WO2000026244A2 - Nouveau membre de la famille des facteurs de necrose des tumeurs, ligand du recepteur de la mort cellulaire (drl) et compositions et procedes correspondants - Google Patents

Nouveau membre de la famille des facteurs de necrose des tumeurs, ligand du recepteur de la mort cellulaire (drl) et compositions et procedes correspondants Download PDF

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WO2000026244A2
WO2000026244A2 PCT/US1999/025954 US9925954W WO0026244A2 WO 2000026244 A2 WO2000026244 A2 WO 2000026244A2 US 9925954 W US9925954 W US 9925954W WO 0026244 A2 WO0026244 A2 WO 0026244A2
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drl
nucleic acid
seq
polypeptide
tag
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PCT/US1999/025954
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WO2000026244A3 (fr
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Michael J. Lenardo
Jin Wang
Di Jiang
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The Government Of The United States Of America, As Represented By The Secretary, Department Of Health And Human Services
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Priority to AU14670/00A priority Critical patent/AU1467000A/en
Publication of WO2000026244A2 publication Critical patent/WO2000026244A2/fr
Publication of WO2000026244A3 publication Critical patent/WO2000026244A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/52—Cytokines; Lymphokines; Interferons
    • C07K14/525—Tumour necrosis factor [TNF]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the invention relates to isolated nucleic acids encoding DRL (death receptor ligand) proteins, to unique fragments of the nucleic acid encoding DRL proteins, and to the polypeptides encoded by those nucleic acids.
  • the invention further relates to antibodies to those polypeptides.
  • the invention also relates to methods of utilizing the nucleic acids, polypeptides and antibodies of the invention for modulating immune responses.
  • Lymphocyte apoptosis is critical for the maintenance of peripheral tolerance.
  • Molecules such as CD95 (Fas/APO-1) and other molecules related to tumor necrosis factor (TNF) and TNF receptor (TNFR) have been shown to play a critical role in lymphocyte apoptosis and peripheral tolerance.
  • Mice deficient in Fas or Fas ligand develop enlarged secondary lymphoid organs, accumulation of CD3+CD4-CD8- T cells, increase in total B cell number and production of autoantibodies.
  • TNF and TNFR type I may also contribute to autoimmunity. This suggests that TNF and TNFR family members play an important role in lymphocyte apoptosis and maintenance of peripheral tolerance.
  • TNF family members are expressed on the cell surface as type II membrane proteins.
  • the extracellular domains of the TNF family members are most conserved among all the members and interact with their receptors.
  • TNFR members are type I membrane proteins with characteristic cysteine-rich repeats in the extracellular domain.
  • the intracellular regions of TNFR members are divergent and mediate different biological signals into the cells.
  • the members of the TNF family are expanding.
  • TNF- ⁇ lymphotoxin- ⁇
  • LT- ⁇ lymphotoxin- ⁇
  • LT- ⁇ lymphotoxin- ⁇
  • TRANCE/RANKL LIGHT
  • TRAIL/APO-2 ligand TWEAK
  • Interactions of TNF family members with their receptors induce diverse biological functions besides apoptosis, including cell proliferation and differentiation.
  • Mice deficient in LT- ⁇ or LT- ⁇ display significant defects in lymph nodes and spleen, suggesting that LT- ⁇ and LT- ⁇ are required for the development of peripheral lymphoid organs.
  • CD40 on B cells and CD40 ligand on T cells has been shown to be important for B cell maturation and activation, and for T cell proper functions.
  • Interactions of TRANCE/RANKL on T cells with its receptors on dendritic cells stimulates both the antigen presentation ability of dendritic cells and T cell growth.
  • Fas ligand and TNF- ⁇ have been shown to be critical for apoptosis of mature lymphocytes. Mice deficient in Fas ligand or its receptor, Fas, display dramatic lymphocyte accumulation in the peripheral lymphoid tissues and autoimmunity.
  • TNFRl deficiency enhances the severity of symptoms resulting from Fas mutation.
  • Fas ligand and TNF appear to be two of the major inducers of apoptosis in the TNF family.
  • TRANCE/RANKL, TRAIL/APO-2 ligand, and TWEAK are capable of inducing apoptosis in certain cell lines, and may induce apoptosis in mature lymphocytes under specific conditions.
  • ligands in the TNF family may have diverse stimulatory and inhibitory functions in lymphocytes as well as other cell- types alone or in combination with other lymphokines, cytokines, chemokines or other stimulatory agents.
  • Various cytokines, chemokines or lymphokine enhancers and inhibitors that are generally known (Paul, W.E. (ed.) Fundamental Immunology (3 rd ed.) New York Raven, 1993) may be employed to enhance or inhibit the activities of DRL-1 or DRL-2.
  • DRL-1 for Jeath receptor /igand-1. It is may also be referred to herein as TNF-gamma or TNF-like ligand (TLL, pronounced "tall”).
  • TLL TNF-like ligand
  • DRL-2 SUMMARY OF THE INVENTION
  • the present invention provides isolated nucleic acids encoding the human DRL- 1 protein and the human DRL-2 protein as set forth in the Sequence Listing as SEQ ID NO: 1 and SEQ ID NO: 3, respectively.
  • the invention also provides a unique fragment of the isolated nucleic acid that encodes amino acid residues 65 to 250 of DRL-1 as set forth in SEQ ID NO:2.
  • the present invention provides an isolated nucleic acid encoding the mouse DRL-1 protein as set forth in the Sequence Listing as SEQ ID NO:5.
  • the invention further provides purified polypeptides encoded by the nucleic acids encoding the DRL protein as forth in SEQ ID NO: 1 , SEQ ID NO:3 and SEQ ID NO:5, purified polypeptides having the sequence set forth in the sequence listing as SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6 and of DRL polypeptides which can be used to stimulate an immune response or induce apoptosis.
  • the invention provides purified antibodies which bind to the
  • DRL protein or fragments thereof can be used to inhibit a T cell response, to inhibit apoptosis or to suppress an autoimmune response. These antibodies can also be used as agonists to stimulate the membrane-bound form of the ligand.
  • Figure 1A shows that amino acid sequence of DRL-1.
  • the amino acid sequence of DRL-1 suggests that it is a type 2 membrane protein.
  • the putative transmembrane domain is underlined and corresponds to amino acids 26-50 of SEQ ID NO:2.
  • the asterisk indicates the position of the predicted N-glycosylation site, corresponding to residue 124 of SEQ ID NO:2.
  • the arrow marks the N-terminus of the extracellular domain, beginning with residue 65 of SEQ ID NO:2, used to generate a soluble version of DRL-1.
  • Figure IB shows a sequence alignment of extracellular receptor-binding regions of DRL-1 and several other membranes of the TNF family. The extracellular domain of DRL-1 with extensive homology to other TNF family members is shown. Alignment was performed using the Megalign software (DNASTAR) and the shading represents identical residues.
  • Figure 2 shows a northern blot analysis of DRL-1.
  • a and B Blots of RNA from a variety of human tissues or PBLs were purchased and detected with DRL-1 or actin probes followed by autoradiography.
  • Figure 3 shows the genomic localization of DRL-1.
  • A DNA prepared from chromosomes 1 to 23, total human DNA (+) or H 2 O (-) was used as template for PCR of DRL-1. The primers are described in the Examples in the section Identification, cloning and sequencing analysis of DRL-1. PCR products were analyzed by agarose gel electrophoresis.
  • B A diagram showing six different chromosome 17 deletion DNA panels. Lines indicate the chromosome 17 sequence that is present in the samples.
  • C PCR products of different chromosome 17 deletion panels shown in (B) or H 2 O (-) using the primer as in (A).
  • Figure 4 shows the purification of soluble DRL-1.
  • DRL-l-Fc purified by anti- Flag column was dialyzed against PBS and 20 ⁇ l of the eluent was used for SDS-
  • Figure 6 shows the predicted protein sequence of DRL-2.
  • the underlined sequence indicates a region likely to be essential for protein function. This fragment corresponds to residues 184-209 of SEQ ID NO:4.
  • the peptide sequence of this region can be used to raise polyclonal and monoclonal antibodies against DRL-2.
  • Figure 7 shows the expression of DRL-2 in spleen and peripheral blood lymphocytes (PBL).
  • a probe specific for DRL-2 sequence was used to probe human mRNA membranes.
  • Figure 8 shows the expression of the cDNAs for DRL-1 and DRL-2.
  • DRL-1 and DRL-2 encode discrete proteins of different sizes.
  • DRL-2 encodes two isoforms.
  • Figure 9 shows the analysis of the protein regions of DRL-1.
  • Figure 10 shows the analysis of the protein regions of DRL-2.
  • Figure 11 illustrates the relationship between DRL-1 and DRL-2 and the relationship of DRL-1 and DRL-2 to other members of the TNF gene family.
  • Figure 12 shows the chromosomal mapping of DRL-2 to chromosome 13.
  • the invention provides an isolated nucleic acid encoding DRL-1, for example, as set forth in the Sequence Listing as SEQ ID NO: 1.
  • SEQ ID NO: 1 This is an example of a cDNA encoding the DRL-1 protein.
  • Another example of a cDNA encoding a DRL-1 protein is set forth in the Sequence Listing as SEQ ID NO:5.
  • SEQ ID NO: 5 is an example of a mouse cDNA of DRL-1.
  • the present invention also provides the gene encoding DRL-1, an example of which is contained within the disclosed sequence corresponding to EMBL/GenBank/DDBJ accession no. AF046888.
  • the term "gene” as used herein means a unit of heredity that occupies a specific locus on a chromosome as well as any sequences associated with the expression of that nucleic acid.
  • the gene includes any introns normally present within the coding region as well as regions preceding and following the coding region. Examples of these non-coding regions include, but are not limited to transcription termination regions, promoter regions, enhancer regions and modulation regions. Since the genomic location of the DRL-1 gene is provided herein, the present invention includes any examples of the DRL-1 gene that occur at that locus.
  • the present invention provides an isolated nucleic acid encoding DRL-2, for example, as set forth in the Sequence Listing as SEQ ID NO:3.
  • DRL-2 This is an example of a cDNA encoding the DRL-2 protein.
  • the present invention also provides the gene encoding DRL-2. Since the genomic location of the DRL-2 gene is provided herein, the present invention includes any examples of the DRL-2 gene that occur at that locus. Similar to DRL-1, DRL-2 is another member of the TNF family, however, the cDNA for DRL-2 provided herein, does not encode an N-terminal hydrophobic stretch that would normally anchor a protein to the membrane as seen in DRL-1 and other TNF family members. Thus, DRL-2 would be directly secreted into the extracellular medium.
  • DRL DRL protein
  • DRL polypeptide DRL cDNA
  • DRL gene can refer to DRL-1 or DRL-2 or both.
  • the term "isolated” refers to a nucleic acid separated or significantly free from at least some of the other components of the naturally occurring organism, for example, the cell structural components commonly found associated with nucleic acids in a cellular environment and/or other nucleic acids.
  • the isolation of the native nucleic acids can be accomplished, for example, by techniques such as cell lysis followed by phenol plus chloroform extraction, followed by ethanol precipitation of the nucleic acids.
  • the nucleic acids of this invention can be isolated from cells according to any of many methods well known in the art.
  • cDNA or cDNA fragments (probes, primers etc.) of a DRL protein provide information with which the genomic nucleic acids corresponding to this cDNA, or genomic variants of this gene can be isolated.
  • primers for amplifying the gene that encodes the DRL-1 protein can be designed using the sequence information disclosed in SEQ ID NO: 1.
  • primers can be designed using the sequence information disclosed in SEQ ID NO: 3 for amplifying the gene that encodes the DRL-2 protein.
  • those same disclosed nucleic acids can be used to design probes for detecting a nucleic acid containing all or part of the genomic nucleic acid in a genomic library.
  • the terms "genomic variant” and "allelic variant” mean a similar gene in another organism of the same species.
  • a nucleic acid from one member of a species can encode for a particular DRL protein while another member of the same species has a different nucleic acid which encodes for that same DRL protein.
  • a similar gene in another species is defined herein as a "homolog" of a DRL protein.
  • a DRL protein from one species can be different than a DRL protein from another species, yet both may have the same function as the exemplified DRL protein.
  • nucleic acid refers to single-or multiple stranded molecules which may be DNA or RNA, or any combination thereof, including modifications to those nucleic acids.
  • the nucleic acid may represent a coding strand or its complement, or any combination thereof.
  • Nucleic acids may be identical in sequence to the sequences which are naturally occurring for any of the novel genes discussed herein, or they may include alternative codons which encode the same amino acid as that which is found in the naturally occurring sequence. These nucleic acids can also be modified from their typical structure.
  • Such modifications include, but are not limited to, methylated nucleic acids, the substitution of a non-bridging oxygen on the phosphate residue with either a sulfur (yielding phosphorothioate deoxynucleotides), selenium (yielding phosphorselenoate deoxynucleotides), or methyl groups (yielding methylphosphonate deoxynucleotides).
  • genes and nucleic acids provided for by the present invention may be obtained in any number of ways.
  • a DNA molecule encoding a DRL protein can be isolated from the organism in which it is normally found.
  • a genomic DNA or cDNA library can be constructed and screened for the presence of the gene or nucleic acid of interest. Methods of constructing and screening such libraries are well known in the art and kits for performing the construction and screening steps are commercially available (for example, Stratagene Cloning Systems, La Jolla, CA).
  • the gene or nucleic acid can be directly cloned into an appropriate vector, or if necessary, be modified to facilitate the subsequent cloning steps.
  • the sequence encoding specific amino acids can be modified or changed at any particular amino acid position by techniques well known in the art.
  • PCR primers can be designed which span the amino acid position or positions and which can substitute any amino acid for another amino acid.
  • a nucleic acid can be amplified and inserted into the wild-type DRL protein coding sequence in order to obtain any of a number of possible combinations of amino acids at any position of the DRL protein.
  • one skilled in the art can introduce specific mutations at any point in a particular nucleic acid sequence through techniques for point mutagenesis. General methods are set forth in Smith, M. "In vitro mutagenesis" Ann. Rev.
  • Another example of a method of obtaining a DNA molecule encoding a specific DRL protein is to synthesize a recombinant DNA molecule which encodes the DRL protein.
  • oligonucleotide synthesis procedures are routine in the art and oligonucleotides coding for a particular protein region are readily obtainable through automated DNA synthesis.
  • a nucleic acid for one strand of a double-stranded molecule can be synthesized and hybridized to its complementary strand.
  • One can design these oligonucleotides such that the resulting double-stranded molecule has either internal restriction sites or appropriate 5 ' or 3' overhangs at the termini for cloning into an appropriate vector.
  • Double-stranded molecules coding for relatively large proteins can readily be synthesized by first constructing several different double-stranded molecules that code for particular regions of the protein, followed by ligating these DNA molecules together.
  • Cunningham, et al "Receptor and Antibody Epitopes in Human Growth Hormone Identified by Homolog-Scanning Mutagenesis," Science, 243:1330-1336 (1989) have constructed a synthetic gene encoding the human growth hormone gene by first constructing overlapping and complementary synthetic oligonucleotides and ligating these fragments together. See also, Ferretti, et al., Proc. Nat. Acad. Sci.
  • the invention also provides for the isolated nucleic acids of SEQ ID NO:l, SEQ ID NO: 3, and SEQ ID NO:5 in a vector suitable for expressing the nucleic acid.
  • a nucleic acid encoding a particular DRL protein of interest, or a region of that nucleic acid is constructed, modified, or isolated, that nucleic acid can then be cloned into an appropriate vector, which can direct the in vivo or in vitro synthesis of that wild- type and/or modified DRL protein.
  • the vector is contemplated to have the necessary functional elements that direct and regulate transcription of the inserted gene, or nucleic acid.
  • These functional elements include, but are not limited to, a promoter, regions upstream or downstream of the promoter, such as enhancers that may regulate the transcriptional activity of the promoter, an origin of replication, appropriate restriction sites to facilitate cloning of inserts adjacent to the promoter, antibiotic resistance genes or other markers which can serve to select for cells containing the vector or the vector containing the insert, RNA splice junctions, a transcription termination region, or any other region which may serve to facilitate the expression of the inserted gene or hybrid gene. (See generally, Sambrook et alf).
  • E. coli ⁇ scherichia coli
  • Other microbial hosts suitable for use include bacilli, such as Bacillus subtilis, and other enterobacteriaceae, such as Salmonella, Serratia, and various Pseudomonas species.
  • bacilli such as Bacillus subtilis
  • enterobacteriaceae such as Salmonella, Serratia, and various Pseudomonas species.
  • prokaryotic hosts one can also make expression vectors, which will typically contain expression control sequences compatible with the host cell (e.g., an origin of replication).
  • any number of a variety of well-known promoters will be present, such as the lactose promoter system, a tryptophan (Tip) promoter system, a beta-lactamase promoter system, or a promoter system from phage lambda.
  • the promoters will typically control expression, optionally with an operator sequence, and have ribosome binding site sequences for example, for initiating and completing transcription and translation.
  • an amino terminal methionine can be provided by insertion of a Met codon 5' and in- frame with the downstream nucleic acid insert.
  • the carboxy-terminal extension of the nucleic acid insert can be removed using standard oligonucleotide mutagenesis procedures.
  • yeast expression can be used. There are several advantages to yeast expression systems. First, evidence exists that proteins produced in a yeast secretion systems exhibit correct disulfide pairing. Second, post-translational glycosylation is efficiently carried out by yeast secretory systems. The Saccharomyces cerevisiae pre-pro-alpha- factor leader region (encoded by the MF"-1 gene) is routinely used to direct protein secretion from yeast. (Brake, et al, A%-F actor-Directed Synthesis and Secretion of Mature Foreign Proteins in Saccharomyces cerevisiae.@ Proc. Nat. Acad. Sci., 81:4642-4646 (1984)).
  • the leader region of pre-pro-alpha- factor contains a signal peptide and a pro-segment which includes a recognition sequence for a yeast protease encoded by the KEX2 gene: this enzyme cleaves the precursor protein on the carboxyl side of a Lys-Arg dipeptide cleavage signal sequence.
  • the nucleic acid coding sequence can be fused in-frame to the pre-pro-alpha- factor leader region. This construct is then put under the control of a strong transcription promoter, such as the alcohol dehydrogenase I promoter or a glycolytic promoter.
  • the nucleic acid coding sequence is followed by a translation termination codon which is followed by transcription termination signals.
  • nucleic acid coding sequences can be fused to a second protein coding sequence, such as Sj26 or ⁇ -galactosidase, used to facilitate purification of the fusion protein by affinity chromatography.
  • a second protein coding sequence such as Sj26 or ⁇ -galactosidase
  • the insertion of protease cleavage sites to separate the components of the fusion protein is applicable to constructs used for expression in yeast. Efficient post translational glycosylation and expression of recombinant proteins can also be achieved in Baculovirus systems.
  • Mammalian cells permit the expression of proteins in an environment that favors important post-translational modifications such as folding and cysteine pairing, addition of complex carbohydrate structures, and secretion of active protein.
  • Vectors useful for the expression of active proteins in mammalian cells are characterized by insertion of the protein coding sequence between a strong viral promoter and a polyadenylation signal.
  • the vectors can contain genes conferring hygromycin resistance, gentamicin resistance, or other genes or phenotypes suitable for use as selectable markers, or methotrexate resistance for gene amplification.
  • the chimeric protein coding sequence can be introduced into a Chinese hamster ovary (CHO) cell line using a methotrexate resistance-encoding vector, or other cell lines using suitable selection markers. Presence of the vector DNA in transformed cells can be confirmed by Southern blot analysis. Production of RNA corresponding to the insert coding sequence can be confirmed by Northern blot analysis.
  • suitable host cell lines capable of secreting intact human proteins have been developed in the art, and include the CHO cell lines, HeLa cells, myeloma cell lines, Jurkat cells, etc.
  • Expression vectors for these cells can include expression control sequences, such as an origin of replication, a promoter, an enhancer, and necessary information processing sites, such as ribosome binding sites, RNA splice sites, polyadenylation sites, and transcriptional terminator sequences.
  • Preferred expression control sequences are promoters derived from immunoglobulin genes, SV40, Adenovirus, Bovine Papilloma Virus, etc.
  • the vectors containing the nucleic acid segments of interest can be transferred into the host cell by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation is commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofectin mediated transfection or electroporation may be used for other cellular hosts.
  • vectors for the expression of genes or nucleic acids in mammalian cells those similar to those developed for the expression of human gamma-interferon, tissue plasminogen activator, clotting Factor VIII, hepatitis B virus surface antigen, protease Nexinl, and eosinophil major basic protein, can be employed.
  • the vector can include CMV promoter sequences and a polyadenylation signal available for expression of inserted nucleic acids in mammalian cells (such as COS-7). Insect cells also permit the expression of mammalian proteins. Recombinant proteins produced in insect cells with baculovirus vectors undergo post-translational modifications similar to that of wild-type proteins.
  • baculovirus vectors useful for the expression of active proteins in insect cells are characterized by insertion of the protein coding sequence downstream of the Autographica californica nuclear polyhedrosis virus (AcNPV) promoter for the gene encoding polyhedrin, the major occlusion protein.
  • AcNPV Autographica californica nuclear polyhedrosis virus
  • Cultured insect cells such as Spodoptera frugiperda cell lines are transfected with a mixture of viral and plasmid DNAs and the viral progeny are plated. Deletion or insertional inactivation of the polyhedrin gene results in the production of occlusion negative viruses which form plaques that are distinctively different from those of wild-type occlusion positive viruses.
  • plaque morphologies allow visual screening for recombinant viruses in which the AcNPV gene has been replaced with a hybrid gene of choice.
  • High quantity expression and production of a DRL protein can also be achieved by transgenic animal technology by which animals can be made to produce DRL in serum, milk, etc in large amounts.
  • the invention also provides for the vectors containing the contemplated nucleic acids in a host suitable for expressing the nucleic acids.
  • the vectors containing the nucleic acid segments of interest can be transferred into host cells by well-known methods, which vary depending on the type of cellular host. For example, calcium chloride transformation, transduction, and electroporation are commonly utilized for prokaryotic cells, whereas calcium phosphate, DEAE dextran, or lipofection mediated transfection or electroporation may be used for other cellular hosts.
  • the genes or nucleic acids of the present invention can be operatively linked to one or more of the functional elements that direct and regulate transcription of the inserted gene as discussed above and the gene or nucleic acid can be expressed.
  • a gene or nucleic acid can be operatively linked to a bacterial or phage promoter and used to direct the transcription of the gene or nucleic acid in vitro.
  • a further example includes using a gene or nucleic acid provided herein in a coupled transcription-translation system where the gene directs transcription and the RNA thereby produced is used as a template for translation to produce a polypeptide.
  • the products of these reactions can be used in many applications such as using labeled RNAs as probes and using polypeptides to generate antibodies or in a procedure where the polypeptides are being administered to a cell or a subject.
  • Expression of the gene or nucleic acid, in combination with a vector can be by either in vivo or in vitro.
  • In vivo synthesis comprises transforming prokaryotic or eukaryotic cells that can serve as host cells for the vector.
  • expression of the gene or nucleic acid can occur in an in vitro expression system.
  • in vitro transcription systems are commercially available which are routinely used to synthesize relatively large amounts of mRNA.
  • the nucleic acid encoding the DRL protein would be cloned into an expression vector adjacent to a transcription promoter.
  • the Bluescript II cloning and expression vectors contain multiple cloning sites which are flanked by strong prokaryotic transcription promoters.
  • kits are available which contain all the necessary reagents for in vitro synthesis of an RNA from a DNA template such as the Bluescript vectors. (Stratagene Cloning Systems, La Jolla, CA). RNA produced in vitro by a system such as this can then be translated in vitro to produce the desired DRL protein. (Stratagene Cloning Systems, La Jolla, CA).
  • nucleic acids from any desired species, preferably mammalian and more preferably human, having 99.9%, 99.7%, 99.5%, 99.3%, 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 76%, 75%, 74%, 73%>, 72%, 71%), 70%, 60%, or 50% overall homology or homology in the region being compared to the same region of a gene comprising a nucleic acid set forth in SEQ ID NO: 1 , SEQ ID NO: 3or SEQ ID NO:5 or to a gene comprising a nucleic acid encoding the polypeptide set forth in SEQ ID NO:2,SEQ ID NO:4 or SEQ ID NO:6 or to an isolated nucleic acid consisting of the nucleic acid set forth in
  • the assessment of homology is preferably based on a base-by-base comparison of the regions being compared.
  • genes and nucleic acids can be synthesized or obtained by the same methods used to isolate homologs, with stringency of hybridization and washing, if desired, reduced accordingly as homology desired is decreased, and further, depending upon the G-C or A-T richness of any area wherein variability is searched for.
  • Allelic variants of any of the present genes and nucleic acids or of their homologs can readily be isolated and sequenced by screening additional libraries following the examples given herein and procedures well known in the art.
  • the gene or nucleic acid encoding any selected protein of the present invention can be any nucleic acid that functionally encodes that protein.
  • a nucleic acid encoding a selected protein can readily be determined based upon the amino acid sequence of the selected protein, and, clearly, many nucleic acids will encode any selected protein.
  • the present invention additionally provides a nucleic acid that selectively hybridizes under stringent conditions with a gene comprising a nucleic acid set forth in SEQ ID NO:l, SEQ ID NO:3 or SEQ ID NO:5.
  • This hybridization can be specific.
  • the degree of complementarity between the hybridizing nucleic acid and the sequence to which it hybridizes should be at least enough to exclude hybridization with a nucleic acid encoding an unrelated protein.
  • a nucleic acid that selectively hybridizes with a nucleic acid of a present protein coding sequence will not selectively hybridize under stringent conditions with a nucleic acid for a different, unrelated protein, and vice versa.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to a labeled nucleic acid of interest and then washed under conditions of different stringencies.
  • Hybridization temperatures are typically higher for DNA-RNA and RNA-RNA hybridizations.
  • the washing temperatures can be used as described above to achieve selective stringency, as is known in the art. (See, for example, Sambrook et al, "Molecular Cloning: A Laboratory Manual” 2nd Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, New York (1989) and Kunkel et al. Methods Enzymol. 1987:154:367 (1987)).
  • Nucleic acid fragments that selectively hybridize to any given nucleic acid can be used, e.g., as primers and or probes for further hybridization or for amplification methods (e.g., polymerase chain reaction (PCR), ligase chain reaction (LCR)).
  • PCR polymerase chain reaction
  • LCR ligase chain reaction
  • the present invention also contemplates polynucleotide probes for detecting a DRL gene, wherein the polynucleotide probe hybridizes to the nucleotide sequence set forth in the Sequence Listing as SEQ ID NO:l, SEQ ID NO:3 or SEQ ID NO:5.
  • polynucleotide probe refers to a nucleic acid fragment that selectively hybridizes under stringent conditions with a nucleic acid comprising a nucleic acid set forth in a sequence listed herein. This hybridization must be specific. The degree of complementarity between the hybridizing nucleic acid and the sequence to which it hybridizes should be at least enough to exclude hybridization with a nucleic acid encoding an unrelated protein.
  • allelic variants can be identified and isolated by nucleic acid hybridization techniques.
  • Probes selective to the nucleic acid set forth in the Sequence Listing as SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:5 can be synthesized and used to probe the nucleic acid from various cells, tissues, libraries etc.
  • High sequence complementarity and stringent hybridization conditions can be selected such that the probe selectively hybridizes to allelic variants of the sequence set forth in the Sequence Listing as SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:5.
  • the selectively hybridizing nucleic acids of the invention can have at least 70%, 71%, 72%o, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%,86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% and 99% complementarity with the segment of the sequence to which it hybridizes.
  • the nucleic acids can be at least 12, 50, 100, 150, 200, 300, 500, 750, or 1000 nucleotides in length.
  • the nucleic acid can be a coding sequence for a DRL protein or fragments thereof that can be used as a probe or primer for detecting the presence of these genes.
  • the invention provides compositions including at least two nucleic acids which hybridize with different regions so as to amplify a desired region.
  • Forward primer, 5'TATCGAATTCACAACCTTCTTCCCTTCTGACCA (SEQ ID NO:7) and reversed primer , 5 ATCGAATTCAATGAAAAGGGAAAAGTGAGGAACG (SEQ ID NO: 8) are provided as examples of amplification primers.
  • target region can range between 70% complementary bases and full complementarity and still hybridize under stringent conditions.
  • the degree of complementarity between the hybridizing nucleic acid (probe or primer) and the sequence to which it hybridizes is at least enough to distinguish hybridization with a nucleic acid from other species.
  • the invention provides examples of nucleic acids unique to SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:5 so that the degree of complementarity required to distinguish selectively hybridizing from nonselectively hybridizing nucleic acids under stringent conditions can be clearly determined for each nucleic acid.
  • washing conditions refers to the washing conditions used in a hybridization protocol.
  • the washing conditions should be a combination of temperature and salt concentration chosen so that the denaturation temperature is approximately 5-20°C below the calculated T m of the nucleic acid hybrid under study.
  • the temperature and salt conditions are readily determined empirically in preliminary experiments in which samples of reference DNA immobilized on filters are hybridized to the probe or protein coding nucleic acid of interest and then washed under conditions of different stringencies.
  • the T m of such an oligonucleotide can be estimated by allowing 2°C for each A or T nucleotide, and 4°C for each G or C. For example, an 18 nucleotide probe of 50% G+C would, therefore, have an approximate T m of 54°C.
  • the present invention also contemplates any unique fragment of these genes or of the nucleic acids set forth in SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:5.
  • the fragment must be of sufficient size to distinguish it from other known sequences, most readily determined by comparing any nucleic acid fragment to the nucleotide sequences of nucleic acids in computer databases, such as GenBank. Such comparative searches are standard in the art.
  • a unique fragment useful as a primer or probe will be at least about 20 to about 25 nucleotides in length, depending upon the specific nucleotide content of the sequence.
  • fragments can be, for example, at least about 30, 40, 50, 75, 100, 200, 210, 211, 212, 213, 214, 215, 216, 220, 230, 240, 245, 246, 247, 248, 249, 250, 300, 350, 400, 500, 600, 700, 800, 900, 1000 nucleotides in length or any number in between. All of the genes, nucleic acids, and fragments of the genes and nucleic acids disclosed and contemplated herein can be single or double stranded, depending upon the purpose for which it is intended.
  • a compound comprising a nucleic acid can be a derivative of a typical nucleic acid such as nucleic acids which are modified to contain a terminal or internal reporter molecule and/or those nucleic acids containing non-typical bases or sugars.
  • reporter molecules include, but are not limited to, isotopic and non- isotopic reporters. Examples include, a FLAG tag or a human IgG Fc. Therefore any molecule which may aid in detection, amplification, replication, expression, purification, uptake, etc. may be added to the nucleic acid construct.
  • the invention also provides for an isolated double-stranded nucleic acid amplified using primer pair 5'-TATCGAATTCACAACCTTCTTCCCTTCTGCACCA (SEQ ID NO:7) and 5' TATCGAATTCAATGAAAAGGGAAAAGTGAGGAACG (SEQ ID NO:8) consisting of 1) single-stranded DNA which has a molecular size of approximately 1.3 Kb and is derived from humans, and 2) a DNA complementary to the single- stranded DNA.
  • RNAs corresponding to the 1.3 Kb single- stranded DNA and the single-stranded 1.3 Kb complementary DNA, respectively.
  • DNA sizes can be measured by several means including, running the nucleic acid on a gel such as an agarose or polyacrylamide gel, or by other molecular size estimates techniques, such as molecular weight filtration. Briefly, the nucleic acid sample is applied to the gel and electrophoresed simultaneously with nucleic acid markers of known molecular size. Upon completion of gel electrophoresis, the size of the nucleic acid sample is estimated by comparing its mobility with the relative mobilities of the nucleic acid markers.
  • the invention provides a polypeptides encoded by the nucleic acid set forth in SEQ ID NO:l, SEQ ID NO:3 and SEQ ID NO:5 and the polypeptides set forth in the Sequence Listing as SEQ ID NO:2, SEQ ID NO:4 and SEQ ID NO:6.
  • the invention also provides fragments of unmodified and modified DRL proteins.
  • the polypeptide fragments of the present invention can be recombinant proteins obtained by cloning nucleic acids encoding the polypeptide in an expression system capable of producing the polypeptide fragments thereof. For example, one skilled in the art can determine the active regions of a DRL protein which can interact with another protein and cause a biological effect associated with the DRL protein.
  • amino acids found to not contribute to either the activity, binding specificity, or other biological effect associated with the DRL protein can be deleted and /or substituted without a loss in the respective activity.
  • the fragments, whether attached to other sequences or not, can also include insertions, deletions, substitutions, or other selected modifications of particular regions or specific amino acid residues, provided the activity of the peptide is not significantly altered or impaired.
  • polypeptides encoded by fragments of the DRL nucleic acids provided herein are polypeptides encoded by fragments of the DRL nucleic acids provided herein.
  • An example of an active fragment of DRL-1 is the extracellular domain of DRL shown in Fig.1.
  • the DRL-1 protein fragment shown in SEQ ID NO:2 is a specific example of a fragment of DRL-1 corresponding to nucleotides 193 to 750, and that other examples of DRL and DRL fragments having slightly different sequences may be found in nature using routine protocols or generated by design.
  • DRL protein fragments encoded by nucleotides 1 to 193 of SEQ ID NO: 1 are also contemplated by this invention as are the protein fragments encoded by nucleotides 1 to 699 or SEQ ID NO: 5 and the protein fragments encoded by nucleotides 1 to 654 or SEQ ID NO:3.
  • DRL-1 fragments comprising or consisting of amino acids 26-50 of SEQ ID NO:2 or a fragment beginning with amino acid 65 of SEQ ID NO:2 are provided.
  • DRL-2 fragments are also provided, including a peptide comprising residues 184-209 of SEQ ID NO:4. Additional fragments of DRL-1 and DRL-2 can be identified based on there correspondence to functional regions of other previously known members of the TNF family. Identification of these regions in DRL is made routine by the provision of exemplary sequences of DRL- land DRL-2 herein and their comparison to published sequences of other members of the family (see Figure IB, for example).
  • active fragment is meant a subpart of a whole DRL protein that exhibits an activity of DRL.
  • the fragment can stimulate and enhance immune responses such as those against viral, bacterial, parasitic and toxic agent.
  • the fragment can also enhance the intensity of immune responses at certain stages to induce or augment antigen-stimulated immune responses or lymphocyte death. The latter may be more important for limiting the intensity or duration of an immune response and/or preventing the development of potential autoimmune reactions.
  • the active fragment can also enhance or stimulate anti-tumor immunity.
  • the DRL fragment can be used in combination with other immune response modifiers to increase or decrease their effect on immune responses.
  • the behavior of any cell bearing an appropriate receptor can be modified by the DRL fragment by contacting said receptor and stimulating a cellular response in a manner that promotes function and preserves or restores health.
  • DRL polypeptides can also be obtained in any of a number of procedures well known in the art.
  • One method of producing a polypeptide is to link two peptides or polypeptides together by protein chemistry techniques.
  • peptides or polypeptides can be chemically synthesized using currently available laboratory equipment using either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., Foster City, CA).
  • Fmoc (9-fluorenylmethyloxycarbonyl) or Boc (tert -butyloxycarbonoyl) chemistry Applied Biosystems, Inc., Foster City, CA.
  • a peptide or polypeptide corresponding to a particular protein can be synthesized by standard chemical reactions.
  • a peptide or polypeptide can be synthesized and not cleaved from its synthesis resin whereas the other fragment of a hybrid peptide can be synthesized and subsequently cleaved from the resin, thereby exposing a terminal group which is functionally blocked on the other fragment.
  • peptide condensation reactions these two fragments can be covalently joined via a peptide bond at their carboxyl and amino termini, respectively, to form a larger polypeptide.
  • the peptide or polypeptide can be independently synthesized in vivo as described above. Once isolated, these independent peptides or polypeptides may be linked to form a larger protein via similar peptide condensation reactions.
  • enzymatic ligation of cloned or synthetic peptide segments can allow relatively short peptide fragments to be joined to produce larger peptide fragments, polypeptides or whole protein domains (Abrahmsen et al. Biochemistry, 30:4151 (1991)).
  • native chemical ligation of synthetic peptides can be utilized to synthetically construct large peptides or polypeptides from shorter peptide fragments. This method consists of a two step chemical reaction (Dawson et al. A Synthesis of Proteins by Native Chemical Ligation,@ Science, 266:776-779 (1994)).
  • the first step is the chemoselective reaction of an unprotected synthetic peptide-%-thioester with another unprotected peptide segment containing an amino-terminal Cys residue to give a thioester-linked intermediate as the initial covalent product. Without a change in the reaction conditions, this intermediate undergoes spontaneous, rapid intramolecular reaction to form a native peptide bond at the ligation site.
  • Application of this native chemical ligation method to the total synthesis of a protein molecule is illustrated by the preparation of human interleukin 8 (IL-8) (Clark-Lewis et al.
  • unprotected peptide segments can be chemically linked where the bond formed between the peptide segments as a result of the chemical ligation is an unnatural (non-peptide) bond (Schnolzer et al. Science, 256:221 (1992)).
  • This technique has been used to synthesize analogs of protein domains as well as large amounts of relatively pure proteins with full biological activity (deLisle Milton et al. ATechniques in Protein Chemistry IV,@ Academic Press, New York, pp. 257-267 (1992)).
  • purified antibodies that selectively or specifically bind to the DRL polypeptides provided and contemplated herein, for example, purified antibodies which selectively or specifically bind to a polypeptide encoded by the nucleic acid set forth in any of SEQ ID NO:l, SEQ ID NO:3 or SEQ ID NO:5, and purified antibodies which selectively or specifically bind to the polypeptide set forth in SEQ ID NO:2, SEQ ID NO:4 or SEQ ID NO:6.
  • the antibody can be raised to any of the polypeptides provided and contemplated herein, both naturally occurring and recombinant polypeptides, and immunogenic fragments, thereof.
  • an anti-DRL-1 antibody can be raised to the extracellular domain of DRL-1, beginning with residue 65 of SEQ ID NO:2, and used herein to generate a soluble version of DRL-1.
  • the antibody can be used in techniques or procedures such as diagnostics, treatment, or vaccination.
  • Anti-idiotypic antibodies and affinity matured antibodies are also considered.
  • the invention provides monoclonal antibodies designated MAbl ,
  • MAb2, MAb3, Mab4 and Mab5 that specifically bind DRL-1.
  • Antibodies against DRL-2 are provided. Antibodies can be raise against the whole DRL-2 molecule or against immunogenic fragments of it. For example, a fragment corresponds to residues 184-209 of SEQ ID NO:4 can be used to raise polyclonal and monoclonal antibodies against DRL-2.
  • the purified antibodies of this invention include monoclonal antibodies which can be used for diagnostic or analytical purposes.
  • the monoclonal antibody could be utilized in a clinical testing kit to monitor levels of DRL in human tissues or secretions.
  • Antibodies can be made by many well-known methods (See, e.g. Harlow and Lane, "Antibodies; A Laboratory Manual” Cold Spring Harbor Laboratory, Cold Spring Harbor, New York, (1988)). Briefly, purified antigen can be injected into an animal in an amount and in intervals sufficient to elicit an immune response. Antibodies can either be purified directly, or spleen cells can be obtained from the animal. The cells can then fused with an immortal cell line and screened for antibody secretion. The antibodies can be used to screen nucleic acid clone libraries for cells secreting the antigen. Those positive clones can then be sequenced. (See, for example, Kelly et al.
  • Humanized and chimeric antibodies are also contemplate in this invention.
  • Heterologous antibodies can be made by well known methods (See, for example, US Patents 5545806, 5569825,5625126, 5633425, 5661016, 5770429, 5789650, and 5814318)
  • the phrase "specifically binds" with the polypeptide refers to a binding reaction which is determinative of the presence of the protein in a heterogeneous population of proteins and other biologies.
  • the specified antibodies bound to a particular protein do not bind in a significant amount to other proteins present in the sample.
  • Selective binding to an antibody under such conditions may require an antibody that is selected for its specificity for a particular protein.
  • a variety of immunoassay formats may be used to select antibodies that selectively bind with a particular protein. For example, solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane "Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that could be used to determine selective binding.
  • This invention also contemplates producing a selected cell line or a non-human transgenic animal model for the analysis of the function of a gene comprising introducing into an embryonic stem cell a vector having a selectable marker which, when the vector is inserted within a gene, the inserted vector can inhibit the expression of the gene, selecting embryonic stem cells expressing the selectable marker, excising the vector from the embryonic stem cells expressing the selectable marker such that host DNA from the gene is linked to the excised vector, sequencing the host DNA in the excised vector, comparing the sequence of the host DNA to known gene sequences to determine which host DNA is from a gene for which a model for the analysis of the function the gene is desired, selecting the embryonic stem cell containing the inhibited gene for which a model for the analysis of gene function is desired, and forming a cell line or a non-human transgenic animal from the selected embryonic stem cell.
  • transgenic animals can be produced which either overproduce the polypeptides of this invention or fail to produce the polypeptides of this invention in a functional form.
  • a transgenic animal which overproduces a DRL of this invention can be produced according to methods well known in the art whereby nucleic acid encoding DRL is introduced into embryonic stem cells, at which stage it is incorporated into the germline of the animal, resulting in the production of DRL in the transgenic animal in increased amounts relative to a normal animal of the same species.
  • One skilled in the art can determine if overproduction or underproduction of DRL results in altered immune responses.
  • transgenic animal in which the expression of DRL, for example, is tissue specific is also contemplated for this invention.
  • transgenic animals that express or overexpress these genes at specific sites such as the liver can be produced by introducing a nucleic acid into the embryonic stem cells of the animal, wherein the nucleic acid is under the control of a specific promoter which allows expression of the nucleic acid in specific types of cells (e.g., a hepatic promoter which allows expression only in hepatic cells.
  • a specific promoter which allows expression of the nucleic acid in specific types of cells
  • a hepatic promoter which allows expression only in hepatic cells.
  • the transgenic animal of this invention can be a "knock out" animal (see, e.g., Willnow et al., 1996), which can be an animal that, for example, normally produces DRL but has been altered to prevent the expression of the animal's nucleic acid which encodes DRL, thereby resulting in an animal which does not produce DRL in a functional form.
  • a "knock out" animal see, e.g., Willnow et al., 1996), which can be an animal that, for example, normally produces DRL but has been altered to prevent the expression of the animal's nucleic acid which encodes DRL, thereby resulting in an animal which does not produce DRL in a functional form.
  • Such an animal may lack the ability to express all of the nucleic acids encoding DRL or the transgenic animal may lack the ability to express some (one or more than one) but not all of the nucleic acids encoding the DRL.
  • the transgenic "knock out” animal of this invention can have the expression of a gene or genes knocked out in specific tissues. This approach obviates viability problems that can be encountered if the expression of a widely expressed gene is completely abolished in all tissues.
  • One skilled in the art could determine whether or not the "knock out” has altered the animal's immune response by comparing the "knock out” animal with control animals that have not had DRL knocked out in a specific tissue.
  • Also contemplated in this invention is a method of stimulating an immune response in a subject by administering DRL and a pharmaceutically acceptable carrier.
  • an active fragment of DRL, or a DRL encoding nucleic acid in an expressable construct can be administered.
  • nucleic acids encoding DRL or its fragments can be administered.
  • DRL-1 and DRL-2 can also be used to prepare highly active trimeric versions of the molecule for clinical use using either a modified leucine zipper motif (Walczak et al. "Tumoricidal activity of tumor necrosis factor-related apoptosis- inducing ligand in vivo" Nat. Med.
  • the subject which can be treated by this method can be any animal.
  • the animal of the present invention is a human.
  • non-human animals which can be treated by the methods of this invention can include, but are not limited to, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils and rabbits.
  • Optimal dosages used will vary according to the individual being treated and the DRL being used.
  • the amount of DRL will also vary among individuals on the basis of age, size, weight, condition, etc.
  • dosages are best optimized by the practicing physician and methods for determining dose amounts and regimens and preparing dosage forms are described, for example, in Remington '_. Pharmaceutical Sciences.
  • suitable doses and dosage regimens can be determined by comparison to agents presently used in the treatment or prevention of autoimmune disorders, allergic disorders, graft rejection and other T cell related disorders.
  • the preferred or optimal dosage is the amount of DRL which results in apoptosis of T cells, in the absence of significant side effects.
  • the DRL of this invention can be administered orally or parenterally in a dosage range of 0.1 to 100 mg/kg of body weight depending on the clinical response that is to be obtained. Administration of DRL can be stopped completely following a prolonged remission or stabilization of disease signs and symptoms and readministered following a worsening of either the signs or symptoms of the disease, or following a significant change in immune status, as determined by routine follow-up immunological studies well known to a clinician in this field.
  • the efficacy of administration of a particular dose of DRL in treating a T cell associated disorder as described herein can be determined by evaluating the particular aspects of the medical history, the signs, symptoms and objective laboratory tests that have a documented utility in evaluating pathophysiological activity of the particular T cell associated disorder being treated. These signs, symptoms and objective laboratory tests will vary depending on the particular disorder being treated, as will be well known to any clinician in this field.
  • a subject's frequency or severity of recurrences is shown to be improved; 2) the progression of the disease or disorder is shown to be stabilized; or 3) the need for use of other immunosuppressive medications is lessened, then a particular treatment can be considered efficacious.
  • clinical parameters and symptoms which can be monitored for efficacy can include the severity and number of attacks; or for continuously progressive disease, the worsening of symptoms and signs; the cumulative development of disability; the number or extent of brain lesions as determined by magnetic resonance imaging; and the use of immunosuppressive medications (65-67).
  • specific signs, symptoms and laboratory tests can be evaluated in accordance with a reduced or discontinued treatment schedule. If a disease activity recurs, based on standard methods of evaluation of the particular signs, symptoms and objective laboratory tests as described herein, DRL treatment can be reinitiated.
  • the efficacy of administration of a particular dose of a peptide ligand in preventing a T cell associated disorder in a subject not known to have a T cell associated disorder, but known to be at risk of developing a T cell associated disorder can be determined by evaluating standard signs, symptoms and objective laboratory tests, known to one of skill in the art, over time. This time interval may be long (i.e., years/decades). The determination of who would be at risk for the development of a T cell associated disorder would be made based on current knowledge of the known risk factors for a particular disorder familiar to clinicians and researchers in this field, such as a particularly strong family history of a disorder or exposure to or acquisition of factors or conditions which are likely to lead to development of a T cell associated disorder.
  • pharmaceutically acceptable a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the selected compound without causing any undesirable biological effects or interacting in a undesirable manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier may depend on the method of administration and the particular patient. Methods of administration can be oral, sublingual, mucosal, inhaled, absorbed, or by injection. It is also noted that not all methods of administering the present DRL polypeptides or nucleic acids require a pharmaceutically acceptable carrier.
  • immunosuppressed describes a condition in which the subject's immune system is unable to mount a protective response.
  • immunosuppression There are several forms of immunosuppression. These include but are not limited to the immune suppression of B cells resulting in the inability of these cells to produce immunoglobulin heavy chains, thereby abrogating antibody production; suppressor T- cell effects on helper and cytotoxic T cells that include inhibition of proliferation, abrogation of lymphokine secretion and clonal anergy. Immunosuppressive effects on cell motility, homing, and cell surface phenotype are also possible.
  • the DRL, DRL antibody, or active fragment can be orally or parenterally administered in a carrier pharmaceutically acceptable to human subjects.
  • Suitable carriers for oral or inhaled administration of DRL can include one or more of the carriers pharmaceutically acceptable to human subjects.
  • Suitable carriers for oral administration of DRL include one or more substances which may also act as a flavoring agents, lubricants, suspending agents, or as protectants.
  • Suitable solid carriers include calcium phosphate, calcium carbonate, magnesium stearate, sugars, starch, gelatin, cellulose, carboxypolymethylene, or cyclodextrans.
  • Suitable liquid carriers may be water, pyrogen free saline, pharmaceutically accepted oils, or a mixture of any of these.
  • the liquid can also contain other suitable pharmaceutical addition such as buffers, preservatives, flavoring agents, viscosity or osmo-regulators, stabilizers or suspending agents.
  • suitable liquid carriers include water with or without various additives, including carboxypolymethylene as a ph-regulated gel.
  • the DRL may be contained in enteric coated capsules that release the polypeptide into the intestine to avoid gastric breakdown.
  • a sterile solution or suspension is prepared in saline that may contain additives, such as ethyl oleate or isopropyl myristate, and can be injected for example, into subcutaneous or intramuscular tissues, as well as intravenously.
  • nucleic acid encoding DRL or functional fragment can be administered.
  • the nucleic acid encoding the peptide of this invention can be placed into a vector and delivered to the cells of a subject either in vivo or ex vivo by standard methods.
  • the cells can be in a subject and the nucleic acid can be administered in a pharmaceutically acceptable carrier.
  • the subject can be any animal in which it is desirable to selectively express a nucleic acid in a cell.
  • the animal of the present invention is a human.
  • non-human animals which can be treated by the method of this invention can include, but are not limited to, cats, dogs, birds, horses, cows, goats, sheep, guinea pigs, hamsters, gerbils and rabbits, as well as any other animal in which selective expression of a nucleic acid in a cell can be carried out according to the methods described herein.
  • DRL-1 and/or DRL-2 could be used to modulate immune cell activity in vitro in the context of gene therapy, bone marrow transplantation, and in cultivating tumor- infiltrating lymphocytes for the treatment of cancer.
  • T cells can be collected from the patient and treated ex vivo with DRL- land/or DRL-2, washed to remove the DRL, then readministered to the patient. This approach offers the advantage of reducing any side-effects of administration of DRL in vivo.
  • Another example of an ex vivo application of the present invention would be the expansion of tumor infiltrating lymphocytes of cytolytic T cells by administering a DRL antibody.
  • Adoptive immunotherapy procedures are known in the art. See, for example, Rosenberg S.A., "Adoptive Immunotherapy for Cancer", Scientific American, pp.62-69, May, 1990 and U.S. Pat. No. 5,229,115, hereby incorporated by reference.
  • the nucleic acids of the present invention can be in the form of naked DNA or the nucleic acids can be in a vector for delivering the nucleic acids to the cells for expression of the nucleic acid inside the cell.
  • the vector can be a commercially available preparation, such as an adenovirus vector (Quantum Biotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleic acid or vector to cells can be via a variety of mechanisms.
  • delivery can be via a liposome, using commercially available liposome preparations such as Lipofectin ® , Lipofectamine ® (GIBCO-BRL, Inc., Gaithersburg, MD), Superfect ® (Qiagen, Inc. Hilden, Germany) and Transfectam ® (Promega Biotec, Inc., Madison, WI), as well as other liposomes developed according to procedures standard in the art.
  • the nucleic acid or vector of this invention can be delivered in vivo by electroporation, the technology for which is available from Genetronics, Inc. (San Diego, CA) as well as by means of a Sonoporation machine (ImaRx Pharmaceutical Corp., Arlington, AZ).
  • vector delivery can be via a viral system, such as a retroviral vector system which can package a recombinant retroviral genome.
  • the recombinant retrovirus can then be used to infect and thereby deliver nucleic acid to the infected cells.
  • the exact method of introducing the nucleic acid into mammalian cells is, of course, not limited to the use of retroviral vectors.
  • Other techniques are widely available for this procedure including the use of adenoviral vectors, adeno-associated viral (AAN) vectors, lentiviral vectors, pseudotyped retroviral vectors, and pox virus vectors, such as vaccinia virus vectors.
  • Physical transduction techniques can also be used, such as liposome delivery and receptor-mediated and other endocytosis mechanism. This invention can be used in conjunction with any of these or other commonly used gene transfer methods.
  • the nucleic acid and the nucleic acid delivery vehicles of this invention can be in a pharmaceutically acceptable carrier for in vivo administration to a subject.
  • pharmaceutically acceptable is meant a material that is not biologically or otherwise undesirable, i.e., the material may be administered to a subject, along with the nucleic acid or vehicle, without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained.
  • the carrier would naturally be selected to minimize any degradation of the active ingredient and to minimize any adverse side effects in the subject, as would be well known to one of skill in the art.
  • the nucleic acid or vehicle may be administered orally, parenterally (e.g., intravenously), by intramuscular injection, by intraperitoneal injection, transdermally, extracorporeally, topically or the like.
  • parenterally e.g., intravenously
  • intramuscular injection by intraperitoneal injection, transdermally, extracorporeally, topically or the like.
  • the exact amount of the nucleic acid or vector required will vary from subject to subject, depending on the species, age, weight and general condition of the subject, the severity or mechanism of any disorder being treated, the particular nucleic acid or vehicle used, its mode of administration and the like.
  • the invention also contemplates a method of suppressing an autoimmune response in a subject by administering an antibody to DRL and a pharmaceutically acceptable earner.
  • an antibody to a functional region of DRL could be administered.
  • DRL-1 enhances T cell responses
  • the blockade of DRL-1 in autoimmune or allergic conditions may lessen disease severity to the extent that the diseases depend on T cell reactions.
  • DRL may be overactive in autoimmune disease and the development of specific antibodies will allow the detection and perhaps inhibition of DRL in these contexts. Similar approaches could be used to suppress allograft rejection to enhance the success of tissue transplantation.
  • suppressing describes the inhibition of the autoimmune response which can range from reduction to complete amelioration of the autoimmune response.
  • Measurement of a suppressed immune response involves determining the level of response of peripheral T cells to a specific antigen or in a mixed lymphocyte reaction, important in the context of a disease by assaying the proliferation, cytokine or lymphokine production, or cytotoxic response. It could also involve measuring the levels of specific antibodies, the degree of inflammation in relevant affected body sites, or decreases in non-specific inflammatory markers that are present in the circulation or in tissues.
  • Truncated or amino-acid substituted versions of DRL-1 or DRL-2 can also be engineered that can bind the receptor but not cause stimulatory activities. These genetically altered variants may have dominant interfering properties.
  • autoimmune response describes a disease state or syndrome whereby a subject's body produces a dysfunctional immune response against the subject's own body components, with adverse effects. This may include production of B cells which produce antibodies with specificity for all antigens, allergens or major histocompatibility (MHC) antigens, or it may include production of T cells bearing receptors that recognize self-components and produce cytokines that cause inflammation.
  • MHC major histocompatibility
  • autoimmune diseases include, but are not limited to, ulcerative colitis, Crohn's disease, multiple sclerosis, rheumatoid arthritis, diabetes mellitus, pernicious anemia, autoimmune gastritis, psoriasis, Bechet's disease, Wegener's granulomatosis, Sarcoidois, autoimmune thyroiditis, autoimmune oophoritis, bullous pemphigoid, phemphigus, polyendocrinopathies, Still's disease, Lambert-Eaton myasthenia syndrome, myasthenia gravis, Goodpasture's syndrome, autoimmune orchitis, autoimmune uveitis, systemic lupus erythematosus, Sjogren's Syndrome and ankylosing spondylitis.
  • an antisense molecule to the mRNA of DRL can be administered.
  • Antisense technology is well known in the art and describes a mechanism whereby a nucleic acid comprising a nucleotide sequence which is in a complementary, "antisense” orientation with respect to a coding or “sense” sequence of an endogenous gene is introduced into a cell, whereby a duplex forms between the antisense sequence and its complementary sense sequence. The formation of this duplex results in inactivation of the endogenous gene.
  • Antisense nucleic acid can be produced for any endogenous gene for which the coding sequence has been or can be determined according to well known methods.
  • Antisense nucleic acid can inhibit gene expression by forming an RNA/RNA duplex between the antisense RNA and the RNA transcribed from a target gene.
  • the precise mechanism by which this duplex formation decreases the production of the protein encoded by the endogenous gene most likely involves binding of complementary regions of the normal sense mRNA and the antisense RNA strand with duplex formation in a manner that blocks RNA processing and translation.
  • Alternative mechanisms include the formation of a triplex between the antisense RNA and duplex DNA or the formation of a DNA-RNA duplex with subsequent degradation of DNA-RNA hybrids by RNAse H.
  • an antisense effect can result from certain DNA-based oligonucleotides via triple-helix formation between the oligomer and double-stranded DNA which results in the repression of gene transcription.
  • T cell responses can include but are not limited to cell activation, cell proliferation and apoptosis.
  • an antisense molecule to the mRNA of DRL can be administered.
  • a method for stimulating the membrane bound form of DRL by administering an antibody to DRL and a pharmaceutically acceptable carrier.
  • the invention also provides a method of inhibiting the T cell responses associated with transplantation rejection or graft versus host disease by administering an antibody to DRL or DRL fragment and a pharmaceutically acceptable carrier.
  • an antisense molecule to the mRNA of DRL can be administered.
  • GvH disease describes a disease state or syndrome whereby an immune response is initiated by grafted cells and is directed against the subject's body with adverse effects.
  • GvH disease include, but are not limited to, acute and chronic GvH disease following bone marrow transplant.
  • Transplantation rejection describes a disease state or syndrome whereby the transplant recipient's body produces an immune response against the engrafted tissue, resulting in rejection. Transplantation rejection can occur, for example, with kidney, heart, lung or liver transplants as well as with any other transplanted tissue.
  • the invention further provides a method of inducing apoptosis in a population of T cells by administering DRL to the cells.
  • DRL can be used to boost or induce T cell reactions against tumors Stimulation of T cells from the TCR induces not only cell activation and proliferation, but also apoptosis under certain conditions. Fas ligand and Fas interactions appear to play a major role in the apoptosis process. TNF is also known to be involved.
  • Fas ligand and TNF do not completely abolish antigen-induced apoptosis in human PBLs. This raised the possibility that other molecules besides Fas ligand and TNF may also play a role in activation-induced apoptosis in PBLs.
  • apoptosis refers to programmed cell death that has distinctive mo ⁇ hological and structural features that are different from those of pathological cell death or necrosis.
  • the apoptotic process is characterized by nuclear fragmentation and cytoplasmic budding that lead to the formation of apoptotic bodies. These bodies are phagocytosed and destroyed by nearby macrophages. The fragmentation of the cells does not lead to the release of cellular contents, and the phagocytosis of the apoptotic bodies does not lead to inflammation. As a result, cell death can occur without damage to adjacent cells or tissues.
  • the invention also provides for a method of inhibiting apoptosis in a population of T cells by administering an antibody to DRL or DRL fragment and a pharmaceutically acceptable carrier.
  • an antisense molecule to the mRNA of DRL can be administered.
  • the invention also provides a modified DRL conjugated to a toxin that may bind and kill tumor cells.
  • toxins include, but are not limited to, diptheria toxin, ricin, and Pseudomonas exotoxin. See, for example, U.S. Pat. No. 4545985 and U.S. Pat. No. 5087616, hereby inco ⁇ orated by reference.
  • the present invention also provides a method of screening for a DRL receptor agonist comprising, contacting cells that express the DRL receptor with a putative agonist, detecting an increase in apoptosis in the cells, whereby an increase in apoptosis indicates a putative agonist having the potential to induce apoptosis; and determining that the putative agonist identified in step (b) induces apoptosis by binding with DRL receptor by competitive binding assays, thereby identifying a DRL receptor agonist.
  • the present invention also provides a method of screening for a DRL receptor antagonist comprising, contacting cells that express the DRL receptor with a putative antagonist in the presence of DRL, detecting a decrease in apoptosis in the cells whereby the decrease in apoptosis indicates a putative antagonist having the potential to inhibit apoptosis; and determining that the putative antagonist identified in step (b) inhibits apoptosis by binding with DRL receptor by competitive binding assays, thereby identifying a DRL receptor antagonist.
  • a novel sequence with homology to the TNF family was identified from the National Center for Biotechnology Information dbEST data base using a consensus amino acid sequence of the TNF family. Further BLAST search of the EST data base with this EST clone resulted in the identification of several other overlapping clones. Based on the sequence of the clones and the sequence of the known TNF family members, a contig of a potential novel TNF-like gene was generated. Full length cDNA of this possible novel gene was therefore cloned from a lymphoma cell line, CEM. Reverse-transcripted cDNAs prepared from CEM cells was prepared. Primers designed at both ends of the contig sequence were used for amplification of the cDNA by PCR.
  • PCR generated a 1.3 kb product which was cloned into pcDNA3 vector. Sequence analysis of multiple clones shows that the cDNA contains a open reading frame encoding a 250 aa protein (Fig. 1 A). The gene was named DRL-1 for death receptor /igand-1. Sequence alignment analysis indicates that DRL-1 is approximately 25%) identical to other TNF family members at the amino acid level (Fig. IB). It contains a putative transmembrane domain near the N-terminus from aa residues 26 to 51, suggesting that it is a type II membrane like other members of the TNF family. DRL-1 contains a possible N-glycosylation region at aa 122 to 125, suggesting that functional form of DRL-1 may be glycosylated.
  • DRL-1 is expressed as a 1.8 kb transcript in heart, placenta, lung, kidney and pancreas (Fig. 2A).
  • DRL-1 is expressed in the peripheral lymphoid organs, including spleen, lymph nodes and peripheral blood lymphocytes (Fig. 2B).
  • DRL-1 transcript is absent from the thymus and fetal liver (Fig. 2B), indicating that DRL-1 is not expressed by immature T cells as well as hemopoietic progenitors in the fetal liver.
  • bone marrow RNA contains abundant amount of DRL-1 (Fig. 2B). What cell types express DRL-1 in the bone marrow is unclear. It is possible that certain hemopoietic progenitor lineages in the bone marrow express DRL-1. Alternatively, this could represent DRL-1 expression by mature lymphocytes that are present in the marrow.
  • TNF family members have the potential to regulate cell growth or apoptosis. Many TNF family genes are up-regulated in T cells after activation, suggesting that they are involved in different aspects of T cell regulation during immune responses.
  • RNA derived from T cells with or without TCR crosslinking was analyzed for DRL-1 expression.
  • Fig. 2C shows that after 2.5 h of TCR crosslinking, DRL-1 is significantly up- regulated.
  • DRL-1 mRNA is completely down-regulated after 5 h of stimulation. This suggests that DRL-1 may be important for T cell functions during active immune responses and its regulation is tightly regulated.
  • PCR method using a pair of specific primers for DRL-1 total human DNA and DNA samples from individual human chromosomes were examined.
  • Fig. 3A shows that PCR of total human DNA generates a 700 bp product.
  • chromosome 17 DNA template gives rise to a PCR product of the same size as total DNA. None of the other Chromosomal DNA samples results in any PCR band.
  • DRL-1 unlike TNF, LT- ⁇ and LT- ⁇ tightly linked to the MHC locus on chromosome 6, is located on chromosome 17.
  • the sizes of the PCR products from genomic DNA are larger in size than that from DRL-1 cDNA, indicating that there is at least one intron between these two primers.
  • PCR analysis using a chromosome 17 deletion panel shows that DRL cannot be amplified from two of the deletion panels, B and E (Fig. 3B and C) but appeared in all the other samples. This indicates that DRL-1 is located on the chromosome 17pl 1.2-12 region. Based on the approximate genomic location of DRL-1, a BAC genomic clone containing DRL-1 was selected and the presence of DRL-1 in this clone was confirmed by PCR analysis. The BAC clone was then used as a probe for a defined localization of DRL by FISH.
  • TNF- ⁇ and TRAIL/APO-2 ligand are sufficient for the biological activities of these proteins. It is therefore possible that the extracellular domain of DRL-1 is biologically active. We therefore fused the DRL-1 extracellular domain from aa 65 to 250 at the N- terminus with human IgG Fc. The construct was expressed in COS cells and the fusion protein was purified from COS cell supernatant with a protein A column. A single band of the 50 KD soluble DRL-l-Fc eluted from the column was observed by SDS- PAGE and coomassie blue staining (Figure 4).
  • DRL-l-Fc Staining of cell lines by DRL-l-Fc
  • the DRL-l-Fc protein was then used to stain cell lines.
  • Fig. 5 shows that the DRL-1 fusion protein binds to lymphoma cell lines H9 and Jurkat but not CEM.
  • DRL-1 also stains a adenocarcinoma cell line, COLO 205 and a transformed human embryonic cell line, 293.
  • DRL also binds to human PBLs. This suggests that DRL- 1 receptor is widely expressed among many cell types. Identification of cell types bearing the receptor for DRL-1 is important because these cells will be important for analysis of DRL-1 functions. These cell types include T cells, B cells, epithelial cells, malignant cells, and other hematopoietic cells.
  • DRL-1 was amplified by PCR from cDNAs derived from a human lymphoma cell line, CEM. PCR primers for DRL-1 amplification are: forward primer, 5'-
  • TATCGAATTCACAACCTTCTTCCCTTCTGCACCA (SEQ ID NO: 3); reversed primer, 5'-TATCGAATTCAATGAAAAGGGAAAAGTGAGGAACG (SEQ ID NO:4).
  • the PCR product was digested with EcoR I and ligated to EcoR I-digested pcDNA3 vector. Multiple clones were sequenced by method. Lasergene softwares (DNASTAR, Madison, WI) were used to analyze DRL-1 for its properties and alignment with other TNF family members.
  • RNA samples were then used for agarose gel (1.4%) electrophoresis and blotted onto a nylon membrane. The blot was probed with DRL-1 probe and actin probes respectively. RNA blots were also obtained from Clontech and probed for DRL-1 and actin as above.
  • DRL-1 ( 1059F+ 1443 reverse) DRL-1 was amplified from genomic DNA using the following primer: 5'-
  • Total human DNA was used as a positive control.
  • the PCR product from genomic DNA is larger than that from DRL-1 cDNA, suggesting that there is at least one intron between these two primers.
  • DRL-1 extracellular domain from amino acid residue 65 to 250 was fused at N- terminus with human IgG Fc containing the CH2 and CH3 domain.
  • the construct was cloned into a pCMVl vector (kodak).
  • the plasmid was then used for transfection of COS cells by DEAE-dextran method (Promega). Seventy two hours after transfection, the supernatant of the COS cells was passed through a 0.45 ⁇ M filterand purified with a protein A column. The column was then washed with TBS and eluted with 0.1 M glycine, pH 3.0.
  • the eluent (5 ml) was dialyzed five times against PBS over a 48 h period and then examined by SDS-PAGE. A single 50 KD band was observed in the eluent by coomassie blue staining.
  • the concentration of the purified DRL-l-Fc was quantitated by the Bio-Rad protein assay method (Bio-Rad, Hercules, CA) using bovine albumin as the standard.
  • Monoclonal antibodies against DRL-1 designated MAbl, MAb2, MAb3, Mab4 and Mab5 were generated by standard methods.
  • a peptide corresponding to the extracellular domain was used as the immunogen to produce the present antibodies. Briefly, the purified immunogen was injected into an animal in an amount and in intervals sufficient to elicit an immune response. Spleen cells were obtained from the animal. The cells were then fused with an immortal cell line and screened for antibody secretion.
  • Antibodies were selected that bind to DRL-1.
  • solid-phase ELISA immunoassays are routinely used to select antibodies selectively immunoreactive with a protein. See Harlow and Lane “Antibodies, A Laboratory Manual” Cold Spring Harbor Publications, New York, (1988), for a description of immunoassay formats and conditions that are commonly used to determine selective binding.
  • FIG. 6 shows the predicted protein sequence of DRL-2.
  • the underlined sequence indicates a region that is likely to be essential for DRL-2 function. In addition to the whole DRL-2 molecule, this underlined sequence can be used to make peptides for the generation of polyclonal and monoclonal antibodies against DRL-2.
  • DRL-2 Northern blot analysis for the expression of DRL-2 was conducted as described above for DRL-1. A probe specific for DRL-2 sequence was used to probe human mRNA.
  • Figure 7 shows the expression of DRL-2 in spleen and peripheral blood lymphocytes (PBL).
  • FIG 8 shows the results of an in vitro translation assay.
  • DRL-1 and DRL-2 encode discrete proteins of different sizes.
  • DRL-2 encodes two isoforms.

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Abstract

L'invention concerne des acides nucléiques codant les protéines DRL, DRL-1 et DRL-2, présentées dans le listage des séquences sous les nos. SEQ ID NO:1 et SEQ ID NO:3, respectivement. L'invention concerne aussi un fragment unique de SEQ ID NO:1 qui code pour les résidus d'acides aminés de 65 à 250 de DRL-1, définis dans SEQ ID NO:2. Elle concerne en outre des polypeptides purifiés codés par les acides nucléiques codant les protéines DRL, présentées sous les nos. SEQ ID NO:1 et SEQ ID NO:3, le polypeptide purifié possédant les séquences présentées dans SEQ ID NO:2, SEQ ID NO:4 et SEQ ID NO:6 et celles des polypeptides de DRL que l'on peut utiliser pour stimuler une réponse immunitaire ou induire l'apoptose. Dans un autre aspect, l'invention concerne des anticorps purifiés qui se lient à la protéine de DRL ou leurs fragments. Ces anticorps peuvent être utilisés pour inhiber une réponse de leucocytes, pour inhiber l'apoptose ou pour supprimer une réponse auto-immune.
PCT/US1999/025954 1998-11-04 1999-11-04 Nouveau membre de la famille des facteurs de necrose des tumeurs, ligand du recepteur de la mort cellulaire (drl) et compositions et procedes correspondants WO2000026244A2 (fr)

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US6716576B1 (en) 1996-10-25 2004-04-06 Human Genome Sciences, Inc. Method of assaying Neutrokine-α mRNA level
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US7083785B2 (en) 1999-08-17 2006-08-01 Biogen Idcc MA Inc. Methods of treatment by administering an anti-BCMA antibody
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US7217788B2 (en) 1996-03-14 2007-05-15 Human Genome Sciences, Inc. Human tumor necrosis factor delta polypeptides
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US8211649B2 (en) 2006-03-31 2012-07-03 Human Genome Sciences, Inc. Methods of diagnosing and prognosing hodgkin's lymphoma
US9168286B2 (en) 2005-10-13 2015-10-27 Human Genome Sciences, Inc. Methods and compositions for use in treatment of patients with autoantibody positive disease
US9545086B2 (en) 1999-01-25 2017-01-17 Biogen Ma Inc. BAFF, inhibitors thereof and their use in the modulation of B-cell response and treatment of autoimmune disorders
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US7217788B2 (en) 1996-03-14 2007-05-15 Human Genome Sciences, Inc. Human tumor necrosis factor delta polypeptides
US6812327B1 (en) 1996-10-25 2004-11-02 Human Genome Sciences, Inc. Neutrokine-alpha polypeptides
US6562579B1 (en) 1996-10-25 2003-05-13 Human Genome Sciences, Inc. Diagnostic methods using antibodies to Neutrokine-alpha
US6635482B1 (en) 1996-10-25 2003-10-21 Human Genome Sciences, Inc. Monoclonal antibodies to membrane neutrokine-α
US6716576B1 (en) 1996-10-25 2004-04-06 Human Genome Sciences, Inc. Method of assaying Neutrokine-α mRNA level
US6881401B1 (en) 1996-10-25 2005-04-19 Human Genome Sciences, Inc. Methods of treatment of immune system related disorders using Neutrokine-alpha
US8231873B2 (en) 1996-10-25 2012-07-31 Human Genome Sciences, Inc. Methods of treatment using antibodies to Neutrokine-alpha
US6403770B1 (en) 1996-10-25 2002-06-11 Human Genome Sciences, Inc. Antibodies to neutrokine-alpha
US8303951B2 (en) 1996-10-25 2012-11-06 Human Genome Sciences, Inc. Neutrokine-alpha antibodies and methods of use thereof
US8071092B1 (en) 1996-10-25 2011-12-06 Human Genome Sciences, Inc. Methods of inhibiting B lymphocytes using antibodies to Neutrokine-alpha
US8173122B2 (en) 1996-10-25 2012-05-08 Human Genome Sciences, Inc. Methods of treatment using antibodies to neutrokine-alpha
US9545086B2 (en) 1999-01-25 2017-01-17 Biogen Ma Inc. BAFF, inhibitors thereof and their use in the modulation of B-cell response and treatment of autoimmune disorders
US6869605B2 (en) 1999-01-25 2005-03-22 Biogen Idec Ma Inc. BAFF, inhibitors thereof and their use in the modulation of B-cell response
US10494416B2 (en) 1999-08-17 2019-12-03 Biogen Ma Inc. Methods of modulating immune responses using BCMA polypeptide
US9650430B2 (en) 1999-08-17 2017-05-16 Biogen, Ma Inc. Methods of treating autoimmune diseases using a B-cell maturation antigen (BCMA)
US7691804B2 (en) 1999-08-17 2010-04-06 Biogen Idec Ma Inc. BAFF receptor (BCMA), an immunoregulatory agent
US8828669B2 (en) 1999-08-17 2014-09-09 Biogen Idec Ma Inc. Methods of screening for a compound that inhibits the interaction between BAFF and BCMA
US7083785B2 (en) 1999-08-17 2006-08-01 Biogen Idcc MA Inc. Methods of treatment by administering an anti-BCMA antibody
US7276241B2 (en) 1999-10-06 2007-10-02 Biogen Idec Ma Inc. Methods of treating a tumor that expresses APRIL by administering BCMA
US7220840B2 (en) 2000-06-16 2007-05-22 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US9187548B2 (en) 2000-06-16 2015-11-17 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US7138501B2 (en) 2000-06-16 2006-11-21 Human Genome Sciences, Inc. Antibodies that immunospecifically bind BLyS
US8101181B2 (en) 2000-06-16 2012-01-24 Human Genome Sciences, Inc. Antibodies that immunospecifically bind to B lymphocyte stimulator protein
US8062906B2 (en) 2000-08-18 2011-11-22 Human Genome Sciences, Inc. B-lymphocyte stimulator binding polypeptides and methods based thereon
WO2002022680A3 (fr) * 2000-09-15 2002-09-12 Klaus Pfizenmaier Activation localement specifique, par l'intermediaire d'anticorps, d'une zytokine pro-apoptose : amaize (antibody-mediated apoptosis inducing zytokine)
US7112421B2 (en) 2000-09-18 2006-09-26 Biogen Idec Ma Inc. Nucleic acids encoding BAFF receptor, chimeric proteins and methods and compositions related thereto
US8026072B2 (en) 2000-09-18 2011-09-27 Biogen Idec Ma Inc. Method of identifying compounds that bind BAFF-R
US7635677B2 (en) 2000-09-18 2009-12-22 Biogen Idec Ma Inc. BAFF receptor polypeptides
US8524672B2 (en) 2000-09-18 2013-09-03 Biogen Idec Ma Inc. Method for inhibiting BAFF-R activity
US7709220B2 (en) 2000-09-18 2010-05-04 Biogen Idec Ma Inc. Methods of monitoring treatment of BAFF-R related disease
US7638327B2 (en) 2000-09-18 2009-12-29 Biogen Idec Ma Inc. BAFF receptor antibodies and methods
US7189820B2 (en) 2001-05-24 2007-03-13 Human Genome Sciences, Inc. Antibodies against tumor necrosis factor delta (APRIL)
US7700317B2 (en) 2003-03-28 2010-04-20 Biogen Idec Ma Inc. Truncated baff receptors
US8022182B2 (en) 2003-03-28 2011-09-20 Biogen Idec Ma Inc. Truncated BAFF receptors
US8303958B2 (en) 2003-03-28 2012-11-06 Biogen Idec Ma Inc. Method of treating immunological disorders by administering truncated BAFF receptors
US8821883B2 (en) 2003-03-28 2014-09-02 Biogen Idec Ma Inc. Method of treating B cell cancers by administering truncated BAFF receptors
EP2333112A2 (fr) 2004-02-20 2011-06-15 Veridex, LLC Pronostics de cancer du sein
EP2332563A2 (fr) 2004-10-13 2011-06-15 The Washington University Utilisation de BAFF pour traiter la sepsie
US9168286B2 (en) 2005-10-13 2015-10-27 Human Genome Sciences, Inc. Methods and compositions for use in treatment of patients with autoantibody positive disease
US9726673B2 (en) 2005-11-23 2017-08-08 Genentech, Inc. Methods and compositions related to B cell assays
US8211649B2 (en) 2006-03-31 2012-07-03 Human Genome Sciences, Inc. Methods of diagnosing and prognosing hodgkin's lymphoma

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