MXPA99007234A - Receptor that binds trail - Google Patents

Abstract

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

Description

RECEIVER THAT JOINS THE LIGANDO INDUCTOR BY APOPTOSIS RELATED TO TNF (LIART) BACKGROUND OF THE INVENTION A protein known as an inducer ligand for TNF-related apoptosis (TRIAL) is a member of the family of tumor necrosis factor-ligands (Wiley et al., Immunity, 3: 673-682, 1995). LIART has demonstrated the ability to induce apoptosis of certain transformed cells including a number of different types of cancer cells as well as virally infected cells (PCT Application WO 97/01633 and Wiley et al., Supra). It could be proved that the identification of the receptor proteins that bind to LIART is useful in a further study of the biological activities of LIART. However, prior to the present invention, no receiver for LIART had been reported. COMPENDIUM OF THE INVENTION The present invention is directed to a novel protein designated LIART receptor (R-LIART), which binds to a protein as an inducer ligand by TNF-related apoptosis (LIART). The DNA encoding the R-LIART and the expression factors comprising said DNA are provided. One method for producing R-LIART polypeptides comprises culturing host cells transformed with a recombinant expression vector encoding R-LIART, under conditions that promote the expression of R-LIART, then recovering the R-LIART polypeptides expressed from | culture. Antibodies that are immunoreactive with R-LIART are also provided. BRIEF DESCRIPTION OF THE FIGURES Figure 1 presents the nucleotide sequence of a fragment of human LIART receptor DNA, as well as the amino acid sequence encoded by it. This DNA fragment is described in Example 3. Figure 2 shows the results of the analysis described in example 7. In the analysis, a soluble fusion protein of R-LIART / Fc blocks the LIART-induced apoptosis of the cells of Jurkat. Figure 3 presents the results of the experiment described in example 8. It was shown that the indicated compounds inhibit the apoptosis of cells expressing the LIART receptor. DETAILED DESCRIPTION OF THE INVENTION In the present, a novel protein designated LIART receptor (R-LIART) is provided. R-LIART binds to the cytokine designated induction ligand for apoptosis related to TNF (LIART). As discussed below, certain uses of the R-LIART come from this ability to join LIART. R-LIART is used to inhibit biological activities of LIART, or for example, to purify LIART by affinity chromatography. The nucleotide sequence in the coding region of a human LIART receptor DNA is presented in SEQ I D NO: 1. The amino acid sequence encoded by the DNA sequence of SEQ I D NO: 1 is shown in SEQ I D NO: 2. This information identifies the LIART receptor protein as a member of the family of tumor necrosis factor receptor (R-TNF) receptors (reviewed in Smith et al., Cell 76: 959-962, 1994). The extracellular domain contains repetitions rich in cysteine; it has been reported that these motifs are important for the binding of ligands in other receptors of this family. R-LIART contains a "dead domain", so-called, in the cytoplasmic region; said domains in certain receptors are associated with the transduction of apoptotic signals. These and other aspects of the protein are discussed in more detail below. The R-LIART protein or myogenic fragments thereof can be used as immunogens to generate antibodies that are immunoreactive therewith. In one embodiment of the invention, the antibodies are monoclonal antibodies. A human R-LIART protein was purified as described in example 1. In Example 2, the amino acid sequence information derived from R-LIART fragments was presented. A method of the invention is directed to a purified human R-LIART protein that is capable of binding to LIART, wherein R-LIART is characterized by comprising the amino acid sequence VPAN EGD (amino acids 327 to 333 of SEQ ID NO: 2). In another embodiment, R-LIART additionally comprises the sequence ETLRQCFDDFADLVPFDSWEPLMRKLGLMDNEIKVAKAEAAGHRDTLXT ML (amino acids 336 to '386 of SEQ ID NO: 2, with an unknown amino acid indicated as X). R-LIART fragments comprising only one of these characterization amino acid sequences were also provided. The nucleotide sequence of a DNA fragment of R-LIART, and the amino acid sequence encoded by it, are presented in Figure 1 (SEQ ID NO: 3 and SEQ ID NO: 4): see example 3. The sequence of amino acids presented in Figure 1 has characteristics of the so-called "dead domains" found in the cytoplasmic region of other receptor proteins. These domains have been reported to be associated with the transduction of apoptotic signals. Dead cytoplasmic domains have been identified in the Fas antigen (Itoh and Nagata, J. Biol. Chem. 268: 10932, 1993), type I TNF receptor (Tartaglia et al., Cell 74: 845, 1993). DR3 (Chinnaiyan et al., Science 274: 990-992, 1996) and CAR-1 (Brojatsch et al., Cell 87: 845-855, 1996). The role of these dead domains to initiate intracellular apoptotic signaling cascades is discussed below. SEQ ID NO: 1 presents the nucleotide sequence of the coding region of a human LIART receptor DNA, including an initiation codon (ATG) and a termination codon (TAA). The amino acid sequence encoded by the DNA of SEQ ID NO: 1 was presented in SEQ ID NO: 2. The fragment described in Figure 1 corresponds to the region of R-LIART that is presented as amino acids 336 to 386 in SEQ I D NO: 2. The R-LIART protein of SEQ ID NO: 2 includes an N-terminal hydrophobic region that functions as a signal peptide, followed by an extracellular domain, a transmembrane region comprising amino acids 21 1 to 231 and a cytoplasmic domain C -terminal. The computer analysis provides that the signal peptide corresponds to residues 1 to 51 of SEQ I D NO: 2. The separation of the signal peptide could thus give a mature protein comprising amino acids 52 to 440 of SEQ I D NO: 2. The calculated molecular weight for a mature protein containing residues 52 to 440 of SEQ I D NO: 2 is approximately 43 kilodaltons. The following most likely computer-predicted signal peptidase sites (in descending order) are presented after amino acids 50 and 58 of SEQ I D NO: 2. In another embodiment of the invention, the N-terminal residue of a mature R-LIART protein is the isoleucine residue at position 56 of SEQ I D NO: 2. The sequences of several fragments of tryptic digestion peptides of R-LIART were determined by a combination of N-terminal sequencing and Nano-ES MS / MS (nano-randomized random mass spectrometry). The N-terminal amino acid of one of the peptide fragments was isoleucine at position 56 of SEQ I D NO: 2. Since this fragment was not preceded by a trypsin separation site, the residue (lie) 56 may correspond to the N-terminal residue resulting from • separation of the signal peptide. A further embodiment of the invention is directed to mature R-LIART having amino acid 54 as the N-terminal residue. In a preparation of R-LIART (a soluble R-LIART / Fc fusion protein expressed in CV1-EBNA cells), the signal peptide was separated after residue 53 of SEQ I D NO: 2. The skilled person will recognize that the molecular weight of particular preparations of the R-LIART protein may differ, according to such factors as in the degree of glycosylation. The glycosylation pattern of a particular preparation of R-LIART may vary according to the type of cells in which, for example, the protein is expressed. In addition, a given preparation may include differentially glycosylated species of the protein. R-LIART polypeptides with or without associated native pattern glycosylation are provided herein. The expression of R-LIART polypeptides in bacterial expression systems, such as E. coli, provides non-glycosylated molecules. In one embodiment, the protein is characterized by a molecular weight within the range of about 50 to 55 kilodaltons, which is the molecular weight determined for a full-length native human R-LIART preparation. The molecular weight can be determined by SDS-polyacrylamide gel electrophoresis (SDS-PAGE).

Example 1 presents a method for purifying a protein from R-LIART. The Jurkat cells are disrupted, and the subsequent purification process includes affinity chromatography (using a chromatography matrix containing LIART) and reverse phase HPLC. The R-LIART polypeptides of the present invention can be purified by any suitable alternative procedure, using known protein purification techniques. In an alternative procedure, the chromatography matrix comprises an antibody that binds to R-LIART. Other cell types expressing R-LIART (eg, PS-1 cells described in Example 2) can be replaced by Jurkat cells. Cells can be altered by any of a number of known techniques, including freeze-thaw cyclization, sound treatment, mechanical alteration, or by the use of cell lysing agents. The degree of purity desired depends on the intended use of the protein. A relatively high degree of purity is desired when the protein is to be administered in vivo, for example. Advantageously, the R-LIART polypeptides are purified so that no protein bands corresponding to other proteins (without R-LIART) are detectable under analysis by SDS-polyacrylamide gel electrophoresis (SDS-PAGE). It will be recognized by the expert in the relevant field that multiple bands corresponding to the R-LIART protein can be visualized by SDS-PAGE, due to the differential glycosylation, differential post-translational processing and the like. R-LIART is most preferably purified at substantial homogeneity, as indicated by a single protein band under analysis by SDS-PAGE. The protein band can be visualized by silver tinsion, Coomassie blue tinsion, or (if the protein is radiolabeled) by autoradiography. The present invention encompasses R-LIART in various forms, including those that occur naturally or that are produced through various techniques such as procedures involving recombinant DNA technology. Said forms of R-LIART include, but are not limited to, fragments, derivatives, variants and oligomers of R-LIART, as well as fusion proteins containing R-LIART or fragments thereof. R-LIART can be modified to create derivatives thereof by forming covalent conjugates or aggregates with other chemical moieties, such as glycosyl groups, lipids, phosphate, acetyl groups and the like. The covalent derivatives of R-LIART can be prepared by linking the chemical moieties to functional groups on the amino acid side chains of R-LIART or at the N-terminus or C-terminus of an R-LIART polypeptide. Conjugates comprising diagnostic agents (detectable) or therapeutic agents linked to R-LIART are contemplated herein, as discussed in more detail below. Other derivatives of R-LIART within the scope of this invention include covalent conjugates or aggregates of R-LIART polypeptides with other proteins or polypeptides, such as by synthesis in recombinant culture as N-terminal or C-terminal fusions. Examples of fusion protein are discussed below in relation to oligomers of R-LIART. In addition, fusion proteins containing R-LIART may comprise aggregated peptides to facilitate the purification and identification of R-LIART. Such peptides include, for example, poly-His or the antigenic identification peptides described in the U.A. No. 5,01 1, 912 and in Hopp et al., Bio / Technology 6: 1204, 1988. One such peptide is the Flag® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, which it is highly antigenic and provides an epitope reversibly linked by a specific monoclonal antibody, allowing rapid analysis and easy purification of the expressed recombinant protein. A murine hybridoma designated R41 1 produces a monoclonal antibody that binds the Flag® peptide in the presence of certain divalent metal cations, as described in the Patent of E. U.A. 5.01 1, 912, incorporated herein by reference. The 4E1 1 hybridoma cell line has been deposited in the American Type Culture Collection under accession number H B 9259. The monoclonal antibodies that bind to the Flag® peptide are available from Eastman Kodak Co., Scientific Imaging Systems Division. New Haven Connecticut. The forms bound to the cell membrane and the soluble (secreted) forms of R-LIART are provided herein. Soluble R-LIART can be identified (and distinguished from counterparts attached to non-soluble membranes) by separating the intact cells expressing a R-LIART polypeptide from the culture medium, e.g. , by centrifugation and analyzing the medium (supernatant) for the presence of the desired protein. The presence of R-LIART in the medium indicates that the protein is secreted from the cells and is therefore a soluble form of the desired protein. Soluble forms of receptor proteins usually lack the transmembrane region that could cause retention of the protein on the cell surface. In one embodiment of the invention, a soluble R-LIART polypeptide comprises the extracellular domain of the protein. A soluble R-LIART polypeptide can include the cytoplasmic domain, or a portion thereof, while the polypeptide is secreted from the cell in which it is produced. An example of a soluble R-LIART is a soluble human R-LIART comprising 52 to 210 amino acids of SEQ I D NO: 2. Other soluble R-LIART polypeptides include, but are not limited to, polypeptides comprising amino acids xa 210 of SEQ ID NO: 2, wherein x is an integer from 51 to 59. Soluble forms of R-LIART have certain advantages on the way of binding of the membrane of the protein. The purification of the protein from the recombinant host cells is facilitated, since the soluble proteins are secreted from the cells. In addition, soluble proteins are generally more suitable for certain applications, v. gr. , for intravenous administration.

The fragments of R-LIART are provided herein. Such fragments can be prepared by any number of conventional techniques. The desired peptide fragments can be chemically synthesized. An alternative involves generating fragments of R-LIART by ezimatic digestion, e.g. , treating the protein with a known enzyme to separate proteins at the sites defined by the particular amino acid residues. Yet another suitable technique involves isolating and amplifying a DNA fragment encoding a desired polypeptide fragment by polymerase chain reaction (PCR). The oligonucleotides defining the desired terminations of the DNA fragment are used as the 5 'and 3' primers in PCR. Examples of fragments are those comprising at least 20, or at least 30, contiguous amino acids of the sequence of SEQ I D NO: 2. The fragments derived from the cytoplasmic domain find use in studies of signal transduction mediated by R-LIART, and in the regulation of cellular processes associated with the transduction of biological signals. Fragments of R-LIART polypeptides can also be used as immunogens to generate antibodies. Particular embodiments are directed to fragments of R-LIART polypeptides that retain the ability to bind LIART. Said fragment may be a soluble R-LIART polypeptide as described above. The variants present in the nature of the R-LIART protein of SEQ I D NO: 2 are provided herein. These variants they include, for example, proteins that result from alternating mRNA cleavage events or from the proteolytic cleavage of the R-LIART protein. The alternating division of mRNA, for example, can produce a truncated but biologically active R-LIART protein, such as a form present in the soluble nature of the protein. Variations that are attributed to proteolysis include, for example, differences in the N or C termini of expression in different types of host cells, due to the proteolytic removal of one or more terminal amino acids of the R-LIART protein (generally from 1-5 terminal amino acids). R-LIART proteins in which differences in amino acid sequence can be attributed to genetic polymorphism (allelic variation between individuals that produce the protein) are also contemplated herein. The skilled person will also recognize that the positions to which the signal peptide is separated may differ from that predicted by the computer program and may vary according to such factors as the type of host cells used to express a recombinant R-LIART polypeptide. A protein preparation can include a mixture of protein molecules that have different N-terminal amino acids, which result from the separation of the signal peptide from more than one site. As discussed above, particular embodiments of mature R-LIART proteins provided herein include, but are not limited to, proteins having the residue at position 51, 52, 54, 56 or 59 of SEQ ID NO: 2 as the N-terminal amino acid. With respect to the present discussion of several domains of the R-LIART protein, the skilled artisan will recognize that the limits described before said regions of the protein are approximate. To illustrate, the boundaries of the transmembrane region (which can be predicted using computer programs available for that purpose) may differ from those described above. Therefore, soluble R-LIART polypeptides in which the C-terminus of the extracellular domain differs from the residue thus identified before contemplated herein. Other R-LIART DNAs present in nature and polypeptides include those derived from non-human species. Human R-LIART homologs of SEQ ID NO: 2, of other mammalian species, are contemplated herein, for example. Probes based on the human DNA sequence of SEQ ID NO: 3 or SEQ ID NO: 1 can be used to screen cDNA libraries derived from other mammalian species, using conventional cross-species hybridization techniques. The R-LIART DNA sequences may vary from the native sequences described herein. Due to the known degeneracy of the genetic code, where more than one codon can encode the same amino acid, a DNA sequence can vary from that shown in SEQ ID NO: 1 and still encode an R-LIART protein having the sequence of amino acids of SEQ I D NO: 2. Said variant DNA sequences may result from silent mutations (e.g., occurring during PCR amplification) or may be the product of deliberate mutagenesis of a native sequence. Therefore, among the DNA sequences provided herein are the native R-LIART sequences (e.g., cDNA comprising the nucleotide sequence presented in SEQ ID NO: 1) and DNA that degenerates as a result of the genetic code to a DNA sequence of R-LIART. Among the R-LIART polypeptides provided herein are variants of native R-LIART polypeptides that retain a biological activity of a native R-LIART. Such variants include polypeptides that are substantially homologous to native R-LIART, but that have an amino acid sequence different from that of a native R-LIART due to one or more deletions, insertions or substitutions. Particular embodiments include, but are not limited to, R-LIART polypeptides comprising from one to ten deletions, insertions or substitutions of amino acid residues, when compared to a native R-LIART sequence. The DNAs encoding R-LIART of the present invention include variants that differ from a native R-LIART sequence due to one or more deletions, insertions or substitutions, but which encode a biologically active R-LIART polypeptide. A biological activity of R-LIART is the ability to bind LIART.

Nucleic acid molecules capable of hybridizing to DNA of SEQ I D NO: 1 or SEQ I D NO: 3 under moderately strict or highly stringent conditions, and which encode a biologically active R-LIART, are provided herein. Such hybridizing nucleic acids include, but are not limited to, variant DNA sequences and DNA derived from non-human species, v. gr. , non-human mammals. Moderately strict conditions include conditions described in, for example, Sambrook et al., Molecular Cloning: A Laboratory Manual, 2nd ed. , Vol 1, pp. 1101-104. Cold Spring Harbor Laboratory Press, 1989. Moderate restriction conditions as defined by Sambrook et al., Include the use of a 5X SSC pre-wash solution, 0.5% SDS, 1 .0 mM EDTA (pH 8.0) and hybridization conditions of approximately 55 ° C, 5X SSC, overnight. Highly stringent conditions include high temperatures of superior hybridization and washing. One embodiment of the invention is directed to the DNA sequences that will hybridize to the DNA of SEQ ID NOS: 1 to 3, under highly stringent conditions, where conditions include hibrization at 68 ° C after washing in 0.1 X SSC / 0.1% SDS at 63-68 ° C. Certain DNA and polypeptides provided herein comprise nucleotide or amino acid sequences, respectively, that are at least 80% identical to a native R-LIART sequence. Also contemplated are embodiments in which an R-LIART DNA or polypeptide comprises a sequence that can be at least 90% identical, at least 95% identical or at least 98% identical to a native R-LIART sequence. The percentage identity can be determined, for example, by comparing the sequence information using the GAP computer program, version 6.0 described by Devereux et al., (Nucí.Aids Res. 12: 387, 1984) and available from University of Wisconsin Genetics Computer. Group (UWGCG). The preferred default parameters for the GAP program include: (1) a unit comparison matrix (containing a value of 1 for identities and 0 for non-identities) for nucleotides and the weighted comparison matrix of Gribskov and Burgess, Nucí. Acids Res. 14: 6745, 1986, as described by Schwartz and Dayhoff, eds. , Atlas of Protein Sequence and Structure, National Biomedical Research Foundation. , p. 353-358, 1979); (2) a penalty of 3.0 for each space and an additional penalty of 0.10 for each symbol in each space; and (3) without penalty for final spaces. In the particular embodiments of the invention, a variant R-LIART polypeptide differs in the amino acid sequence of a native R-LIART, but is substantially equivalent to a native R-LIART in a biological activity. An example is an R-LIART variant that binds LIART that essentially has the same binding affinity as a native R-LIART. The binding affinity can be measured by conventional methods, v. gr. , as described in the Patent of E. U.A. do not. 5,512,457.

The variant amino acid sequences may comprise conservative substitutions, meaning that one or more amino acid residues of native R-LIART are replaced by a different residue, but that the conservatively substituted R-LIART polypeptide retains a desired biological activity of the protein native (eg, the ability to join LIART). A given amino acid can be replaced by a residue that has similar physiochemical characteristics. Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Me, Val, Leu or Ala for another, or substitutions for one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. Other conservative substitutions, eg. , which involve substitutions of all regions that have similar hydrophobicity characteristics, are well known. In another example of variants, the sequences encoding Cys residues that are not essential for biological activity can be altered to cause the Cys residues to be suppressed or replaced with other amino acids, preventing formation in incorrect intramolecular disulfide bridges under the re-naturalization Certain receptors of the TN F-R family contain repeat motifs rich in cysteine in their extracellular domains (Marsters et al., J. Biol. Chem. 267: 5747-5750, 1992). These repeats are thought to be important for ligand binding. To illustrate, Marsters et al., Supra, reported that soluble TN F-R type 1 peptides lacking one of the repeats exhibited a tenfold reduction in binding affinity for TNFα and TNFβ; deletion of the second repeat resulted in a complete loss of the detectable binding of the ligands. The human R-LIART of SEQ ID NO: 2 contains two such cysteine-rich repeats, the first including residues 94 to 137, the second including residues 138 to 178. The cysteine residues within these domains rich in cysteine remain advantageously unchanged in the R-LIART variants, when retention of LIART binding activity is desired. Other variants were prepared by modifying adjacent dibasic amino acid residues, to increase expression in yeast systems in which the KEX2 protease activity is present. EP 212, 914 discloses the use of site-specific mutagenesis at inactive KEX2 protease processing sites in a protein. KEX2 protease processing sites are inactivated by deletion, addition or substitution of residues to alter the pairs of Arg-Arg, Arg-Lys and Lys-Arg to eliminate the presentation of these adjacent basic residues. Mature human R-LIART contains said pairs of adjacent basic residues at amino acids 72-73, 154- 155, 322-323, 323-324 and 359-360 of SEQ ID NO: 2. The Lys-Lys pairs are considerably less susceptible to the separation of KEX2 and the conversion of Arg-Lys or Lys-Arg to Lys-Lys represents a conservative and preferred approach to inactivate KEX2 sites.

The R-LIART polypeptides, including variants and fragments thereof, can be tested for biological activity in any suitable assay. The ability for an R-LIART polypeptide to bind LIART can be confirmed by conventional binding assays, examples of which are described below. Expression Systems The present invention also provides recombinant expression and cloning vectors containing R-LIART DNA, as well as host cells containing the recombinant vectors. Expression vectors comprising R-LIART DNA can be used to prepare R-LIART polypeptide encoded by DNA.

A method for producing R-LIART polypeptide comprises culturing host cells transformed with a recombinant expression vector encoding R-LIART, under conditions that promote the expression of R-LIART, then recovering polypeptides from R-LIART expressed from the crop. The skilled artisan will recognize that the procedure for purifying the expressed R-LIART will vary according to such factors as the type of host cells employed and wherein R-LIART is bound to a membrane or is a soluble form that is secreted from the host cell. Any suitable expression system can be used.

The vectors include a DNA encoding a polypeptide of R-LIART, operably linked to suitable transcriptional or translational regulatory nucleotide sequences, such like those derived from a gene of a mammal, microbial, vira! or of insects. Examples of regulatory sequences include transcriptional promoters, operators or enhancers, a ribosomal binding site of mRNA and appropriate sequences that control the initiation and termination of transcription and translation. The nucleotide sequences are operably linked when the regulatory sequence functionality refers to the DNA sequence of R-LIART. Therefore, a promoter nucleotide sequence is operably linked to an R-LIART DNA sequence if the sequence of nucleotide promoters controls the transcription of the R-LIART DNA sequence. An origin of replication that confers the ability to replicate in the desired host cells and a selection gene by which transformants are identified is generally incorporated in the expression vector. In addition, a sequence encoding an appropriate signal peptide (native or heterologous) can be incorporated into expression vectors. A DNA sequence for a signal peptide (secretory leader) can be fused in frame to the R-LIART sequence so that R-LIART is initially translated as a fusion protein comprising a signal peptide. A signal peptide that is functional in the intended host cells promotes the extracellular secretion of the R-LIART polypeptide. The signal peptide is separated from the R-LIART polypeptide by secreting R-LIART from the cell.

Suitable host cells for the expression of R-LIART polypeptides include prokaryotic, yeast or higher eukaryotic cells. Mammalian or insect cells are generally preferred for use as host cells. Expression cloning vectors suitable for use with cellular hosts of fungal, yeast and mammalian bacteria are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, Elsevier, New York, (1985). Cell-free translation systems can also be employed to produce R-LIART peptides using the RNAs derived from the DNA constructs described herein. Prokaryotes include gram negative or gram positive organisms, for example E. coli or Bacilli. Prokaryotic host cells suitable for transformation include, for example, E. coli, Bacillus substilis, Salmonellla typhymurium and several other different species within the genera Pseudomonas, Streptomyces and Staphylococcus. In a prokaryotic host cell, such as E. coli, a R-LIART polypeptide may include an N-terminal methionine residue to facilitate the expression of recombinant polypeptide in the prokaryotic host cell. The N-terminal Met can be separated from the expressed recombinant R-LIART polypeptide. Expression vectors for use in prokaryotic host cells generally comprise one or more phenotypic selectable marker genes. A phenotypic selectable marker gene is, for example, a gene that encodes a protein that confers antibiotic resistance or that provides an autotrophic requirement. Examples of useful expression vectors for prokaryotic host cells include those derived from commercially available plasmids such as the cloning vector pBR322 (TCC 37017). pBR322 contains genes for ampicillin and tetracycline resistance and therefore provides simple means to identify transformed cells. An appropriate promoter and a DNA sequence of R-LIART is inserted into the vector pBR322. Other commercially available vectors include, for example, pKK233-3 (Pharmacia Fine Chemicals, Uppsala, Sweden) and pGEMI (Promega Biotec, Madison, Wl, USA). Promoter sequences commonly used for the expression vectors of recombinant prokaryotic host cells include β-lactamase (penicillinase), the lactose promoter system (Chang et al., Nature 275: 615, 1978; and Goeddel et al., Nature 281: 544, 1979), tryptophan (trp) promoter system (Maniatis, Molecular cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, page 412, 1982). A particularly useful prokaryotic host cell expression system employs a phage P promoter. and a thermolabile sequence of c1857ts. Plasmid vectors available from the American Type Culture Collective incorporating Phage P-promoter derivatives. they include the plasmid pH U B2 (resident in the E. coli strain JMB9, ATCC 37092) and pPLc28 (resident in E. coli RR 1, ATCC 53082).

Alternatively, R-LIART can be expressed in yeast host cells, preferably of the genus Saccharomyces (e.g., S. cerevisiae). Other yeast genera, such as Pichia or Kluyveromyces, may also be employed. Yeast vectors often contain a replication sequence origin of a 2μ yeast plasmid, an autonomously replicating sequence (SRA), a promoter region, sequences for polyadenylation, sequences for transcription termination and a selectable marker gene. Suitable promoter sequences for yeast vectors include, among others, promoters for metallothionein, 3-phosphoglycerate kinase (Hitzeman et al., J. Biol. Chem. 255: 2073, 1980) or other glycolytic enzymes (Hess et al., J. Adv. Enzime Reg. 7: 149, 1968; and Holland et al., Biochem, 17: 4900, 1978), such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate, decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase , 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate, shampoo, phosphoglucose, and glucokinase. Other vectors and promoters suitable for use in expression of yeast are further described in Hitzeman, EPA-73,657. Another alternative is the repressible DH2 promoter with glucose described by Russell et al. (J. Biol. Chem. 258: 2674, 1982) and Beier et al., (Nature 300: 724, 1982). Shuttle vectors replicable in both yeast and E. coli can be constructed by inserting DNA sequences from pBR322 for selection and replication in E. coli (Ampr gene and origin of replication) in the yeast vectors described above. The leader sequence of the yeast a factor can be used to direct the secretion of the LIART polypeptide. The leader sequence of the a factor is often inserted between the promoter sequence and the sequence of structural genes. See, for example, Kurjan et al., Cell 30: 933, 1982 and Bitter et al., Proc. Nati Acad. Sci. USA 81: 5330, 1984. Other suitable leader sequences for facilitating the secretion of recombinant polypeptides from yeast hosts are known to those skilled in the art. A leader sequence can be modified near its 3 'end to contain one or more restriction sites. This will facilitate the leader sequence function to the structural gene. The yeast transformation protocols are known to those skilled in the art. One such protocol is described by Hinnen and others, Proc. Nati Acad. Sci. USA 75: 1929, 1978. The protocol of Hinnen et al. Selects Trp + transformants in a selective medium, wherein the selective medium consists of 0.67% yeast nitrogen base, 0.5% casamino acids, 2% glucose, 10 μg / ml uracil. Yeast host cells transformed by vectors containing an ADH2 promoter sequence can be developed by inducing expression in a "rich" medium. An example of a rich medium is one consisting of 1% yeast extract, 2% peptone and 1% glucose supplemented with 80 μg / ml of adenine and 80 μg / ml uracil. The derepression of the ADH2 promoter occurs when glucose is depleted from the medium. Mammalian or insect host cell culture systems can also be used to express recombinant R-LIART polypeptides. Baculovirus systems for the production of heterologous proteins in insect cells are reviewed by Luckow and Summers, Bio / Technology 6:47 (1988). The established cell lines of mammalian origin can also be used. Examples of suitable mammalian host cell lines include the COS-7 line of monkey kidney cells (ATCC CRL 1651) (Gluzman et al., Cell 23: 175, 1981), L cells, C127 cells, 3T3 cells (ATCC CCL 163), Chinese hamster ovary (OHC), HeLa cells and BHK cell lines (ATCC CRL 10) and the cell line of CV1 / EBNA derived from the African green monkey CVI kidney cell line (ATCC CCL 70) as described by McMahan et al., (EMBO J. 10: 2821, 1991). Transcriptional and translational control sequences for expression vectors of mammalian host cells can be excised from viral genomes. The commonly used promoter sequences and modifier sequences are derived from the Polyoma virus. Adenovirus 2, Simian Virus 40 (SV40) and human cytomegalovirus. DNA sequences derived from the SV40 viral genome, eg, SV40 origin sites, the early and late promoter enhancer, cleavage and polyadenylation can provide other different genetic elements for the expression of the sequence of structural genes in a mammalian host cell. The early and late viral promoters are particularly useful since they are readily obtained from a viral genome as a fragment that may also contain a viral origin of refolding (Fiers et al., Nature 273: 13, 1978). Smaller or larger SV40 fragments can also be used, as long as the approximately 250 bp sequence extending from the Hind I I I site to the Bgl I site located in the SV40 viral origin of the replication site is included. Expression vectors for use in mammalian host cells can be constructed as described by Okayama and Berg (Mol Cell. Biol. 3: 280, 1993), for example. A useful system for expressing stable high level mammalian cDNA in murine C127 mammary epithelial cells can be constructed substantially as described by Cosman et al., (Mol.immunol.23: 935, 1986). A high expression vector, PMLSV N1 / N4, described by Cosman et al., Nature 312: 768, 1984 has been deposited as ATCC 39890. Additional mammalian expression vectors were described in EP-A-0367566 and in WO 91 / 18982. As an alternative, the vector of a retrovirus can be derived. Overexpression of full-length R-LIART has resulted in membrane inclusion and nuclear condensation of transfected CV-1 / EBNA cells, indicating that the mechanism of cell death was apoptosis. For host cells in which such R-LIART-mediated apoptosis occurs, A suitable inhibitor of apoptosis can be included in the expression system. To inhibit R-LIART-induced apoptosis of host cells expressing recombinant R-LIART, the cells can be co-transfected with an expression vector encoding a polypeptide that functions as an inhibitor of apoptosis. The expression vectors encoding said polypeptides can be prepared by conventional methods. Another approach involves adding an inhibitor of apoptosis to the culture medium. The use of CrmA poxvirus, P35 baculovirus, a C-terminal fragment of FADD, the tripeptide derivative zVAD-fmk, to reduce death of host cells is illustrated in examples 6 and 8. zVAD-fmk (benzyloxycarbonyl-Val-Ala -fluoromethyl ketone) is a compound based on tripeptides, available from Enzyme System Products, Dublin, California. As illustrated in example 8, zVAD-fmk can be added to the medium in which cells expressing R-LIART are cultured. The protein derived from 38 kiiodaltons of cow pox virus that was subsequently designated CrmA was described in Pickup et al. (Proc. Nati. Acad. Sci. USA 83: 7698-7702, 1986; incorporated herein by reference). The sequence information for CrmA was presented in Figure 4 of Pickup et al., Supra. An approach for producing and purifying the CrmA protein is described in Ray et al (Cell, 69: 597-607, 1992, incorporated herein by reference).

A 35 kilodalton protein encoded by Autographa californica nuclear polyhedrosis virus, a baculovirus, was described in Friesen and Miller (J. Virol 61: 2264-2272, 1987, incorporated herein by reference). The sequence information for this protein, designated p35 baculovirus in the present, was presented in Figure 5 of Friesen DNA Miller, supra. The cytoplasmic protein containing dead domain FADD (also known as MORTI) is described in Boldin et al. (J. Biol. Chem. 270: 7795-7798, 1995, incorporated herein by reference). It was reported that FADD is directly or indirectly associated with the cytoplasmic dead domain of certain receptors that mediate apoptosis (Boldin et al., Cell 85: 803-815, June 1996; Hsu et al., Cel! 84: 299-308, January 1996). ). In one embodiment of the present invention, the truncated FADD polypeptides that include the dead domain (located in the C-terminal portion of the protein), but lacking the N-terminal region for which the effect functions have been attributed of apoptosis, are used to reduce apoptosis. The use of certain FADD mutant polypeptides, truncated at the N-terminus, to inhibit the death of cells expressing other receptors that induce apoptosis, was described in Hsu et al. (Cell 84: 299-308, 1996, incorporated herein by reference). ). This approach is illustrated in Example 8, which employs a suitable dominant FADD negative polypeptide (FADD-DN), having an amino acid sequence corresponding to amino acids 1 17 to 245 of the MORTI amino acid sequence presented in Boldin et al. (J. Biol. Chem. 270: 7795-7798, 1995). In Example 8, the cells were co-transfected with an expression vector encoding R-LIART and with an expression vector encoding the Flag® peptide described above, fused with the N-terminus of the FADD-DN polypeptide. Although one does not wish to be bound by a theory, one possible explanation is that the C-terminal fragments of FADD are associated with the intracellular dead domain of the receptor, but that it lacks the N-terminal portion of the protein that is necessary to effect apoptosis. (Hsu et al., Cell 84: 299-308, January 1996, Boldin et al., Cell 85: 803-815, June 1996). Therefore, truncated FADD can block the association of endogenous full-length FADD with the dead domain of the receptor; consequently, the apoptosis that could be initiated by said endogenous FADD is inhibited. Other apoptosis inhibitors useful in expression systems can be identified in conventional analysis procedures. One such assay, in which the compounds for the ability to reduce apoptosis of cells expressing R-LIART are tested, was described in example 8. Poxvirus CrmA, baculovirus P35, and zVAD-fmk are caspase-viral inhibitors. Other viral caspase inhibitors can be tested for the ability to reduce cell death mediated by R-LIART.

The use of CrmA, p35 baculovirus, and certain peptide derivatives (including zVAD-fmk) as inhibitors of apoptosis, in particular cells / systems, are discussed in Sarin et al. (J. Exp. Med. 184: 2445-2450, Dec. 1996, incorporated herein by reference). The role of proteases in the enzyme family that converts interleukin-1β (ICE) into cascades of signal transduction leading to the death of programmed cells and the use of inhibitors of these proteases to block apoptosis is discussed in Sarin et al., Supra, and Muzo et al., Cell 85: 817-827, 1996). Apoptosis inhibitors generally do not need to be employed for the expression of R-LIART polypeptides lacking cytoplasmic domain (ie lacking the region of the protein involved in signal transduction). Therefore, expression systems for producing soluble R-LIART polypeptides comprising only the extracellular domain (or a fragment thereof) need not include one of the apoptosis inhibitors described above. With respect to the signal peptides that can be used to produce R-LIART, the native signal peptide of R-LIART can be replaced by a heterologous signal peptide or leader sequence, if desired. The choice of signal or leader peptide may depend on factors such as the type of host cells in which the recombinant R-LIART is to be produced. To illustrate, examples of heterologous signal peptides that are functional in mammalian host cells include signal sequences for interleukin-7 (I L-7) described in U.S. Patent No. 4,965, 195, the signal sequence for the interleukin-2 receptor described in Cosman et al., Nature 312: 768 (1984); the signal peptide of the interleukin-4 receptor described in EP 367, 566; the interleukin-1 type I receptor signal peptide described in the U.A. 4,968,607; and the interleukin-1 receptor type I I signal peptide described in EP 460,846. Oligomeric Forms of R-LIART Oligomers containing R-LIART polypeptides are encompassed within the present invention. The R-LIART oligomers may be in the form of covalently linked or non-covalently bound dimers, trimers or oligomers. One embodiment of the invention is directed to oligomers comprising multiple R-LIART polypeptides joined via covalent or non-covalent interactions between the peptide portions fused to the R-LIART peptides. Said peptides may be peptide linkers (separators), or peptides having the property of promoting oligomerization. Leucine-binding agents and certain antibody-derived polypeptides are among the peptides that can promote the oligomerization of R-LIART polypeptides bound thereto, as described in detail below. In particular embodiments, the oligomers comprise two to four R-LIART polypeptides. The R-LIART portions of the oligomer may be soluble polypeptides as described above.

As an alternative, an R-LIART oligomer was prepared using polypeptides derived from immunoglobulins. The preparation of fusion proteins comprising certain heterologous polypeptides fused to various portions of polypeptides derived from antibodies (including the Fe domain), e.g., by Ashkenazi et al. (PNAS USA 88: 10535, 1991); Byrn et al. (Nature 344: 677, 1990); and Hollenbaugh and Aruffo ("Construction of Immunogiobulin Fusion Proteins," in Current Protocols in Immunology, Suppl 4, pp. 10.19.1 - 10.19.11, 1992). One embodiment of the present invention is directed to an R-LIART dimer comprising two fusion proteins created by fusing R-LIART to the Fe region of an antibody. A fusion of genes encoding the R-LIART / Fc fusion protein is inserted into an appropriate expression vector. The R-LIART / Fc fusion proteins were expressed in host cells transformed with the recombinant expression vector and very similar antibody molecules were allowed to assemble, whereby the disulfide bond between the chains is formed between the portions of Fe for give R-LIART divalent. Here, fusion proteins comprising an R-LIART polypeptide fused to an Fe polypeptide derived from an antibody are provided. DNA encoding said fusion proteins is also provided, as well as dimers containing two fusion proteins linked via disulfide linkages between the Fe portions thereof. The term "Fe polypeptide" as used in the present, includes native and mutein forms of polypeptides derived from the Fe region of an antibody. Also included are truncated forms of said polypeptides that contain the connecting region that promotes dimerization. A suitable Fe polypeptide, described in the PCT application WO 93/10151 (incorporated herein by reference), is a single polypeptide chain extending from the N-terminal binding region to the C-terminus native to the Fe region of a human IgG 1 antibody. Another useful Fe polypeptide is the Fe mutein described in the U. U.A. Patent. No. 5,457, 035 and in Baum et al., (EMBO J. 13: 3992-4001, 1994). The amino acid sequence of this mutein is identical to the native Fe sequence presented in WO 93/10151, except that amino acid 19 had been changed from Leu to Ala, amino acid 20 had been changed from Leu to Glu, and amino acid 22 He had changed from Gly to Ala. The mutein exhibits reduced affinity for Fe receptors. In other modalities, R-LIART can be substituted for the variable portion of a heavy or light chain antibody. If the fusion proteins are made with heavy and light chains of an antibody, it is possible to form an oligomer of R-LIART with as many as four extracellular regions of R-LIART. Alternatively, the oligomer is a fusion protein comprising multiple R-LIART polypeptides, with or without peptide linkers (spacer peptides). Among the suitable peptide linkers are those described in the Patents of E.U.A. 4,751, 180 and 4,935,233, which are incorporated herein by reference. A DNA sequence encoding a desired peptide linker can be inserted between, and in the same reading frame as, the DNA sequences encoding R-LIART, using any suitable conventional technique. For example, an oligonucleotide synthesized chemically encoding DNA sequences encoding a ligand can be ligated between the sequences encoding R-LIART. In particular embodiments, a fusion protein comprises two to four soluble R-LIART polypeptides, separated by peptide linkers. Another method for preparing oligomeric R-LIART involves the use of a leucine compactor. The leucine compactor domains are peptides that promote the oligomerization of the proteins in which they are found. Leucine compactors are originally identified in several DNA binding proteins (Landschulz et al., Science 240: 1759, 1988) and, therefore, have been found in a variety of different proteins. Among the known leucine compactors are the peptides present in nature and derivatives thereof which are dimerized or trimerized. Examples of leucine compactor domains suitable for producing soluble oligomeric proteins are described in PCT application WO 94/10308 and leucine compactor derived from lung surfactant protein D (ATP) described in Hoppe et al., (FEBS Letters 344: 191, 1994), incorporated herein by reference. The use of a compactor Modified leucine that allows stable trimerization of a heterologous protein fused thereto was described in Fanslow et al. (Semin.Immunol.6: 267-278, 1994). Recombinant fusion proteins comprising a soluble R-LIART polypeptide fused to a leucine compactor peptide is expressed in suitable host cells and the soluble oligomeric R-LIART that is formed is recovered from the culture supernatant. The oligomeric R-LIART has the property of bivalent, trivalent binding sites, etc. , for LIART. The fusion proteins described above that comprise portions of Fe (and oligomers formed therewith) offer the advantage of easy purification by affinity chromatography on the Protein A or Protein G columns. The DNA sequences encoding oligomeric R-LIART , or coding fusion proteins useful for preparing oligomers of R-LIART, is provided herein. Analysis R-LIART proteins (including fragments, variants, oligomers or other forms of R-LIART) can be tested for the ability to bind LIART in a suitable analysis, such as a conventional binding analysis. To illustrate, R-LIART can be labeled with a detectable reagent (e.g., a radionuclide, chromophore, enzyme that catalyzes a colorimetric or fluorometric reaction and the like). The labeled R-LIART is contacted with cells expressing LIART. The cells are then washed to remove unbound labeled R-LIART and the presence of the brand bound to the cells is determined by any suitable technique, chosen according to the nature of the label. An example of a joining analysis procedure is as follows. A recombinant expression vector containing LIART cDNA is constructed, e.g. , as described in PCT application WO 97/01633, incorporated herein by reference. The DNA and amino acid sequence information of human and mouse LIART was presented in WO 97/016333. LIART comprises an N-terminal cytoplasmic domain, a transmembrane region and a C-terminal extracellular domain. The CVI-EBNA cells in 10 cm2 plates are transfected with the recombinant expression vector. CV-1 / EBNA cells (ATCC CRL 10478) constitutively express EBV nuclear antigen-1 derived from the CMV immediate-early enhancer / promoter. CV1-EBNA-1 was derived from cell line of the African Green Monkey CV-1 kidney (ATCC CCL70), as described by McMahan et al. (EMBO J. 10: 2821, 1991). The transfected cells were cultured for 24 hours and the cells in each plate were divided into a 24-well plate. After culturing for an additional 48 hours, the transfected cells (approximately 4 x 10 4 cells / wells) were washed with BM-N FDM, which is the binding medium (RPMI 1640 containing 45 mg / ml bovine serum albumin , 2 mg / ml of sodium azide, 20 mM of Hepes pH 7.2) to which 50 mg / ml of non-fat dry milk was added. The cells were then incubated for 1 hour at 37 ° C with various concentrations of a soluble fusion protein of R-LIART / Fc.

The cells were then washed and incubated with a constant saturation concentration of mouse anti-human IgG 125l in binding medium, with gentle shaking for 1 hour at 37 ° C. After extensive washing, the cells were released via trypsinization. The mouse anti-human IgG used above was directed against the human IgG Fe region and can be obtained from Jackson Immunoresearch Laboratories, Inc., West Grove, PA. The antibody was radioiodinated using the normal chloramine-T method. The antibody will bind to the Fe portion to any R-LIART / Fc protein that is bound to the cells. In all analyzes, the non-specific binding of the antibody of 1251 in the absence of R-LIART / Fc, as well as in the presence of R-LIART / Fc and a 200-fold molar excess of anti-human IgG antibody were not analyzed. unmarked mouse The 2SI antibody bound to the cell was quantified in a Packard Autogamma counter. Affinity calculations (Scatchard, Ann. N. Y. Acad. Sci. 51: 660, 1949) were generated in RS / 1 (BBN Software, Boston, MA) operated on a Microvax computer. Another type of suitable binding analysis is a competitive binding analysis. To illustrate, the biological activity of a variant of R-LIART can be determined by analyzing the ability of the variant to compete with a native R-LIART to join LIART. Competitive union analyzes can be carried out by conventional methodology. Reagents that can be used in competitive binding assays include radiolabeled R-LIART and intact cells expressing LIART (endogenous or recombinant) on the cell surface. For example, a radiolabeled soluble R-LIART fragment can be used to compete with a soluble R-LIART variant to bind it to the LIART cell surface. In place of the intact cells, a soluble LIART / Fc fusion protein bound to a solid phase could be substituted through the interaction of Protein A or Protein G (on the solid phase) with the Fe portion. The chromatography columns containing Protein A and Protein G include those available from Pharmacia Biotech, Inc., Piscataway, NJ. Another type of competitive binding analysis uses radiolabeled soluble LIART such as a soluble LIART / Fc fusion protein and intact cells expressing R-LIART. Qualitative results can be obtained by competitive autoradiographic plate binding analysis, while Scatchard plots (Scatchard, Ann.N.A. Acad.Sci. 51: 660 1949) can be used to generate quantitative results. Another type of analysis for biological activity involves testing an R-LIART polypeptide for the ability to block LIART-mediated apoptosis of target cells, such as the human leukemic T-cell line known as, for example, Jurkat cells. LIART-mediated apoptosis of the cell line designated clone E6-1 of Jurkat (ATCC TI B 152) was demonstrated in assay methods described in PCT application WO 97/01633, incorporated herein by reference. Uses of R-LIART The uses of R-LIART include, but are not limited to, the following. Certain of these uses of R-LIART originate from your ability to join LIART. R-LIART is used as a protein purification reagent. The R-LIART polypeptides can be attached to a solid support material and used to purify LIART proteins by affinity chromatography. In particular embodiments, a R-LIART polypeptide (in any form described herein that is capable of binding to LIART) is attached to a solid support by conventional methods. As an example, chromatography columns containing functional groups that will react with functional groups on amino acid side chains of proteins (Pharmacia Biotech, Inc., Píscataway, NJ) are available. In an alternative, an R-LIART / Fc protein binds to chromatography columns containing Protein A or Protein G through interaction with the Fe portion. R-LIART proteins are also used to measure biological activity of LIART proteins in terms of their binding affinity for R-LIART. The R-LIART proteins, therefore, can be used by those who carry "quality control" studies v.gr. , to monitor the storage life and stability of the LIART protein under different conditions. To illustrate, R-LIART can be used in a binding affinity study to measure the biological activity of a LIART protein that is to be stored at different temperatures or will be produced in different Cell types. R-LIART can also be used to determine if biological activity was retained after modification of a LIART protein (eg, modification, truncation, chemical mutation, etc.). The binding affinity of the LIART protein modified for R-LIART is compared to that of an unmodified LIART protein in order to detect any adverse impact of the modifications on the biological activity of LIART. The biological activity of a LIART protein, therefore, for example, can be specified before it is used in a research study. R-LIART is also used to purify or identify cells that express LIART on the cell surface. The R-LIART polypeptides are attached to a solid phase such as a column chromatography matrix or a similar suitable substrate. For example, the magnetic microspheres can be coated with R-LIART and kept in an incubation vessel through a magnetic field. Suspensions of cell mixtures containing cells expressing LIART are contacted with the solid phase having R-LIART thereon. Then the cells expressing LIART on the cell surface are bound to fixed R-LIART and unbound cells. Alternatively, R-LIART can be conjugated to a detectable portion, after being incubated with cells to be tested for LIART expression. After incubation, unbound labeled R-LIART is removed and the presence or absence of the detectable portion on the cells is determined.

In a further alternative, mixtures suspected of containing LIART cells are incubated with biotinylated R-LIART. Incubation periods are normally at least one hour to ensure specific binding. The resulting mixture is then passed through a column packed with beads coated with avidin, whereby the high affinity of biotin for avidin provides for the binding of the desired cells to the beads. Methods for using avidin-coated beads are known (see Berenson, et al., J. Cell, Biochem., 10D: 239, 1989). Washing to remove unbound material and release of bound cells is carried out using conventional methods. The R-LIART polypeptides can also be used as carriers to deliver agents bound thereto to cells having LIART. Cells that express LIART include those identified in Wiley et al. (Immunity, 3: 673-682, 1995). The R-LIART proteins can therefore be used to deliver diagnostic or therapeutic agents to said cells (or to other cell types found to express LIART on the cell surface) in in vitro or in vivo procedures. Detectable (diagnostic) and therapeutic agents, which can bind to the R-LIART polypeptide include, but are not limited to, toxins, other cytotoxic agents, drugs, radionuclides, chromophores, enzymes that catalyze a colorimetric or fluorometric reaction and the like , with the particular agent being chosen according to the intended application. Between the toxins, are ricin, abrin, diphtheria toxin, Pseudomonas aeruginosa exotoxin A, ribosomal activation proteins, mycotoxins such as trichothecenes and derivatives and fragments thereof (eg, chains alone). Suitable radionuclides include, but are not limited to, 123 l '131 l, 99mTc, 11 ln and 7βBr. Examples of suitable radionuclides for therapeutic use are 131 l, 21 1At, 77 Br, 186 D Re_, 1 88 D R? E, 212 r P-, b, 21 2 D B; i, 109 D Pyd_ ?, 64 Clu, "and 67 C | u ,. Said agents can be linked to R-LIART by any suitable conventional procedure. R-LIART, being a protein, comprises functional groups on side chains of amino acids that can be reacted with functional groups on a desired agent to form, for example, covalent linkages. Alternatively, the protein or agent can be derived to generate or bind to a desired reactive functional group. Derivatization may involve the binding of one of the available bifunctional coupling reagents to bind several molecules to proteins (Pierce Chemical Company, Rockford, Illinois). A number of techniques for radiolabelling proteins are known. The radionuclide metals can be linked to R-LIART using any suitable bifunctional chelating agent, for example. Conjugates comprising R-LIART and a suitable diagnostic or therapeutic agent (preferably covalently linked) are then prepared. The conjugates are administered or in some way employed in an amount appropriate for the particular application.

The R-LIART DNA and polypeptides of the present invention can also be used in developmental treatments for any disorder mediated (directly or indirectly) by defective or insufficient amounts of R-LIART. The R-LIART polypeptides can be administered to any mammal suffering from said disorder. Alternatively, a gene therapy approach can be taken. The present description of nucleotide sequences of native R-LIART allows the detection of defective R-LIART genes and the replacement thereof with genes encoding normal R-LIART. Defective genes can be detected in in vitro diagnostic assays and by comparing a nucleotide sequence of native R-LIART described herein with that of an R-LIART gene derived from a person suspected of having a defect in this gene. Another use of the protein of the present invention is a research tool for studying biological effects resulting from the inhibition of LIART / R-LIART interactions on different cell types. The R-LIART polypeptides can also be used in in vitro assays to detect LIAR or R-LIART or their interactions. R-LIART can also be used to inhibit a biological activity of LIART, in in vitro or in vivo procedures. A purified R-LIART polypeptide can be used to inhibit the binding of LIART to the endogenous cell surface of R-LIART. The biological effects that result from the binding of LIART to endogenous receptors they are inhibited like this. Several forms of R-LIART can be employed; including, for example, fragments of R-LIART described above, oligomers, derivatives, and variants that are capable of binding LIART. In one embodiment, a soluble R-LIART is used to inhibit a LIART biological activity, v. gr. , to inhibit LIART-mediated apoptosis of particular cells. R-LIART can be administered to a mammal to treat a LIART-mediated disorder. Such disorders mediated by LIART include conditions caused (directly or indirectly) or augmented by LIART. R-LIART can be useful to test thrombotic microangiopathies. One such disorder is thrombotic thrombocytopenic purpura (TTP) (Kwaan, H .C. Semin.Hymatoi., 24: 71, 1987, Thompson et al., Blood, 80: 1890, 1992). The mortality regimes associated with increasing TTP have been reported by U .S. Centers for Disease Control (Torok et al., Am. J. Hematoi, 50:84, 1995). The plasma of patients suffering from TTP (including patients with HIV4"and VI H") induces apoptosis of human endothelial cells of dermal microvascular origin, but not of large vessel origin (Laurence et al., Blood, 87: 3245, April 15, 1996). The plasma of patients with TTP, therefore, is thought to contain one or more factors that directly or indirectly induce apoptosis. As described in the PCT application WO 97/01633 (incorporated herein by reference), LIART is present in the serum of patients with TTP and may play a role in inducing endothelial cell apoptosis. Another thrombotic microangiopathy is hemolytic-uremic syndrome (HUS) (Moake, JL Lancet, 343: 393, 1994, Melnyk et al., (Arch. Intern. Med. 155: 2077, 1995, Thompson et al., Supra). of the invention is directed to the use of R-LIART to treat the condition frequently referred to as "adult HUS" (although it may also occur in children.) A disorder known as HUS associated with infantile diarrhea differs with adult HUS etiology. Other conditions characterized by coagulation of small blood vessels can be treated using R-LIART, which includes, but is not limited to, the following: Heart problems observed in approximately 5-10% of pediatric patients with AIDS, involves the coagulation of small blood vessels.The rupture of the microvasculature in the heart has been reported in patients with multiple sclerosis.As an additional example, the treatment of systemic lupus erythematosus (SLE) is contemplated. In one embodiment, a patient's blood or plasma is contacted with R-LIART ex vivo. R-LIART can be attached to a suitable chromatography matrix by conventional methods. The patient's blood or plasma flows through a chromatography column containing R-LIART bound to the matrix, before returning to the patient. The immobilized receiver is binds LIART, thereby removing the LIART protein from the patient's blood. Alternatively, R-LIART can be administered in vivo to a patient suffering from thrombotic microangiopathy. In one embodiment, a soluble form of R-LIART is administered to the patient. The present invention, therefore, provides a method for treating a thrombotic microangiopathy, involving the use of an effective amount of R-LIART. An R-LIART polypeptide can be used in in vivo or ex vivo procedures to inhibit LIART mediated damage (eg, apoptosis d) microvascular endothelial cells. R-LIART can be used in conjunction with other agents useful in treating a particular disorder. In an in vivo study reported by Laurence et al. (Blood 87: 3245, 1996), some reduction of plasma-mediated apoptosis of TTP from microvascular endothelial cells was achieved using an anti-Fas blocking antibody, an aurintricarboxylic acid, or normal plasma. exhausted by cryoprecipitate. Therefore, a patient can be treated with an agent that inhibits mediated apoptosis by Fas ligand or endothelial cells, in combination with a people that inhibits endothelial cell LIART mediated apoptosis. In one embodiment, R-LIART and an anti-FAS block antibody are administered to a patient suffering from a disorder characterized by thrombotic mycroangiopathy, such as TTP or H US. Examples of monoclonal antibodies of Blocks directed against Fas antigen (CD95) were described in PCT publication application number WO 95/10540, incorporated herein by reference. Another embodiment of the present invention is directed to the use of R-LIART to reduce LIART-mediated death of T cells in HIV-infected patients. The role of T cell apoptosis in the development of SI DA has been subjected to a number of studies (see, for example Meyaard et al., Science 257: 217-219, 1992; Groux et al., J. Exp. Med. 175 : 331, 1992, and Oyaizu et al., In Cell Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-1 14). Some researchers have studied the role of Fas-mediated apoptosis; the involvement of interleukin-1β conversion enzyme (ICE) has also been explored (Estaquier et al., Blood 87: 4959-4966, 1996; Mitra et al., Immunology 87: 581-585, 1996; Katsikis et al., J. Exp. Med. 181: 2029-2036, 1995). It is possible that apoptosis of T cells occurs through multiple mechanisms. At least some of the T cell death observed in patients with VI H is thought to be mediated by LIART. While not wishing to be bound by a theory, said T cell death mediated by LIART is thought to occur through a mechanism known as cell death induced by activation (MCIA).

Activated human T cells are induced to undergo programmed cell death (apoptosis) under the drive through the CD3 / T cell receptor complex, a process called induced cell death activated (AICD). ICD of CD4 + T cells isolated from asymptomatic individuals infected with HIV has been reported (Groux et al., Supra). Therefore, AICD may play a role in the depletion of CD4 + T cells and in the progression of SI DA in HIV-infected individuals. The present invention provides a method for inhibiting LIART mediated T cell death in HIV + patients, comprising administering R-LIART (preferably, a soluble R-LIART polypeptide) to patients. In one modality, the patient is asymptomatic when the treatment against R-LIART begins. If desired, prior to treatment peripheral blood T cells can be extracted from a patient with HIV + and tested for susceptibility to cell death mediated by LIART by conventional methods. In one embodiment, the patient's blood or plasma is contacted with R-LIART ex vivo. R-LIART can be attached to a suitable chromatography matrix by conventional methods. The patient's blood or plasma flows through a chromatography column containing R-LIART bound to the matrix, before returning to the patient. R-LIART immobilized binds to LIART, thereby removing the LIART protein from the patient's blood.

To treat patients with VI H +, R-LIART can be used in combination with other inhibitors of T cell apoptosis. Fas-mediated apoptosis has also been implicated in the loss of T cells in individuals with HIV + (Katsikis et al., J. Exp. Med. 181: 2029-2036, 1995). Therefore, a patient who is susceptible to T-cell death mediated by the Fas (L-Fas) ligand and mediated by LIART can be treated with an agent that blocks the interaction of LIART / R-LIART and an agent that blocks the interaction of Fas-L / Fas. Suitable agents that block the binding of Fas-L to Fas include, but are not limited to, soluble Fas polypeptides; Oligomeric forms of soluble Fas polypeptides (e.g., sFas / Fc dimers); anti-Fas antibodies that bind to Fas without transduction the biological signal that results in apoptosis; anti-Fas-L antibodies that block the binding of Fas-L to Fas; and Fas-L muteins that bind Fas but do not transduce the biological signal that results in apoptosis. Preferably, the antibodies used in the monoclonal antibody method. Examples of suitable agents for blocking Fas-L / Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, incorporated herein by reference. Compositions comprising an effective amount of an R-LIART polypeptide of the present invention, in combination with other components such as a physiologically acceptable diluent, carrier or excipient, are provided herein. R-LIART can be formulated according to known methods used for preparing pharmaceutically useful compositions. R-LIART may be combined in admixture, either as the active material alone or with other known active materials suitable for a given indication, with pharmaceutically acceptable diluents (eg, saline, Tris-HCl, acetate and phosphate buffered solutions). ), preservatives (e.g., thimerosal, benzyl alcohol, parabens), emulsifiers, solubilizers, adjuvants and / or vehicles. Suitable formulations for pharmaceutical compositions include those described in Temington's Pharmaceutical Sciences, 16th ed. 1980, Mack Publishing Company, Easton. PA. In addition, said compositions may contain R-LIART complexed with polyethylene glycol (PEG), metal ions, or incorporated into polymeric compounds such as polyacetic acid, polyglycolic acid, hydrogels, dextran, etc. , or incorporated in liposomes, microemulsions, microspheres, unilamellar or multilamellar vesicles, images of erythrocytes or sphenoblasts. Said compositions will influence the physical state, solubility, stability, in vivo release regimen and in vivo elimination regime of R-LIART, and are therefore chosen in accordance with the intended application. R-LIART expressed on the surface of a cell can also be used. The compositions of the present invention may contain an R-LIART polypeptide in any form described herein, such as native proteins, variants, derivatives, oligomers and biologically active fragments. In modalities In particular, the composition comprises a soluble R-LIART polypeptide or an oligomer comprising soluble R-LIART polypeptides. R-LIART can be administered in any suitable form, v. gr. , topically, parenterally or by inhalation. The term "parenteral" includes injection, v. gr. , by subcutaneous, intravenous or intramuscular routes, also including localized administration, v.gr. , in a site of illness or injury. The sustained release of implants is also contemplated. One of skill in the relevant art will recognize that adequate dosages will vary, depending on factors such as the nature of the disorder to be treated, the body weight, age and general condition of the patient, and the route of administration. Preliminary dosages can be determined according to animal tests, and the dose increase for human administration is carried out in accordance with accepted practices in the art. Also contemplated are compositions comprising R-LIART nucleic acids in physiologically acceptable formulations. The R-LIART DNA can be formulated, for example, by injection. Antibodies Antibodies that are immunoreactive with R-LIART polypeptides are provided herein. Said antibodies bind specifically to R-LIART, since the bodies bind to R-LIART via the antigen binding sites of the antibody (as opposed to the specific binding).

The R-LIART protein prepared as described in example 1, can be used as an immunogen to produce antibodies unreactive with them. Alternatively, another form of R-LIART, such as a fragment or fusion protein, is used as the immunogen. Polyclonal and monoclonal antibodies can be prepared by conventional techniques. See, for example, Monoclonal Antibodies, Hybridomas: A New Dimension in Biological Analyzes, Kennet et al. (Eds.), Plenum Press, New York (1980); and Antibodies: A Laboratory Manual, Harlow and Land (eds.), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (1988). The production of monoclonal antibodies directed against R-LIART is further illustrated in example 4. Antigen binding fragments of said antibodies, which can be produced by conventional techniques, are also encompassed by the present invention. Examples of such fragments include, but are not limited to, Fab and F (ab ') 2 fragments. Fragments of antibodies and derivatives produced by genetic engineering techniques are also provided. The monoclonal antibodies of the present invention include chimeric antibodies, v. gr. , humanized versions of monoclonal antibodies of mice. Said engineered antibodies can be prepared by known techniques, and offer the advantage of reduced immunogenicity when the antibodies are administered to human beings. In one modality, an antibody humanized monoclonal comprises a variable region of a murine antibody (or only the antigen binding site thereof) and a constant region derived from an antibody of humans. Alternatively, a humanized antibody fragment can comprise the antigen binding site of a murine monoclonal antibody and a fragment of the variable region (which lacks the antigen binding site) derived from a human antibody. Methods for the production of chimeric and genetically-treated monoclonal antibodies include those derived in Riechmann et al. (Nature 332: 323, 1988), Liuy others (PNAS 84: 3439, 1987), Larrick et al. (Bio / Technology 7: 934). , 1989) and Winter and Harris (TIPS 74: 139, May 1993). Among the uses of the antibodies is the use in analysis to detect the presence of R-LIART polypeptides, either in vitro or in vivo. The antibodies can also be employed by purifying R-LIART protein by immunoaffinity chromatography. Antibodies that can block the binding additionally of R-LIART to LIART can be used to inhibit a biological activity resulting from such binding. Such block antibodies can be identified using any suitable assay method, such as by testing antibodies for the ability to inhibit LIART binding to cells expressing R-LIART. Examples of said cells are Jurkat cells and PS1 cells described in example 2 below. Alternatively, block antibodies can be identified with the analysis for ability to inhibit a biological effect that results from the binding of LIART to target cells. The antibodies can be analyzed for the ability to inhibit, for example, LIART mediated lysis of Jurkat cells. Said antibodies can be used in an in vitro procedure or administered in vivo to inhibit a biological activity mediated by R-LIART. The disorders caused or exacerbated (directly or indirectly) by the interaction of LIART with the LIART receptor on the cell surface, then can be treated. A therapeutic method involves the in vivo administration of a block antibody to a mammal in an amount effective to inhibit a biological activity mediated by LIART. The disorders caused or augmented by LIART, directly or indirectly, are thus treated. Monoclonal antibodies are generally preferred for use in such therapeutic methods. In a modality, an antigen-binding antibody fragment is used. A blocking antibody directed against R-LIART can be substituted for R-LIART in the method described above for treating thrombotic microangiopathy, e.g. , to treat TTP or HUS. The antibody was administered in vivo, to inhibit LIART mediated damage to microvascular endothelial cells (e.g., apoptosis). Antibodies raised against R-LIART can be screened for agonistic (i.e., ligand mimicking) properties. These antibodies, when bound to cell surface R-LIART, induce biological effects (e.g., transduction of biological signals) similar to the biological effects induced when LIART binds to R-LIART from the cell surface. Agonist antibodies can be used to induce apoptosis of certain cancer cells or virally infected cells, as reported for LIART. LIART's ability to kill cells (including but not limited to leukemia, lymphoma and melanoma cells) and virally infected cells are described in Wiley et al., (Immunity 3: 673-682, 1995); and in the PCT application WO 97/01633. Compositions comprising an antibody that is directed against R-LIART, and a physiologically acceptable diluent, excipient or carrier, are provided herein. Suitable components of said compositions are as described above for compositions containing R-LIART proteins. Also herein are provided conjugates comprising a detectable (eg, diagnostic) or therapeutic agent, linked to an antibody directed against R-LIART. Examples of said agents were presented before. The conjugates find use in in vitro or in vivo procedures. Nucleic Acids The present invention provides R-LIART nucleic acids. Said nucleic acids include, but are not limited to, DNA encoding the peptide described in example 2. Said DNA can be identified from the knowledge of genetic code. Other nucleic acids of the present invention include isolated DNAs that they comprise the nucleotide sequence presented in SEQ ID NO: 1 or SEQ ID NO: 3. The present invention provides isolated nucleic acids useful in the production of R-LIART polypeptides, v. gr. , in the recombinant expression systems discussed above. Such nucleic acids include, but are not limited to, the human R-LIART DNA of SEQ I D NO: 1. The nucleic acid molecules of the present invention include R-LIART DNA in the form of a single strand, as well as the RNA complement thereof. R-LIART DNA includes, for example, cDNA, genomic DNA, chemically synthesized DNA, PCR amplified DNA and combinations thereof. Genomic DNA can be isolated by conventional techniques, e.g. , using the cDNA of SEQ I D NO: 1 or 3, or a suitable fragment thereof as a probe. DNA encoding R-LIART is provided in any of the forms contemplated herein (e.g., full-length R-LIART or fragments thereof). Particular embodiments of R-LIART that encode DNA include a DNA encoding full-length human R-LIART of SEQ ID NO: 2 (including the N-terminal signal peptide) and a DNA encoding a mature human R-LIART full length Other embodiments include DNA encoding a soluble R-LIART (e.g., which encodes the extracellular domain of the SEQ ID NO: 2 protein, either with or without the signal peptide).

One embodiment of the invention is directed to fragments of nucleotide sequences of R-LIART comprising at least about 17 contiguous nucleotides in an R-LIART DNA sequence. In other embodiments, a DNA fragment comprises at least 30, or at least 60 contiguous nucleotides of an R-LIART DNA sequence. The nucleic acids provided herein, include DNA and RNA supplements of said fragments, together with single-stranded and double-stranded forms of the R-LIART DNA. Among the uses of R-LIART nucleic acid fragments is the use as probes or primers. Using the knowledge of the genetic code in combination with the amino acid sequences exhibited in example 2, sets of degenerating oligonucleotides can be prepared. Said oligonucleotides can be used as initiators, e.g. , in polymerase chain reactions (PCR). So the DNA fragments of R-LIART are isolated and amplified. Other useful fragments of R-LIART nucleic acids include sense or sense oligonucleotides comprising a single-stranded nucleic acid sequence (either RNA or DNA) capable of binding mRNA sequences of white R-LIART (sense) or R-LIART DNA (contradictory). The sense or sense oligonucleotides according to the present invention comprise a fragment of the DNA coding region of R-LIART. Said fragment generally comprises at least about 14 nucleotides, preferably from about 14 to about 30 nucleotides. The ability to derive an antisense or sense oligonucleotide, based on a cDNA sequence encoding a given protein, is described in, for example, Stein and Cohen (Cancer Res. 48: 2659, 1988) and van der Krol et al. (BioTechniques 6 : 958, 1988). Binding of the sense or sense oligonucleotides to the target nucleic acid sequences results in the formation of duplexes that block the transcription or translation of the target sequence by one of several means, including increased degradation of the duplexes, premature termination of transcription or translation, or by other means. The antisense oligonucleotides can then be used to block the expression of R-LIART proteins. The sense or sense oligonucleotides further comprise oligonucleotides having modified sugar-base phosphodiester structures (or other sugar ligatures, such as those described in WO 91/06629) and wherein sugar ligatures are resistant to endogenous nucleases. . Such oligonucleotides with resistant sugar ligations are stable in vivo (ie, they are capable of resisting enzymatic degradation) but retain the sequence specificity to be able to bind to the target nucleotide sequences.

Other examples of sense or antisense oligonucleotides include those oligonucleotides that are covalently bound to organic portions, such as those described in WO 94/10448 and other portions that increase the affinity of the oligonucleotides for a target nucleic acid sequence, such as poly- (L-lysine). In addition intercalary agents, such as ellipticine and alkylating agents or metal complexes can be attached to sense or antisense oligonucleotides to modify the binding specificities of the sense or sense oligonucleotide for the target nucleotide sequence. The sense or sense oligonucleotides can be introduced into a cell containing a target nucleic acid sequence by any method of gene transfer, including, for example, transfection of CaPO-mediated DNA, electroporation or using gene transfer vectors such as Epstein-Barr virus. In a preferred procedure, a sense or sense oligonucleotide is inserted into a suitable retroviral vector. A cell containing the target nucleic acid sequence is contacted with the recombinant retroviral vector, either in vivo or ex vivo. Suitable retroviral vectors include, but are not limited to, those derived from the murine retroviruses M-MuLV, N2 (a retrovirus derived from M-MulV), or the double copy of vectors designated DCT5A, DCT5B and DECT5C (see WO 90 / 13641). Sense or contradictory oligonucleotides can also be introduced into a cell containing the target nucleotide sequence by the formation of a conjugate with a ligand binding molecule, as described in WO 91/04753.

Suitable ligand binding molecules include, but are not limited to, cell surface receptors, growth factors, other cytokines, or other ligands that bind to cell surface receptors. Preferably, the conjugation of the ligand binding molecule does not substantially interfere with the ability of the ligand binding molecule to bind to its corresponding molecule or receptor, or block the entry of the sense or sense nucleotide oligoonucleotide or its conjugated version into the cell. Alternatively, a sense or nonsense nucleotide oligonucleotide can be introduced into a cell containing the target nucleic acid sequence by forming a complex of oligonucleotide oligonucleotide complexes, as described in WO 90/1 0448. lipoides of oligonucleotides of sense or contradictory is preferably dissociated within the cell by an endogenous lipase. The following examples are provided to further illustrate the particular embodiments of the invention and should not be construed as limiting the scope of the present invention. MPLO AXIS 1: Purification of the R-LIART Protein A human LIART receptor protein (R-LIART) was prepared by the following procedure. R-LIART was isolated from the cellular membranes of cells or those of J urkat, a cell line of leukemia T ag uda h umana. The cells of J urkat were chosen given that a specific band can be precipitated by affinity of the Jurkat biotinylated surface cells, using Flag®-LIART covalently coupled to affi-gel beads (Bíorad Laboratories, Richmond, CA). The precipitated band has a molecular weight of approximately 52 kD. A specific band less than about 42 kD was also present, possibly taking into account a proteolytic decomposition product or a less glycosylated form of R-LIART. Approximately 50 billion Jurkat cells were recovered by centrifugation (80 ml of cell pellets), washed once with PBS, then frozen by shock in liquid nitrogen. The plasma membranes were isolated according to the number three method described in Maeda et al., Biochim., Et Biophys. Acta. 731: 115, 1983; incorporated herein by reference) with five modifications: 1. The following protease inhibitors were included in all solutions at the indicated concentrations: Aprotinin, 150 nM; EDTA, 5mM; Leupeptin, 1 μM; pA-PMSF, 20 μM; Pefabloc, 500 μM; Pepstatin A, 1 μM; PMSF, 500 μM. 2. Ditp'otreitol was not used. 3. DNase was not used in the homogenization solution. 4. 1.25 ml of homogenization buffer was used per ml of cell pellets. 5. The homogenization was achieved by five passages through a ground glass homogenizer.

From the isolation of the cell membranes, the proteins were solubilized by resuspending the isolated membranes in 50 ml of PBS containing 1% of octylglucoside and all protease inhibitors mentioned above at the concentrations indicated above. The resulting solution was stirred repeatedly during an incubation of thirty minutes at 4 ° C. The solution was then centrifuged at 20,000 rpm in a SW28 rotor in a Beckman LE-80 ultracentrifuge (Beckman I nstruments, I nc., Palo Alto, CA) at 4 ° C for 30 minutes to obtain the supernatant (the extract from membrane). Chromatography The first purification step of R-LIART outside the membrane extract prepared before was affinity chromatography. The membrane extract was first applied to a column of Affi-gel anti-FIag® M2 (10 mg of monoclonal antibody M2 coupled with 2 ml of affi-gel beads) to absorb any material that is not specifically bound. The step flow was then applied to the Affi-gel Flag®-LIART column (10 mg of recombinant protein coupled with 1 ml of affi-gel beads) The Affi-gel support is a N-oxysuccinimide ester of a pearl of derivatized crosslinked agarose gel (available from Biorad Laboratoires, Richmond, CA) As discussed above, the Flag® peptide, Asp-Tyr-Lys-Asp-Asp-Asp-Asp-Lys, provides a reversibly bound epitope by specific monoclonal antibodies, allowing rapid analysis and easy purification of the protein expressed recombinant. M2 is a monoclonal antibody that binds to Flag®. Monoclonal antibodies that bind to Flag® peptide, as well as other reagents for making and using Flag® fusion proteins, are available from Eastman Kodak Co., Scientific Imaging Systems Division, New Haven, Connecticut. The preparation of the LIART Flag® fusion proteins (comprising Flag® fused to a soluble LIART polypepide) was further described in the PCT application of WO 97/01633, incorporated herein by reference. The column was washed with 25 ml of each of the following pH buffer solutions in the indicated order: 1. PBS containing 1% octyl glycoside 2. PBS 3. PBS containing an additional 200 mM NaCl 4. PBS The bound material was eluted with 50 mM Na Citrate (pH3) in 1 ml fractions and neutralized immediately with 300 μl of 1 M Tris-HCl (pH 8.5) per fraction. The LIART binding activity of each fraction was determined by ELISA specific for R-LIART as described above. Fractions with high LIART binding activity were combined, brought to 0.1% Trifluoroacetic acid (TFA) and subsequently chromatographed on a capillary reverse phase HPLC column [500 μm x 25 cm internal diameter of silicon capillary column fused packaged with 5 μm of Vydac C4 material (Vydac, Hesperia, CA)] using a linear gradient (2% per minute) from 0% to 100% acetonitrile in water containing 0.1% TFA. Fractions containing high LIART binding activity were determined as above, combined and, if desired, lyophilized. Specific ELISA for R-LIART Serial dilutions of samples containing R-LIART (in 50 mM NaHCO3, brought to pH 9 with NaOH), were coated in microtitre plates of E. I .A. 96-well flat bottom of Linbro / Titertek (ICN Biomedicals Inc., Aurora, OH) at 100 μl / well. After incubation at 4 ° C for 16 hours, the wells were washed six times with 200 μl of PBS containing 0.05% Tween 20 (PBS-Tween). The wells were then incubated with Flag®-LIART at 1 μg / ml in PBS-Tween with 5% fetal calf serum (FCS) for 90 minutes (100 μl per well), followed by washing as before, then, each well was incubated with the anti-Flag® M2 monoclonal antibody at 1 μg / ml in PBS-Tween containing 5% SBF for 90 minutes (100 μl per well), followed by washing as before. Subsequently, the wells were incubated with a polyclonal goat anti-m IgG 1 horseradish peroxidase-conjugated antibody (a 1: 5000 dilution of the commercial raw material in PBS-Tween containing 5% FCS) for 90 minutes (100 μl per well). Antibody conjugated to H RP was obtained from Southern Biotechnology Associates, Inc., Birmingham, Alabama. The wells were then washed six times, as before.

For the development of ELISA, a substrate mix was added to the wells [100 μl per well of a 1: 1 pre-mix of the TMB Peroxidase substrate and Peroxidase Solution B (Kerkegaard Perry Laboratoires, Gaithersburg, Maryland)] . After the reaction with sufficient color, the enzymatic reaction was terminated by addition of 2 N H2SO4 (50 μl per well). The color intensity (indicating the LIART binding activity) was determined by measuring the extraction at 450 nm in a V Max plate reader (Molecular Devices, Sunnyvale, CA). EXAMPLE 2: Amino Acid Sequence (a) R-LIART Purified from Jurkat Cells The R-LIART protein isolated from Jurkat cells was digested with trypsin, using standard procedures. The amino acid sequence analysis was carried out in one of the peptide fragments produced by tryptic digestion. The fragment was found to contain the following sequence, which corresponds to amino acids 327 to 333 of the sequence presented in SEQ ID NO: 2. VPANEGD. (b) R-LIART purified from PS-1 cells The R-LIART protein was also isolated from PS-1 cells.

PS-1 is a line of human B cells that arise spontaneously after the lethal irradiation of human peripheral blood lymphocytes (PBL). The R-LIART protein was digested with trypsin, using conventional procedures. The analysis of amino acid sequences was carried out in fragments of peptides that resulted from the digestion of triptychs. It was found that one of the fragments contains the following sequence, which, like the fragment presented in (a), corresponds to amino acids 327 to 333 of the sequence presented in SEQ ID NO: 2: VPANEGD. EXAMPLE 3: DNA and Amino Acid Sequences The amino acid sequences of the extra tryptic digestion peptide fragments of R-LIART were determined. Degenerate oligonucleotides were prepared, based on the amino acid sequence of two of the peptides. A fragment of the R-LIART DNA was isolated and amplified by the polymerase chain reaction (PCR), using the degenerate oligonucleotides as the 5 'and 3' primers. The PCR was carried out according to conventional procedures, using cDNA derived from the PS-1 cell line described in Example 2 as the standard. The nucleotide sequence of the isolated R-LIART DNA fragment (excluding portions corresponding to the part of the primers) and the sequence of the amino acids encoded by it, is presented in Figure 1 (SEQ ID NOS: 3 and 4 ). The sequence of the entire R-LIART DNA fragment isolated by PCR corresponds to nucleotides 988 to 1 164 of SEQ ID NO: 1, which encode amino acids 330 to 388 of SEQ I D NO: 2. The amino acid sequence in SEQ ID NO: 4 has significant homology with the so-called dead domains found in certain receptors. The cytoplasmic region of Fas and the type I TNF receptor each contains a dead domain, which is associated with the transduction of an apoptotic signal (Tartaglia et al., Cell 74: 845, 1993, Itoh and Nagata, J. Biol. Chem. 268: 10932, 1993). Therefore, it is thought that the sequence presented in SEQ I D NO: 4 was not found within the cytoplasmic domain of R-LIART. A probe derived from the isolated fragment before was used to screen a cDNA library (cDNA derived from human anterior skin fibroblasts in the vector? Gt10) and a human R-LIART cDNA was isolated. The nucleotide sequence of the coding region of this cDNA is presented in SEQ I D NO: 1 and the amino acid sequence encoded therein is shown in SEQ I D NO: 2. EXAMPLE 4: Monoclonal Antibodies Binding to R-LIART This example illustrates a method for preparing the monoclonal antibodies that bind R-LIART. Suitable immunogens that can be used to generate said antibodies include, but are not limited to, a purified R-LIART protein or an immunogenic fragment thereof such as the extracellular domain or fusion proteins containing R-LIART (e.g. , a soluble fusion protein of R-LIART / Fc). Purified R-LIART can be used to generate monoclonal antibodies immunoreactive therewith, using conventional techniques such as those described in the US Pat. No. 4,41 1, 993. Briefly, mice are immunized with R-LIART immunogen emulsified in complete Freund's adjuvant and injected in amounts ranging from 10-100 μg subcutaneously or intraperitoneally. Ten to twelve days later, the Immunized animals were boosted with additional R-LIART emulsified with incomplete Freund's adjuvant. The mice were periodically boosted on an immunization schedule of one to two weeks. Serum samples were taken periodically by retro-orbital bleeding or removal of the tip of the tail to test R-LIART antibodies, by spot spot analysis, ELISA (Enzyme-Linked Immunosorbent Assay) or inhibition of LIART binding. After detection of an appropriate antibody titre, the animals are provided with an intravenous injection of R-LIART in saline. Three to four days later, the animals were sacrificed, spleen cells recovered, and the spleen cells were fused to a murine myeloma cell line, e.g., NS1 or preferably P3x63Ag8.653 (ATCC CRL 1580) . The fusions generate hybridoma cells, which are seeded in multiple microtiter plates in a selective medium of HAT (hypoxanthine, aminopterin and thymidine) to inhibit the proliferation of unfused cells, myeloma hybrids and spleen cell hybrids. Hybridoma cells were screened by ELISA for reactivity against R-LIART purified by adaptations of the techniques described in Engvall et al., Immunochem, 8: 871, 1971 and in the US Patent. No. 4,703,004. A preferred screening technique is the antibody capture technique described in Beckmann et al., (J. Immunol., 144: 4212, 1990). The cells of Positive hybridomas can be injected intraperitoneally into syngeneic BALB / c mice to produce ascites containing high concentrations of R-LIART monoclonal antibodies. Alternatively, the hybridoma cells can be grown in vitro in flasks or roller bottles by various techniques. Monoclonal antibodies produced in mouse ascites can be purified by precipitation of ammonium sulfate, followed by gel exclusion chromatography. Alternatively, affinity chromatography based on the binding of antibodies to Protein A or Protein G can also be used, such as affinity chromatography based on the binding of R-LIART. EXAMPLE 5: Northern Blot Analysis The tissue distribution of R-LIART mRNA was investigated by Northern blot analysis, as follows. An aliquot of a radiolabelled probe (the same radiolabeled probe used to screen the cDNA library in Example 3) was added to two different Northern blot analyzes of human multiple tissue (Clontech, Palo Alto, CA; Biochain, Palo Alto, CA). Hybridization was carried out overnight at 63 ° C in 50% formamide as previously described (March et al., Nature 315: 641-647, 1985). The spot analyzes were then washed with 2X SSC, 0.1% SDS at 68 ° C for 30 minutes. A specific probe for glycerol-aldehyde phosphate dehydrogenase (GAPDH) was used to standardize RNA loads.

A single 4.4 kilobase (kb) transcript was present in all tissues examined, including basal, thymus, peripheral blood lymphocytes (LSP), prostate, testes, ovaries, uterus, placenta and multiple tissues throughout the gastrointestinal tract ( including esophagus, stomach, duodenum, jejunum / ileum, colon, rectum and small intestine). The cells and tissues with the higher levels of R-LIART mRNA are LSP, spleen and ovaries, as shown by comparison to control hybridizations with a specific GAPDH probe. EXAMPLE 6: Full length human R-LIART binding analysis was expressed and tested for the ability to bind LIART. The binding analysis was carried out in the following manner. A fusion protein comprising a leucine compaction peptide fused to the N-terminus of a soluble LIART polypeptide (LZ-LIART) was used in the analysis. An expression construct was prepared, essentially as described for the preparation of the expression construct of Flag®-LIART in Wiley et al. (Immunity, 3: 673-682, 1995; incorporated herein by reference), except that DNA encoding the Flag® peptide was replaced with a sequence encoding a modified leucine compactor that allows the trimerization. The construct, in the expression vector pDC409, encoded a leader sequence derived from human cytomegalovirus, followed by the compaction portion of leucine fused to the N-terminus. of a soluble LIART polypeptide. The LIART polypeptide comprised amino acids 95-281 of human LIART (a fragment of the extracellular domain), as described in Wiley et al., (Supra). LZ-LIART was expressed in CHO cells and purified from the culture supernatant. The expression vector designated pDC409 is a mammalian expression vector derived from vector pDC406 described in McMahan et al. (EMBO J. 10: 2821-2832, 1991, incorporated herein by reference). The features added to pDC409 (compared to pDC406) include additional unique restriction sites in the multiple cloning site (scm); three stop codons (one in each reading frame) placed downstream of the scm and a T7 polymerase promoter, downstream of the scm, which facilitates sequencing of the DNA inserted into the scm. For the expression of the full-length human R-LIART protein, the entire coding region (ie, the DNA sequences presented in SEQ ID NO: 1) was amplified by polymerase chain reaction (PCR). The pattern used in the PCR was the cDNA clone isolated from a cDNA bank of human anterior skin fibroblasts, as described in example 3. The isolated and amplified DNA was inserted into the expression vector pDC409, to give a construct designated pDC409-R-LIART. The CrmA protein was used to inhibit apoptosis of host cells expressing recombinant R-LIART, as discussed above and in Example 8. An expression vector designated pDC409- CrmA contained pox virus CrmA encoding DNA in pDC4Q9 = The protein derived from pox of a 38 kilodalt cow that was subsequently designated CrmA was described in Pickiup et al., (Proc. Nati. Acad. Sci. USA 83: 7698-7702, 1986, incorporated herein by reference). The CV-1 / EBNA cells were co-transfected with pDC409-R-LIART together with pDC409-CrmA, or with pDC409-CrmA alone. The cells were cultured in DMEM supplemented with 10% fetal bovine serum, penicillin, streptomycin and glutamine. 48 hours after transfection, cells were removed non-enzymatically and incubated with LZ-LIART (5 μg / ml), a biotinylated anti-LZ monoclonal antibody (5 μg / ml) and streptavidin conjugated with phycoerythrin (1: 400), before analysis, by fluorescence activated cell scanning (FACS). The cytometric analysis was carried out in FACscan (Beckton Dickinson, San José, CA). CV-1 / EBNA cells co-transfected with vectors encoding R-LIART and CrmA showed significantly increased binding of LZ-LIART, compared to cells transfected with the CrmA coding vector alone. EXAMPLE 7: R-LIART Blocks LIART-induced Apoptosis of White Cells R-LIART was tested for the ability to block LIART-induced apoptosis of Jurkat cells. R-LIART used in the analysis took the form of a fusion protein designated R-LIART / Fc, which comprises the extracellular domain of R-LIART human, fused to the N-terminus of a Fe polypeptide derived from human IgG1. The CV1-EBNA cells were transfected with a recombinant expression vector comprising a gene fusion encoding the R-LIART / Fc protein, in the vector pDC409 described in example 6, and cultured to allow the expression of the protein of fusion. The R-LIART / Fc fusion protein was recovered from the culture supernatant. Methods for fusing DNA encoding a lgG 1 Fe polypeptide to DNA encoding a heterologous protein were described in Smíth et al (Cell 73: 1349-1360, 1993); in the present, analogous procedures were employed. A fusion protein designated TN F-R2-Fc, used as a control, comprised the extracellular domain of the TNF receptor protein known as p75 or p80 TNF-4 (Smith et al., Science 248: 1019-1023, 1990; Smith et al., Cell 76: 959-962, 1994), fused to a Fe polypeptide. A monoclonal mouse antibody which is a blocking antibody directed against human LIART was also used in the assay. Jurkat cells were incubated with variable or constant concentrations of LZ-LIART (the LZ-LIART fusion protein described in Example 6), in the absence or presence of varying concentrations of R-LIART-Fc, TN F-R2- Fc, or the monoclonal antibody specific for LIART. Cell death was quantified using a viability assay of MTT cells (MTT is 3- [4,5-dimethylthiazol-2-yl] -2,4-diphenyltetrazolium bromide), as previously described (Mosmann, J. Immunol, Methods 65: 55-63, 1983). The results are shown in Figure 2, which shows the percentage death of Jurkat cells that were not treated (?) Or that were treated with variable (A) or constant (O, •,, M,) concentrations of LZ-LIART (13 ng / ml) in the absence (•) or presence of variable concentrations of LIART-R2-Fc (M). TN F-R2-Fc (Q), or the anti-LIART antibody (o). The variable concentrations for all substances were initiated at 10 μg / ml and serially diluted. The anti-LIART monoclonal antibody and R-LIART / Fc block each apoptosis induced by LIART in a dose-dependent manner, while TN FR2-Fc does not. Therefore, the extracellular domain of R-LIART is capable of binding to LIART and of inhibiting apoptosis mediated by LIART. EXAMPLE 8: R-LIART-induced apoptosis is blocked by caspase and FADD-DN inhibitors CV-1 / EBNA cells were transfected, by the DEAE-dextran method with expression plasmids for R-LIART (pDC409-R- LIART), together with an excess of three times the expression vector (pDC409) in the presence or absence of z-VAD-fmk (10 μM: in the culture medium), or together with a three-fold excess of the expression vector pDC409-CrmA, pDC409-p35 or pDC409-FADD-DN. In addition, 400 ng / portlet of an expression vector of the E. coli lacz gene was co-transfected together with all DNA mixtures. The Transfected cells were grown in cameras mounted in porta porta objects. The expression vector pDC409 of mammals and the vector pDC409-R-LIART encoding full-length human R-LIART, was described in example 6. The tripeptide derivative of zVAD-fmk (benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone) is available from Enzyme System Products, Dublin, California. The 38 kilodalton cow pox virus-derived protein subsequently designated CrmA was described in Pickup et al. (Proc. Nati, Acad. Sci. USA 83: 7698-7702, 1986, incorporated herein by reference). The sequence information for CrmA was presented in Figure 4 of Pickup et al., Supra. A 35 kilodalton protein encoded by the nuclear polyhedrosis virus of Autographa californica, a baculovirus, was described in Friesen and Miller (J. Viro !. 61: 2264-2272, 1987, incorporated herein by reference). The sequence information for this protein, designated p35 baculovirus in the present, was presented in Figure 5 of Friesen and Miller, supra. FADD (also referred to as MORTI) was described in Boldin et al. (J. Biol. Chem. 270: 7795-7798, 1995, incorporated herein by reference). The preferred protein called FADD-DN (Dominant negative FADD) in a C-terminal fragment of FADD that includes the dead domain. The DNA encoding FADD-DN, fused to an N-terminal Flag® epitope tag (described above), was inserted into the expression vector pDC409 described in FIG. example 6, to form pDC409-FADD-DN. The FADD-DN polypeptide corresponds to amino acids 1 17 to 245 of the MORTI amino acid sequence presented in Boldin et al., Supra. 48 hours after transfection, the cells were washed with PBS, fixed with glutaraldehyde and incubated with X-gal (5-bromo-4-chloro-3-indoxyI-β-D-galactopyranoside). Cells that express β-galactosidase stain blue. A decrease in the percentage of stained cells indicates the loss of β-galactosidase expression and correlates with the death of cells expressing the proteins co-transfected with the lacz gene. The results are presented in Figure 3, where the plotted values represent the mean and normal deviation of at least three separate experiments. Poxvirus CrmA, baculovirus p36, FADD-DN and z-VAD-fmk can effectively reduce the death of transfected cells expressing R-LIART.

LIST OF SEQUENCES (1) GENERAL INFORMATION (i) Applicant: Rauchs, Charles Walczak, Henning (i) TITLE OF THE INVENTION: RECEIVER WHO JOINS THE LINKING INDUCTOR FOR TNF-related APOPTOSIS (LIART) (ii) NUMBER OF SEQUENCES: 5 (iv) ADDRESS OF CORRESPONDENCE: (A) ADDRESS: Kathryn A. Anderson, Immunex Corporation (B) STREET: 51 University Street (C) CITY : Seattle, (D) STATE: WA (E) COUNTRY: USA (F) CP: 98101 (v) COMPUTER LEADABLE FORM: (A) TYPE OF MEDIA: Flexible Disk (B) COMPUTER: IBM Compatible PC (C) OPERATING SYSTEM: MS-DOS / Windows 95 (D) SOFTWARE: Word for Windows 95, 7.0a (vi) CURRENT REQUEST DATA: (A) APPLICATION NUMBER: -by being assigned- (B) SUBMISSION DATE: FEB 11-1998 (C) CLASSIFICATION: (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 883,036 (B) SUBMISSION DATE: JUNE 26, 1997 (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 869,852 (B) SUBMISSION DATE: 04-JUN-1997 (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 829,536 (B) SUBMISSION DATE: MARCH 28, 1997 (vii) PREVIOUS APPLICATION DATA : (A) APPLICATION NUMBER: US 08 / 815,255 (B) SUBMISSION DATE: MARCH 12, 1997 (vii) PREVIOUS APPLICATION DATA: (A) APPLICATION NUMBER: US 08 / 799,861 (B) SUBMISSION DATE: 13-FEB-1997 (viii) POWDER / AGENT INFORMATION: (A) NAME: Anderson, Kathryn A. (B) REGISTRATION NUMBER: 32,172 (C) REFERENCE / CASE NUMBER: 2625-WO (ix) TELECOMMUNICATION INFORMATION: (A) TELEPHONE : (206) 587-0430 (B) TELEFAX: (206) 233-0644 (C) TELEX: 75622 (2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 1323 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: single (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) CONTRASTING: NO (vií) IMMEDIATE SOURCE: (B) CLON: huTrail-R (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 1..1323 (x¡) DESCRIPTION OF SEQUENCE: SEQ ID NO: 1 ATG GAA CAA CGG GGA CAG AAC GCC "CCG GCC GCT TCG GGG GCC CGG AAA 4 Met Glu Gln Arg Gly Gln Asn Wing Pro Wing Wing Ser Gly Wing Arg Lys 1 5 10 15 AGG CAC GGC CCA GGA CCC AGG GAG GCG CGG GGA GCC AGG CCT GGG CCC 96 Arg His Gly Pro Gly Pro Arg Glu Wing Arg Gly Wing Arg Pro Gly Pro 20 25 30 CGG GTC CCC AAG ACC CTT GTG CTC GTT GTC GCC GCG GTC CTG CTG TTG 144 Arg Val Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45 GTC TCA GCT GAG TCT GCT CTG ATC ACC CA CA CA GAC CTA GCT CCC CAG 192 Val Ser Ala Glu Be Ala Leu lie Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60 CAG AGA GCG GCC CCA CAA CAA AAG AGG TCC AGC CCC TCA GAG GGA TTG 240 Gln Arg Ala Wing Gln Gln Lys Arg Ser Ser Pro Glu Gly Leu 65 70 75 80 TGT CCA CCT GGA CAC CAT ATC TCA GAA GAC GGT AGA GAT TGC ATC TCC 288 Cys Pro Pro Gly His His lie Ser Glu Asp Gly Arg Asp Cys He Ser 85 90 95 TGC AAA TAT GGA CAG GAC TAT AGC ACT CAC TGG AAT GAC CTC CTT TTC 336 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110TGC TTG CGC TGC ACC AGG TGT GAT TGA GGT GAA GTG GAG CTA AGT CCG 384 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 TGC ACC ACG ACC AGA AAC AC GTG TGT CAG TGC GAA GAA GGC ACC TTC 432 Cys Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 CGG GAA GAA GAT TCT CCT GAG ATG TGC CGG AAG TGC CGC AC GGG TGT 480 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 CCC AGA GGG ATG GTC AAG GTC GGT GAT TGT AC CCC TGG AGT GAC ATC 528 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp He 165 170 175 GAA TGT GTC CAC AAA GAA TCA GGT AC AAG CAC AGT GGG GAA GCC CCA 576 Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180 185 190 GCT GTG GAG GAG ACG GTG ACC TCC AGC CC? GGG? CT CCT GCC TCT CCC 624 Wing Val Glu Glu Thr Val Thr Ser Ser Pro Pro Gly Thr Pro Wing Pro Pro 195 200 205 TGT TCT CTC TCA GGC ATC ATC ATA GGA GTC ACÁ GTT GCA GCC GTA GTC 672 Cys Ser Leu Ser Gly He He He Gly Val Thr Val Wing Wing Val Val 210 215 220 TTG ATT GTG GCT GTG TTT GTT TGC AAG TCT TTA CTG TGG AAG AAA GTC 720 Leu He Val Wing Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 CTT CCT TAC CTG AAA GGC ATC TGC TCA GGT GGT GGT GGG GAC CCT GAG 768 Leu Pro Tyr Leu Lys Gly He Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 CGT GTG GAC AGA AGC TCA CA CGA CCT GGG GCT GAG GAC AAT GTC CTC 816 Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Wing Glu Asp Asn Val Leu 260 265 270 AAT GAG ATC GTG AGT ATC TTG CAG CCC ACC CAG GTC CCT GAG CAG GAA 864 Asn Glu He Val Ser He Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280 285 ATG GAA GTC CAG GAG CCA GCA GAG CAC AC GGT GTC AAC ATG TTG TCC 912 Met Glu Val Gln Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 290 295 300 CCC GGG GAG TCA GAG CAT CTG CTG GAA CCG GCA GAA GCT GAA AGG TCT 960 Pro Gly Glu Ser Glu His Leu Leu Glu Pro Wing Glu Wing Glu Arg Ser 305 310 315 320 CAG AGG AGG CTG CTG GTT CCA GCA AAT GAA GGT GAT CCC ACT GAG 1008 Gln Arg Arg Arg Leu Leu Val Pro Wing Asn Glu Gly Asp Pro Thr Glu 325 330 335 ACT CTG AGA CAG TGC TTC GAT GAC TTT GCA GAC TTG GTG CCC TTT GAC 1056 Thr Leu Arg Gln Cys Phe Asp Asp Phe Wing Asp Leu Val Pro Phe Asp 340 345 350 TCC TGG GAG CCG CTC ATG AGG AAG TTG GGC CTC ATG GAC AAT GAG ATA 1104 Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu He 355 360 365 AAG GTG GCT AAA GCT GAG GCA GCG GGC CAC AGG GAC ACC TTG TAC ACG 1152 Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Tyr Thr 370 375 380 ATG CTG ATA AAG TGG GTC AAC AAA ACC GGG CGA GAT GCC TCT GTC CAC 1200 Met Leu He Lys Trp Val Asn Lys Thr Gly Arg Asp Ala Ser Val His 385 390 395 400 ACC CTG CTG GAT GCC TTG GAG ACG CTG GGA GAG AGA CTT GCC AAG CAG 1248 Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln 405 410"415 AAG ATT GAG GAC CAC TTG TTG AGC TCT GGA AAG TTC ATG TAT CTA GAA 1296 Lys He Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430 GGT A? T GCA G? C TCT GCC ATG TCC TAA 1323 Gly Asn Ala Asp Ser Ala Met Ser * 435 440 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 440 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) DESCRIPTION OF SEQUENCE: SEQ ID NO: 32 Met Glu Gln Arg Gly Gln Asn Wing Pro Wing Wing Ser Gly Wing Arg Lye 1 5 10 15 Arg His Gly Pro Gly Pro Arg Glu Wing Arg Gly Wing Arg Pro Gly Pro 20 25 30 Arg Val Pro Lys Thr Leu Val Leu Val Val Wing Wing Val Leu Leu Leu 35 40 45 Val Ser Wing Glu Wing Ala Leu He Thr Gln Gln Asp Leu Ala Pro Gln 50 55 60 Gln Arg Ala Ala Pro Gln Gln Lys Arg Ser Ser Pro Ser Glu Gly Leu 65 70 75 80 Cys Pro Pro Gly His His Ser Glu Asp Gly Arg Asp Cys He Ser 85 90 95 Cys Lys Tyr Gly Gln Asp Tyr Ser Thr His Trp Asn Asp Leu Leu Phe 100 105 110 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 Cys Thr Thr Arg Asn Thr Val Cys Gln Cys Glu Glu Gly Thr Phe 130 135 140 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 160 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp He 165 170 175 Glu Cys Val His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Wing Pro 180 185 190 Wing Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro 'Wing Ser Pro 195 200 205 Cys Ser Leu Ser Gly He He He Gly Val Thr Val Ala Wing Val Val 210 215 220 Leu He Val Wing Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 Leu Pro Tyr Leu Lys Gly He Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 Arg Val Asp Arg Ser Ser Gln Arg Pro Gly Wing Glu Asp Asn Val Leu 260 265 270 Asn Glu He Val Ser He Leu Gln Pro Thr Gln Val Pro Glu Gln Glu 275 280. 285 Met Glu Val Gln Glu Pro Wing Glu Pro Thr Gly Val Asn Met Leu Ser 290 295 300 Pro Gly Glu Ser Glu His Leu Leu Glu Pro Wing Glu Wing Glu Arg Ser 305 310 315 320 Gln Arg Arg Arg Leu Leu Val Pro Wing Asn Glu Gly Asp Pro Thr Glu 325 330 335 Thr Leu Arg Gln Cys Phe Asp Asp Phe Wing Asp Leu Val Pro Phe Asp 340 345 '350 Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu He 355 360 365 Lys Val Wing Lys Wing Wing Glu Wing Wing His Arg Asp Thr Leu Tyr Thr 370 375 380 Met Leu He Lys Trp Val Asn Lys Thr Gly Arg Asp Wing Ser Val His 385 390 395 400 Thr Leu Leu Asp Ala Leu Glu Thr Leu Gly Glu Arg Leu Ala Lys Gln 405 410 415 Lys He Glu Asp His Leu Leu Being Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430 GlV Asn Ala Asp Ser Ala Met Ser 435 440 (2) INFORMATION FOR SEQ ID NO: 3: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 157 base pairs (B) TYPE: nucleic acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: cDNA (iii) HYPOTHETICAL: NO (iv) CONTRASTING: NO (v) TYPE OF FRAGMENT: internal (vii) IMMEDIATE SOURCE: (B) ) CLON: huTrail-R frag (ix) FEATURE: (A) NAME / KEY: CDS (B) LOCATION: 3.155 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: CT GAO ACT CTG AGA CAG TGC TTC GAT GAC TTT GCA GAC TTG GTG CCC 47 Glu Thr Leu Arg Gln Cys Phe Asp Asμ Phe Wing Asp Leu Val Pro 1 5 10 15 TTT GAC TCC TGG GAG CCG CTC ATG AGG AAG TTG GGC CTC ATG GAC AAT 95 Phe Asp Ser Trp Glu Pro Leu MOL Arg Lys Leu Gly Leu Met Asp Asn 20 25 30 GAG ATA AAG GTG GCT AAA GCT GAG GCA GCG GGC CAC AGG GAC ACC TTG 14 i Glu He Lys Val Wing Lys Wing Glu Wing Wing Gly His Arg Asp Thr Leu 35 40 45 TNC ACN ATG CTG AT '157 Xaa Thr Met Leu 50 (2) INFORMATION FOR SEQ ID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 51 amino acids (B) TYPE: amino acid (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE: protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: Glu Thr Leu Arg Gln Cys Phe Asp Asp Phe Wing Asp Leu Val Pro Phe 1 5 10 15 Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn Glu 20 25 30 He Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Xaa 35 40 45 Thr Met Leu 50 (2) INFORMATION FOR SEQ ID NO: 5: (i) SEQUENCE CHARACTERISTICS (A) LENGTH: 8 amino acids (B) TYPE: amino acid (C) THREAD FORM: simple (D) TOPOLOGY: linear (ii) TYPE OF MOLECULE : peptide (iii) HYPOTHETICAL: NO (iv) CONTRASTING: NO (vii) IMMEDIATE SOURCE: (B) CLON: FLAG peptide (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 5: Asp Tyr Lys Asp Asp Asp Asp Lys 1 5

Claims (39)

1. CLAIMS 1. A purified LIART receptor (R-LIART) polypeptide that is capable of binding to LIART, wherein R-LIART is characterized by comprising the VPANEGD amino acid sequence.
2. 2. An R-LIART polypeptide of claim 1, wherein said polypeptide is further characterized by a molecular weight of about 50 to 55 kilodaltons.
3. 3. An R-LIART polypeptide of claim 1, wherein the polypeptide is further characterized in that it comprises the amino acid sequence ETLRQCFDDFADLVPFDSWEPLMRK LGLMDNEIKVAKAEAAGHRDTLXTML.
4. 4. An R-LIART polypeptide of claim 3, wherein said polypeptide is further characterized by a molecular weight of about 50 to 55 kilodaltons.
5. 5. A purified R-LIART polypeptide selected from the group consisting of: a) the R-LIART polypeptide of SEQ ID NO: 2 and b) a fragment of the polypeptide of (a), wherein said fragment is capable of binding to LIART.
6. 6. An R-LIART polypeptide of claim 6, wherein the polypeptide comprises amino acids of xa 440 of SEQ ID NO: 2, wherein x represents an integer from 51 to 59.
7. 7. A R-LIART polypeptide of Claim 6, wherein the polypeptide comprises amino acids 54 to 440 of SEQ ID NO: 2.
8. 8. An R-LIART polypeptide of claim 5, wherein said fragment is a soluble R-LIART comprising an extracellular domain of the R-LIART protein of SEQ ID NO: 2.
9. 9. A purified R-LIART polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence presented in SEQ ID NO: 2.
10. 10. An R-LIART polypeptide of claim 9, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence presented in SEQ ID NO: 2.
11. 11. An R-LIART polypeptide of claim 10, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence presented in SEQ ID NO: 2.
12. 12. An R-LIART polypeptide of claim 9, wherein the polypeptide is present in nature.
13. 13. An oligomer comprising two to four R-LIART polypeptides of claim 5.
14. 14. An oligomer comprising two to four R-LIART polypeptides of claim 8.
15. 15. A composition comprising the R polypeptide. -LIART of claim 5, and a physiologically acceptable diluent, excipient, or vehicle.
16. 16. A composition comprising the R-LIART polypeptide of claim 8, and a physiologically acceptable diluent, excipient, or vehicle.
17. 17. An isolated R-LIART DNA, wherein DNA comprises the nucleotide sequence presented in Figure 1.
18. 18. An isolated R-LIART DNA, wherein DNA encodes a polypeptide selected from the group consisting of: a) the R-LIART polypeptide of SEQ ID NO: 2 and b) a fragment of the polypeptide of (a), wherein the fragment is capable of binding to LIART.
19. 19. An R-LIART DNA of claim 18, wherein the DNA encodes amino acids 1 to 440 of SEQ ID NO: 2.
20. 20. An R-LIART DNA of claim 18, wherein the polypeptide comprises the amino acids of xa 440 of SEQ ID NO: 2, wherein x represents an integer from 51 to 59.
21. 21. A DNA of R-LIART of claim 20, wherein the polypeptide comprises amino acids 54 to 440 of SEQ ID NO: 2.
22. 22. An R-LIART DNA of claim 18, wherein the fragment is a soluble R-LIART comprising the extracellular domain of the R-LIART protein of SEQ ID NO: 2.
23. 23. An isolated R-LIART DNA, wherein DNA encodes a polypeptide comprising an amino acid sequence that is at least 80% identical to the amino acid sequence presented in SEQ ID NO: 2.
24. 24. A R-LIART DNA of claim 23, wherein the polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence presented in SEQ ID NO: 2.
25. 25. An R-LIART DNA of claim 24, wherein the polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence presented in SEQ ID NO: 2.
26. 26. An R-LIART DNA of claim 23, wherein the polypeptide is present in nature.
27. 27. An expression vector comprising a DNA according to claim 18.
28. 28. An expression vector comprising a DNA according to claim 19.
29. 29. An expression vector comprising a DNA according to claim 20.
30. 30. An expression vector comprising a DNA according to claim 22.
31. 31. An expression vector comprising a DNA according to claim 23.
32. 32. A host cell transformed with an expression vector of claim 27.
33. 33. A host cell transformed with an expression vector of claim 28.
34. 34. A host cell transformed with an expression vector of claim 29.
35. 35. A host cell transformed with an expression vector of claim 30.
36. 36. A host cell transformed with an expression vector of claim 31.
37. 37. An isolated R-LIART DNA comprising at least 60 nucleotides of the SEQ I D NO: 1 sequence, or the DNA or RNA complement thereof.
38. 38. An antibody that is directed against an R-LIART polypeptide of claim 5, or an antigen-binding fragment of the antibody.
39. 39. An antibody of claim 38, wherein the antibody is a monoclonal antibody.