains of proteins (Pharmacia Biot).
ech, Inc. , Piscataway, NJ). As another option, TRAIL-
The R / Fc protein is bound to a protein A or protein G containing chromatography column by interaction with its FC residue.

TRAIL-R proteins are also used in measuring their biological activity with respect to the binding affinity of TRAIL proteins to TRAIL-R. Thus, TRAIL-R proteins can be used, for example, by those conducting "quality assurance" experiments that monitor the shelf life and stability of TRAIL proteins under various conditions. Specifically, TRAIL-R can be used in binding affinity experiments to measure the biological activity of TRAIL proteins stored at different temperatures or produced in different cell types. TRAIL-R can also be used to determine whether biological activity is retained after modification (eg, chemical modification, truncation, mutation, etc.) of the TRAIL protein. Modified TRAIL protein T
The binding affinity for RAIL-R is compared to that of the unmodified TRAIL protein to detect any adverse effects of those modifications on the biological activity of TRAIL. Thus, the biological activity of a TRAIL protein can be determined, for example, before using it in a laboratory study.

TRAIL-R is also used to purify or identify cells that express TRAIL on the cell surface. The TRAIL-R polypeptide is bound to a solid phase such as a column chromatography matrix or similar suitable substrate. For example, magnetic microspheres can be coated with TRAIL-R and held in a magnetic field in an incubation vessel. A suspension of the cell mixture containing TRAIL-expressing cells is contacted with the solid phase having TRAIL-R on the solid phase. Cells expressing TRAIL on the cell surface bind to the immobilized TRAIL-R, and subsequently wash away unbound cells.

Alternatively, TRAIL-R can be coupled to a detectable residue and then incubated with cells for testing for TRAIL expression. After incubation, unbound labeled TRAIL-R is removed and the presence or absence of detectable residues on those cells is confirmed.

In yet another case, a mixture of cells suspected of containing TRAIL ⁺ cells is incubated with biotinylated TRAIL-R. The incubation time is typically at least one hour in a continuous fashion to ensure sufficient binding. The desired cells are then bound to the beads by passing the resulting mixture through a column packed with avidin-coated beads due to the high affinity of biotin for avidin. Procedures using avidin-coated beads are known (Berenso
n et al. Cell. Biochem. , 10D: 239, 1986). Washing to remove unbound material and release of bound cells is performed using conventional methods.

[0079] TRAIL-R polypeptides are also used as carriers for delivering agents bound to them to TRAIL-containing cells. Cells expressing TRAIL include those identified by Wiley et al. (Immunity, 3: 673-682, 1995). Thus, the TRAIL-R protein may be used to deliver diagnostic or therapeutic agents to such cells (or to other cell types known to express TRAIL on the cell surface) in in vitro or in vivo procedures. Can be used.

Detectable (diagnostic) and therapeutic agents that can bind to the TRAIL-R polypeptide include toxins, other cytotoxic agents, drugs, radionuclides, chromophores, and colorimetric or fluorometric reactions. Examples include, but are not limited to, enzymes and the like, and specific agents are selected for their intended use. Among the toxins, ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa
There are exotoxin A, ribosome-inactivating proteins, mycotoxins such as trichothecene, and derivatives and fragments thereof (eg, single chain). 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 therapeutic use are ^{13 1 I, 211 At, 77} Br, 186 Re, 188 Re, 212 Pb, 212 Bi, 109 Pd, 64 C u and ⁶⁷ Cu.

[0081] Such agents can be linked to TRAIL-R by any suitable conventional method. TRAIL-R, a protein, contains a functional group on the amino acid side chain that can react with a functional group on the desired drug, for example, to form a covalent bond. Alternatively, the protein or agent can be derivatized to produce or attach the desired reactive functionality. The derivatization may involve the attachment of one of the bifunctional coupling reagents available to attach various molecules to the protein (Pierce Chemical Company, Rockford, Ill.). Numerous techniques for radiolabeling proteins are known. Radionuclide metals can be obtained, for example, by using a suitable bifunctional chelating agent to obtain TRA.
It can bind to IL-R.

Thus, a (preferably covalently) conjugate comprising TRAIL-R and a suitable diagnostic or therapeutic agent is produced. The conjugates are administered or otherwise used in an amount appropriate for the particular use.

The TRAIL-R DNAs and polypeptides of the present invention develop a method for treating any disease mediated (directly or indirectly) by incomplete or insufficient amounts of TRAIL-R. Can be used in some cases. A TRAIL-R polypeptide can be administered to a mammal suffering from such a disease. Alternatively, a gene therapy approach can be employed. The disclosure herein of the native TRAIL-R nucleotide sequence allows for the detection of incomplete TRAIL-R genes and their replacement with normal TRAIL-R encoding genes. The incomplete gene has been identified in in vitro diagnostic assays and with the native TRAIL-R nucleotide sequence disclosed herein, and the sequence of the TRAIL-R gene from a person who is believed to be defective in this gene. Can be detected by comparing

Another use of the proteins of the present invention is in TRAIL /
As a test tool to study biological effects resulting from inhibition of TRAIL-R interaction. TRAIL-R polypeptide is TRAIL or TRAIL-R
Alternatively, it can also be used in an in vitro assay for detecting their interaction.

[0085] Purified TRAIL-R polypeptides can be used to bind TRAIL, and thus can block TRAIL binding to endogenous cell surface TRAIL receptors. Such TRAIL receptors include the TR disclosed herein.
AIL-R, and further, TRAIL-binding proteins different from TRAIL-R of the present invention are included. Several ligands of the TNF family, of which TRAIL is a member, have been reported to bind to two or more different cell surface receptor proteins. TRAIL can also bind to a number of cell surface proteins. A receptor protein, reportedly DR4, which binds TRAIL but differs from TRAIL-R of the present invention, is described by Pan et al. (Science
276: 111-113, 1997; incorporated herein by reference).

[0086] TRAIL-R can be used in in vitro or in vivo procedures.
Can be used to inhibit the biological activity of By inhibiting the binding of TRAIL to cell surface receptors, TRAIL-R consequently inhibits the biological effects resulting from TRAIL binding to endogenous receptors. Various forms of the TRAIL-R fragments, oligomers, derivatives and TRAIs described above, for example
TRAIL-R including mutants capable of binding L can be used. In a preferred embodiment, soluble TRAIL-R is used to inhibit the biological activity of TRAIL, eg, to prevent such apoptosis in cells susceptible to TRAIL-mediated apoptosis.

[0087] TRAIL-R can be administered to a mammal to treat a TRAIL-mediated disease. Such TRAIL-mediated diseases include conditions caused or exacerbated (directly or indirectly) by TRAIL.

TRAIL-R may be useful for treating thrombotic microangiopathy. One such disease is thrombotic thrombocytopenic purpura (TTP) (Kwaan,
H. C. , Semin. Hematol. , 24:71, 1987; Thomp.
Son et al., Blood, 80: 1890, 1992). The increase in mortality associated with TTP is described in US Pat. S. Centers for Disease Control
(Torok et al., Am. J. Hematol. 50:84, 1995).

Plasma from patients suffering from TTP (including HIV ⁺ and HIV ⁻ patients)
Induces apoptosis of human endothelial cells of skin microvascular origin but not of macrovascular origin (Laurence et al., Blood, 87: 3245, April 1996).
March 15). Thus, the plasma of TTP patients may contain one or more factors that directly or indirectly induce apoptosis. As described in PCT application WO 97/01633 (incorporated herein),
RAIL 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) (Mo
ake, J .; L. , Lancet, 343: 393, 1994; Melnyk et al. (Arch. Intern. Med., 155: 2077, 1995);
mpson et al., supra). One embodiment of the present invention is a TRAI for treating a condition often referred to as "adult HUS" (even if it may affect children).
It relates to the use of LR. A disease known as childhood / diarrhea-related HUS is adult H
Etiology is different from US.

Other conditions characterized by small blood vessel thrombus formation can be treated with TRAIL-R. Such conditions include, but are not limited to: Cardiac problems seen in about 5-10% of pediatric AIDS patients are thought to involve small vessel thrombus formation. Breakage of microvessels in the heart has been reported in multiple sclerosis patients. Another example is the treatment of systemic lupus erythematosus (SLE).

[0092] In one embodiment, the patient's blood or plasma is ex vivo TRA
Contact with IL-R. The TRAIL-R can be bound to a suitable chromatography matrix by conventional methods. The patient's blood or plasma
Before returning to the patient, it is passed through a chromatography column containing TRAIL-R bound to the matrix. As the immobilized receptor binds TRAIL, TRAIL protein is removed from the patient's blood.

[0093] Alternatively, TRAIL-R may be administered to patients suffering from thrombotic microangiopathy in vivo.
Can be administered. In one embodiment, the soluble form of TRAIL-R
Is administered to the patient.

Accordingly, the present invention provides a method of treating thrombotic microangiopathy, comprising using an effective amount of TRAIL-R. A TRAIL-R polypeptide
It can be used in in vivo or ex vivo procedures to block TRAIL-mediated damage (eg, apoptosis) to microvascular endothelial cells.

[0095] TRAIL-R can be used in conjunction with other agents that are useful in treating certain diseases. Laurance et al. (Blood 87: 3245, 1996).
), The TT of microvascular endothelial cells in an in vitro study reported by
Some reduction in P plasma-mediated apoptosis was achieved by using normal plasma from which anti-Fas blocking antibodies, aurin tricarboxylic acid, or cryoprecipitate had been removed.

Thus, patients can be treated with a combination of an agent that inhibits Fas-Licand-mediated apoptosis of endothelial cells and an agent that elicits TRAIL-mediated apoptosis of endothelial cells. In one embodiment, TRAIL-R and anti-Fas
Both blocking antibodies are administered to a patient suffering from a disease characterized by thrombotic microangiopathy such as TTP or HUS. Examples of blocking monoclonal antibodies directed against the Fas antigen (CD95) are described in PCT Application Publication No. WO95, incorporated herein by reference.
/ 10540.

[0097] Another embodiment of the present invention relates to the use of TRAIL-R to reduce TRAIL-mediated cell death of T cells in HIV infected patients. The role of T cell apoptosis in the development of AIDS has been the subject of numerous studies (eg, Me
yaard et al., Science 257: 217-219, 1992; Grou.
x et al. Exp. Med. , 175: 331, 1992; and Oyaizu
Et al., Cell Activation and Apoptosis in H.
IV Infection, Supervised by Andrieu and Lu, Plenum
Press, New York, 1995, pp. 101-114).
. Several studies have investigated the role of Fas-mediated apoptosis, and the involvement of interleukin-1β converting enzyme (ICE) has also been investigated (Estaquie).
r et al., Blood 87: 4959-4966, 1996; Mitra et al., Im.
Munology 87: 581-585, 1996; Katsikis et al., J.
. Exp. Med. 181: 2029-2036, 1995). It is believed that T cell apoptosis can occur by a number of mechanisms.

It is believed that at least some of the T cell deaths seen in HIV ⁺ patients are mediated by TRAIL. I do not want to be bound by theory, but such a TR
AIL-mediated T cell death is thought to occur by a mechanism known as activation-induced cell death (AICD).

Active human T cells are induced to undergo programmed cell death (apoptosis) by triggering by the CD3 / T cell receptor complex,
This is a process called activation-induced cell death (AICD). AICD of CD4 ⁺ T cells isolated from asymptomatic individuals infected with HIV has been reported (Groux et al., Supra). Thus, AICD may play a role in the depletion of CD4 ⁺ T cells and in the development of AIDS in HIV-infected individuals.

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 those patients. provide. In one embodiment, the patient has
She is asymptomatic at the start of treatment with TRAIL-R. If desired, before treatment
Peripheral blood T cells can be extracted from HIV ⁺ patients and tested for susceptibility to TRAIL-mediated cell death by conventional methods.

[0101] In one embodiment, the patient's blood or plasma is ex vivo TRA
Contact with IL-R. The TRAIL-R can be bound to a suitable chromatography matrix by conventional methods. The patient's blood or plasma
Before returning to the patient, it is passed through a chromatography column containing TRAIL-R bound to the matrix. Because the immobilized TRAIL-R binds TRAIL, TRAIL protein is removed from the patient's blood.

When treating HIV ⁺ patients, TRAIL-R can be used in combination with other inhibitors of T cell apoptosis. Fas-mediated apoptosis has also been implicated in T cell depletion in HIV ⁺ individuals (Katsikis et al., J. Exp. Me.
d. 181: 2029-2036, 1995)). Thus, patients susceptible to both Fas ligand (Fas-L) -mediated and TRAIL-mediated T cell death will require agents that block TRAIL / TRAIL receptor interaction and Fas-L /
It can be treated with both agents that block the Fas interaction. Agents suitable for blocking the binding of Fas-L to Fas include soluble Fas polypeptides; soluble Fas polypeptides in oligomeric form (eg, sFas / Fc dimers); Anti-Fas antibody that binds Fas without transmitting a specific signal; anti-Fas-L antibody that blocks binding of Fas-L to Fas; and binds Fas but does not transmit a biological signal that triggers apoptosis Fas-L
Muteins, but are not limited to. Preferably, the antibodies used in the method are monoclonal antibodies. Examples of suitable agents for blocking Fas-L / Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, which is incorporated herein by reference.

[0103] 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 by known methods used to produce pharmaceutically useful compositions. TRAIL-R is a pharmaceutically acceptable diluent (e.g., saline, Tris-HCl, acetate), either alone or with other known actives suitable for a given indication. And phosphate buffered solutions), preservatives (eg, thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and / or carriers and can be combined in a mixture. Formulations suitable for pharmaceutical compositions include Remington's
Pharmaceutical Sciences, 16th edition, 1980, Mac
k Publishing Company, Easton, PA.

Further, such compositions may be complexed with polyethylene glycol (PEG), metal ions, or included in polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextrans, and the like. Alternatively, it can contain TRAIL-R contained in liposomes, microemulsions, micelles, unilamellar or multilamellar vesicles, erythrocyte ghosts or spheroblasts. Such a composition has a T
It is selected for its intended use as it affects the physical state, solubility, stability, rate of in vivo release, and rate of in vivo clearance of RAIL-R. TRAIL-R expressed on the surface of cells can also be used.

The compositions of the present invention contain a TRAIL-R polypeptide in any of the forms disclosed herein, including native proteins, variants, derivatives, oligomers and biologically active fragments. sell. In a specific embodiment, 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, for example, topically, parenterally, or by inhalation. The term "parenteral" includes injection by, for example, subcutaneous, intravenous or intramuscular routes, including topical administration. Sustained release from the implant is also contemplated. Those of skill in the art will understand that appropriate dosages will vary depending upon factors such as the nature of the condition being treated, the weight, age and general condition of the patient, and the route of administration. Preliminary test doses can be determined by animal studies, and increasing or decreasing the dosage administered to humans is accomplished by pharmaceutical arts.

[0107] Compositions comprising the TRAIL-R nucleic acid in a physiologically acceptable formulation are also contemplated. TRAIL-R DNA can be formulated, for example, for injection. Antibodies that bind an antibody TRAIL-R polypeptide are provided herein. The TRAIL-R protein of FIG. 1 or 2 (SEQ ID NO: 2 or 4) can be used as an immunogen when producing antibodies immunoreactive with it. Alternatively, another form of TRAIL-R, such as a fragment or a fusion protein, is used as the immunogen.

The present invention therefore provides an antibody obtained by immunizing an animal with TRAIL-R of FIG. 1 or 2, or an immunogenic fragment thereof. A method of producing an antibody comprises immunizing an animal with a TRAIL-R polypeptide, thereby generating an antibody directed against the TRAIL-R in the animal. The desired antibody can be purified, for example, from the serum of the animal by conventional techniques.

Some procedures for producing polyclonal and monoclonal antibodies include Mo.
noclonal Antibodies, Hybridomas: A New
Dimension in Biological Analyses, Ke
nnet et al. (supervised), Plenum Press, New York (1980);
And Antibodies: A Laboratory Manual, Ha
rrow and Land (supervised), Cold Spring Harbor L
laboratory Press, Cold Spring Harbor, NY (1
988). The production of monoclonal antibodies directed against TRAIL-R is described in more detail in Example 4.

The antigen-binding fragments of such antibodies, which can be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include Fa
b, and F (ab ') ² fragments, but are not limited thereto. Antibody fragments and derivatives produced by genetic engineering techniques are also provided.

The monoclonal antibodies of the present invention include chimeric antibodies, for example, humanized murine monoclonal antibodies. Such humanized antibodies can be produced by known techniques, which confer the advantage of low immunogenicity when administered to humans. In one embodiment, a humanized monoclonal antibody comprises the variable region of a murine antibody (or just the antigen binding site thereof) and a constant region derived from a human antibody. Alternatively, a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable region fragment derived from a human antibody (lacking the antigen binding site). Procedures for producing chimeric and further engineered monoclonal antibodies include those described by Riechmann et al. (Nature 332: 323, 1988).
), Liu et al. (PNAS 84: 3439, 1987), Larrick et al.
io / Technology 7: 934, 1989), and those described by Winter and Harris (TIPS 14: 139, May 1993).

In one embodiment, the antibodies are specific for a TRAIL-R of the invention and do not cross-react with other (non-TRAIL-R) proteins. Screening procedures that can identify such antibodies are well known and may include, for example, immunoaffinity chromatography.

[0113] Hybridoma cell lines that produce monoclonal antibodies specific for TRAIL-R are also contemplated herein. Such high-fridoma can be produced and identified by conventional techniques. One method of producing such a hybridoma cell line is to immunize an animal with TRAIL-R; harvest spleen cells from the immunized animal; fuse the spleen cells to a myeloma cell line. Generating hybridoma cells; and identifying a hybridoma cell line that produces a monoclonal antibody that binds TRAIL-R. These monoclonal antibodies can be recovered by conventional techniques.

In the use of these antibodies, the presence of the TRAIL-R polypeptide is determined in vivo.
There are uses in assays that detect either tro or in vivo. These antibodies are
It can also be used when purifying TRAIL-R protein by immunoaffinity chromatography.

In one embodiment, the antibodies further comprise the TRAIL TRAIL-R
Can be prevented from binding. Such an antibody may be, for example, a cell surface TR of TRAIL.
It can be used to block binding to AIL-R. Blocking antibody (Block
ing antibodies) can be identified using conventional assays.

[0116] Such antibodies may be used in in vitro procedures, or may be administered in vivo, to inhibit TRAIL-R mediated biological activity. Accordingly, diseases caused or exacerbated (directly or indirectly) by the interaction of TRAIL with cell surface TRAIL-R can be treated. Therapeutic methods comprise administering to the mammal a blocking antibody in an amount effective to inhibit TRAIL-R mediated biological activity to the mammal. Therefore, TRA
Diseases caused or exacerbated directly or indirectly by IL are treated. Monoclonal antibodies are generally preferred for use in such treatments. In one embodiment, an antigen binding antibody fragment is used.

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

Further provided herein is a conjugate comprising a detectable (diagnostic) or therapeutic agent conjugated to an antibody directed against TRAIL-R. Examples of such agents are set forth above. The conjugates can be in vitro or in v
Used in the ivo procedure. Nucleic acids The present invention provides TRAIL-R nucleic acids. Such nucleic acids include, but are not limited to, the human TRAIL-R DNA of FIGS. 1 and 2 (SEQ ID NOs: 1 and 3). The nucleic acid molecules of the present invention include both single- and double-stranded forms of TRAIL-R DNA, as well as its RNA complement. TRAI
LR DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PC
DNA amplified by R, and combinations thereof. Genomic DNA
Can be isolated by conventional techniques, for example, using the cDNA of FIG. 1 or 2 or a suitable fragment thereof as a probe.

[0119] In specific embodiments, the nucleic acids are useful in the production of TRAIL-R polypeptides, for example, in the recombinant expression systems discussed above. Any TRAIL-R in the form contemplated herein (e.g., full length TRAI
LR or a fragment thereof). TRAI
Specific embodiments of the LR-encoding DNA include the full-length human TRAIL-R encoding DNA (including the N-terminal signal peptide) and the full-length mature human TRAIL-R of FIG. Includes encoding DNA. Other embodiments encode a soluble TRAIL-R (e.g., encode an extracellular domain of the protein with or without a signal peptide).
Contains DNA.

The TRAIL-R nucleic acid fragments provided herein have uses, including but not limited to use as probes or primers. Oligonucleotides derived from the nucleic acid sequences disclosed herein can be used, for example, in polymerase chain reaction (PCR) as 3 'and 5' primers, thereby isolating TRAIL-R DNA fragments and Amplified.

A specific fragment of the TRAIL-R nucleotide sequence is TRAIL-R
It comprises at least 30 or at least 60 contiguous nucleotides of the DNA sequence. The nucleic acids provided herein are based on the DNA and R
NA complement is included along with both single-stranded and double-stranded forms of TRAIL-R DNA.

Other useful fragments of TRAIL-R nucleic acids include target TRAIL-R m
Antisense or sense oligonucleotides comprising single-stranded nucleic acid sequences (RNA or DNA) capable of binding to RNA (sense) or TRAIL-R DNA (antisense) sequences are included. An antisense or sense oligonucleotide according to the present invention may comprise a fragment of the coding region of the TRAIL-R DNA of FIG. 1 or 2. Such fragments generally have at least about 1
It comprises 4 nucleotides, preferably about 14 to about 30 nucleotides. The ability to derive antisense or sense oligonucleotides based on a cDNA sequence encoding a given protein is described, for example, by Stein and Cohen.
(Cancer Res. 48: 2659, 1988) and van der.
(BioTechniques 6: 958, 1988).

The binding of the antisense or sense oligonucleotide to the target nucleic acid sequence can be accomplished by one of several means, including increased duplex disassembly, immature termination of transcription or translation, or by other means. Results in the formation of a double helix that blocks the transcription or translation of DNA. Thus, these antisense oligonucleotides can be used to block TRAIL-R protein expression. Antisense or sense oligonucleotides further include oligonucleotides having a modified sugar-phosphate diester backbone (or other sugar linkages such as those described in WO 91/06629), in which case such Large sugar chains are resistant to endogenous nucleases. Such oligonucleotides with sugar-chain resistance include:
It is stable in vivo (ie, resistant to enzymatic degradation), but retains sequence specificity capable of binding to a target nucleotide sequence.

Other examples of sense or antisense oligonucleotides include WO90 / 10
Oligonucleotides, such as those described in US Pat. No. 448, and oligonucleotides covalently linked to other residues that increase the affinity of the oligonucleotide for a target nucleic acid sequence, such as poly (L-lysine), are included. . Still further, intercalating agents such as ellipticine and alkylating agents or metal complexes can be attached to sense or antisense oligonucleotides to alter the binding specificity of the antisense or sense oligonucleotide to a target nucleotide sequence.

The antisense or sense oligonucleotide can be isolated from the target nucleic acid by any gene transfer, including, for example, CaPO ₄ -mediated DNA transfection, electroporation, or by using a gene transfer vector such as Epstein-Barr virus. It can be introduced into cells containing the sequence.
In a preferred procedure, the 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 the murine retrovirus M-MuLV,
N2 (retrovirus derived from M-MuLV), or DCT
5A, DCT5B and DCT5C, a double copy vector (WO90 /
13641), but is not limited thereto.

A sense or antisense oligonucleotide can be introduced into a cell containing the target nucleotide sequence by formation of a conjugate with a ligand binding molecule, as described in WO 91/04753. Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, binding of the ligand binding molecule substantially impairs the ability of the ligand binding molecule to bind to the corresponding molecule or receptor, or to block the entry of a sense or antisense oligonucleotide or its bound form into a cell. Absent.

Alternatively, the sense or antisense oligonucleotide is WO90 / 104.
As described in No. 48, it can be introduced into cells containing the target nucleic acid sequence by formation of an oligonucleotide-lipid complex. The sense or antisense oligonucleotide-lipid complex is preferably dissociated intracellularly by endogenous lipase.

The following examples are provided to further illustrate specific embodiments of the present invention, and are not to be construed as limiting the scope of the invention. Example 1: TRAIL-R cDNA clone TRAIL-R clone was isolated from two types of cDNA libraries. The first cDNA library was derived from human foreskin fibroblasts and the second was from human peripheral blood lymphocytes (PBL).

The TRAIL-R cDNA isolated from PBL was found to contain the nucleotide sequence shown in FIG. 2 (SEQ ID NO: 3). Both forms of TRAIL-R (sequences shown in FIG. 1 or FIG. 2) were shown in clones isolated from a human foreskin fibroblast library.

The nucleotide sequences in FIG. 1 (SEQ ID NO: 1) and FIG. 2 (SEQ ID NO: 3) differ in two places. In FIG. 1, the nucleotide at position 145 is C, whereas nucleotide 145 in FIG. 2 is T; the nucleotide at position 971 in FIG. 1 is C, but is T in FIG. The amino acid sequence also differs in two places. Residue 35 is Pro in FIG. 1 (SEQ ID NO: 2) and Ser in FIG. 2 (SEQ ID NO: 4);
Ser in FIG. 1 and Leu in FIG. One possible explanation is that FIGS. 1 and 2
TRAIL receptors are allelic variants.

[0131] The TRAIL-R protein of Figure 1 (SEQ ID NO: 2) and Figure 2 (SEQ ID NO: 4) contains an N-terminal hydrophobic region that serves as a signal peptide. One signal peptide cleavage site predicted by computational analysis is similar to amino acid 55 in FIGS.
Comes next. Thus, cleavage of the signal peptide (amino acids 1-55) is believed to result in a mature protein that includes amino acids 56-386. The protein has an extracellular domain (amino acids 56-211), a transmembrane region (up to amino acids 212-232), and a C-terminal cytoplasmic domain (amino acids 233-386).
Up to).

The two amino acids in which the proteins of FIGS. 1 and 2 differ are signal peptide (3
5) and the cytoplasmic domain (position 310). Thus, the extracellular domains of the proteins of FIGS. 1 and 2 are identical. Example 2 Binding Assay The ability of TRAIL-R to bind TRAIL was examined in a slide binding assay. The DNA encoding the full length TRAIL-R of FIG. 1 was inserted into a mammalian expression vector called pDC409. pDC409 is McMahan
(EMBO J. 10: 2821-22832, 1991; incorporated herein by reference). Features added to pDC409 (compared to pDC406) include additional unique restriction sites in multiple cloning sites (mcs); three stop codons located downstream of mcs (one in each reading frame). ); And mc to facilitate sequencing of the DNA inserted into the mcs.
s downstream of the T7 polymerase promoter.

CV1-EBNA cells were transfected with the recombinant expression vector and cultured on glass slides to express TRAIL-R. The slide binding assay is generally described in Gearing et al. (EMBO J.8, incorporated herein by reference.
: 3667, 1989); McMahan et al. (EMBO J. 10: 2821,
1991) and Goodwin et al. (Eur. J. Immunol. 23:26).
31, 1993). Briefly, the transfected cells were transformed with the LZ-TRAIL fusion protein (
(See below). Next, the slides were washed and a ¹²⁵ I-labeled antibody specific for the leucine zipper (LZ) residue of the fusion protein was added. After incubation with the antibodies, the slides were washed, fixed, and immersed in a photographic emulsion. The assay showed that the TRAIL-R protein binds TRAIL.

The LZ-TRAIL used in the assay was the soluble TRAI used in the assay.
A fusion protein containing a leucine zipper fused to the N-terminus of the L polypeptide (L
Z-TRAIL). Expression constructs consist essentially of Wiley et al. (Except that the DNA encoding the Flag® peptide has been replaced with a sequence encoding a modified leucine zipper that allows trimerization. Immunit
y, 3: 673-682, 1995; incorporated herein by reference) as described for the production of the Flag®-TRAIL expression construct. The construct encoded a leader sequence from human cytomegalovirus followed by a leucine zipper fused to the N-terminus of the soluble TRAIL polypeptide in the expression vector pDC409. The TRAIL polypeptide is Wil
It contained amino acids 95-281 of human TRAIL (a fragment of the extracellular domain) as described in ey et al. (supra). The LZ-TRAIL was expressed in CHO cells and purified from culture supernatant. Example 3 Production of Soluble TRAIL-R An expression vector encoding a soluble TRAIL-R fusion protein was constructed as follows. The fusion protein comprised a soluble TRAIL-R fusion protein fused to the N-terminus of the IgG1 Fc region polypeptide mutein.

The DNA fragment encoding amino acids 1-208 (SEQ ID NO: 2) of FIG. 1 was isolated by polymerase chain reaction (PCR). Oligonucleotides defining the desired termini of the DNA fragment were used as 3 'and 5' primers in the PCR and the cDNA clone shown in FIG. 1 (SEQ ID NO: 1)
Was used as a template.

The Fc residue of the fusion protein was a human IgG1 Fc region polypeptide mutein. DNA and amino acid sequence information on this Fc mutein
No. 5,457,035 and Baum et al. (E.
MBOJ. 13: 3992-4001, 1994).

The procedure for producing an expression vector containing a TRAIL-R / Fc gene fusion comprises:
In general, Smith et al. (Cell 73: 1349-1, incorporated herein by reference).
360, 1993) and Fanslow et al. (J. Immunol. 149: 6).
55-660, 1992). The expression vector pDC409 described in Example 2 was used. CV1 / EBN was obtained using the obtained recombinant expression vector.
A cells were transfected and cultured to express and secrete TRAIL-R / Fc from the cells. Example 4 Monoclonal Antibody that Binds TRAIL-R This example details a method for making a monoclonal antibody that binds TRAIL-R. Suitable immunogens that can be used to raise such antibodies include purified TRAIL-R protein or immunogenic fragments thereof, such as the extracellular domain, or fusion proteins containing TRAIL-R (eg, soluble TRAIL-R / Fc fusion protein), but is not limited thereto.

[0138] Purified TRAIL-R can be used to raise monoclonal antibodies immunoreactive therewith, using conventional techniques as described in US Pat. No. 4,411,993. Briefly, mice are immunized with TRAIL-R immunogen emulsified in Freund's complete adjuvant and injected subcutaneously or intraperitoneally in an amount of 10-100 μg. Ten to twelve days later, the immunized animals are boosted with additional TRAIL-R emulsified in Freund's incomplete adjuvant. Thereafter, mice are periodically boosted with a weekly-twicely week immunization schedule. Serum samples are collected periodically by retro-orbital bleeding or tail excision and tested by dot blot assay, ELISA (enzyme linked immunosorbent assay) or TRAIL for TRAIL-R antibodies.
Determined by inhibition of binding.

After detection of appropriate antibody titers, positive animals are given a single last intravenous injection of TRAIL-R in saline. Three to four days later, the animals are sacrificed, spleen cells are harvested, and the spleen cells are transferred to a murine myeloma cell line, eg, NS1 or preferably
Ag8.653 (ATCC CRL 1580). The fusion results in hybridoma cells, which are plated in HAT (hypoxanthine, aminopterin and thymidine) selective medium in a number of microtiter plates to prevent the growth of unfused cells, myeloma hybrids and spleen cell hybrids I do.

The hybridoma cells were prepared according to the method of Engval et al., Immunochem. 8
: 871,1971 and U.S. Patent No. 4,703,004 by ELISA for reactivity against purified TRAIL-R. A preferred screening method is described in Beckmann et al.
Immunol. 144: 4212, 1990). Positive hybridoma cells can be injected intraperitoneally into syngeneic BALB / c mice to generate ascites containing high concentrations of anti-TRAIL-R monoclonal antibodies. Alternatively, the hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies generated in mouse ascites can be purified by gel exclusion chromatography after ammonium sulfate precipitation. Alternatively, affinity chromatography based on binding of the antibody to protein A or protein G can be used in the same manner as affinity chromatography based on binding to TRAIL-R.

[Sequence list]

[Brief description of the drawings]

FIGS. 1A and 1B show the nucleotide sequence of the human TRAIL receptor cDNA and the amino acid sequence encoded thereby.

FIGS. 2A and 2B show the nucleotide and encoded amino acid sequences of another human TRAIL receptor cDNA clone. The amino acid sequences of FIGS. 2A-2B differ from the sequences shown in FIGS. 1A-1B at two locations.

──────────────────────────────────────────────────続 き Continued on the front page (51) Int.Cl. ⁷ Identification code FI Theme coat ゛ (Reference) A61P 43/00 111 C07K 16/28 C07K 14/715 16/46 16/28 C12P 21/02 C 16/46 21/08 C12P 21/02 C12N 15/00 ZNAA // C12P 21/08 A61K 37/02 F term (reference) 4B024 AA01 AA20 BA44 BA63 CA04 CA07 DA02 DA06 DA12 EA04 FA15 GA03 GA11 HA01 HA15 4B064 AG20 AG27 CA02 CA06 CA10 CA19 CA20 CC24 DA13 4C084 AA01 AA07 AA13 BA01 BA22 BA41 BA44 CA53 DA39 DA45 ZA362 ZA532 ZB212 4C085 AA13 AA14 BB11 DD63 DD88 4H045 AA10 AA11 BA10 BA41 CA40 DA51 DA76 EA50 FA72 FA74

Claims (27)

[Claims] 1. An isolated DNA comprising a nucleotide sequence encoding a TRAIL receptor polypeptide (TRAIL-R), wherein the TRAIL-R comprises: (a) the TRAIL-R polypeptide of FIG. 1; b) a TRAIL-R polypeptide of FIG. 2; and (c) a fragment of the polypeptide of (a) or (b), wherein
The above DNA selected from the group consisting of said fragments capable of binding L. 2. The DNA according to claim 1, wherein said fragment is a soluble TRAIL-R polypeptide. 3. The method according to claim 1, wherein the soluble TRAIL-R polypeptide is selected from the group consisting of FIG.
The DNA according to claim 2, comprising the extracellular domain of TRAIL-R. 4. The DNA according to claim 1, wherein the TRAIL-R lacks a transmembrane region. 5. The TRAIL-R polypeptide comprises the amino acid sequence of residues xy of FIG. 1 or 2, wherein x is an integer from 1-98 and y is an integer from 181-386. The DNA according to claim 1, which is: 6. x is an integer selected from the group consisting of 1 and 56;
The DNA according to claim 5, wherein y is an integer selected from the group consisting of 181, 208, 211 and 386. 7. An isolated DNA encoding a TRAIL-R polypeptide, wherein the polypeptide comprises: (a) residues 1-386 of FIG. 1; (b) residues 1-386 of FIG. (C) residues 56-386 of FIG. 1; (d) residues 56-386 of FIG. 2; (e) residues 1-211 of FIG. 1; (G) the isolated DNA comprising an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of residues 56-211 of FIG. 8. The polypeptide comprises: (a) residues 1-386 of FIG. 1; (b) residues 1-386 of FIG. 2; (c) residues 56-386 of FIG. 1; (E) residues 1-211 of FIG. 1; (f) residues 1-211 of FIG. 2; and (g) residues 56-211 of FIG. The DNA according to claim 7, which comprises an amino acid sequence of any of the above. 9. An isolated DNA comprising at least 60 adjacent nucleotides of the nucleotide sequence of FIG. 1 or FIG. 10. An expression vector comprising the DNA according to claim 1, claim 2, claim 3, claim 5, claim 7, or claim 8. 11. A method for producing a TRAIL-R polypeptide, comprising culturing a host cell containing the vector according to claim 10 under conditions that promote TRAIL-R expression. The above method comprising recovering the peptide. 12. A purified TRAIL-R polypeptide, comprising: (a) a mature TRAIL-R polypeptide of FIG. 1; (b) a mature TRAIL-R polypeptide of FIG. 2; and (c) ( A fragment of the polypeptide of a) or (b), which comprises TRAI
Such a purified TRAIL-R polypeptide selected from the group consisting of said fragments capable of binding L. 13. The TRAIL-R of claim 12, wherein said fragment is a soluble TRAIL-R polypeptide. 14. The TRAIL-R of claim 13, wherein said soluble TRAIL-R polypeptide comprises the extracellular domain of TRAIL-R of FIG. 1 or FIG.
. 15. The TRAIL-R lacks a transmembrane region.
2. TRAIL-R according to 2. 16. The TRAIL-R polypeptide comprises the amino acid sequence of residues xy of FIG. 1 or 2, wherein x is an integer from 1-98, and y
The TRAIL-R according to claim 12, wherein is an integer of 181-386. 17. The TRAIL- according to claim 16, wherein x is an integer selected from the group consisting of 1 and 56, and y is an integer selected from the group consisting of 181, 208, 211 and 386. R. 18. A purified TRAIL-R polypeptide, comprising: (a) residues 56-386 of FIG. 1; (b) residues 56-386 of FIG. 2; and (c) residue 56-386 of FIG. 211. The above purified TRAIL-R polypeptide comprising an amino acid sequence at least 90% identical to an amino acid sequence selected from the group consisting of 211. 19. The polypeptide as defined by the group consisting of: (a) residues 56-386 of FIG. 1; (b) residues 56-386 of FIG. 2; and (c) residues 56-211 of FIG. 19. The TRAIL- according to claim 18, comprising the selected amino acid sequence.
R. 20. An oligomer comprising at least two TRAIL-R polypeptides according to claim 12, claim 13, claim 18 or claim 19. 21. The oligomer of claim 20, wherein said oligomer comprises 2-4 soluble TRAIL-R polypeptides. 22. A fusion protein comprising a TRAIL-R polypeptide and an antibody-derived Fc polypeptide, wherein the TRAIL-R polypeptide is a soluble fragment of the TRAIL-R protein of FIG. 1 or FIG. The above fusion protein, which can bind TRAIL. 23. A dimer comprising the two fusion proteins according to claim 22. 24. A composition comprising the polypeptide of claim 12 and a physiologically acceptable diluent, excipient or carrier. 25. A composition comprising the oligomer according to claim 20 and a physiologically acceptable diluent, excipient or carrier. 26. An antibody directed against the TRAIL-R polypeptide of claim 12, or an antigen-binding fragment of said antibody. 27. The antibody according to claim 26, wherein said antibody is a monoclonal antibody.