IL183048A - Trail receptor polypeptide and dna encoding same - Google Patents

Description

TRAIL TRAIL RECEPTOR POLYPEPTIDE AND DNA ENCODING SAME IMMUNEX CORPORATION The present application is a divisional application of Israel patent application OF THE INVENTION A protein known as is a of the tumor necrosis factor family of ligands et TRAIL has the ability to induce apoptosis of certain transformed including a number of different types of cancer cells as well as infected cells application and Identification of receptor that bind TRAIL would prove useful in study of the biological activities of prior to the present no receptor for TRAIL had been SUMMARY OF THE INVENTION The present invention is directed to a novel protein designated TRAIL receptor which binds to a protein known as ligand DNA and expression vectors comprising such are A method for producing polypeptides comprises culturins host cells transformed with a recombinant expressio vector encorling TRAIL under promote expression of recovering the expressed polypeptides from the BRIEF DESCRIPTION OF THE FIGURES Figure 1 presents the nucleotide sequence of a TRAEL receptor DNA as well as the amino acid sequence encoded This DNA fragment is described in Example Figure 2 presents the esults soluble Figure 3 presents Notice Under Circular dated April Inasmuch as the invention is defined in the appended it will be apparent that the portions of the present which fall outside the scope of the do not relate directly to the claimed This Notice is not meant to disclaim any legitimate rights to which the Patentee is legally especially any rights in accordance with Section 49 of the Israel Patent la DETAILED DESCRIPTION OF THE INVENTION A protein TRAIL receptor is provided binds to the cytokine designated ligand Certain uses of flow from this ability to bind as discussed further finds use in inhibiting biological activities of or in purifying TRAIL by affinity for The nucleotide sequence of the region of a human TRAIL receptor DNA is presented in SEQ ID The ino sequence encoded A of 10 This e i c re c discussed protein or immunogenic fragments thereof may be employed as immunogens to generate antibodies that are In one embodiment of the the antibodies are monoclonal A human protein was purified as described in example In example amino acid sequence information derived from fragments of is One embodiment of the invention is directed to a purified human protein that is capable of binding wherein the is characterized as comprising amino acid sequence VPANBGD acids In another embodiment the comprises the sequence ETLROCFDDFADL FDS GLMDNElKVAKAEAAGHFJJTLXTML Also provided are fragments comprising one of these characterizing amino acid The sequence of a DNA and the acid sequence encoded are presented in Figure 1 O see example The amino acid sequence presented in Figure 1 has characteristics of the found in the cytoplasmic region of certain other receptor 35 Such domains have been reported to be associated with transduction of a optotic signals Fas and J j sign i cascades I J presents the nucleotide of the coding region of a human 5 TRAIL receptor including initiation codon and a termination codon The acid sequence encoded bv the DNA of NO is presented in The fragment depicted in Figure 1 corresponds to the region of that is presented as amino acids to in A The protein of includes an hydrophobic region that functions as a signal followed bv an extracellular a transmembrane region comprising amino acids through and a cytoplasmic Computer analysis predicts that the signal peptide corresponds to residues 1 to 51 of SEP ID Cleavage of the signal peptide thus would yield a mature protein comprising amino acids 52 through 440 of SEP The calculated molecular weight for a mature protein containing residues 52 to 440 of SEP about 43 t is the se gest me ts of a i on fusion C peptide was cjeayed 30 The skilled artisan will recognize that the molecular weight of preparations of protein may according to factors as the degree of The glycosylation pattern of a particular preparation of may vary according to the type of cells in which the protein is for a given preparation may include multiple differentially glycosylated species of the 35 with or without associated glycosylation are In protein is characterized by a molecular weight within the range of about 50 to which is the molecular weight determined for a preparation of full human MoJecular weight can be determined by gel electrophoresis Example 1 presents one method for purifying a cells are and the subsequent purification process includes affinity chromatography a chromatography matrix containing and reversed phase polypeptides of the present invention may be purified by any suitable alternative using known purification In one alternative 10 chromatography matrix instead comprises an antibody that binds Other cell expressing the cells described in example can be substituted for the Jurkat The cells can be disrupted by any of the numerous known including mechanical or by use of lysing 15 The desired degree of purity depends on the intended use of the A relatively high degree of purity is desired when the protein is to be administered in for polypeptides are purified such that no protein bands to other proteins are detectable upon analysis by gel electrophoresis It be recognized by one 20 skilled in the pertinent field that multiple bands corresponding to protein may be visualized by due to differential differential translational and the most preferably is purified to substantial as indicated by a single protein band upon analysis by The protein band may be visualized by silver Coomassie the 25 protein is by The present invention encompasses in various naturally occurring or produced procedures recombinant D Such forms of limited and oligomers of as 30 well as containing or fragments may be modified to create derivatives thereof by covalent or aggregative with other chemical such as acetyl groups and the Covalent of may be prepared by the chemical moieties functional groups on amino acid side 35 chains or at the or of a Conjugates comprising diagnostic or therapeutic attached to as discussed in more detail Other derivatives of within the scope of this invention include covalent or aggregative conjugates of polypeptides with other proteins or such as by synthesis in recombinant culture as or C Examples of fusion proteins are discussed below in connection with TRAIL fusion proteins can comprise peptides added to facilitate purification and identification of Such peptides for or the antigenic identification peptides described in Patent 1 and in Hopp et One such peptide is the which is highly antigenic and provides an epitope bound by a specific monoclonal enabling rapid assay and facile purification expressed recombinant A murine hybridoma designated 4E1 produces a monoclonal antibody that binds the peptide in the presence of certain divalent metal as described in Patent hereby incorporated by The hybridoma cell has been deposited with the American Type 15 Culture Collection under accession Monoclonal antibodies that the peptide are available Eastman Kodak Scientific Imaging Systems New Both cell and soluble forms of are provided Soluble may be identified distinguished from 20 by separating intact cells expressing a polypeptide from the culture by and assaying the medium for the presence of the The presence of the medium indicates the protein was secreted from the cells and thus is a soluble form of the desired 25 Soluble forms of receptor proteins typically lack the transmembrane region that would cause retention of the protein on the cell In one embodiment of the a soluble polypeptide the extracellular domain the A so p lypeptid u d cytoplasmic do a as One example of a soluble is a soluble human amino acids 52 to of are olypeptides com x of ID Soluble forms of possess certain advantages over the 35 form of Purification of the protein from recombinant host cells is since the soluble proteins are secreted from the soluble proteins are generally more suitable for certain for intravenous Such fragments may be prepared by any of a number of conventional Desired peptide fragments may be chemically An alternative involves generating fragments by enzymatic by treating the protein with an enzyme known to cleave proteins at sites 5 defined by particular amino acid are the and the i e of y as to described Naturally occurring variants of the protein of are provided Such variants for proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the Alternate 20 splicing of mRNA for yield a truncated but biologically active such as a naturally occurring soluble form of the Variations attributable to proteolysis for differences in the or expression different types of host due to proteolytic removal of one or more terminal amino acids from the protein from terminal proteins in which differences in am inn acid sequence are to genetic polymorphism variation among individuals producing the are also contemplated The skilled artisan will also recognize that the at which the signal peptide is cleaved may differ from that predicted bv computer and mav 30 according to such factors as the host cells employed in expressing a recombinant A protein may include res from of ski ms of the 6 5 herd uman A of S other mammalian are contemplated for Probes based on the human DNA sequence of or may be used to screen cDNA libraries derived from other mammalian using conventional h vbri d izati o DNA native disci 15 enc 20 and degenerate o a DNA ti ides i that bu which 35 a d ds I b a e and from p 7 of ft As ti d am mo 15 described et ffl 20 N of 8 ί of bridges receptors o c motifs soluble remain unaltered in when retention of activity is yeast j protease a i t 20 adj basic residue at 25 3 and can be for The present invention also provides recombinant cloning and expression vectors 35 as as host containing the recombinant vectors comprising DNA mav be used to prepare polypeptides encoded bv the A method for producing polypeptides comprises cuituring host cells transformed with a recombinant expression encoding under conditions that promote expression of then recovering the expressed polypeptides from the The skilled artisan 5 will recognize that the procedure for purifying the expressed according to such factors as the type of host cells and whether the is or a soluble form that is secreted from the host suitable expression system be The vectors include a DNA a operablv linked to suitable transcriptional or 10 nucleotide such as those derived a mammalian viral or insect Examples of regulatory sequences include transcriptional or an mRNA ribosomal binding and appropriate sequences which control transcription and translation initiation and Nucleotide sequences are linked the regulatory sequence functionally 15 relates to the DNA a promoter nucleotide sequence is operablv linked to an DNA sequence the promoter nucleotide sequence controls the transcription of the DNA An origin of replication that confers the ability to replicate in the desired host and a selection gene bv which are are generally incorporated into the expression 20 In a sequence encoding an appropriate signal peptide fnative can be incorporated into A DNA sequence for a peptide fsecretorv be fused in frame to the sequence so that the is initially translated as a fusion protein comprising the signal A signal peptide that is functional the intended promotes extracellular secretion 25 of the The signal peptide is cleaved from the polypeptide upon of from the Suitable host cells for expression of polypeptides include or higher Mammalian or cells are generally preferred for cloning and expression vectors for use with 30 and mammalian cellular hosts are in Pouwels et A Laboratory New translation 10 could also be employed to produce polvnentides using RNAs DNA constructs disclosed include gram or gram positive for F coli or Suitable prokarvotic host cells for for 5 Bacillus Salmonella and various other species within the genera and In a prokarvotic host as a polypeptide may include an methionine residue to facilitate of the recombinant polypeptide in the prokarvotic host The terminal Met be cleaved from the expressed recombinant 10 Expression vectors for use in prokarvotic host cells generally comprise one ητ more phenotvpic selectable marker A selectable marker gene for a gene encoding a that confers antibiotic resistance or that supplies an autotrophic Examples of useful expression vectors for prokarvotic host cells include those derived from commercially available such as the cloning vector 15 nBR322 37017V PBR322 contains genes for amnicillin and tetracycline resistance and thus provides simple means for identifying transformed An appropriate promoter and a TRAILER DNA sequence are inserted into the PPJR Other commercially available vectors for Fine and 20 Promoter sequences commonly used for recombinant prokaryotic host cell expression vectors include lactose promoter system f et Nature and et Nature 2 tryptophan promoter system fGoeddel et Acids and and 25 tac promoter Molecular At Laboratory Cold Spring Harbor A particularly useful prokarvotic host cell expression system employs a λ a cI857ts thermolabile repressor PI asm id vectors available from the American Type Culture Collection which incorporate derivatives of the λ promoter include PHUB2 Resident in coli strain 30 ATCC and Resident in E coli ATCC aitematiyelv may be expressed in yeast host preferably from the Other genera of such as Pichia or 1 1 mav also be Yeast vectors often contain an of replication sequence from a veast an replicating a promoter for sequences for transcription and a selectable marker Suitable promoter sequences for veast vectors 5 among promoters for fHitzeman et Biol 801 or other glycolytic fHess et and Holland et pyruvate 10 triosephosphate and Other suitable vectors and promoters for use in expression are further described in Another alternative is the promoter described et Biol 19 2 and 19821 Shuttle vectors in both veast and cob 5 constructed bv inserting DNA sequences from DBR322 for selection and replication in coli gene and origin of into the veast The veast leader sequence be employed to direct secretion of the TRAIL The leader sequence is often inserted between the promoter sequence and the structural gene et Cell and Bitter et USA 1 Other leader sequences suitable for facilitating secretion of recombinant polypeptides from hosts are known to those of skill in the A leader seouence mav be modified near its end to contain one or more restriction This will facilitate of the leader sequence to the structural Yeast transformation protocols are known to those of skill in the One is described bv Hinnen et Natl USA The Hitmen et protocol selects for transformants selective wherein the selective medium consists of veast nitrogen casamiho adenine and 20 Yeast host transformed bv vectors containing an promoter seouence mav be grown for inducing expression in a An example of a rich medium is one consisting of 1 veast and 1 supplemented with 80 12 adenine and 80 of the promoter occurs when glucose is exhausted from the Mammalian or insect host cell culture svstems also mav he employed to recombinant virus systems for production of heterologous proteins in insect cells are reviewed bv Luckow and Established cell lines of mammalian also he Examples of suitable mammalian host cell lines include the line of mnnkev kidnev cells fATCC CRL 1 6511 et Cell L CI 3T3 cells CCL 1 Chinese hamster ovary f HeLa 10 and fATCC CRL 101 cell and the CV1 EBNA derived from the African monkev kidnev cell line fATCC CCL as described bv McMahan 1 Transcriptional and translational control sequences for mammalian host cell expression vectors mav be excised from viral Commonly used promoter 15 sequences and enhancer sequences are derived from Polyoma Adenovirus Simian Virus 40 and human DNA sequences derived from the SV40 viral for SV40 and late and polvadenylation sites mav he used to provide other genetic elements for expression of a structural gene sequence in a mammalian host Viral earlv late promoters are 20 particularly useful because both are easily obtained from a viral genome as a fragment which mav also contain a viral origin of replication fFiers et Nature 1 Smaller or larger SV40 fragments mav also he provided the approximately 250 bp sequence extending from the Hind site toward the I ocated in the SV40 viral ori in of replication site is 25 vectors for use mammalian host cells can be constructed as disclosed by Okavama and Cell for A useful system for stable fevel expression of mammalian cDNAs in murine mammary epithelial cells can be constructed substantially as described bv Cosman et A high expression hv 30 Cosman et Nature has been deposited Additional mammalian expression vectors are described in and in WO As one the vector mav be derived from of 13 of As are hereb n in i in reference Seq ted 30 14 5 the d seg 0 be One Poxvirus ba cu j 0 virus a re viral i ba cascades te ases to siK 15 the the J 5 ofthe native peptide of mav be replaced bv a heterologous signal peptide leader if The choice of signal peptide or leader depend on factors such as the vpe of host cells in which the recombinant to be To examples of heterologous signal peptides that are functional in mammalian host cells include the signal sequence for described in United States Patent the signal sequence for receptor described in Cosman et Nature the receptor signal peptide described in the I receptor signal peptide described in Patent and the II receptor signal peptide described in EP Oli pom eric Forms of Encompassed by the present invention are oligomers that contain oligomers may be in the form of or or higher embodiment of the invention is directed to oligomers comprising multiple polypeptides joined via covalent or interactions between peptide moieties fused to the Such peptides may be peptide linkers or peptides that have the property of promoting Leucine zippers and certain polypeptides derived from antibodies are among the peptides that can promote oligomerization of polypeptides attached as described in detail particular the oligomers comprise from two to four The moieties of the oligomer may be soluble as As one a oligomer is prepared using polypeptides derived from Preparation of fusion comprising certain heterologous polypeptides fused to various portions of polypeptides the Fc has been by Ashkenazi et USA 16 st and and Aruffo of Immunoglobulin Fusion in Current Protocols pages 1 One embodiment of the present invention is directed to a dimer 5 comprising two fusion proteins created by fusing to the Fc region of an A gene fusion encoding the Fc fusion protein is inserted into an appropriate expression fusion proteins are expressed in host cells transformed with the recombinant expression and allowed to assemble much like antibody whereupon interchain disulfide bonds form between the Fc moieties 0 to yield divalent Provided herein are fusion proteins comprising a polypeptide fused to an Fc polypeptide derived from an DNA encoding such fusion as well as dimers containing two fusion proteins joined via disulfide bonds between the Fc moieties are also The term as used herein includes 5 native and mutein forms of polypeptides derived from the Fc region of an Truncated forms of such polypeptides containing the hinge region that promotes are also One suitable Fc described in PCT application WO incorporated by is a single chain polypeptide extending from the hinge region to the native of the Fc region of a human Another useful Fc polypeptide is the Fc mutein described in Patent and i Baum et The amino acid sequence of mutein is identical to that of the native Fc sequence presented in WO except that amino acid has been changed from Leu to amino acid 20 has been changed from Leu to and acid 22 has been changed from to The mutein exhibits reduced affinity for Fc In other may be substituted for the variable portion of an antibody heavy or light If fusion proteins are made with both heavy and light chains of an it is possible to form a oligomer with as many as four the oligomer is a fusion rotein comprising multiple with or without peptide linkers Among the suitable peptide linkers are those described in Patents and which are hereby incorporated by A DNA sequence encoding a desired peptide linker may be and in the same reading frame the DNA sequences encoding using any suitable conventional For a chemically synthesized oligonucleotide encoding the linker may be ligated between sequences 17 encoding In particular a fusion protein comprises two four soluble separated by peptide Another method for preparing oligomeric involves use of a leucine Leucine zipper domains are peptides that promote of the proteins in which they are Leucine zippers were identified in several binding proteins et Science and have since been found in a variety of different Among the known leucine zippers are naturally occurring peptides and derivatives thereof that dimerize or of leucine zipper domains suitable for producing soluble oligomeric proteins are described in PCT application WO and the leucine zipper derived lung surfactant protein D described in Hoppe et Letters 191 hereby incorporated The a fusion proteins comprising a soluble TRAIL R polypeptide fused to a leucine zipper peptide are expressed in suitable host and the soluble oligomeric that forms is recovered from the culture Oligomeric has the property of binding sites for The fusion proteins comprising Fc moieties oligomers formed offer the advantage of facile purification by affinity over Protein A or Protein G DRA iuences or roteins and other forms of may be tested for the ability to bind TRAIL in any suitable such as conventional binding To may be labeled with a detectable reagent a enzyme that catalyzes a colorimetric or fluorometric and the The labeled is contacted with cells expressing The cells then are washed to remove unbound labeled and the presence of label is determined by a suitable chosen according the nature of the One example of a binding assay procedure is as A recombinant expression vector containing TRAIL cDNA is as described in in PCT application WO hereby incorporated by DNA and amino acid sequence information for human and mouse TRAIL is presented in WO TRAIL comprises an cytoplasmic a transmembrane and a terminal extracellular cells in cm2 dishes are transfected with S the recombinant expression cells CRL 1 express EBV nuclear from the CMV CVl was derived from the African Green Monkey kidney cell line CCL as described by McMahan et 5 The transfected cells are cultured for 24 and the ceils in each dish then are split into a After culturing additional 48 the transfected cells 4 x are washed with which is binding medium containing 25 bovine serum 2 sodium 20 mM pH to which 50 nonfat dry milk has been The cells then are incubated for 1 hour at with various concentrations of a soluble fusion Cells then are washed and incubated with a constant saturating concentration of a mouse in binding with gentle agitation for 1 hour at After extensive ceils are released via The mouse IgG employed above is directed against the Fc region of human IgG and can be obtained from Jackson Immunoresearch West The antibody is radioiodinated using the standard The antibody will bind to the Fc portion of any protein that has bound to the In binding of is assayed in the absence of as well as in the presence of Fc and a molar excess of unlabeled mouse IgG is quantified on a Packard Autogamma Affinity calculations Set 52 are generated on run on a Microvax Another type of suitable binding assay is a competitive binding To biological activity of a variant may be determined by assaying for the ability to compete with a native binding to Competitive binding assays can be performed by conventional that may be employed in competitive binding assays include radiolabeled and intact cells expressing TRAIL or on the cell For a radiolabeled soluble fragment can be used to compete with a soluble variant for binding to cell surface Instead of intact could substitute a soluble TRAIL Fc fusion protein bound to a solid phase through the interaction of Protein A or Protein G the solid with the Fc Chromatography columns that contain Protein A and Protein G include those available from Pharmacia Another type of competitive binding assay utilizes radiolabeled soluble such as a soluble fusion 19 and intact cells expressing Qualitative results be obtained by competitive autoradiographic plate binding while Scatchard plots 5 may be utilized to generate quantitative Another type of assay for biological activity involves testing a 5 polypeptide for the to block TRAIL apoptosis of target such as the human leukemic line known as Jurkat for apoptosis of the cell line designated Jurkat clone TIB is demonstrated in assay procedures described in PCX application WO hereby incorporated by 0 Uses of Uses of but are not limited the Certain of these uses of flow from its ability to bind finds use as a protein purification polypeptides may 5 be attached to a solid support material and used to purify TRAIL proteins by affinity In particular a polypeptide any form described herein that is capable of binding is attached to a solid support by conventional As one chromatography columns containing functional groups that will react with functional groups on amino acid side chains of proteins are available In an a Fc is attached to Protein or Protein chromatography columns through interaction with Fc proteins also find use in measuring the biological activity of TRAIL proteins in terms of their binding affinity for proteins thus may be employed by those conducting to monitor shelf life and stability of TRAIL protein unde different To may be employed in a binding affinity study to measure the biological activity of a TRAIL protein that has been stored at different or produced in different cell may be used to determine whether biological activity is retained after modification of a TRAIL protem chemical The binding affinity of the modified TRAIL protein for is compared to that of an unmodified TRAIL protein to detect any adverse impact the modifications on biological activity of The biological activity of a TRAIL protein thus can be ascertained before it is used in a research for also finds use in purifying or identifying cells that express TRAIL on the cell polypeptides are bound to a solid phase such as a column chromatography matrix or a similar suitable For magnetic 20 microspheres be with and held in an incubation vessel through a magnetic Suspensions of cell mixtures containing cells are contacted with the solid phase having Cells expressing TRAIL on the cell surface bind to the fixed and unbound cells then are washed 5 can be conjugated to a detectable then incubated with cells to be tested for TRAIL After labeled R is removed and the presence or absence of the detectable moiety on the cells is In a further mixtures of cells suspected of containing cells are 10 incubated with biotinylated Incubation periods are typically at least one hour in duration to ensure sufficient The resulting rnixture then is passed through a column packed with whereby the high affinity of biotin for avidin provides bmding of the desired cells to the Procedures for using beads are known et J Cell Washing to remove unbound and the release of the bound are performed using polypeptides also find use as carriers agents attached thereto to cells bearing Cells expressing TRAIL those identified in Wiley et proteins thus can be used to deliver G diagnostic therapeutic agents to such cells to other cell types found to express TRAIL on the cell in in vitro or in vivo Detectable and therapeutic agents that may be attached to a polypeptide but are not other cytotoxic enzymes that catalyze a colorimetric or fluorometric and the with the agent being chosen according to the Among the toxins are diphtheria aeruginosa exotoxin inactivating mycotoxins such as and derivatives and fragments single Radionuclides suitable for diagnostic use but are not limited and Examples of radionuclides suitable for therapeutic use are Such agents may be attached to the by any suitable conventional being a comprises functional groups on amino acid side chains that can be reacted functional groups on a desired agent to form covalent for the protein or agent may be derivatized to generate or attach a desired reactive The derivatization may involve attachment one of the bifunctional reagents available for attaching various molecules to proteins Chemical A number of techniques for radiolabeling proteins are Radionuclide metals may be attached to using a suitable bifunctional chelating for Conjugates comprising and a suitable diagnostic or therapeutic agent 5 covalently are thus The conjugates are administered or otherwise employed in an amount appropriate for the particular TRAILER of the present invention may be used in developing treatments for any disorder mediated or by or amounts polypeptides may be administered to a mammal afflicted with such a of defective TRA ne Another use of the protein of present invention is as a research tool for studying the biological effects that from inhibiting interactions on different cell polypeptides also may be employed in in vitro assays for detecting TRAIL or of the interactions may be employed in inhibiting a biological activity of in in vitro or in vivo purified polypeptide may be used to inhibit binding of TRAIL to endogenous cell surface Biological effects that result from the binding of TRAIL to endogenous receptors thus are Various forms of may be for the and variants that are capable of binding In one a soluble is employed to inhibit a biological of to inhibit apoptosis of particular may be administered to a mammal to treat a Such disorders include conditions caused or or exacerbated by may be useful for treating thrombotic One disorder is thrombotic thrombocytopenic purpura Thompson Increasing mortality rates have by the Centers for Disease Control et Plasma from patients afflicted with ΓΤΡ and induces apoptosis of human endothelial cells of dermal microvascular not 22 large vessel origin et Plasma of TTP patients thus is thought to contain one or more factors that directly or indirectly induce As described in application WO incorporated by TRAIL is present in the serum of TTP and may play a role in 5 apoptosis of microvascular endothelial Another thrombotic microangiopathy is syndrome Melnyk et Thompson et One embodiment of the invention is directed to use of R to treat the condition that is often to as though it can strike children as A disorder known as HUS differs in etiology from adult Other conditions characterized by clotting of small blood vessels be treated using Such conditions include but are not limited to the Cardiac problems seen in about of pediatric patients are believed to involve clotting of small blood Breakdown of the vasculature in the heart has been in multiple sclerosis As a further treatment of systemic lupus erythematosus is In one a blood or plasma is contacted with ex The may be bound to a suitable chromatography matrix by conventional The blood or plasma flows through a chromatography column containing bound to before being returned to the The immobilized receptor binds thus removing TRAIL protein from the may be administered in vivo to a patient afflicted with a thrombotic In one a soluble form of is administered to the The present invention provides a method for treating a thrombotic involving use of effective amount of A polypeptide may be employed in in νίνο ex vivo to inhibit damage to apoptosis microvascular endothelial be in conjunction with other agents useful in treating a particular In an in vitro study reported by Laurence some reduction of TTP apoptosis of microvascular endothelial cells was achieved by using an blocking aurintricarboxylic or normal plasma depleted of a patient may be treated with an agent that inhibits apoptosis of endothelial in combination with an agent that inhibits 23 In one and an blocldng antibody are both administered to a patient afflicted with a disorder characterized by thrombotic such as TTP or Examples of blocking monoclonal antibodies directed against Fas antigen described in PCT application 5 publication number WO hereby incorporated by Another of T cells of 7 Certai of ej 5 is apoptosis At T to bv T cells death ί triggering xe eptor termed activated cells isolated 2 mdi death m 30 wt If max an jested co ex 35 24 TRAIL s ed i oilier inhibitor of T 5 d is et ith agents me e Fas o p vpeptides biological of of that in hod i es of ng i Compositions comprising an effective amount of a polypeptide of the present in combination with other components such as a physiologically acceptable or are provided can be formulated 20 according to known methods used to prepare pharmaceutically useful can be combined in either as the sole active material or with other known active materials suitable for a given with pharmaceutically acceptable diluents and phosphate buffered preservatives benzyl adjuvants 25 Suitable formulations for pharmaceutical compositions include those described in Pharmaceutical Mack Publishing In such compositions can contain complexed with polyethylene metal incorporated into polymeric compounds such as 30 polyacetic or incorporated into unilamellar or multilamellar erythrocyte ghosts or Such compositions will influence the physical rate of in vivo and of in vivo clearance of and are thus chosen according to the intended expressed on the surface of a cell 35 may find as Compositions of present invention may contain a polypeptide in any form described such as and 25 biologically In particular the composition comprises a soluble TRAIL polypeptide or an oligomer comprising soluble can be administered in any suitable or by The term includes by 5 or intramuscular also including localized at a site of disease or Sustained release from implants is also One skilled in the pertinent art will recognize that suitable dosages will depending upon such factors as the nature of the disorder to be the body and general and the route of Preliminary doses can be determined according to animal and the scaling of dosages for human administration are performed according to Compositions comprisin i i Antibodies Antibodies that are immunoreactive with polypeptides are provided Such antibodies specifically bind in that the antibodies bind to R via the binding sites of the antibody opposed to The protein prepared as described in example 1 may be employed as an immunogen in producing antibodies immunoreactive another form of as a fragment or fusion is employed as the Polyclonal and monoclonal antibodies may be prepared conventional for Monoclonal A New Dimension Biological Kennet et Plenum New York and A Laboratory Manual Harlow and Land Cold Spring Harbor Laboratory Cold Spring Production of antibodies directed against is further illustrated in example fragments of which be produced by conventional are also encompassed by resent Examples of fragments but are not limited and Antibody fragments and derivatives produced by genetic engineering techniques are also The monoclonal antibodies of the present invention include chimeric humanized versions of murine monoclonal Such humanized antibodies may prepared by known and offer the advantage of reduced immunogenicity when the are administered to In one a humanized monoclonal antibody comprises the variable region of a murine antibody 26 just the antigen binding site and a constant region derived from a human a humanized antibody fragment may comprise the antigen binding site of a murine monoclonal antibody and a variable fragment the derived from a human Procedures for the production of 5 chimeric and further engineered monoclonal antibodies include those described in et Liu et Larrick et and Winter and Harris Among the uses of the antibodies is use in assays to detect the presence of either in vitro or in The antibodies also may be employed in purifying proteins by that additionally can block binding of to TRAIL may be used to inhibit a biological activity that results from such Such blocking antibodies may be identified using any suitable assay such as by testing antibodies for the ability to inhibit binding of TRAIL to cells expressing Examples of such cells are the Jurkat cells and PS cells described in example 2 blocking antibodies may be identified in assays for the ability to inhibit a biological effect that results binding of TRAIL to target Antibodies may be assayed for the ability to inhibit lysis of Jurkat for Such an antibody may be employed in an in vitro or administered in vivo to inhibit a biological Disorders caused or exacerbated or by the interaction of TRAIL with cell surface TRAIL receptor thus may be A therapeutic method involves in vivo a rmiriistration of a blocking antibody to a mammal an amount effective in inhibiting a biological Disorders caused or exacerbated are thus Monoclonal antibodies are generally preferred for use in such therapeutic In one an antigenrbinding antibody frajgment is A blocking antibody directed against may be substituted for in the method of treating thrombotic in treating TTP or The antibody is administered in to inhibit damage to apoptosis microvascular endothelial upo surface J r 27 CornpositionE an antibody that is directed against and a physiologically acceptable or are provided Suitable 5 components of such compositions are as described above for compositions containing Also provided herein are conjugates comprising a detectable or therapeutic attached to an antibody directed against Examples of such agents are presented The conjugates find use in in vitro or in vivo 10 The present invention provides nucleic Such nucleic acids but are not limited DNA encoding the peptide described in example Such DNAs can be identified from knowledge of the genetic in ude c the n i v stems c 20 Nucleic of the both amp ified isolated i of N en any of th c the signal pe One embodiment of the invention is directed to fragments of nucleotide sequences comprising at least 5 se D at or at contiguous DNA Nucleic acids provided 28 A of said along with both and forms of the TRAIL Among the uses of nucleic acid fragments is use as probes or Using laiowledge of the genetic code in combination with the amino acid sequences set 5 forth in example sets of degenerate can be oligonucleotides find use as in chain reactions whereby DNA fragments are isolated and 10 comprise a of the a 15 re fom or or The splipd i ester backbones 30 29 s c nucleotide b formal of The following examples are provided to further illustrate particular embodiments of the and are not to be construed the scope of the present EXAMPLE Purification of Protein A human TRAIL receptor protein was prepared by the following was isolated the cell membranes of Jurkat a human acute T leukemia cell Jurkat cells were chosen because a specific band can be affinity Jurkat using covalently The precipitated band has a molecular weight of about 52 A minor specific band of about 42 kD also was possibly accounting for a proteolytic breakdown or a glycosylated of Approximately 50 billion Jurkat cells were harvested by centrifugation ml of washed once with then frozen on liquid Plasma membranes were isolated according to method number three described in Maeda et et hereby incorporated by with five The following protease inhibitors were included in all solutions at the indicated 5 1 μ 500 Dithiothreitol was not 5 DNAase was not used in the ml of homogenization buffer was used per ml of cell The homogenization was accomplished by five passages through a ground glass dounce After isolation of the cell proteins were solubilized by resuspending 10 the isolated membranes in 50 ml PBS containing octylglucoside and all of the above mentioned protease inhibitors at the above indicated The resulting solution was then repeatedly vortexed during a incubation at The solution was then centrifuged at rpm in an SW28 rotor in an Beclcman Palo at for 30 minutes to 5 obtain the sup ernatant membrane Chromatography The first step of purification of T out of the membrane extract prepared above was affinity The membrane extract was first applied to an 0 M2 column of monoclonal antibody M2 coupled to 2 ml of to adsorb any nonspecifically The was then applied to a column mg of recombinant protein coupled to 1 of The support is an ester of a crosslinked agarose gel bead from Biorad As discussed the provides an epitope reversibly bound by specific monoclonal enabling rapid assay and facile purification of expressed recombinant is a monoclonal antibody binds Monoclonal antibodies that the as well as other reagents for preparing and are available from Eastman Kodak Scientific Imaging Systems New of fusion proteins fused to a soluble TRAIL is further described in PCT application WO incorporated by The column was washed with 25 ml of each of the following in the 31 PBS containing octylglucoside PBS PBS an additional 200 mM NaCl PBS 5 The bound was eluted with 50 mM Na Citrate in 1 ml fractions and immediately neutralized with 300 of 1 M per The activity of each fraction was determined by a ELISA as described Fractions with high activity were brought to TrifJuoroacetic acid and subsequently chromatographed on a capillary column internal diameter X 25 cm fused silicone column packed with 5 Vydac material using a linear gradient per from to acetonitrile in water containing Fractions containing high activity are then determined as if Serial dilutions of samples 50 mM brought to pH 9 with coated onto Linbro Titertek 96 well flat bottom microtitration plates Biomedicals at After incubation at for the wells were washed six times with 200 PBS containing The wells were then incubated with at 1 in with fetal calf serum for 90 rninutes per followed by washing as each well was incubated the antibody M2 at containing for 90 minutes per wed by as wells were with a polyclonal goat horseradish antibody dilution of the commercial stock in containing for 90 minutes μΐ per The antibody was obtained from Southern Biotechnology Wells men were washed as For development of the substrate mix μΐ per well of premix of the TMB Peroxidase Substrate and Peroxidase Solution B Perry was added to the After sufficient color the enzymatic reaction was terminated addition of 2 N per Color intensity was determined b measuring extinction at 450 on a Y Max plate reader 32 EXAMPLE Amino Acid Sequence purified from cells protein isolated from Jurkat cells was digested with using conventional Amino acid sequence analysis was conducted on one of the 5 peptide fragments produced by the tryptic The fragment was found to contain the purified from cells protein was also isolated from is a human cell line that spontaneously arose after lethal irradiation of human peripheral blood lymphocytes The protein was digested with using conventional acid sequence analysis was conducted on peptide fragments that resulted from the tryptic One of the fragments was found to contain the Like the fragment presented in corresponds acids 327 333 of the sequence 15 EXAMPLE DNA and Amino Acid Sequences The amino acid sequence of additional trvptic digest peptide fragments of R was Degenerate based upon the amino acid sequence of two of The were A DNA fragment was isolated and amplified polymerase chain reaction using the degenerate oligonucleotides as and 3 The PGR was conducted according to conventional using derived from the cell line described in example 2 as the The nucleotide sequence of the isolated DNA fragment 25 corresponding to part of and the amino acid sequence encoded are presented in Figure 1 TP and The sequence of the entire DNA The amino acid sequence in I significant homology to the called domains found certain other The cytoplasmic region of Fas and TNF receptor I each contain a death which is associated with transduction of an signal et Cell and Biol he sequence in SEQ ID is believed to be within the cytoplasmic domai of 35 fro the fragment isolated above was used to screen a cDNA i foreskin cDNA in and a human R cDNA was The nucleotide sequence of the coding region of this 33 the acid sequence encoded thereby shown in EXAMPLE Monoclonal Antibodies That Bind 5 This example illustrates a for preparing monoclonal antibodies that bind Suitable immunogens that may be employed in generating such antibodies but are not limited purified protein or an immunogenic fragment thereof such as the extracellular or fusion proteins containing a soluble fusion Purified can be used to generate monoclonal antibodies irnmunoreacti e using conventional techniques such as those described in Patent mice are immunized with immunogen emulsified in complete and injected in amounts ranging from subcuianeousJy or Ten to twelve days the immunized animals are 15 boosted with additional emulsified in incomplete Mice periodically boosted thereafter on a weekly to immunization Serum samples are periodically taken by bleeding or excision to test for antibodies by dot ELISA Immunosorbent or inhibition of TRAIL 20 Following detection of an appropriate antibody positive animals are provided one last intravenous inj ection of in Three to four days animals are spleen cells and cells are fused to a murine myeloma cell NS1 or preferably CRL Fusions generate hybridoma are plated in multiple microtiter plates in a HAT 25 aminopterin and selective inhibit proliferation of myeloma and cell The hybridoma cells are screened by ELISA against purified R by adaptations of the techniques disclosed in et 1971 and in Patent A preferred screening technique is the antibody capture 30 technique described in Beckmann et Positive hybridoma cells can be inj ected intraperitoneally into syngeneic mice to roduce containing high concentrations of monoclonal hybridoma cells can be grown in vitro in flasks roller bottles by various 34 Monoclonal antibodies produced in ascites can be purified by ammonium sulfate followed by gel exclusion affinity chromatography based upon binding of antibody to Protein A or Protein G can also be as can affinity chromatography based upon binding to 5 d p tissues highest by rison to wi th ec pro leucine the of a essentially as o s r 35 soluble The ypepTid a ids J of 5 expression vector derived three of th and a 15 react the PGR was from a human 20 pp A 25 a ivl ated ntibod v s c F 36 5 10 the of an derived human 15 DN a i e protein protein 20 was the assay as 25 deat i treated with or constant of or of ar the ex I n A J and hi C by with of encoding human The from incorporated S inf Tf to i and sfec 39 SEQUENCE LISTING 1 GENERAL FORMATIO Charles Henning TITLE OF Receptor That Binds TRAIL NUMBER OF CORRESPONDENCE athryn Corporat 51 University Street A US 98101 COMPUTER READABLE MEDIUM TYPE Flopp atible PRIOR APPLICATION ATION US FILING PRIOR APPLICATION APPLICATION US DATE PRIOR APPLICATION US FILING APPLICATION APPLICATIO US FILING APPLICATION APPLICATION US FILING Kathryn REGISTRATION 2 TELECOMMUNICATION 40 756822 INFORMATION FOR SEQ ID SEQUENCE 1323 base pairs nucleic acid single MOLECULE NO NO IMMEDIATE SOURCE CDS SEQUENCE SEQ ID ATG GAA CAA CGG GGA CAG AAC GCC CCG GCC TCG GGG GCC CGG AAA 48 Met Glu Gin Arg Gly Gin Asn Ala Pro Ala Ala Ser Gly Ala 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 Ala Arg Gly Ala Arg Pro Gly Pro 20 25 30 CGG GTC CCC AAG ACC CTT GTG GTT GTC GCC GTC CTG CTG TTG 144 Arg Val Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 40 45 GTC TCA GCT GAG TCT GCT CTG ATC ACC CAA CAA GAC GCT CCC 192 Ser Ala Glu Ser Thr Leu Ala Gin 50 55 60 CAG AGA GCG GCC CCA CAA CAA AAG AGG TCC AGC CCC TCA GAG GGA TTG Gin Arg Ala Pro Gin Lys Arg Ser Pro Ser Glu Gly Leu 75 80 TGT CCA CCT GGA CAC CAT ATC TCA GAA GAC GGT AGA GAT TGC ATC TCC 28 Cys His Asp Gly Arg Asp Cys Ser 85 90 TGC AAA TAT GGA CAG GAC TAT AGC ACT CAC TGG GAC TTC Cys Tyr Ser His Trp Asn Asp Leu Leu 100 105 TGC TTG CGC TGC ACC AGG TGT GAT TCA GGT GAA GTG GAG CTA CCG 384 Cys Cys Thr Arg Cys Asp Ser Gly Val Leu Ser Pro 115 125 41 TGC ACC ACG ACC AGA AAC ACA GTG TGT CAG TGC GAA GAA GGC ACC TTC 432 Cys Thr Thr Asn Thr Cys Cys Glu Gly Phe 130 135 CGG GAA GAA GAT TCT ATG TGC CGG AAG TGC CGC ACA GGG TGT 480 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Thr Gly Cys 145 150 155 CCC AGA GGG ATG GTC AAG GTC GGT GAT TGT ACA CCC TGG AGT GAC ATC 528 Pro Arg Gly Met Val Lys Val Gly Asp Cys Thr Pro Trp Ser Asp e 165 170 TGT GTC CAC AAA GAA TCA GGT ACA AAG CAC AGT GAA GCC CCA 576 Glu Cys Val His Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180 1S5 190 GCT GTG GAG GAG ACG GTG ACC TCC AGC CCA GGG ACT CCT GCC CCC 624 Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro i95 200 205 TGT TCT CTC TCA GGC ATC ATC ATA GGA GTC ACA GTT GCA GCC GTA GTC 672 Cys Ser Leu Ser Gly Gly Val Thr Val Ala Ala Val Val 210 215 220 TTG ATT GTG GCT GTG TTT GTT TGC AAG TCT CTG TGG AAG AAA GTC 720 Leu He Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 CTT CCT TAG CTG AAA GGC ATC TGC TCA GGT GGT GGT GGG GAC CCT GAG 768 Leu Pro Tyr Leu Lys Gly Cys Ser Gly Gly Gly Gly Asp Pro Glu 245 250 255 CGT GTG GAC AGA AGC TCA CAA CGA CCT GGG GCT GAG GAC AAT CTC B16 Arg Val Asp Ser Arg Pro Gly Glu Asp Asn Val Leu 265 270 AAT GAG GTG AGT ATC TTG CCC ACC CAG GTC CCT GAG CAG GAA B64 Asn Glu He Val Ser He Leu Gin Pro Thr Gin Val Pro Gin Glu 2B0 AAC ATG TTG TCC 912 Asn Met Leu Ser CCC GGG GAG TCA GAG CAT CTG CTG GAA CCG GCA GCT GAA AGG 960 Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Ala Glu Arg Ser 305 315 320 CAG AGG AGG CTG CTG GTT GCA AAT GAA GGT GAT CCC ACT GAG 1008 Arg Leu Val Pro Ala Asn Glu Gly Asp Glu 330 335 CTG AGA CAG TGC TTC GAC GCA GAC TTG GTG CCC TTT GAC Leu Gin Cys Phe Asp Asp Phe Asp Leu Pro Phe Asp 45 GAG CCG TG AGG AAG TTG GGC CTC ATG GAC GAG ATA 1104 Ser Trp Glu Pro Leu Met Leu Gly Leu Met Asn Glu 355 360 365 AAG GTG GCT AAA GC GCA GCG GGC CAC AGG GAC TTG TAC ACG 1152 42 Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Tyr Thr 375 390 ATG CTG ATA AAG TGG GTC AAC AAA ACC GGG CGA GAT GCC TCT GTC CAC 1200 Met Leu Lys Trp Val Asn Lys Thr Gly Arg Asp Ala Ser Val His 3B5 390 395 400 ACC CTG CTG GAT GCC TTG GAG ACG CTG GGA GAG AGA CTT GCC AAG CAG 1248 Thr Leu Leu Asp Thr Leu Gly Glu Arg Leu Ala Lys Gin 405 410 415 AAG ATT GAG GAC CAC TTG TTG AGC TCT GGA AAG TTC ATG TAT CTA GAA 1296 Lys Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 GGT AAT GCA GAC TCT GCC ATG TCC TAA 1323 Gly Asn Ala Asp Ser Ala Met Ser 435 440 INFORMATION FOR SEQ ID SEQUENCE CHARACTERISTICS 440 amino acids amino acid linear MOLECULE protein SEQUENCE SEQ ID Met Glu Arg Gly Gin Asn Ala Pro Ala Ala Ser Gly Ala Arg Lys 1 5 10 15 Arg His Gly Pro Gly Pro Arg Glu Ala Arg Gly Arg Pro Gly Pro 20 30 Arg Pro Lys Thr Leu Val Leu Val Val Ala Ala Val Leu Leu Leu 35 40 45 Val Ser Ala Glu Ser Ala Leu Gin Asp Leu Ala Pro 50 55 60 Ala Ala Pro Gin Lys Arg Ser Ser Pro Ser Glu Gly 75 Cys Pro Pro Gly His His Glu Gly Arg Asp Cys Ser 85 95 Asp Tyr Ser Thr His Trp Asn Asp Leu Phe 110 Cys Leu Arg Cys Thr Arg Cys Asp Ser Gly Glu Val Glu Leu Ser Pro 115 120 125 Cys Thr Thr Thr Arg Asn Thr Val Cys Glu Gly Phe 135 Arg Glu Glu Asp Ser Pro Glu Met Cys Arg Lys Cys Arg Gly Cys 145 155 160 43 Pro Arg Gly Met Val Lys Val Gly Asp Cys Pro Trp Ser Asp Glu Cys His Lys Glu Ser Gly Thr Lys His Ser Gly Glu Ala Pro 180 185 190 Ala Val Glu Glu Thr Val Thr Ser Ser Pro Gly Thr Pro Ala Ser Pro 195 200 205 Cys Ser Leu Ser Gly Gly Val Thr Ala Ala 210 215 220 Leu Val Ala Val Phe Val Cys Lys Ser Leu Leu Trp Lys Lys Val 225 230 235 240 Leu Pro Tyr Leu Lys Gly Cys Ser Gly Gly Gly Gly Glu 255 Arg Val Asp Arg Ser Ser Gin Arg Pro Gly Ala Glu Asp Asn Val Leu 260 270 Asn Glu Val Ser Leu Pro Thr Gin Val Pro Glu Gin Glu 275 280 285 Wet Glu Val Gin Glu Pro Ala Glu Pro Thr Gly Val Asn Met Leu Ser 300 Pro Gly Glu Ser Glu His Leu Leu Glu Pro Ala Glu Glu Arg Ser 310 315 320 Arg Arg Arg Leu Leu Val Pro Ala Asn Glu Gly Asp Thr Glu 325 330 335 Thr Leu Arg Gin Cys Phe Asp Ala Asp Leu Val Pro Phe Asp 340 350 Trp Glu Pro Leu Met Arg Lys Gly Leu Met Asp Asn Glu 355 360 Lys Val Al Lys Ala Glu Ala Ala Gly His Arg Thr Leu Thr 370 375 380 Trp Val Asn Lys Gly Arg Asp Ala Ser Val His 395 400 Thr Leu Leu Asp Ala Leu Glu Thr Leu Gin 405 Lys Glu Asp His Leu Leu Ser Ser Gly Lys Phe Met Tyr Leu Glu 420 425 430 Gly Asn Ala Ala Met Ser 440 44 INFORMATION FOR SEQ ID SEQUENCE 157 base pairs nucleic acid STRANDEDNESS single linear MOLECULE TYPE cDNA NO NO FRAGMENT internal IMMEDIATE frag FEATURE CDS SEQUENCE SEQ ID CT GAG ACT CTG AGA CAG TGC TTC GAT GAC TTT GCA GAC TTG GTG CCC 47 Glu Thr Leu Arg Cys Phe Asp Asp Phe Ala Asp Leu Val Pro 1 5 10 15 TTT GAC GAG CCG CTC ATG AGG AAG TTG GGC CTC ATG GAC AAT 95 Phe Asp Ser Trp Glu Pro Leu Met Arg Lys Leu Gly Leu Met Asp Asn 25 GAG ATA AAG GTG GCT AAA GCT GAG GCA GCG GGC CAC AGG GAC ACC TTG 143 lie Lys Val Ala Ala Glu Ala Ala Gly Arg Asp Thr Leu 35 40 45 TNC ACN ATG CTG AT Xaa Thr Met 50 INFORMATION FOR SEQ ID SEQUENCE 51 amino acids amino acid linear TYPE protein SEQUENCE SEQ ID Glu Thr Arg Cys Phe Asp Asp Phe Asp Leu Val Pro Phe 1 10 15 sp Ser Trp Glu Pro Leu Met Leu Gly Asp Asn Glu 20 25 30 45 Lys Val Ala Lys Ala Glu Ala Ala Gly His Arg Asp Thr Leu Xaa 35 10 Thr Met Leu 50 INFORMATION FOR SEQ ID NO SEQUENCE CHARACTERISTICS B amino acids amino acid STRANDEDNESS single linear MOLECULE peptide NO NO IMMEDIATE FLAG peptide SEQUENCE SEQ ID Asp Tyr Lys Asp Asp Asp Asp Lys 1 5 46 insufficientOCRQuality

Claims (38)

183048/3 CLAIMS:

1. A purified TNF-related apoptosis inducing ligand receptor (TRAIL-R) polypeptide that binds TRAIL, wherein the TRAIL-R is characterized as comprising the amino acid sequence VPANEGD.

2. A TRAIL-R polypeptide of claim \, wherein said polypeptide is further characterized by a molecular weight of 50 to 55 kilodaltons.

3. A TRAIL-R polypeptide of claim 1, wherein said polypeptide is further characterized by comprising the amino acid sequence ETLRQCFDDFADLWFDSWEPLMR LGLMDNEIKVAKAEAAGHRDTLXTML.

4. A TRAIL-R polypeptide of claim 3, wherein said polypeptide is further characterized by a molecular weight of 50 to 55 kilodaltons.

5. A purified TRAIL-R polypeptide of claim 1 wherein the TRAIL-R polypeptide is the TRAIL-R polypeptide of SEQ ID NO:2.

6. A TRAIL-R polypeptide of claim 5, wherein said polypeptide comprises amino acids x to 440 of SEQ ID N0:2, wherein x represents an integer from 51 through 59.

7. A TRAIL-R polypeptide of claim 6, wherein said polypeptide comprises amino acids 54 to 440 of SEQ ID NO:2.

8. A TRAIL-R polypeptide of claim 5, wherein said fragment is a soluble TRAIL-R comprising the extracellular domain of the TRAIL-R protein of SEQ ID N0:2.

9. A purified TRAIL-R polypeptide of claim 1 comprising an amino acid sequence that is at least 80% identical to the amino acid sequence presented in SEQ ID N0:2.

10. A TRAIL-R polypeptide of claim 9, wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid 47 183048/2 sequence presented in SEQ ID NO:2.

11. A TRAIL-R polypeptide of claim 10, wherein said polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence presented in SEQ ID NO:2.

12. A TRAIL-R polypeptide of claim 9 wherein said polypeptide is naturally occurring.

13. An oligomer comprising from two to four TRAIL-R polypeptides of claim 5.

14. An oligomer comprising from two to four TRAIL-R polypeptides of claim 8.

15. A composition comprising a TRAIL-R polypeptide of claim 5, and a physiologically acceptable diluent, exdpient, or earner.

16. A composition comprising a TRAIL-R polypeptide of daim 8, and a physiologically acceptable diluent, exdpient, or carrier.

17. An isolated DNA encoding the TRAIL receptor (TRAIL-R) polypeptide according to claim 1.

18. . An isolated TRAIL-R DNA of claim 17, wherein said DNA comprises the nudeotide sequence presented in Figure 1.

19. An isolated TRAIL-R DNA of claim 17, wherein said DNA encodes a TRAIL-R polypeptide of SEQ ID N0:2.

20. A TRAIL-R polypeptide of claim 19, wherein, said DNA encodes arnino adds 1 to 440 of SEQ ID N0:2.

21. A TRAIL-R DNA of claim 19, wherein said polypeptide comprises arnino adds x to 440 of SEQ ID N0:2, wherein x represents an integer from 51 through 48 183048/3 59.

22. A TRAIL-R DNA of claim 21, wherein said polypeptide comprises amino acids 54 to 440 of SEQ ID NO:2.

23. A TRAIL-R DNA of claim 19, wherein the polypeptide comprises the extracellular domain of the TRAIL-R protein of SEQ ID NO:2.

24. An isolated TRAIL-R DNA, wherein said DNA encodes a polypeptide according to claim 9.

25. A TRAIL-R DNA of claim 24, wherein said polypeptide comprises an amino acid sequence that is at least 90% identical to the amino acid sequence presented in SEQ ID N0:2.

26. A TRATL-R DNA of claim 25, wherein said polypeptide comprises an amino acid sequence that is at least 95% identical to the amino acid sequence presented in SEQ ID NO:2.

27. A TRAIL-R DNA of claim 24, wherein said polypeptide is naturally occurring.

28. An expression vector comprising a DNA according to claim 19.

29. An expression vector comprising a DNA according to claim 20.

30. An expression vector comprising a DNA according to claim 21.

31. An expression vector comprising a DNA according to claim 22.

32. An expression vector comprising a DNA according to claim 23.

33. A host cell transformed with an expression vector of claim 28.

34. A host cell transformed with an expression vector of claim 29. 49 183048/2

35. A host cell transformed with an expression vector of claim 30.

36. A host cell transformed with an expression vector of claim 31.

37. A host cell transformed with an expression vector of claim 32.

38. An isolated TRAIL-R DNA of claim 17 comprising at nucleotides of the sequence of SEQ ID NO: 1. For the Applicant, Sanford T. Colb &c Co. O61940 50