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Definitions

• the present invention relates to a novel human gene encoding a polypeptide which is a member of the TNF receptor family, and has now been found to bind TRAIL. More specifically, an isolated nucleic acid molecule is provided encoding a human polypeptide named tumor necrosis factor receptor-5 , sometimes referred to as "TNFR-5" or “TR5,” and now referred to hereinafter as "TRAIL receptor without intracellular domain” or "TRID.” TRID polypeptides are also provided, as are vectors, host cells, and recombinant methods for producing the same. The invention further relates to screening methods for identifying agonists or antagonists of TRID polypeptide activity. Also provided are diagnostic and therapeutic methods utilizing such compositions.
• cytokines Many biological actions, for instance, response to certain stimuli and natural biological processes, are controlled by factors, such as cytokines. Many cytokines act through receptors by engaging the receptor and producing an intracellular response.
• tumor necrosis factors (TNF) alpha and beta are cytokines, which act through TNF receptors to regulate numerous biological processes, including protection against infection and induction of shock and inflammatory disease.
• the TNF molecules belong to the "TNF-ligand” superfamily, and act together with their receptors or counter-ligands, the "TNF-receptor” superfamily. So far, nine members of the TNF ligand superfamily have been identified and ten members of the TNF-receptor superfamily have been characterized.
• TNF- lymphotoxin-o.
• LT- ⁇ also known as TNF- ⁇
• LT- ⁇ found in complex heterotrimer LT- 2- ⁇ , FasL, CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).
• the superfamily of TNF receptors includes the p55TNF receptor, p75TNF receptor, TNF receptor-related protein. FAS antigen or APO-1, CD40, CD27, CD30, 4-1BB, OX40, low affinity p75 and NGF-receptor (Meager, A., Biologicals 22:291-295 (1994)).
• TNF-ligand superfamily Many members of the TNF-ligand superfamily are expressed by activated T-cells, implying that they are necessary for T-cell interactions with other cell types which underlie cell ontogeny and functions (Meager, A., supra).
• TNF and LT- ⁇ are capable of binding to two TNF receptors (the 55- and 75-kd TNF receptors).
• TNF and LT- ⁇ are involved in the pathogenesis of a wide range of diseases, including endotoxic shock, cerebral malaria, tumors, autoimmune disease, AIDS and graft-host rejection (Beutler, B. and Non Huffel,
• Apoptosis or programmed cell death, is a physiologic process essential for the normal development and homeostasis of multicellular organisms (H.
• Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C.B. Thompson, Science
• TNF receptor family Both are members of the TNF receptor family which also include TNFR-2, low affinity NGFR, CD40, and CD30, among others (CA. Smith et al. , Science 248, 1019-23 (1990); M. Tewari et al, in Modular Texts in Molecular and Cell Biology M. Purton, Heldin, Carl, Ed. (Chapman and Hall, London, 1995). While family members are defined by the presence of cysteine-rich repeats in their extracellular domains, Fas/APO-1 and TNFR-1 also share a region of intracellular homology, appropriately designated the "death domain", which is distantly related to the Drosophila suicide gene, reaper (P. Golstein, et al, Cell 81.
• TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kB (L.A. Tartaglia etal, Immunol Today 13, 151-3 (1992)).
• TNFR-1 recruits the multivalent adapter molecule TRADD, which like FADD, also contains a death domain (H. Hsu et al, Cell 81, 495-504 (1995); H. Hsu, et al, Cell 84, 299-308 (1996)).
• TRADD can signal both apoptosis and NF-kB activation (H. Hsu et al, Cell 84, 299-308 (1996); H. Hsu, et al, Immunity 4, 387-396 (1996)).
• TRAIL acts independently from the Fas ligand (Wiley et al, supra). It has also been shown that TRAIL activates apoptosis rapidly, within a time frame that is similar to death signalling by Fas/Apo-IL, but much faster than TNF-induced apoptosis.
• TRAIL may interact with a unique receptor(s).
• TNF family ligands and TNF family receptors are varied and influence numerous functions, both normal and abnormal, in the biological processes of the mammalian system. There is a clear need, therefore, for identification and characterization of such receptors and ligands that influence biological activity, both normally and in disease states. In particular, there is a need to isolate and characterize additional novel receptors that bind TRAIL. Summary of the Invention
• the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding the TRID polypeptide having the amino acid sequence shown in SEQ ID NO:2, or the amino acid sequence encoded by the cDNA clone deposited as ATCC Deposit Number 97798 on November 20, 1996.
• the nucleotide sequence determined by sequencing the deposited TRID clone, which is shown in SEQ ID NO: 1 contains an open reading frame encoding a polypeptide of about 259 amino acid residues, with a leader sequence of about 26 amino acids.
• the present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and methods for using them for production of TRID polypeptides or peptides by recombinant techniques.
• the invention further provides an isolated TRID polypeptide having an amino acid sequence encoded by a polynucleotide described herein.
• the present invention also provides diagnostic assays such as quantitative and diagnostic assays for detecting levels of TRID protein.
• diagnostic assays such as quantitative and diagnostic assays for detecting levels of TRID protein.
• TRID or soluble form thereof, may be used to detect the ability of normal tissue to withstand or be protected from the deleterious effects of TRAIL, such as TRAIL-induced apoptosis.
• Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes.
• Cellular response to TNF-family ligands include not only normal physiological responses, but also diseases associated with increased apoptosis or the inhibition of apoptosis.
• Apoptosis - programmed cell death - is a physiological mechanism involved in the deletion of peripheral T lymphocytes of the immune system, and its dysregulation can lead to a number of different pathogenic processes.
• Diseases associated with increased cell survival, or the inhibition of apoptosis include cancers, autoimmune disorders, viral infections, inflammation, graft vs. host disease, acute graft rejection, and chronic graft rejection.
• Diseases associated with increased apoptosis include AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic injury, toxin-induced liver disease, septic shock, cachexia and anorexia.
• the invention further provides a method for enhancing apoptosis induced by a TNF-family ligand, such as TRAIL, which involves administering to a cell which expresses the TRID polypeptide an effective amount of an antagonist capable of decreasing TRID's ability to bind TRAIL.
• a TNF-family ligand such as TRAIL
• TRID binding is decreased to treat a disease wherein decreased apoptosis is exhibited.
• the present invention is directed to a method for inhibiting apoptosis induced by a TNF-family ligand, such as TRAIL, which involves administering to a cell an effective amount of TRID or an agonist capable of increasing TRID activity.
• TRID activity is increased to treat a disease wherein increased apoptosis is exhibited.
• Whether any candidate "agonist” or “antagonist” of the present invention can enhance or inhibit apoptosis can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below.
• a screening method is provided for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand, such as TRAIL.
• the method involves contacting cells which co-expresses the TRID polypeptide and a second TNFR with a candidate compound and a TNF-family ligand (e.g., TRAIL), assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is a TRID antagonist and a decreased cellular response compared to the standard indicates that the candidate compound is TRID agonist.
• a cell expressing the TNFR polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand, such as TRAIL.
• Figure 1 A-B shows the nucleotide sequence (SEQ ID NO : 1 ) and deduced amino acid sequence (SEQ ID NO:2) of TRID.
• Figure 2A-P shows an alignment created by the Clustal method using the
• TNFR-like the amino acid sequences of TNFR-5 (now called “TRID,” denoted as “TNFR-like” in the figure), with other TNF receptors, as follows: TNFR1 (SEQ ID NO:3); TNFR2 (SEQ ID NO:4); NGFR (SEQ ID NO:5) LTbR (SEQ ID NO:6); FAS (SEQ ID NO:7); CD27 (SEQ ID NO:8); CD30 (SEQ ID NO:9); CD40 (SEQ ID NO.10); 4-lBB (SEQ ID NO:3); TNFR2 (SEQ ID NO:4); NGFR (SEQ ID NO:5) LTbR (SEQ ID NO:6); FAS (SEQ ID NO:7); CD27 (SEQ ID NO:8); CD30 (SEQ ID NO:9); CD40 (SEQ ID NO.10); 4-lBB (SEQ ID NO:
• Figure 3 shows an analyses of the TRID amino acid sequences.
• Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown, as predicted for the amino acid sequence depicted in SEQ ID NO:2 using the default parameters of the recited computer program.
• the "Antigenic Index - Jameson- Wolf ' graphs, indicate the location of the highly antigenic regions of the proteins, i.e., regions from which epitope-bearing peptides of the invention may be obtained.
• Figure 4 shows the nucleotide sequence of gene fragments related to the TRID gene of the present invention, including: HPRCB54R (SEQ ID NO: 15), HSJAU57RA (SEQ IDNO:16), HELBP70R (SEQ ID NO: 17), and HUSCB54R (SEQ ID NO: 18) all of which are related to SEQ ID NO: 1.
• Figure 5A is an immunoblot showing that TRID-Fc (as well as DR4 and
• FIG. 5A shows the input Fc-fusions present in the binding assays.
• Figure 5B is a bar graph showing that TRID-Fc blocked the ability of TRAIL to induce apoptosis.
• Figure 5C is a bar graph showing that TRID-Fc had no effect on TNF ⁇ -induced apoptosis under conditions where TNFRl-Fc completely abolished TNF ⁇ killing.
• Figure 6 is a bar graph showing that MCF7 cells expressing TRID were protected from TRAIL-induced apoptosis, as were cells expressing the virally encoded caspase inhibitor CrmA.
• the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide encoding a TRID polypeptide, having the amino acid sequence shown in SEQ ID NO:2. which was determined by sequencing a cloned cDNA.
• the nucleotide sequence shown in SEQ ID NO: 1 was obtained by sequencing the HPRCB54 clone, which was deposited on November 20, 1996 at the American Type Culture Collection, 10801 University Boulevard, Manassas, Virginia, 20110-2209, and given accession number ATCC
• the deposited clone is inserted in the pBluescript SK(-) plasmid (Stratagene, La Jolla, CA).
• the TRID protein of the present invention has an amino acid sequence which is 21.7% identical to and shares multiple conserved cysteine rich domains with the translation product of the human nerve growth factor (hNGF) mRNA
• TRID was also found to be homologous to the cysteine-rich domain in CAR1, a chicken TNF receptor family member with amino acid identities ranging from 42-48% (J. Brojatsh et al, Cell 87: 1 (1996)).
• a potential protective role for TRID was suggested by the finding that its transcript was detectable in many normal human tissues but not in most transformed cell lines.
• TRID has an extracellular TRAIL binding domain and a transmembrane domain but, surprisingly, lacks a putative intracellular signalling domain, in keeping with the possibility that this receptor does not signal following ligand binding. Given the absence of an intracellular domain, this receptor was termed "TRID" for TRAIL Receptor Without an Intracellular Domain.
• nucleotide sequences determined by sequencing a DNA molecule herein were determined using an automated DNA sequencer (such as the Model 373 from Applied Biosystems, Inc., Foster City, CA), and all amino acid sequences of polypeptides encoded by DNA molecules determined herein were predicted by translation of a DNA sequence determined as above. Therefore, as is known in the art for any DNA sequence determined by this automated approach, any nucleotide sequence determined herein may contain some errors. Nucleotide sequences determined by automation are typically at least about 90% identical, more typically at least about 95% to at least about 99.9% identical to the actual nucleotide sequence of the sequenced DNA molecule. The actual sequence can be more precisely determined by other approaches including manual DNA sequencing methods well known in the art.
• a single insertion or deletion in a determined nucleotide sequence compared to the actual sequence will cause a frame shift in translation of the nucleotide sequence such that the predicted amino acid sequence encoded by a determined nucleotide sequence will be completely different from the amino acid sequence actually encoded by the sequenced DNA molecule, beginning at the point of such an insertion or deletion.
• nucleotide sequence of a nucleic acid molecule or polynucleotide is intended, for a DNA molecule or polynucleotide, a sequence of deoxyribonucleotides. and for an RNA molecule or polynucleotide, the corresponding sequence of ribonucleotides (A, G. C and U), where each thymidine deoxyribonucleotide (T) in the specified deoxyribonucleotide sequence is replaced by the ribonucleotide uridine (U).
• a nucleic acid molecule of the present invention encoding a TRID polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
• standard cloning and screening procedures such as those for cloning cDNAs using mRNA as starting material.
• NO: 1 was discovered in a cDNA library derived from prostate tissue. Additional clones of the same gene were also identified in cDNA libraries from the following tissues: endothelial cells, stimulated monocytes, and kerotinocytes.
• the determined nucleotide sequence of the TRID cDNA of SEQ ID NO: 1 contains an open reading frame encoding a protein of about 259 amino acid residues, with an initiation codon at nucleotide positions 183-185 of the nucleotide sequences in SEQ ID NO: 1.
• the open reading frame of the TRID gene shares sequence homology with the translation product of the human mRNA for NGFR, including the following conserved domains : (a) a soluble extracellular domain of about 214 amino acids
• amino acid residues from about 27 to about 240 in SEQ ID NO:2 amino acid residues from about 27 to about 240 in SEQ ID NO:2
• transmembrane domain of about 19 amino acids amino acid residues from about 241 to about 259 in SEQ ID NO:2
• cysteine rich domain of about 97 amino acids (amino acid residues from about 53 to about 150 in SEQ ID NO:2).
• the actual open reading frames may be anywhere in the range of ⁇ 20 amino acids, more likely in the range of ⁇ 10 amino acids, of that predicted from the first methionine codon from the N-terminus shown in SEQ ID NO:l, which is in-frame with the translated sequences shown in each respective figure.
• the exact "address" of the extracellular, cysteine rich, and transmembrane domain(s) of the TNFR polypeptides may differ slightly from the predicted positions above.
• the exact location of the extracellular domain, cysteine-rich domain, and transmembrane domain in SEQ ID NO:2 may vary slightly (e.g.
• the address may "shift" by about 1 to about 20 residues, more likely about 1 to about 5 residues) depending on the criteria used to define the domain.
• the beginning of the transmembrane domain and the end of the extracellular domain were predicted on the basis of the identification of the hydrophobic amino acid sequence in the above indicated positions, as shown in Figure 3.
• the invention further provides polypeptides having various residues deleted from the N-terminus of the complete TRID, including polypeptides lacking one or more amino acids from the N-terminus of the extracellular domain described herein, which constitute soluble forms of the extracellular domain of the
• the amino acid sequence of the TRID protein includes a leader sequence and a mature protein, as shown in SEQ ID NO:2. More in particular, the present invention provides nucleic acid molecules encoding mature forms of the TRID protein.
• proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the complete polypeptide to produce a secreted "mature" form of the protein.
• Most mammalian cells and even insect cells cleave secreted proteins with the same specificity. However, in some cases, cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein.
• the present invention provides a nucleotide sequence encoding a mature TRID polypeptide having the amino acid sequence encoded by a cDNA clone identified as ATCC Deposit No. 97798.
• mature TRID polypeptide having the amino acid sequence encoded by a cDNA clone in ATCC Deposit No. 97798 is meant the mature form(s) of the protein produced by expression in a mammalian cell (e.g. , COS cells, as described below) of the complete open reading frame encoded by the human DNA sequence of the clone contained in the deposited plasmid.
• Methods for predicting whether a protein has a secretory leader as well as the cleavage point for that leader sequence are available. For instance, the method ofMcGeoc (Virus Res. 5:271-286 (1985)) uses the information from a short N- terminal charged region and a subsequent uncharged region of the complete (uncleaved) protein. The method of von Heinje (Nucleic Acids Res. 14:4683- 4690 (1986)) uses the information from the residues surrounding the cleavage site, typically residues -13 to +2 where +1 indicates the amino terminus of the mature protein.
• the deduced amino acid sequence of the complete TRID polypeptide was analyzed by a computer program "PSORT.” See, K. Nakai and M. Kanehisa, Genomics 14:891-91 1 (1992).
• PSORT is an expert system for predicting the cellular location of a protein based on the amino acid sequence. As part of this computational prediction of localization, the methods of McGeoch and von Heinje are incorporated.
• the analysis by the PSORT program predicted the cleavage sites between amino acids 26 and 27 in SEQ ID NO:2. Thereafter, the complete amino acid sequences were further analyzed by visual inspection, applying a simple form of the (-1,-3) rule of von Heinje. von Heinje, supra.
• the leader sequence for the TRID protein is predicted to consist of amino acid residues from about 1 to about 26, underlined in SEQ ID NO:2, while the mature TRID protein is predicted to consist of residues from about 27 to about 259 in
• the mature TRID polypeptide encoded by the deposited cDNA comprises about 233 amino acids, but may be anywhere in the range of about 223 to about 243 amino acids, and the predicted leader sequence of this protein is about 26 amino acids, but may be anywhere in the range of about 16 to about 36 amino acids.
• nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNA, including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
• RNA such as mRNA
• DNA including, for instance, cDNA and genomic DNA obtained by cloning or produced synthetically.
• DNA may be double-stranded or single-stranded.
• Single-stranded DNA or RNA may be the coding strand, also known as the sense strand, or it may be the non-coding strand, also referred to as the anti-sense strand.
• isolated nucleic acid molecule(s) is intended a nucleic acid molecule, DNA, or RNA, which has been removed from its native environment.
• recombinant DNA molecules contained in a vector are considered isolated for the purposes of the present invention.
• Further examples of isolated DNA molecules include recombinant DNA molecules maintained in heterologous host cells or purified (partially or substantially) DNA molecules in solution.
• Isolated RNA molecules include in vivo or in vitro RNA transcripts of the DNA molecules of the present invention. Isolated nucleic acid molecules according to the present invention further include such molecules produced synthetically.
• a nucleic acid molecule contained in a clone that is a member of a mixed clone library e.g., a genomic or cDNA library
• a mixed clone library e.g., a genomic or cDNA library
• a chromosome isolated or removed from a cell or a cell lysate e.g. , a "chromosome spread", as in a karyotype
• Isolated nucleic acid molecules of the present invention include DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in SEQ ID NO: 1 ; DNA molecules comprising, or alternatively consisting of, the coding sequence for the mature TRID protein; and DNA molecules which comprise, or alternatively consist of, a sequence substantially different from those described above, but which, due to the degeneracy of the genetic code, still encode the TRID protein.
• ORF open reading frame
• the invention provides nucleic acid molecules having nucleotide sequences related to extensive portions of SEQ ID NO: 1 , which have been determined from the following related cDNA clones: HELBP70R (SEQ ID NO: 1 , which have been determined from the following related cDNA clones: HELBP70R (SEQ ID NO: 1 ), which have been determined from the following related cDNA clones: HELBP70R (SEQ ID NO: 1
• the invention provides isolated nucleic acid molecules encoding the TRID polypeptide having an amino acid sequence as encoded by the cDNA clone contained in the plasmid deposited as ATCC Deposit No. 97798.
• nucleic acid molecules are provided that encode the mature TRID polypeptide or the full length TRID polypeptide each lacking the N-terminal methionine.
• the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ID NO:l or the nucleotide sequence of the TRID cDNA contained in the above-described deposited clone, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
• Such isolated molecules, particularly DNA molecules are useful as probes for gene mapping, by in situ hybridization with chromosomes, and for detecting expression of the TRID gene in human tissue, for instance, by Northern blot analysis.
• the present invention is further directed to fragments of the isolated nucleic acid molecules described herein.
• a fragment of an isolated nucleic acid molecule having the nucleotide sequence of the deposited cDNA (the cDNA contained in the plasmid deposited as ATCC Deposit No.97798) or the nucleotide sequence shown in SEQ ID NO: 1 are intended DNA fragments at least 20 nt, and more preferably at least 30 nt in length, and even more preferably, at least about 40, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1 100, 1150, 1200, 1250, 1300, 1350, 1400, 1450, or 1500 nt in length, which are useful as DNA probes as discussed herein.
• DNA fragments corresponding to most, if not all, of the nucleotide sequence shown in SEQ ID NO: 1 are also useful as DNA probes.
• a fragment at least 20 nt in length for example, is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO: l .
• “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini.
• TRID polynucleotide fragments of the invention include, for example, fragments that comprise, or alternatively, consist of, a sequence from about nucleotide 1 to 50, 51 to 100, 101 to 150, 151 to 182, 183 to 260, 261 to 300, 301 to 350, 351 to 400, 401 to 450, 451 to 500, 501 to
• the polynucleotide fragments of the invention encode a polypeptide which demonstrates a TRID functional activity.
• a polypeptide demonstrating a TRID “functional activity” is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) TRID protein.
• Such functional activities include, but are not limited to, biological activity (e.g., binding TRAIL), antigenicity [ability to bind (or compete with a TRID polypeptide for binding) to an anti-TRID antibody], immunogenicity (ability to generate antibody which binds to a TRID polypeptide), ability to form multimers with TRID polypeptides of the invention, and ability to bind to a receptor or ligand for a TRID polypeptide (e.g., TRAIL).
• biological activity e.g., binding TRAIL
• antigenicity ability to bind (or compete with a TRID polypeptide for binding) to an anti-TRID antibody
• immunogenicity ability to generate antibody which binds to a TRID polypeptide
• ability to form multimers with TRID polypeptides of the invention ability to form multimers with TRID polypeptides of the invention
• a receptor or ligand for a TRID polypeptide e.g., TRAIL
• various immunoassays known in the art can be used, including but not limited to, competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric assays, gel diffusion precipitation reactions, immunodiffusion assays, in situ immunoassays (using colloidal gold, enzyme or radioisotope labels, for example), western blots, precipitation reactions, agglutination assays (e.g., gel agglutination assays, hemagglutination assays), complement fixation assays, immunofluorescence assays, protein A assays, and immunoelectrophoresis assays, etc.
• competitive and non-competitive assay systems using techniques such as radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoradiometric
• antibody binding is detected by detecting a label on the primary antibody .
• the primary antibody is detected by detecting binding of a secondary antibody or reagent to the primary antibody.
• the secondary antibody is labeled.
• binding can be assayed, e.g. , by means well-known in the art, such as, for example, reducing and non-reducing gel chromatography, protein affinity chromatography, and affinity blotting. See generally, Phizicky, E., et al, Microbiol Rev. 59:94-123 (1995).
• physiological correlates of TRID binding to its substrates can be assayed.
• TRID polypeptides and fragments, variants derivatives and analogs thereof to elicit TRID related biological activity (e.g. , to block TRAIL induced apoptosis in vitro or in vivo).
• Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding: epitope-bearing portions of the TRID polypeptide as identified in Figure 3 and described in more detail below.
• the invention provides polynucleotides having a nucleotide sequence representing the portion of SEQ ID NO: l, which consist of positions 183-959 of SEQ ID NO:l .
• polynucleotides encoding TRID polypeptides which lack an amino terminal methionine.
• One such preferred polynucleotide of the invention encodes a full-length TRID polypeptide lacking the nucleotides encoding the amino-terminal methionine (e.g.
• nucleotides 186-959 in SEQ ID NO: 1 as it is known that the methionine is cleaved naturally and such sequences maybe useful in genetically engineering TRID expression vectors.
• Polypeptides encoded by such polynucleotides are also, provided, such as polypeptides comprising, or alternatively consisting of, an amino acid sequence at positions 2-259 of SEQ ID NO:2, or the polypeptide sequence encoded by the clone deposited with the ATCC as Deposit No. 97798 lacking an amino terminal methionine.
• Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding a member selected from the group: a polypeptide comprising, or alternatively consisting of, the TRID extracellular domain (amino acid residues from about 27 to about 240 in SEQ ID NO:2); a polypeptide comprising or alternatively consisting of, the TRID cysteine rich domain (amino acid residues from about 53 to about 150 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, the TRID transmembrane domain
• polypeptide comprising, or alternatively consisting of, one, two, three, four or more, epitope bearing portions of the TRID receptor protein.
• polynucleotide fragments of the invention encode a polypeptide comprising, or alternatively consisting of, any combination of 1, 2, 3, 4, or all 5 of the above-encoded polypeptide embodiments. Since the location of these domains have been predicted by computer graphics, one of ordinary skill would appreciate that the amino acid residues constituting these domains may vary slightly (e.g., by about 1 to 15 residues) depending on the criteria used to define each domain.
• polypeptides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of amino acid residues 53 to 110, and/or 11 1 to 150 of SEQ ID NO:2, as disclosed in Figures 1A-B.
• polynucleotides encoding TRID polypeptides of the invention comprise, or alternatively consist of both of the extracellular cysteine rich motifs disclosed in Figures 1A-B. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the polynucleotides of the invention encode functional attributes of TRID.
• Preferred embodiments of the invention in this regard include fragments that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet forming regions ("beta-regions”), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions
• foil-regions hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of TRID.
• the data presented in columns VIII, IX, XIII, and XIV of Table I can be used to determine regions of TRID which exhibit a high degree of potential for antigenicity . Regions of high antigenicity are determined from the data presented in columns VIII, IX, XIII, and/or XIV by choosing values which represent regions of the polypeptide which are likely to be exposed on the surface of the polypeptide in an environment in which antigen recognition may occur in the process of initiation of an immune response.
• Certain preferred regions in these regards are set out in Figure 3, but may, as shown in Table I, be represented or identified by using tabular representations of the data presented in Figure 3.
• the DNA* STAR computer algorithm used to generate Figure 3 was used to present the data in Figure 3 in a tabular format (See Table I).
• the tabular format of the data in Figure 3 may be used to easily determine specific boundaries of a preferred region.
• the above-mentioned preferred regions set out in FIG. 3 and in Table I include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence set out in SEQ ID NO:2. As set out in FIG.
• such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (columns I, III, V, and VII in Table I), Chou-Fasman alpha-regions, beta-regions, and turn-regions (columns II, IV, and VI in Table I), Kyte-Doolittle hydrophilic regions (column VIII in Table I), Hopp- Woods hydrophobic regions (column IX in Table I), Eisenberg alpha- and beta-amphipathic regions (columns X and XI in Table I), Karplus-Schulz flexible regions (column XII in Table I), Jameson- Wolf regions of high antigenic index (column XIII in Table I), and Emini surface-forming regions (column XIV in
• polypeptides comprising, or alternatively consisting of, regions of TRID that combine several structural features, such as several (e.g.. 1, 2, 3, or 4) of the same or different region features set out above.
• Vdl 34 A C 2 23 -0 73 * F 1 10 2 37
• nucleic acid fragments of the present invention further include nucleic acid molecules encoding a polypeptide comprising, or alternatively consisting of, one, two, three, four, five, or more epitope-beanng portions of the TRID protein
• a polypeptide comprising, or alternatively consisting of, amino acid residues from aboutH ⁇ s-58 to about Cys-66 in SEQ ID NO 2
• polynucleotides of the invention comprise, or alternatively consist of, at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TRID coding sequence, but consist of less than or equal to 1000 kb, 500 kb, 250 kb, 200 kb, 150 kb, 100 kb, 75 kb, 50 kb, 30 kb, 25 kb, 20 kb, 15 kb, 10 kb, or 5 kb of genomic DNA that flanks the 5' or 3' coding nucleotide set forth in Figures 1 A-D
• polynucleotides of the invention comprise, or alternatively consist of, at least 15, at least 30, at least 50, at least 100, or at least 250, at least 500, or at least 1000 contiguous nucleotides of TRID coding sequence, but do not comprise all or a portion of any TRID intron.
• nucleic acid comprising, or alternatively consisting of,
• TRID coding sequence does not contain coding sequences of a genomic flanking gene (i.e., 5' or 3' to the TRID gene in the genome).
• the polynucleotides of the invention do not contain the coding sequence of more than 1000, 500, 250, 100, 50, 25, 20, 15, 10, 5, 4, 3, 2, or 1 genomic flanking gene(s).
• the invention includes a polynucleotide comprising, or alternatively consisting of, any portion of at least about 30 nucleotides, preferably at least about 50 nucleotides, of SEQ ID NO: 1 from residue 183 to 959.
• the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under stringent hybridization conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, a cDNA clone contained in ATCC Deposit No. 97798.
• stringent hybridization conditions is intended overnight incubation at 42° C in a solution comprising, or alternatively consisting of,: 50% formamide, 5x SSC (750 mM NaCl, 75 mMtrisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in O.lx SSC at about 65° C.
• Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention.
• the invention provides an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which hybridizes under lower stringency conditions to a portion of the polynucleotide in a nucleic acid molecule of the invention described above, for instance, a cDNA clone contained in ATCC Deposit No. 97798.
• lower stringency conditions is intended overnight incubation at 35° C or 42° C in a solution comprising, or alternatively consisting of: 50% formamide, 5x SSC (750 mM NaCl, 75 mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10% dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNA, followed by washing the filters in 3x, 2x, lx, or 0.5x SSC at about 35° C, 45° C, 55° C, or 65° C. Polypeptides encoded by these nucleic acid molecules are also encompassed by the invention.
• a polynucleotide which hybridizes to a "portion" of a polynucleotide is intended a polynucleotide (either DNA or RNA) hybridizing to at least about 15 nucleotides (nt), and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably about 30-70 (e.g., 50) nt of the reference polynucleotide.
• “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2, or 1) nucleotides, at either terminus or at both termini. These have uses, which include, but are not limited to, as diagnostic probes and primers as discussed above and in more detail below.
• a portion of a polynucleotide of "at least 20 nt in length ' for example, is intended 20 or more contiguous nucleotides from the nucleotide sequence of the reference polynucleotide (e.g., a deposited cDNA or the nucleotide sequence as shown in SEQ ID NO:l).
• a polynucleotide which hybridizes only to a poly A sequence such as the 3' terminal poly(A) tract of the TRID cDNA shown in SEQ ID NO:l), or to a complementary stretch of T (or U) residues, would not be included in a polynucleotide of the invention used to hybridize to a portion of a nucleic acid of the invention, since such a polynucleotide would hybridize to any nucleic acid molecule containing a poly (A) stretch or the complement thereof (e.g., practically any double-stranded cDNA clone).
• nucleic acid molecules of the present invention which encode a TRID polypeptide may include, but are not limited to the coding sequence for the mature polypeptide, by itself; the coding sequence for the mature polypeptide and additional sequences, such as those encoding a leader or secretary sequence, such as a pre-, or pro- or prepro- protein sequence; the coding sequence of the mature polypeptide, with or without the aforementioned additional coding sequences, together with additional, non-coding sequences, including for example, but not limited to introns and non-coding 5' and 3' sequences, such as the transcribed, non-translated sequences that play a role in transcription, mRNA processing - including splicing and polyadenylation signals, for example - ribosome binding and stability of mRNA; additional coding sequence which codes for additional amino acids, such as those which provide additional functionalities.
• the polypeptide may be fused to a marker sequence, such as a peptide, which facilitates purification of the fused polypeptide.
• the marker sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (Qiagen, Inc.), among others, many of which are commercially available.
• hexa- histidine provides for convenient purification of the fusion protein.
• the "HA” tag is another peptide useful for purification which corresponds to an epitope derived from the influenza hemagglutinin protein, which has been described by Wilson et al, Cell 57:767-778(1984).
• other such fusion proteins include the TRID receptor fused to Fc at the N- or C- terminus.
• the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode portions, analogs, or derivatives of the TRID receptor.
• Variants may occur naturally, such as a natural allelic variant.
• allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Genes II, Lewin, B., ed., John Wiley & Sons, New York (1985).
• Non-naturally occurring variants may be produced using art-known mutagenesis techniques.
• Such variants include those produced by nucleotide substitutions, deletions or additions. The substitutions, deletions or additions may involve one or more nucleotides.
• the variants may be altered in coding regions, non-coding regions, or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions or additions. Especially preferred among these are silent substitutions, additions and deletions, which do not alter the properties and activities of the TRID polypeptide or portions thereof. Also especially preferred in this regard are conservative substitutions.
• nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%,
• TRID polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97798; (g) a nucleotide sequence encoding the TRID extracellular domain having the amino acid sequence at positions from about 27 to about 240 in SEQ ID NO:2; (h) a nucleotide sequence that encoding the TRID extracellular domain having the amino acid sequence encoded by the cDNA contained in ATCC Deposit No.
• nucleotide sequence that encodes a fragment of the polypeptide of (e) or (f) having TRID functional activity (e.g., antigenic or biological activity); and (n) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (1) or (m) above.
• “about” includes the particularly recited size, larger or smaller by several (5, 4, 3, 2 or 1) nucleotides, at either terminus or at both termini.
• inventions include isolated nucleic acid molecules that comprise, or alternatively consist of, a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j), (k), (1) or (m) above.
• This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues.
• An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising, or alternatively consisting of, a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a TRID polypeptide having an amino acid sequence in (a), (b), (c), (d), (e), (f), (g), (h), (i), 0), (k), (1) or (m) above.
• a polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a TRID polypeptide is intended that the nucleotide sequence of the polynucleotide is identical to the reference sequence except that the polynucleotide sequence may include up to five mismatches per each 100 nucleotides of the reference nucleotide sequence encoding the TRID polypeptide.
• a polynucleotide having a nucleotide sequence at least 95% identical to a reference nucleotide sequence up to 5% of the nucleotides in the reference sequence may be deleted or substituted with another nucleotide, or a number of nucleotides up to 5% of the total nucleotides in the reference sequence may be inserted into the reference sequence.
• These mutations of the reference sequence may occur at the 5' or 3' terminal positions of the reference nucleotide sequence or anywhere between those terminal positions, interspersed either individually among nucleotides in the reference sequence or in one or more contiguous groups within the reference sequence.
• the reference (query) sequence may be the entire TRID encoding nucleotide sequence shown in SEQ ID NO: l or any TRID polynucleotide fragment (e.g. , a polynucleotide encoding the amino acid sequence of any of the TRID N- and/or C- terminal deletions described herein), variant, derivative or analog, as described herein.
• nucleic acid molecule is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the nucleotide sequence shown in SEQ ID NO: 1 , or to the nucleotide sequence of the deposited cDNA clone can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for
• Bestfit uses the local homology algorithm of Smith and Waterman, Advances in Applied Mathematics 2:482-489 (1981), to find the best segment of homology between two sequences.
• Bestfit or any other sequence alignment program to determine whether a particular sequence is, for instance, 95% identical to areference sequence according to the present invention, the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference nucleotide sequence and that gaps in homology of up to 5% of the total number of nucleotides in the reference sequence are allowed.
• the identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:231-245 (1990)).
• the percent identity is corrected by calculating the number of bases of the query sequence that are 5' and 3' of the subject sequence, which are not matched/aligned, as a percent of the total bases of the query sequence. A determination of whether a nucleotide is matched/aligned is determined by results of the FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This corrected score is what is used for the purposes of this embodiment.
• nucleic acid sequence shown in SEQ ID NO : 1 or to the nucleic acid sequence of the deposited cDNA, irrespective of whether they encode a polypeptide having TRID activity. This is because even where a particular nucleic acid molecule does not encode a polypeptide having TRID activity, one of skill in the art would still know how to use the nucleic acid molecule, for instance, as a hybridization probe or a polymerase chain reaction (PCR) primer.
• PCR polymerase chain reaction
• nucleic acid molecules of the present invention that do not encode a polypeptide having TRID activity include, inter alia: (1 ) isolating a TRID gene or allelic variants thereof in a cDNA library; (2) in situ hybridization (e.g., "FISH") to metaphase chromosomal spreads to provide precise chromosomal location of the TRID gene, as described in Verma et al., Human Chromosomes: A Manual of Basic Techniques, Pergamon Press, New York (1988); and Northern Blot analysis for detecting TRID mRNA expression in specific tissues.
• a polypeptide having TRID receptor activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the TRID receptor of the invention (either the full length protein or preferably the mature protein or extracellular domain alone), as measured in aparticular biological assay.
• the TNF family ligands include TRAIL
• a cellular response to a TNF-family ligand is intended any genotypic, phenotypic, and/or morphological change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand.
• cellular responses include not only normal physiological responses to TNF-family ligands, but also diseases associated with increased cell proliferation or the inhibition of increased cell proliferation, such as by the inhibition of apoptosis.
• nucleic acid molecules having a sequence at least 90%, 95%, 96%, 97%, 98%, or 99% identical to the nucleic acid sequence of a deposited cDNA or the nucleic acid sequence shown in SEQ ID NO: 1 will encode a polypeptide "having TRID protein activity.”
• degenerate variants of these nucleotide sequences all encode the same polypeptide, this will be clear to the skilled artisan even without performing the above described comparison assay. It will be further recognized in the art that, for such nucleic acid molecules that are not degenerate variants, a reasonable number will also encode a polypeptide having TRID protein activity.
• This invention is also related to the use of the TRID polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of TRID associated with a dysfunction will provide a diagnostic tool that can add or define a diagnosis of a disease or susceptibility to a disease which results from under-expression over-expression or altered expression of TRID or a soluble form thereof, such as, for example, tumors or autoimmune disease. Individuals carrying mutations in the TRID gene may be detected at the
• Nucleic acids for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva, tissue biopsy and autopsy material.
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR prior to analysis. (Saiki et al, Nature J2J: 163-166 (1986)).
• RNA or cDNA may also be used in the same ways.
• PCR primers complementary to the nucleic acid encoding TRID can be used to identify and analyze TRID expression and mutations. For example, deletions and insertions can be detected by a change in size of the amplified product in comparison to the normal genotype.
• Point mutations can be identified by hybridizing amplified DNA to radiolabeled TRID RNA or alternatively, radiolabeled TRID antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
• DNA segments may be employed as probes to detect specific DNA segments.
• the sensitivity of such methods can be greatly enhanced by appropriate use of PCR or another amplification method.
• a sequencing primer is used with double-stranded PCR product or a single-stranded template molecule generated by a modified PCR.
• the sequence determination is performed by conventional procedures with radiolabeled nucleotide or by automatic sequencing procedures with fluorescent-tags.
• DNA sequence differences may be achieved by detection of alteration in electrophoretic mobility of DNA fragments in gels, with or without denaturing agents. Small sequence deletions and insertions can be visualized by high resolution gel electrophoresis. DNA fragments of different sequences may be distinguished on denaturing formamide gradient gels in which the mobilities of different DNA fragments are retarded in the gel at different positions according to their specific melting or partial melting temperatures (see. e.g., Myers et al, Science 230: 1242 (1985)).
• Sequence changes at specific locations also may be revealed by nuclease protection assays, such as RNase and SI protection or the chemical cleavage method (e.g., Cotton et al, Proc. Natl. Acad. Sci. USA 85: 4397-4401 (1985)).
• the detection of a specific DNA sequence may be achieved by methods such as hybridization, RNase protection, chemical cleavage, direct DNA sequencing or the use of restriction enzymes, (e.g., restriction fragment length polymorphisms ("RFLP”) and Southern blotting of genomic DNA.
• restriction enzymes e.g., restriction fragment length polymorphisms ("RFLP") and Southern blotting of genomic DNA.
• mutations also can be detected by in situ analysis.
• the present invention also relates to vectors which include the isolated DNA molecules of the present invention, host cells which are genetically engineered with the recombinant vectors of the invention and the production of
• TRID polypeptides or fragments thereof by recombinant techniques.
• Host cells can be genetically engineered to incorporate nucleic acid molecules and express polypeptides of the present invention.
• the polynucleotides may be introduced alone or with other polynucleotides. Such other polynucleotides may be introduced independently, co-introduced or introduced joined to the polynucleotides of the invention.
• the vector may be, for example, a plasmid vector, a single or double-stranded phage vector, a single or double-stranded RNA or DNA viral vector.
• Such vectors may be introduced into cells as polynucleotides, preferably DNA, by well known techniques for introducing DNA and RNA into cells.
• Viral vectors may be replication competent or replication defective. In the latter case viral propagation generally will occur only in complementing host cells.
• vectors are those for expression of polynucleotides and polypeptides of the present invention.
• such vectors comprise c/-s"-acting control regions effective for expression in a host operatively linked to the polynucleotide to be expressed.
• Appropriate trans-acting factors either are supplied by the host, supplied by a complementing vector or supplied by the vector itself upon introduction into the host.
• vectors can be used to express a polypeptide of the invention.
• Such vectors include chromosomal, episomal and virus-derived vectors e.g., vectors derived from bacterial plasmids, from bacteriophage, from yeast episomes, from yeast chromosomal elements, from viruses such as baculoviruses, papova viruses, such as S V40, vaccinia viruses, adenoviruses, fowl pox viruses, pseudorabies viruses and retroviruses, and vectors derived from combinations thereof, such as those derived from plasmid and bacteriophage genetic elements, such as cosmids and phagemids, all may be used for expression in accordance with this aspect of the present invention.
• any vector suitable to maintain, propagate or express polynucleotides to express a polypeptide in a host may be used for expression in this regard.
• the DNA sequence in the expression vector is operatively linked to appropriate expression control sequence(s)), including, for instance, a promoter to direct mRNA transcription.
• appropriate expression control sequence(s) include the phage lambda PL promoter, the E. coli lac, trp and tac promoters, the SV40 early and late promoters and promoters of retroviral LTRs, to name just a few of the well-known promoters.
• expression constructs will contain sites for transcription, initiation and termination, and, in the transcribed region, a ribosome binding site for translation.
• the coding portion of the mature transcripts expressed by the constructs will include a translation initiating AUG at the beginning and a termination codon (U AA, UGA or U AG) appropriately positioned at the end of the polypeptide to be translated.
• the constructs may contain control regions that regulate as well as engender expression. Generally, such regions will operate by controlling transcription, such as repressor binding sites and enhancers, among others.
• Vectors for propagation and expression generally will include selectable markers. Such markers also may be suitable for amplification or the vectors may contain additional markers for this purpose.
• the expression vectors preferably contain one or more selectable marker genes to provide a phenotypic trait for selection of transformed host cells. Preferred markers include dihydrofolate reductase or neomycin resistance for eukaryotic cell culture, and tetracycline or ampicillin resistance genes for culturing E. coli and other bacteria.
• the vector containing the appropriate DNA sequence as described elsewhere herein, as well as an appropriate promoter, and other appropriate control sequences, may be introduced into an appropriate host using a variety of well known techniques suitable to expression therein of a desired polypeptide.
• hosts include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
• bacterial cells such as E. coli, Streptomyces and Salmonella typhimurium cells
• fungal cells such as yeast cells
• insect cells such as Drosophila S2 and Spodoptera Sf9 cells
• animal cells such as CHO, COS and Bowes melanoma cells
• plant cells include bacterial cells, such as E. coli, Streptomyces and Salmonella typhimurium cells; fungal cells, such as yeast cells; insect cells such as Drosophila S2 and Spodoptera Sf9 cells; animal cells such as CHO, COS and Bowes melanoma cells; and plant cells.
• vectors preferred for use in bacteria are pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNHl ⁇ a, pNH18A, pNH46A, available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
• preferred eukaryotic vectors are pWLNEO, pSV2CAT, pOG44, pXTl and pSG available from Stratagene; and pSVK3, pBPV, pMSG and pSVL available from Pharmacia. These vectors are listed solely by way of illustration of the many commercially available and well known vectors available to those of skill in the art.
• the present invention also relates to host cells containing the above- described vector constructs described herein, and additionally encompasses host cells containing nucleotide sequences of the invention that are operably associated with one or more heterologous control regions (e.g. , promoter and/or enhancer) using techniques known of in the art.
• the host cell can be a higher eukaryotic cell, such as a mammalian cell (e.g. , a human derived cell), or a lower eukaryotic cell, such as a yeast cell, or the host cell can be a prokaryotic cell, such as a bacterial cell.
• the host strain may be chosen which modulates the expression of the inserted gene sequences, or modifies and processes the gene product in the specific fashion desired.
• Expression from certain promoters can be elevated in the presence of certain inducers; thus expression of the genetically engineered polypeptide may be controlled.
• different host cells have characteristics and specific mechanisms for the translational and post-translational processing and modification (e.g.. phosphorylation, cleavage) of proteins. Appropriate cell lines can be chosen to ensure the desired modifications and processing of the foreign protein expressed.
• Introduction of the construct into the host cell can be effected by calcium phosphate transfection, DEAE-dextran mediated transfection, cationic lipid-mediated transfection, electroporation, transduction, infection or other methods. Such methods are described in many standard laboratory manuals, such as Davis et al, Basic Methods In Molecular Biology (1986).
• the invention also encompasses primary, secondary, and immortalized host cells of vertebrate origin, particularly mammalian origin, that have been engineered to delete or replace endogenous genetic material (e.g., TRID coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with TRID polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous TRID polynucleotides.
• genetic material e.g., heterologous polynucleotide sequences
• TRID receptor polynucleotides and polypeptides may be used in accordance with the present invention for a variety of applications, particularly those that make use of the chemical and biological properties of TRID.
• applications in treatment of tumors, resistance to parasites, bacteria and viruses, to induce proliferation of T-cells, endothelial cells and certain hematopoietic cells, to treat restenosis, graft vs. host disease, to regulate anti-viral responses and to prevent certain autoimmune diseases after stimulation of TRID by an agonist or by a TRAIL binding facilitator.
• Additional applications relate to diagnosis and to treatment of disorders of cells, tissues and organisms. These aspects of the invention are discussed further below.
• the proteins of the invention can also be expressed in transgenic animals.
• mice Animals of any species, including, but not limited to, mice, rats, rabbits, hamsters, guinea pigs, pigs, micro-pigs, goats, sheep, cows and non-human primates, e.g., baboons, monkeys, and chimpanzees may be used to generate transgenic animals.
• transgene i.e., nucleic acids of the invention
• transgene i.e., nucleic acids of the invention
• Such techniques include, but are not limited to, pronuclear microinjection (Paterson etal, Appl. Microbiol Biotechnol 40:691-698 (1994); Carver et al, Biotechnology (NY) 77:1263-1270 (1993); Wright et al, Biotechnology (NY) 9:830-834 (1991); and Hoppe et al., US Patent Number 4,873,191 (1989)); retrovirus mediated gene transfer into germ lines (Van der Putten et al, Proc. Natl Acad. Sci., USA 52:6148-6152 (1985)).
• blastocysts or embryos or embryos; gene targeting in embryonic stem cells (Thompson et al, Cell 56:313- 321 ( 1989)); electroporation of cells or embryos (Lo, Mol Cell. Biol. 3:1803-1814 (1983)); introduction of the polynucleotides of the invention using a gene gun (see, e.g., Ulmer et al, Science 259:1145 (1993); introducing nucleic acid constructs into embryonic pleuripotent stem cells and transferring the stem cells back into the blastocyst; and sperm-mediated gene transfer (Lavitrano et al, Cell 57:1X1-123 (1989); etc.
• transgenic clones containing polynucleotides of the invention for example, nuclear transfer into enucleated oocytes of nuclei from cultured embryonic, fetal, or adult cells induced to quiescence (Campell et al, Nature 380:64-66 (1996); Wilmut et al, Nature
• the present invention provides for transgenic animals that carry the transgene in all their cells, as well as animals which carry the transgene in some, but not all their cells, i. e. , mosaic animals or chimeric animals.
• the transgene may be integrated as a single transgene or as multiple copies such as in concatamers, e.g, head-to-head tandems or head-to-tail tandems.
• the transgene may also be selectively introduced into and activated in a particular cell type by following, for example, the teaching of Lasko et al. (Proc. Natl. Acad. Sci. USA ⁇ 9:6232-6236 ( 1992)).
• the regulatory sequences required for such a cell-type specific activation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art.
• gene targeting is preferred.
• vectors containing some nucleotide sequences homologous to the endogenous gene are designed for the purpose of integrating, via homologous recombination with chromosomal sequences, into and disrupting the function of the nucleotide sequence of the endogenous gene.
• the transgene may also be selectively introduced into a particular cell type, thus inactivating the endogenous gene in only that cell type, by following, for example, the teaching of Gu et al. (Science 265: 103-106 ( 1994)).
• the regulatory sequences required for such a cell-type specific inactivation will depend upon the particular cell type of interest, and will be apparent to those of skill in the art. The contents of each of the documents recited in this paragraph is herein incorporated by reference in its entirety.
• the expression of the recombinant gene may be assayed utilizing standard techniques. Initial screening may be accomplished by Southern blot analysis or PCR techniques to analyze animal tissues to verify that integration of the transgene has taken place. The level of mRNA expression of the transgene in the tissues of the transgenic animals may also be assessed using techniques which include, but are not limited to, Northern blot analysis of tissue samples obtained from the animal, in situ hybridization analysis, and reverse transcriptase-PCR (rt-PCR). Samples of transgenic gene- expressing tissue may also be evaluated immunocytochemically or immunohistochemically using antibodies specific for the transgene product.
• founder animals may be bred, inbred, outbred, or crossbred to produce colonies of the particular animal.
• breeding strategies include, but are not limited to: outbreeding of founder animals with more than one integration site in order to establish separate lines; inbreeding of separate lines in order to produce compound transgenics that express the transgene at higher levels because of the effects of additive expression of each transgene; crossing of heterozygous transgenic animals to produce animals homozygous for a given integration site in order to both augment expression and eliminate the need for screening of animals by DNA analysis; crossing of separate homozygous lines to produce compound heterozygous or homozygous lines; and breeding to place the transgene on a distinct background that is appropriate for an experimental model of interest.
• Transgenic and "knock-out" animals of the invention have uses which include, but are not limited to, animal model systems useful in elaborating the biological function of TRID polypeptides, studying conditions and/or disorders associated with aberrant TRID expression, and in screening for compounds effective in ameliorating such conditions and/or disorders.
• cells that are genetically engineered to express the proteins of the invention, or alternatively, that are genetically engineered not to express the proteins of the invention are administered to a patient in vivo. Such cells may be obtained from the patient (i.e..
• an MHC compatible donor can include, but are not limited to fibroblasts, bone marrow cells, blood cells (e.g., lymphocytes), adipocytes, muscle cells, endothelial cells, etc.
• the cells are genetically engineered in vitro using recombinant DNA techniques to introduce the coding sequence of polypeptides of the invention into the cells, or alternatively, to disrupt the coding sequence and/or endogenous regulatory sequence associated with the polypeptides of the invention, e.g. , by transduction
• the coding sequence of the polypeptides of the invention can be placed under the control of a strong constitutive or inducible promoter or promoter/enhancer to achieve expression, and preferably secretion, of the polypeptides of the invention.
• the engineered cells which express and preferably secrete the polypeptides of the invention can be introduced into the patient systemically, e.g., in the circulation, or intraperitoneally.
• the cells can be incorporated into a matrix and implanted in the body, e.g. , genetically engineered fibroblasts can be implanted as part of a skin graft; genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft.
• genetically engineered fibroblasts can be implanted as part of a skin graft
• genetically engineered endothelial cells can be implanted as part of a lymphatic or vascular graft.
• the cells to be administered are non-autologous or non-MHC compatible cells, they can be administered using well known techniques which prevent the development of a host immune response against the introduced cells.
• the cells may be introduced in an encapsulated form which, while allowing for an exchange of components with the immediate extracellular environment, does not allow the introduced cells to be recognized by the host immune system.
• the polypeptides of the present invention are preferably provided in an isolated form.
• a recombinantly produced version of the TRID polypeptide can be substantially purified by the one-step method described in Smith and Johnson, Gene 67:31-40 (1988).
• the invention further provides an isolated TRID polypeptide having the amino acid sequences encoded by the deposited cDNA, or the amino acid sequences in SEQ ID NO:2. or a peptide or polypeptide comprising, or alternatively consisting of, a portion of the above polypeptides.
• Polypeptide fragments of the present invention include polypeptides comprising or alternatively, consisting of, an amino acid sequence contained in SEQ ID NO:2, encoded by the cDNA contained in the deposited clone, or encoded by nucleic acids which hybridize (e.g., under stringent hybridization conditions) to the nucleotide sequence contained in the deposited clone, or shown in SEQ ID NO: 1 or the complementary strand thereto. Protein fragments may be "free-standing,” or comprised within a larger polypeptide of which the fragment forms a part or region, most preferably as a single continuous region.
• Representative examples of polypeptide fragments of the invention include, for example, fragments that comprise or alternatively, consist of from about amino acid residues: 1 to 26, 27 to 50, 51 to 100, 151 to 200, 201 to 240, and/or 241 to
• polypeptide fragments of the invention include, for example, fragments that comprise, or alternatively consisting of, from about amino acid residues: 1-60, 1 1-70, 21 -80, 31-90, 41-100, 51-1 10, 61 -120, 71-130, 81-140, 91-150, 101-160, 1 1 1-170, 121 -180, 131-190, 141-200, 151-210. 161-220, 171-230, 181-240, 191-250, and/or 201-249 of SEQ ID NO:2, as well as isolated polynucleotides which encode these polypeptides.
• polypeptide fragments can be at least 10, 20, 30, 40. 50, 60, 70, 80, 90, 100, 1 10, 120, 130, 140, 150, 175 or 200 amino acids in length. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• polypeptide fragments of the invention comprise, or alternatively consist of, amino acid residues: 1-259, 27-259, 27-240, 53-150, and/or 241-259, of TRID as depicted in SEQ ID NO:2.
• Polynucleotides encoding these polypeptides are also encompassed by the invention.
• polypeptide fragments of the invention comprise, or alternatively consist, of one or more TRID domains.
• Preferred polypeptide fragments of the present invention include a member selected from the group: (a) a polypeptide comprising or alternatively, consisting of, the TRID transmembrane domain (predicted to constitute amino acid residues from about 241 to about 259 of SEQ ID NO:2); (b) a polypeptide comprising or alternatively, consisting of, the TRID receptor extracellular domain (predicted to constitute amino acid residues from about 27 to about 240 of SEQ ID NO:2); (c) a polypeptide comprising or alternatively, consisting of, the TRID cysteine rich domain (predicted to constitute amino acid residues from about 53 to about 150 of SEQ ID NO:2); (d) a polypeptide comprising or alternatively, consisting of, fragment of the predicted mature TRID polypeptide, wherein the fragment has a TRID functional activity (e.g.,
• polypeptide fragments of the invention comprise, or alternatively consist of, any combination of (a), (b), (c), (d), or (e) of the above members. Polynucleotides encoding these polypeptides are also encompassed by the invention. As discussed above, it is believed that one or both of the extracellular cysteine rich motifs of TRID is important for interactions between TRID and its ligands (e.g. , TRAIL). Accordingly, in preferred embodiments, polypeptide fragments of the invention comprise, or alternatively consist of amino acid residues 53 to 1 10, and/or 1 1 1 to 153 of SEQ ID NO:2. In a specific embodiment the polypeptides of the invention comprise, or alternatively consist of both of the extracellular cysteine rich motifs disclosed in SEQ ID NO:2. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• fragments of the invention are fragments characterized by structural or functional attributes of TRID.
• Such fragments include amino acid residues that comprise alpha-helix and alpha-helix forming regions ("alpha-regions"), beta-sheet and beta-sheet-forming regions ("beta- regions”), turn and turn-forming regions ("turn-regions”), coil and coil-forming regions ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, surface forming regions, and high antigenic index regions (i.e., containing four or more contiguous amino acids having an antigenic index of greater than or equal to 1.5, as identified using the default parameters of the Jameson- Wolf program) of complete (i.e., full-length) TRID (SEQ ID NO:2).
• Certain preferred regions are those set out in Figure 3 and include, but are not limited to, regions of the aforementioned types identified by analysis of the amino acid sequence depicted in SEQ ID NO:2, such preferred regions include; Garnier-Robson predicted alpha-regions, beta-regions, turn- regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, and turn-regions; Kyte-Doolittle predicted hydrophilic and Hopp- Woods hydrophobic regions; Eisenberg alpha and beta amphipathic regions; Emini surface-forming regions; and Jameson- Wolf high antigenic index regions, as predicted using the default parameters of these computer programs. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present invention encompasses TRID proteins containing the polypeptide sequence encoded by the polynucleotides of the invention.
• the TRID proteins of the invention may be in monomers or multimers (/. e. , dimers, trimers, tetramers, and higher multimers).
• the present invention relates to monomers and multimers of the TRID proteins of the invention, their preparation, and compositions (preferably, pharmaceutical compositions) containing them.
• the polypeptides of the invention are monomers, dimers, trimers or tetramers.
• the multimers of the invention are at least dimers, at least trimers, or at least tetramers.
• Multimers encompassed by the invention may be homomers or heteromers.
• the term homomer refers to a multimer containing only TRID proteins of the invention (including TRID fragments, variants, and fusion proteins, as described herein). These homomers may contain TRID proteins having identical or different polypeptide sequences.
• a homomer of the invention is a multimer containing only TRID proteins having an identical polypeptide sequence.
• a homomer of the invention is a multimer containing TRID proteins having different polypeptide sequences.
• the multimer of the invention is a homodimer (e.g.
• the homomeric multimer of the invention is at least a homodimer, at least a homotrimer, or at least a homotetramer.
• heteromer refers to a multimer containing heterologous proteins (i.e., proteins containing only polypeptide sequences that do not correspond to a polypeptide sequences encoded by the TRID gene) in addition to the TRID proteins of the invention.
• the multimer of the invention is a heterodimer, a heterotrimer, or a heterotetramer.
• the heteromeric multimer of the invention is at least a heterodimer, at least a heterotrimer, or at least a heterotetramer. Multimers of the invention may be the result of hydrophobic, hydrophilic, ionic and/or covalent associations and/or may be indirectly linked, by for example, liposome formation.
• multimers of the invention such as, for example, homodimers or homotrimers
• multimers of the invention are formed when proteins of the invention contact one another in solution.
• heteromultimers of the invention such as, for example, heterotrimers or heterotetramers, are formed when proteins of the invention contact antibodies to the polypeptides of the invention (including antibodies to the heterologous polypeptide sequence in a fusion protein of the invention) in solution.
• multimers of the invention are formed by covalent associations with and/or between the TRID proteins of the invention.
• covalent associations may involve one or more amino acid residues contained in the polypeptide sequence of the protein (e.g., the polypeptide sequence recited in SEQ ID NO:2 or the polypeptide encoded by the deposited cDNA clone).
• the covalent associations are cross-linking between cysteine residues located within the polypeptide sequences of the proteins which interact in the native (i.e., naturally occurring) polypeptide.
• the covalent associations are the consequence of chemical or recombinant manipulation.
• such covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in a TRID fusion protein.
• covalent associations are between the heterologous sequence contained in a fusion protein of the invention (see, e.g., US Patent Number 5,478,925).
• the covalent associations are between the heterologous sequence contained in a TRID-Fc fusion protein of the invention (as described herein).
• covalent associations of fusion proteins of the invention are between heterologous polypeptide sequences from another TNF family ligand/receptor member that is capable of forming covalently associated multimers, such as for example, oseteoprotegerin (see, e.g. , International Publication No. WO 98/49305, the contents of which are herein incorporated by reference in its entirety).
• the multimers of the invention may be generated using chemical techniques known in the art.
• proteins desired to be contained in the multimers of the invention may be chemically cross-linked using linker molecules and linker molecule length optimization techniques known in the art (see, e.g. , US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• multimers of the invention may be generated using techniques known in the art to form one or more inter-molecule cross-links between the cysteine residues located within the polypeptide sequence of the proteins desired to be contained in the multimer (see, e.g., US Patent Number 5,478,925, which is herein incorporated by reference in its entirety).
• proteins of the invention may be routinely modified by the addition of cysteine or biotin to the C terminus or N-terminus of the polypeptide sequence of the protein and techniques known in the art may be applied to generate multimers containing one or more of these modified proteins (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety). Additionally, techniques known in the art may be applied to generate liposomes containing the protein components desired to be contained in the multimer of the invention (see, e.g. , US
• Patent Number 5,478,925 which is herein inco ⁇ orated by reference in its entirety).
• multimers of the invention may be generated using genetic engineering techniques known in the art.
• proteins contained in multimers of the invention are produced recombinantly using fusion protein technology described herein or otherwise known in the art (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• polynucleotides coding for a homodimer of the invention are generated by ligating a polynucleotide sequence encoding a polypeptide of the invention to a sequence encoding a linker polypeptide and then further to a synthetic polynucleotide encoding the translated product of the polypeptide in the reverse orientation from the original C-terminus to the N-terminus (lacking the leader sequence) (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• recombinant techniques described herein or otherwise known in the art are applied to generate recombinant polypeptides of the invention which contain a transmembrane domain and which can be incorporated by membrane reconstitution techniques into liposomes (see, e.g., US Patent Number 5,478,925, which is herein inco ⁇ orated by reference in its entirety).
• TRID polypeptide protein engineering may be employed.
• Recombinant DNA technology known to those skilled in the art can be used to create novel mutant proteins or "muteins" including single or multiple amino acid substitutions, deletions, additions or fusion proteins.
• modified polypeptides can show, e.g., enhanced activity or increased stability.
• they may be purified in higher yields and show better solubility than the corresponding natural polypeptide, at least under certain purification and storage conditions.
• proteins including the extracellular domain of a membrane associated protein or the mature form(s) of a secreted protein, it is known in the art that one or more amino acids may be deleted from the N- terminus or C-terminus without substantial loss of biological function.
• Polypeptides having further N-terminal deletions including the C-53 residue in SEQ ID NO:2, would not be expected to retain such biological activities because it is known that these residues in a TRID-related polypeptide are required for forming a disulfide bridge to provide structural stability which is needed for ligand binding.
• deletion of one or more amino acids from the N-terminus of a protein results in modification or loss of one or more biological functions of the protein
• other functional activities e.g., biological activities, ability to multimerize, ability to bind TRIAL ligand
• the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature form of the TRID protein generally will be retained when less than the majority of the residues of the complete protein or extracellular domain are removed from the N-terminus.
• TRID muteins with a large number of deleted N-terminal amino acid residues are expected to retain some biological or immunogenic activities. In fact, peptides composed of as few as six TRID amino acid residues may often evoke an immune response.
• the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the amino acid sequence shown in SEQ ID NO:2, up to the cysteine residue in each which is at position number 53, and polynucleotides encoding such polypeptides.
• the present invention provides TRID polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n'-259 of SEQ ID NO:2 where n 1 is an integer in the range of 1-53 where 53 is the position of the first cysteine residue from the N-terminus of the complete TRID polypeptide (shown in SEQ ID NO:2) believed to be required for activity of the TRID protein.
• the invention provides polynucleotides encoding polypeptides having the amino acid sequence of residues: M-1 to V-259; A-2 to V-259; R-3 to V-259; 1-4 to V-259; P-5 to V-259; K-6 to V-259; T-7 to V-259; L-8 to V-259; K-9 to V-259; F-10 to V-259; V-l 1 to V-259; V-12 to V-259; V- 13 to V-259; 1-14 to V-259; V-15 to V-259; A-16 to V-259; V-17 to V-259; L-
• the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above.
• the invention is further directed to nucleic acid molecules comprising, or alternatively consisting of, polynucleotide sequences which encode polypeptides that are at least 80%, 85%,
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Similarly, the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the TRID amino acid sequence shown in SEQ ID NO:2, up to the leucine residue at position number 255 and polynucleotides encoding such polypeptides.
• the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n 2 -259 of SEQ ID NO:2, where n 2 is an integer from 2 to 254 corresponding to the position of the amino acid residue in SEQ ID NO:2.
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues of A-2 to V-259; R-3 to V-259; 1-4 to V-259; P-5 to V-259; K-6 to
• V-259 T-27 to V-259 T-28 to V-259 A-29 to V-259; R-30 to V-259 Q-31 to V- 259 E-32 to V-259 E-33 to V-259 V-34 to V-259; P-35 to V-259 Q-36 to V- 259 Q-37 to V-259 T-38 to V-259 V-39 to V-259; A-40 to V-259 P-41 to V- 259 Q-42 to V-259 Q-43 to V-259 Q-44 to V-259; R-45 to V-259: H-46 to V- 259 S-47 to V-259 F-48 to V-259 K-49 to V-259; G-50 to V-259 E-51 to V- 259 E-52 to V-259 C-53 to V-259 P-54 to V-259; A-55 to V-259 G-56 to V- 259 S-57 to V-259 H-58 to V-259 R-59 to V-259; S-60 to V-259 E-61
• the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%,
• the invention is further directed to nucleic acid molecules comprising, or alternatively consisting of, polynucleotide sequences which encode polypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptides described above.
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• C-terminal deletion muteins are known. For instance, interferon gamma shows up to ten times higher activities by deleting 8-10 amino acid residues from the carboxy terminus of the protein (Dobeli et al, J. Biotechnology 7: 199-216 (1988)).
• the protein of the invention is a member of the TNFR polypeptide family, deletions of C-terminal amino acids up to the cysteine at position 149 of SEQ ID NO:2, may retain some biological activity such as regulation of proliferation and apoptosis of lymphoid cells.
• Polypeptides having further C-terminal deletions including the cysteine at position 149 of SEQ ID NO:2 would not be expected to retain such biological activities because it is known that this residue in TNF receptor-related polypeptides is required for forming a disulfide bridge to provide structural stability which is needed for ligand binding. Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of a protein results in modification or loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind TRID ligand) may still be retained.
• other functional activities e.g., biological activities, ability to multimerize, ability to bind TRID ligand
• the ability of the shortened protein to induce and/or bind to antibodies which recognize the complete or mature form of the protein generally will be retained when less than the majority of the residues of the complete or mature form protein are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete protein retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. TRID muteins with a large number of deleted C-terminal amino acid residues are expected to retain some biological or immunogenic activities. In fact, peptides composed of as few as six TRID amino acid residues may often evoke an immune response.
• the present invention further provides polypeptides having one or more residues from the carboxy terminus of the amino acid sequence of TRID shown in SEQ ID NO:2 up to the cysteine at position 149 of SEQ ID NO:2
• polypeptides having the amino acid sequence of residues 1 -m 1 of the amino acid sequence in SEQ ID NO:2, where m' is any integer in the range of 149-259. Polynucleotides encoding these polypeptides also are provided.
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: M-1 to V-259; M-1 to F-258; M-1 to V-257; M-1 to 1-256; M-1 to L-255; M-1 to L-254; M-1 to V-253; M-1 to 1-252; M-1 to L-251 ; M-1 to V-250; M-1 to 1-249; M-1 to 1-248; M-1 to G-247; M-1 to V-246; M-1 to 1-245; M-1 to T-244; M-1 to C-243; M-1 to S-242; M-1 to L-241 ; M-1 to Y-240; M-1 to H-
• M-1 to S-238 M-1 to S-237; M-1 to A-236; M-1 to P-235; M-1 to T-234; M-1 to G-233; M-1 to P-232; M-1 to S-231 ; M-1 to T-230; M-1 to T-229; M-1 to M-228; M-1 to T-227; M-1 to E-226; M-1 to E-225; M-1 to A-224; M-1 to A- 223; M-1 to P-222; M-1 to A-221; M-1 to P-220; M-1 to T-219; M-1 to G-218; M-1 to P-217; M-1 to S-216; M-1 to T-215; M-1 to T-214; M-1 to M-213; M-1 to T-212; M-1 to E-21 1 ; M-1 to E-210; M-1 to A-209; M-1 to A-208; M-1 to P- 207; M-1 to A-206; M-1 to P-205; M-1 to T-204; M-1 to
• the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%,
• the invention is further directed to nucleic acid molecules comprising, or alternatively consisting of, polynucleotide sequences which encode polypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptides described above.
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the
• polypeptide shown in SEQ ID NO:2 up to the lysine residue at position number 6, and polynucleotides encoding such polypeptides.
• the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 1 -nr of SEQ ID NO:2, where m 2 is an integer from 6 to 258 corresponding to the position of the amino acid residue in SEQ ID NO:
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: M-1 to F-258; M-1 to V-257; M-1 to 1-256; M-1 to L-255; M-1 to L-254; M-1 to V-253; M-1 to 1-252; M-1 to L-251 ; M-1 to V-250; M-1 to 1-249;
• M-1 to A-221 M-1 to P-220; M-1 to T-219; M-1 to G-218; M-1 to P-217; M-l to S-216; M-1 to T-215; M-1 to T-214; M-1 to M-213; M-1 to T-212; M-1 to E-211; M-1 to E-210; M-1 to A-209; M-1 to A-208; M-1 to P-207; M-1 to A- 206; M-1 to P-205; M-1 to T-204; M-1 to G-203; M-1 to P-202; M-1 to S-201; M-1 to T-200; M-1 to T-199; M-1 to M-198; M-1 to T-197; M-1 to E-196; M-1 to E-195; M-1 to A-194; M-1 to A-193; M-1 to P-192; M-1 to A-191; M-1 to P- 190; M-1 to T-189; M-1 to G-188; M-1 to P-187; M-1 to
• the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polynucleotide sequences encoding the polypeptides described above.
• the invention is further directed to nucleic acid molecules comprising, or alternatively consisting of, polynucleotide sequences which encode polypeptides that are at least 80%, 85%,
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also encompassed by the invention. Similarly, the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus ofthe amino acid sequence ofthe extracellular domain ofthe TRID polypeptide shown in SEQ ID NO:2, up to the glutamine residue at position number 33, and polynucleotides encoding such polypeptides. In particular, the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues
• the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues: T-27 to F-258; T-27 to V-257; T-27 to 1-256; T-27 to L-255; T-27 to L-254; T-27 to V-253; T-27 to 1-252; T-27 to L-251 ; T-27 to V-250; T-27 to 1-249; T-27 to 1-248 T-27 to G-247; T-27 to V-246; T-27 to 1-245; T-27 to T-
• T-27 to S-238 T-27 to S-237; T-27 to A-236; T-27 to P-235; T-27 to T-
• T-27 to G-233 T-27 to P-232; T-27 to S-231; T-27 to T-230; T-27 to T-
• T-27 to M-228 T-27 to T-227; T-27 to E-226; T-27 to E-225; T-27 to A-
• T-27 to A-208 T-27 to P-207; T-27 to A-206; T-27 to P-205; T-27 to T-
• T-27 to G-203 T-27 to P-202; T-27 to S-201; T-27 to T-200; T-27 to T-
• T-27 to G- 188 T-27 to P-187; T-27 to S-186; T-27 to T-185; T-27 to N-
• T-27 to A- 178 T-27 to P-177; T-27 to A-176; T-27 to P-175; T-27 to T-
• T-27 to A-163 T-27 to P-162; T-27 to T-161; T-27 to E-160; T-27 to V-
• T-27 to T-158 T-27 to A-157; T-27 to N-156; T-27 to A-155; T-27 to G-
• T-27 to Q-148 T-27 to 1-147; T-27 to D-146; T-27 to D-145: T-27 to W-
• T-27 to S-143 T-27 to T-142; T-27 to C-141; T-27 to N-140; T-27 to S-
• T-27 to V-138 T-27 to Q-137; T-27 to V-136; T-27 to E-135; T-27 to G-
• T-27 to S-133 T-27 to P-132; T-27 to C-131; T-27 to R-130; T-27 to S-
• T-27 to C-128 T-27 to K-127; T-27 to R-126; T-27 to C-125; T-27 to M-
• T-27 to E-l 23 T-27 to P-122; T-27 to S-121; T-27 to N-120; T-27 to E-
• T-27 to E-l 13 T-27 to K-l 12; T-27 to C-lll; T-27 to Q-110; T-27 to C-
• T-27 to V-108 T-27 to V-108; T-27 to T-107; T-27 to D-106; T-27 to R-l05; T-27 to T-
• T-27 to M-103 T-27 to T-102; T-27 to C-101; T-27 to S-100; T-27 to S-99;
• Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%,
• the invention is further directed to nucleic acid molecules comprising, or alternatively consisting of, polynucleotide sequences which encode polypeptides that are at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98%, or 99% identical to the polypeptides described above.
• the present invention also encompasses the above polynucleotide sequences fused to a heterologous polynucleotide sequence. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
• the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini, which may be described generally as having residues n'-m 1 , n 2 -m', n'-m 2 , n 2 -m ⁇ n'-m 3 , or n 2 -m 3 of SEQ ID NO:2, where n 1 , n 2 , m 1 , m ⁇ and m 3 are integers as described above.
• nucleotide sequence encoding a polypeptide consisting of a portion of a complete TRID amino acid sequence encoded by a cDNA clone contained in ATCC Deposit No. 97798, where this portion excludes from 1 to about 49 amino acids from the amino terminus of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97798, or from 1 to about 1 10 amino acids from the carboxy terminus ofthe complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97798, or any combination of the above amino terminal and carboxy terminal deletions, of the complete amino acid sequence encoded by the cDNA clone contained in ATCC Deposit No. 97798.
• Polynucleotides encoding all of the above deletion mutant polypeptide forms also are provided.
• the invention further includes variations ofthe TRID polypeptide, which show substantial TRID polypeptide activity or which include regions of TRID protein such as the protein portions discussed below.
• Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
• the fragment, derivative, or analog of the polypeptide of SEQ ID NO:2, or that encoded by the deposited cDNA may be: (i) one in which one or more of the amino acid residues are substituted with a conserved or non- conserved amino acid residue (preferably a conserved amino acid residue(s), and more preferably at least one but less than ten conserved amino acid residue(s)), and such substituted amino acid residue(s) may or may not be one encoded by the genetic code; or (ii) one in which one or more ofthe amino acid residues includes a substituent group;or (iii) one in which the mature or soluble extracellular polypeptide is fused with another compound, such as a compound to increase the half-life ofthe polypeptide (for example, polyethylene glycol); or (iv) one in which the additional amino acids are fused to the mature polypeptide, such as an IgG Fc fusion region peptide or leader or secretory sequence or a sequence which is employed for purification of the mature polypeptide
• the TRID ofthe present invention may include one or more amino acid substitutions, deletions or additions, either from natural mutations or human manipulation. As indicated, changes are preferably of a minor nature, such as conservative amino acid substitutions that do not significantly affect the folding or activity of the protein (see Table 2).
• the number of substitutions, additions or deletions in the amino acid sequence of SEQ ID NO:2 and/or any of the polypeptide fragments described herein is 75, 70, 60, 50, 40, 35, 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1 or 30-20, 20-15, 20-10, 15-10, 10-1, 5-10, 1-5, 1-3 or 1-2.
• Amino acids in the TRID protein ofthe present invention that are essential for function can be identified by methods known in the art, such as site-directed mutagenesis or alanine-scanning mutagenesis (Cunningham and Wells, Science 244: 1081 - 1085 (1989)). The latter procedure introduces single alanine mutations at every residue in the molecule. The resulting mutant molecules are then tested for biological activity such as receptor binding or in vitro proliferative activity.
• the replacement of amino acids can also change the selectivity of binding of a ligand to cell surface receptors.
• Ostade et al Nature 361:266- 268 (1993) describes certain mutations resulting in selective binding of TNF- ⁇ to only one of the two known types of TNF receptors.
• Sites that are critical for ligand-receptor binding can also be determined by structural analysis such as crystallization, nuclear magnetic resonance or photoaffinity labeling (Smith et al. , J. Mol Biol 224:899-904 (1992) and de Vos et al, Science 255:306-312 (1992)).
• Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g., Carter et al, Nucl. Acids Res. 75:4331 (1986); and Zoller et al, Nucl. Acids Res. 10:6481 (1982)), cassette mutagenesis (see e.g., Wells et al , Gene 54:315 (1985)), restriction selection mutagenesis (see e.g. , Wells et al , Philos. Trans. R. Soc. london Ser A 577:415 (1986)).
• art-known mutagenesis techniques include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see e.g.,
• the invention also encompasses TRID derivatives and analogs that have one or more amino acid residues deleted, added, or substituted to generate TRID polypeptides that are better suited for expression, scale up, etc., in the host cells chosen.
• cysteine residues can be deleted or substituted with another amino acid residue in order to eliminate disulfide bridges; N-linked glycosylation sites can be altered or eliminated to achieve, for example, expression of a homogeneous product that is more easily recovered and purified from yeast hosts which are known to hyperglycosylate N-linked sites.
• the TRID polypeptide is a 259 residue protein exhibiting two main structural domains.
• the extracellular TRAIL ligand binding domain was identified within residues from about 27 to about 240 in SEQ ID NO:2.
• the transmembrane domain was identified within residues from about 241 to about 259 in SEQ ID NO:2.
• TRID su ⁇ risingly lacks a putative intracellular signalling domain, thus, the name "TRID" (TRAIL Receptor Without an Intracellular Domain").
• polypeptides ofthe present invention include the polypeptide encoded by the deposited cDNA including the leader; the mature polypeptide encoded by the deposited the cDNA minus the leader (i. e. , the mature protein); a polypeptide comprising, or alternatively consisting of, amino acids about 1 to about 259 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acids about 2 to about 259 in SEQ ID NO:2 as well as polypeptides which are at least 80% identical, more preferably at least 90% or 95% identical, still more preferably at least 96%, 97%, 98%, or 99% identical to the polypeptides described above, and also include portions of such polypeptides with at least 30 amino acids and more preferably at least 50 amino acids.
• a polypeptide having an amino acid sequence at least, for example, 95% "identical" to a reference amino acid sequence of a TRID polypeptide is intended that the amino acid sequence of the polypeptide is identical to the reference sequence except that the polypeptide sequence may include up to five amino acid alterations per each 100 amino acids ofthe reference amino acid ofthe TRID polypeptide.
• up to 5% of the amino acid residues in the reference sequence may be deleted or substituted with another amino acid, or a number of amino acids up to 5% ofthe total amino acid residues in the reference sequence may be inserted into the reference sequence.
• These alterations ofthe reference sequence may occur at the amino or carboxy terminal positions of the reference amino acid sequence or anywhere between those terminal positions, interspersed either individually among residues in the reference sequence or in one or more contiguous groups within the reference sequence.
• any particular polypeptide is at least 90%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the amino acid sequence shown in SEQ ID NO:2, or to the amino acid sequence encoded by the deposited cDNA clone, can be determined conventionally using known computer programs such the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics Computer Group, University Research Park, 575 Science Drive, Madison, WI 53711).
• the parameters are set, of course, such that the percentage of identity is calculated over the full length of the reference amino acid sequence and that gaps in homology of up to 5% ofthe total number of amino acid residues in the reference sequence are allowed.
• identity between a reference (query) sequence (a sequence of the present invention) and a subject sequence is determined using the FASTDB computer program based on the algorithm of Brutlag et al. (Comp. App. Biosci. 6:231-245 (1990)).
• the percent identity is corrected by calculating the number of residues ofthe query sequence that are N- and C-terminal ofthe subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent ofthe total bases ofthe query sequence.
• a determination of whether a residue is matched/aligned is determined by results ofthe FASTDB sequence alignment. This percentage is then subtracted from the percent identity, calculated by the above FASTDB program using the specified parameters, to arrive at a final percent identity score. This final percent identity score is what is used for the purposes of this embodiment.
• the 10 unpaired residues represent 10% of the sequence (number of residues at the N- and C- termini not matched/total number of residues in the query sequence) so 10% is subtracted from the percent identity score calculated by the FASTDB program. If the remaining 90 residues were perfectly matched the final percent identity would be 90%.
• a 90 residue subject sequence is compared with a 100 residue query sequence. This time the deletions are internal deletions so there are no residues at the N- or C-termini ofthe subject sequence which are not matched/aligned with the query. In this case the percent identity calculated by FASTDB is not manually corrected.
• polypeptide ofthe present invention have uses which include, but are not limited to, as sources for generating antibodies that bind the polypeptides of the invention, and as a molecular weight marker on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
• the present application is also directed to proteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to the TRID polypeptide sequence set forth herein as n'-259, n 2 -259,l-m', 1-m 2 , l-m J , n'-m', n 2 -m', n'-m 2 , n 2 -m 2 , n'-m 3 , or n 2 -m 3 of SEQ ID NO:2, where n', n 2 , m 1 , m 2 , and m 3 are integers as described above.
• the application is directed to proteins containing polypeptides at least 90%, 95%, 96%, 97%, 98% or 99% identical to polypeptides having the amino acid sequence ofthe specific TRID N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• TRID proteins of the invention comprise, or alternatively consist of, fusion proteins as described herein wherein the TRID polypeptides are those described as n'-259, n 2 -259, 1-m', 1-m 2 , l-m 3 , n'- m 1 , n 2 -m', n'-m 2 , n 2 -m 2 , n'-m 3 , or n 2 -m 3 of SEQ ID NO:2, where n', n 2 , m', m 2 , and m 3 are integers as described above.
• the application is directed to nucleic acid molecules at least 90%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences encoding polypeptides having the amino acid sequence of the specific N- and C-terminal deletions recited herein. Polynucleotides encoding these polypeptides are also encompassed by the invention.
• the present invention encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2, or an epitope ofthe polypeptide sequence encoded by a polynucleotide sequence contained in the cDNA assigned ATCC Accession No. 97798, encoded by a polynucleotide that hybridizes to the complement of the sequence of SEQ ID NO : 1 , or contained in the cDN A assigned ATCC Accession No. 97798 under stringent hybridization conditions or lower stringency hybridization conditions as defined supra.
• the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence ofthe invention (such as, for example, the sequence disclosed in SEQ ID NOT), polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope ofthe invention, and polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
• epitopes refers to portions of a polypeptide having antigenic or immunogenic activity in an animal, preferably a mammal, and most preferably in a human.
• the present invention encompasses a polypeptide comprising an epitope, as well as the polynucleotide encoding this polypeptide.
• An "immunogenic epitope,” as used herein, is defined as a portion of a protein that elicits an antibody response in an animal, as determined by any method known in the art, for example, by the methods for generating antibodies described infra. (See, for example, Geysen et al. , Proc.
• antigenic epitope is defined as a portion of a protein to which an antibody can immunospecifically bind its antigen as determined by any method well known in the art, for example, by the immunoassays described herein. Immunospecific binding excludes non-specific binding but does not necessarily exclude cross- reactivity with other antigens. Antigenic epitopes need not necessarily be immunogenic.
• Fragments that function as epitopes may be produced by any conventional means. (See, e.g., Houghten, Proc. Natl. Acad. Sci. USA 52:5131-5135 (1985), further described in U.S. Patent No. 4,631,21 1). Antigenic epitope-bearing peptides and polypeptides ofthe invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide ofthe invention. See, for instance, Wilson et al, Cell 57:767-778 (1984) at 777.
• antigenic epitopes preferably contain a sequence of at least 4, at least 5, at least 6, at least 7, more preferably at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, and. most preferably, between about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide ofthe invention.
• Preferred polypeptides comprising immunogenic or antigenic epitopes are at least 10, 15. 20, 25, 30, 35, 40, 45, 50, 55. 60, 65, 70,
• Antigenic epitopes are useful, for example, to raise antibodies, including monoclonal antibodies, that specifically bind the epitope.
• Antigenic epitopes can be used as the target molecules in immunoassays. (See, for instance, Wilson et al, Cell 37:767-778 ( 1984); Sutcliffe et al. , Science 219:660-666 (1983)). Polynucleotides encoding these polypeptides are also encompassed by the invention.
• immunogenic epitopes can be used, for example, to induce antibodies according to methods well known in the art. (See, for instance, Sutcliffe et al, supra; Wilson et al, supra; Chow et al, Proc. Natl. Acad. Sci. USA 82:910-914; and Bittle et al, J. Gen. Virol. 66:2347-2354 (1985).
• a preferred immunogenic epitope includes the secreted protein.
• the polypeptides comprising one or more immunogenic epitopes may be presented for eliciting an antibody response together with a carrier protein, such as an albumin, to an animal system (such as, for example, rabbit or mouse), or, if the polypeptide is of sufficient length (at least about 25 amino acids), the polypeptide may be presented without a carrier.
• a carrier protein such as an albumin
• immunogenic epitopes comprising as few as 8 to 10 amino acids have been shown to be sufficient to raise antibodies capable of binding to, at the very least, linear epitopes in a denatured polypeptide (e.g., in Western blotting).
• Epitope-bearing polypeptides of the present invention may be used to induce antibodies according to methods well known in the art including, but not limited to, in vivo immunization, in vitro immunization, and phage display methods. See, e.g., Sutcliffe et al, supra; Wilson et al, supra, and Bittle et al, J. Gen. Virol, (5(5:2347-2354 (1985).
• animals may be immunized with free peptide; however, anti-peptide antibody titer may be boosted by coupling the peptide to a macromolecular carrier, such as keyhole limpet hemacyanin (KLH) or tetanus toxoid.
• KLH keyhole limpet hemacyanin
• peptides containing cysteine residues may be coupled to a carrier using a linker such as maleimidobenzoyl-N-hydroxysuccinimide ester (MBS), while other peptides may be coupled to carriers using a more general linking agent such as glutaraldehyde.
• Animals such as, for example, rabbits, rats, and mice are immunized with either free or carrier-coupled peptides, for instance, by intraperitoneal and/or intradermal injection of emulsions containing about 100 micrograms of peptide or carrier protein and Freund's adjuvant or any other adjuvant known for stimulating an immune response.
• booster injections may be needed, for instance, at intervals of about two weeks, to provide a useful titer of anti-peptide antibody that can be detected, for example, by ELISA assay using free peptide adsorbed to a solid surface.
• the titer of anti-peptide antibodies in serum from an immunized animal may be increased by selection of anti-peptide antibodies, for instance, by adso ⁇ tion to the peptide on a solid support and elution ofthe selected antibodies according to methods well known in the art.
• peptides or polypeptides bearing an antigenic epitope i.e., that contain a region of a protein molecule to which an antibody can bind
• relatively short synthetic peptides that mimic part of a protein sequence are routinely capable of eliciting an antiserum that reacts with the partially mimicked protein. See, for instance, Sutcliffe, J. G., et al.,
• Antibodies That React With Predetermined Sites on Proteins Science, 219:660-666 (1983).
• Peptides capable of eliciting protein-reactive sera are frequently represented in the primary sequence of a protein, can be characterized by a set of simple chemical rules, and are confined neither to immunodominant regions of intact proteins (i.e., immunogenic epitopes) nor to the amino or carboxyl terminals.
• Antigenic epitope-bearing peptides and polypeptides ofthe invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide ofthe invention. See, for instance, Wilson et al, Cell 37:161-118 (1984) at 777.
• Non-limiting examples of antigenic polypeptides or peptides that can be used to generate TRID-specific antibodies include: a polypeptide comprising, or alternatively consisting of, amino acid residues from about Gln-42 to about Glu-52 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about His-58 to about Cys-66 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of.
• polypeptide comprising, or alternatively consisting of, amino acid residues from about Pro-68 to about Thr-76 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Ser-79 to about Cys-85 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Cys-91 to about Thr- 102 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Gln-1 10 to about Pro-122 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about Arg- 126 to about Val- 136 in SEQ ID NO:2; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about Thr-142 to about Gin- 148 in SEQ ID NO:2.
• the inventors have determined that the above polypeptide fragments are antigenic regions of theTRI
• the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means *See, e.g., Houghten, R. A. "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids. " Proc. Natl.
• U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site) of an antibody of interest. More generally, U.S. Patent No. 4,433,092 to Geysen (1989) describes a method of detecting or determining a sequence of monomers which is a topographical equivalent of a ligand which is complementary to the ligand binding site of a particular receptor of interest. Similarly, U.S. Patent No. 5,194,392 to Geysen (1990) describes a general method of detecting or determining the sequence of monomers (amino acids or other compounds) which is a topological equivalent ofthe epitope (i.e., a "mimotope") which is complementary to a particular paratope (antigen binding site)
• TRID receptor polypeptides ofthe present invention and the epitope-bearing fragments thereof described herein above e.g. , corresponding to a portion ofthe extracellular domain, such as, for example, polypeptide sequence comprising, or alternatively, consisting of, amino acid residues 1 to 240, 27 to 240, 30 to 240, 35 to 240, 40 to 240 and 50 to 240 of SEQ ID NO:2 fused to other polypeptide sequences.
• polypeptides ofthe present invention may be fused with the constant domain of immunoglobulins (IgA, IgE, IgG, IgM), or portions thereof (CHI, CH2, CH3, or any combination thereof and portions thereof) resulting in chimeric polypeptides.
• immunoglobulins IgA, IgE, IgG, IgM
• CHI CH2, CH3, or any combination thereof and portions thereof
• Nucleic acids encoding the above epitopes can also be recombined with a gene of interest as an epitope tag (e.g. , the hemagglutinin ("HA") tag or flag tag) to aid in detection and purification ofthe expressed polypeptide.
• an epitope tag e.g. , the hemagglutinin ("HA") tag or flag tag
• HA hemagglutinin
• a system described by Janknecht et al. allows for the ready purification of non-denatured fusion proteins expressed in human cell lines (Janknecht et al , 1991 , Proc. Natl. Acad. Sci. USA 88:8972- 897).
• the gene of interest is subcloned into a vaccinia recombination plasmid such that the open reading frame of the gene is translationally fused to an amino-terminal tag consisting of six histidine residues.
• the tag serves as a matrix-binding domain for the fusion protein. Extracts from cells infected with the recombinant vaccinia virus are loaded onto Ni 2+ nitriloacetic acid-agarose column and histidine-tagged proteins can be selectively eluted with imidazole-containing buffers.
• DNA shuffling may be employed to modulate the activities of polypeptides of the invention, such methods can be used to generate polypeptides with altered activity, as well as agonists and antagonists of the polypeptides. See, generally, U.S. Patent Nos. 5,605,793; 5,81 1,238; 5,830,721 ; 5,834,252; and 5,837,458, and Patten et al,
• alteration of polynucleotides corresponding to SEQ ID NO: 1 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
• DNA shuffling involves the assembly of two or more DNA segments by homologous or site-specific recombination to generate variation in the polynucleotide sequence.
• polynucleotides of the invention, or the encoded polypeptides may be altered by being subjected to random mutagenesis by error-prone PCR, random nucleotide insertion or other methods prior to recombination.
• one or more components, motifs, sections, parts, domains, fragments, etc., of a polynucleotide coding a polypeptide of the invention may be recombined with one or more components, motifs, sections, parts, domains, fragments, etc. of one or more heterologous molecules.
• TRID polypeptides ofthe present invention and the epitope-bearing fragments thereof described herein can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
• IgG immunoglobulins
• Fusion proteins facilitate purification and show an increased half-life in vivo. This has been shown, e.g., for chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains of the constant regions of the heavy or light chains of mammalian immunoglobulins (EPA 394,827; Traunecker et al, Nature 331:84- 86 (1988)). Fusion proteins that have a disulfide-linked dimeric structure due to the IgG part can also be more efficient in binding and neutralizing other molecules than the monomeric TRID protein or protein fragment alone (Fountoulakis et al, J Biochem 270:3958-3964 (1995)).
• the epitope-bearing peptides and polypeptides ofthe invention may be produced by any conventional means. Houghten, R.A., "General method for the rapid solid-phase synthesis of large numbers of peptides: specificity of antigen-antibody interaction at the level of individual amino acids," Proc. Natl. Acad. Sci. USA 52:5131-5135 (1985). This "Simultaneous Multiple Peptide Synthesis (SMPS)" process is further described in U.S. Patent No. 4,631,21 1 to Houghten et al. (1986).
• SMPS Simultaneous Multiple Peptide Synthesis
• the polypeptide may be expressed in a modified form, such as a fusion protein, and can include not only secretion signals but also additional heterologous functional regions.
• a region of additional amino acids, particularly charged amino acids can be added to the N-terminus of the polypeptide to improve stability and persistence in the host cell, during purification or during subsequent handling and storage.
• peptide moieties can be added to the polypeptide to facilitate purification. Such regions can be removed prior to final preparation of the polypeptide.
• the addition of peptide moieties to polypeptides to engender secretion or excretion, to improve stability and to facilitate purification, among others, are familiar and routine techniques in the art.
• a preferred fusion protein comprises a heterologous region from immunoglobulin that is useful to solubilize proteins.
• EP-A-O 464 533 (Canadian counte ⁇ art 2045869) discloses fusion proteins comprising various portions of constant region of immunoglobin molecules together with another human protein or part thereof.
• the Fc part in a fusion protein is thoroughly advantageous for use in therapy and diagnosis and thus results, for example, in improved pharmacokinetic properties (EP-A 0232262).
• Fc portion proves to be a hindrance to use in therapy and diagnosis, for example, when the fusion protein is to be used as an antigen for immunizations.
• human proteins such as the hIL5-receptor
• Fc portions for the purpose of high-throughput screening assays to identify antagonists of hIL-5. See, D. Bennett et al. , Journal of Molecular Recognition 8:52-58 (1995) and K. Johanson et al. , The Journal of Biological Chemistry 270: 16:9459-9471 (1995).
• TRID receptor polypeptides of the present invention and the epitope-bearing fragments thereof described herein above e.g. , corresponding to a portion of the extracellular domain, such as, for example, polypeptide sequence comprising, or alternatively, consisting of, amino acid residues 1 to 240, 27 to 240, 30 to 240, 35 to 240, 40 to 240 and 50 to 240 of
• SEQ ID NO:2 can be combined as a fusion protein with a polypeptide having intracellular signaling activity which is activated upon ligand binding.
• a TRID polypeptide of the present invention can be coupled with the intracellular activation domain of a heterologous TNF-family receptor.
• Such a fusion protein when expressed in a host cell, would, when bound to TRAIL, activate a detectable signal, such as, but not limited to, apoptosis or NF-kB activation .
• a fusion protein is useful for screening for ligand binding, or screening for agonists and/or antagonists of a TRID polypeptide.
• proteins ofthe invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y. (1983), and Hunkapiller, M., et al, Nature 570: 105-1 1 1 (1984)).
• a peptide corresponding to a fragment ofthe TRID polypeptides ofthe invention can be synthesized by use of a peptide synthesizer.
• nonclassical amino acids or chemical amino acid analogs can be introduced as a substitution or addition into the TRID polypeptide sequence.
• Non-classical amino acids include, but are not limited to, to the D-isomers ofthe common amino acids, 2,4-diaminobutyric acid, a-amino isobutyric acid, 4-aminobutyric acid, Abu, 2-amino butyric acid, g-Abu, e-Ahx, 6-amino hexanoic acid, Aib, 2-amino isobutyric acid, 3-amino propionic acid, ornithine, norleucine, norvaline, hydroxyproline, sarcosine, citrulline, homocitrulline, cysteic acid, t-butylglycine, t-butylalanine, phenylglycine, cyclohexylalanine, b-alanine, fluoro-amino acids, designer amino acids such as b- methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
• Non-naturally occurring variants may be produced using art-known mutagenesis techniques, which include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.g., Carter et al, Nucl. Acids Res. 75:4331 (1986); and Zoller et al, Nucl. Acids Res. 10:6481 (1982)), cassette mutagenesis (see, e.g., Wells et al, Gene 34:3 5 (1985)), restriction selection mutagenesis (see, e.g., Wells etal,
• the TRID polypeptides ofthe invention can be recovered and purified by well-known methods including ammonium sulfate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography. Most preferably, high performance liquid chromatography (“HPLC”) is employed for purification.
• HPLC high performance liquid chromatography
• Polypeptides ofthe present invention include naturally purified products, products of chemical synthetic procedures, and products produced by recombinant techniques from a prokaryotic or eukaryotic host, including, for example, bacterial, yeast, higher plant, insect and mammalian cells. Depending upon the host employed in a recombinant production procedure, the polypeptides of the present invention may be glycosylated or non-glycosylated. In addition, polypeptides of the invention may also include an initial modified methionine residue, in some cases as a result of host-mediated processes.
• the invention additionally, encompasses TRID polypeptides which are differentially modified during or after translation, e.g., by glycosylation, acetylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to an antibody molecule or other cellular ligand, etc. Any of numerous chemical modifications may be carried out by known techniques, including but not limited to, specific chemical cleavage by cyanogen bromide, trypsin, chymotrypsin, papain, V8 protease, NaBH 4 acetylation, formylation, oxidation, reduction, metabolic synthesis in the presence of tunicamycin, etc.
• Additional post-translational modifications encompassed by the invention include, for example, e.g. , N-linked or O-linked carbohydrate chains, processing of N-terminal or C-terminal ends), attachment of chemical moieties to the amino acid backbone, chemical modifications of N-linked or O-linked carbohydrate chains, and addition or deletion of an N-terminal methionine residue as a result of procaryotic host cell expression.
• the polypeptides may also be modified with a detectable label, such as an enzymatic, fluorescent, isotopic or affinity label to allow for detection and isolation ofthe protein.
• TRID chemically modified derivatives of TRID which may provide additional advantages such as increased solubility, stability and circulating time ofthe polypeptide, or decreased immunogenicity (see
• the chemical moieties for derivitization may be selected from water soluble polymers such as polyethylene glycol, ethylene glycol/propylene glycol copolymers, carboxymethylcellulose, dextran, polyvinyl alcohol and the like.
• the polypeptides may be modified at random positions within the molecule, or at predetermined positions within the molecule and may include one, two, three or more attached chemical moieties.
• the polymer may be of any molecular weight, and may be branched or unbranched.
• the preferred molecular weight is between about 1 kDa and about 100 kDa (the term "about” indicating that in preparations of polyethylene glycol, some molecules will weigh more, some less, than the stated molecular weight) for ease in handling and manufacturing.
• Other sizes may be used, depending on the desired therapeutic profile (e.g. , the duration of sustained release desired, the effects, if any on biological activity, the ease in handling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol to a therapeutic protein or analog).
• the polyethylene glycol may have an average molecular weight of about 200, 500, 1000, 1500, 2000, 2500,
• polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains ofthe protein.
• attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference (coupling PEG to G-CSF), see also Malik et al, Exp. Hematol 20:1028-1035
• polyethylene glycol may be covalently bound through amino acid residues via a reactive group, such as, a free amino or carboxyl group.
• Reactive groups are those to which an activated polyethylene glycol molecule may be bound.
• the amino acid residues having a free amino group may include ly sine residues and the
• N-terminal amino acid residues those having a free carboxyl group may include aspartic acid residues glutamic acid residues and the C-terminal amino acid residue.
• Sulfhydryl groups may also be used as a reactive group for attaching the polyethylene glycol molecules. Preferred for therapeutic purposes is attachment at an amino group, such as attachment at the N-terminus or lysine group.
• polyethylene glycol may be attached to proteins via linkage to any of a number of amino acid residues.
• polyethylene glycol can be linked to a proteins via covalent bonds to lysine, histidine, aspartic acid, glutamic acid, or cysteine residues.
• reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g.. lysine, histidine, aspartic acid, glutamic acid, or cysteine) ofthe protein or to more than one type of amino acid residue (e.g. , lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) ofthe protein.
• specific amino acid residues e.g.. lysine, histidine, aspartic acid, glutamic acid, or cysteine
• amino acid residues e.g. lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof
• polyethylene glycol as an illustration ofthe present composition, one may select from a variety of polyethylene glycol molecules (by molecular weight, branching, etc.), the proportion of polyethylene glycol molecules to protein (or peptide) molecules in the reaction mix, the type of pegylation reaction to be performed, and the method of obtaining the selected N-terminally pegylated protein.
• the method of obtaining the N-terminally pegylated preparation i.e., separating this moiety from other monopegylated moieties if necessary
• Selective proteins chemically modified at the N-terminus modification may be accomplished by reductive alkylation which exploits differential reactivity of different types of primary amino groups (lysine versus the N-terminal) available for derivatization in a particular protein. Under the appropriate reaction conditions, substantially selective derivatization ofthe protein at the N-terminus with a carbonyl group containing polymer is achieved.
• pegylation of the proteins of the invention may be accomplished by any number of means.
• polyethylene glycol may be attached to the protein either directly or by an intervening linker.
• Linkerless systems for attaching polyethylene glycol to proteins are described in Delgado et al, Crit. Rev. Thera. Drug Carrier Sys. 9:249-304 (1992); Francis et al, Intern. J. of Hematol 65: 1-18 (1998); U.S. Patent No. 4,002,531 ; U.S. Patent No. 5,349,052; WO 95/06058; and WO 98/32466, the disclosures of each of which are incorporated herein by reference.
• One system for attaching polyethylene glycol directly to amino acid residues of proteins without an intervening linker employs tresylated MPEG, which is produced by the modification of monmethoxy polyethylene glycol (MPEG) using tresylchloride (ClSO 2 CH,CF 3 ).
• MPEG monmethoxy polyethylene glycol
• ClSO 2 CH,CF 3 tresylchloride
• polyethylene glycol is directly attached to amine groups ofthe protein.
• the invention includes protein-polyethylene glycol conjugates produced by reacting proteins ofthe invention with a polyethylene glycol molecule having a 2,2,2-trifluoreothane sulphonyl group.
• Polyethylene glycol can also be attached to proteins using a number of different intervening linkers.
• U.S. Patent No. 5,612,460 discloses urethane linkers for connecting polyethylene glycol to proteins.
• Protein-polyethylene glycol conjugates wherein the polyethylene glycol is attached to the protein by a linker can also be produced by reaction of proteins with compounds such as MPEG- succinimidylsuccinate, MPEG activated with l,l'-carbonyldiimidazole, MPEG- 2,4,5-trichloropenylcarbonate, MPEG-p-nitrophenolcarbonate, and various combinations thereof.
• MPEG-succinate derivatives A number additional polyethylene glycol derivatives and reaction chemistries for attaching polyethylene glycol to proteins are described in WO 98/32466, the entire disclosure of which is incorporated herein by reference. Pegylated protein products produced using the reaction chemistries set out herein are included within the scope ofthe invention.
• the number of polyethylene glycol moieties attached to each protein ofthe invention may also vary.
• the pegylated proteins ofthe invention may be linked, on average, to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 12, 15, 17, 20, or more polyethylene glycol molecules.
• the average degree of substitution within ranges such as 1-3, 2-4, 3-5, 4-6, 5-7, 6-8,
• TRID proteins of the invention may be modified by either natural processes, such as posttranslational processing, or by chemical modification techniques which are well known in the art. It will be appreciated that the same type of modification may be present in the same or varying degrees at several sites in a given TRID polypeptide.
• TRID polypeptides may be branched, for example, as a result of ubiquitination, and they may be cyclic, with or without branching. Cyclic, branched, and branched cyclic TRID polypeptides may result from posttranslation natural processes or may be made by synthetic methods.
• Modifications include acetylation, acylation, ADP-ribosylation, amidation, covalent attachment of flavin, covalent attachment of a heme moiety, covalent attachment of a nucleotide or nucleotide derivative, covalent attachment of a lipid or lipid derivative, covalent attachment of phosphotidylinositol, cross- linking, cyclization, disulfide bond formation, demethylation, formation of covalent cross-links, formation of cysteine, formation of pyroglutamate, formylation, gamma-carboxylation, glycosylation, GPI anchor formation, hydroxylation, iodination, methylation, myristoylation, oxidation, pegylation, proteolytic processing, phosphorylation, prenylation, racemization, selenoylation, sulfation, transfer-RNA mediated addition of amino acids to proteins such as arginylation, and ubiquitination.
• TRID-protein specific antibodies for use in the present invention can be raised against the intact TRID proteins or an antigenic polypeptide fragment thereof, which may be presented together with a carrier protein, such as an albumin, to an animal system (such as rabbit or mouse) or, if it is long enough (at least about 25 amino acids), without a carrier.
• a carrier protein such as an albumin
• the present invention further relates to antibodies and T-cell antigen receptors (TCR) which specifically bind the polypeptides ofthe present invention.
• TCR T-cell antigen receptors
• the antibodies ofthe present invention include IgG (including IgG 1 , IgG2, IgG3 , and IgG4), IgA (including IgAl and IgA2), IgD, IgE, or IgM, and IgY.
• antibody or “monoclonal antibody” (Mab) is meant to include intact molecules (e.g., whole antibodies), as well as antibody fragments including single-chain whole antibodies, and antigen-binding fragments thereof
• Fab and F(ab')2 fragments which are capable of specifically binding to a TNFR protein.
• Fab and F(ab')2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)). Thus, these fragments are preferred.
• the antibodies are human antigen-binding antibody fragments ofthe present invention and include, but are not limited to, Fab, Fab' and F(ab')2, Fd, single-chain Fvs (scFv), single-chain antibodies, disulfide-linked Fvs (sdFv) and fragments comprising either a V L or V H domain.
• Antigen-binding antibody fragments, including single-chain antibodies may comprise the variable region(s) alone or in combination with the entirety or a portion ofthe following: hinge region, CHI, CH2, and CH3 domains. Also included in the invention are antigen-binding fragments also comprising any combination of variable region(s) with a hinge region, CHI, CH2, and CH3 domains.
• the antibodies of the invention may be from any animal origin including birds and mammals.
• the antibodies are human, murine, donkey, ship rabbit, goat, guinea pig, camel, horse, or chicken.
• "human” antibodies include antibodies having the amino acid sequence of a human immunoglobulin and include antibodies isolated from human immunoglobulin libraries or from animals transgenic for one or more human immunoglobulin and that do not express endogenous immunoglobulins, as described infra and, for example in, U.S. Patent No. 5,939,598 by Kucherlapati et al
• the antibodies ofthe present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide ofthe present invention or may be specific for both a polypeptide of the present invention as well as for heterologous compositions, such as a heterologous polypeptide or solid support material. See, e.g., WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt, A. et al. J. Immunol 147:60-69 (1991); US Patents 5,573,920, 4,474,893, 5,601 ,819. 4,714,681. 4,925,648; Kostelny, S.A. et al. J. Immunol. 148: 1541- 553 (1992).
• Antibodies ofthe present invention may be described or specified in terms ofthe epitope(s) or portion(s) of a polypeptide ofthe present invention which are recognized or specifically bound by the antibody.
• the epitope(s) or polypeptide portion(s) may be specified as described herein, e.g. , by N-terminal and C-terminal positions, by size in contiguous amino acid residues, or listed in the Tables and
• Antibodies which specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides ofthe present invention, and allows for the exclusion ofthe same. Antibodies ofthe present invention may also be described or specified in terms of their cross-reactivity. Antibodies that do not bind any other analog, ortholog, or homolog ofthe polypeptides ofthe present invention are included. Antibodies that do not bind polypeptides with less than 95%, less than 90%, less than 85%, less than 80%, less than 75%, less than 70%, less than 65%, less than 60%, less than 55%.
• antibodies which only bind polypeptides encoded by polynucleotides which hybridize to a polynucleotide of the present invention under stringent hybridization conditions are also included in the present invention.
• Antibodies ofthe present invention may also be described or specified in terms of their binding affinity.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5X10 "2 M, 10 "2 M, 5X10 “3 M, 10 “3 M, 5X10 “4 M, 10 '4 M, 5X10 "5 M, 10 "5 M, 5X10 “6 M, 10 “6 M, 5X10 “7 M, 10 “7 M, 5X10 “8 M, 10 “8 M, 5X10 “9 M, 10 “9 M, 5X10-
• the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope ofthe invention as determined by any method known in the art for determining competitive binding, for example, the immunoassays described herein.
• the antibody competitively inhibits binding to the epitope by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50%.
• Antibodies ofthe present invention may act as agonists, TRAIL binding facilitators, or antagonists of the polypeptides of the present invention.
• the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides ofthe invention either partially or fully.
• the invention features both receptor-specific antibodies and ligand-specific antibodies.
• the invention also features receptor-specific antibodies which do not prevent ligand binding but prevent receptor activation.
• Receptor activation i.e., signaling
• signaling may be determined by techniques described herein or otherwise known in the art.
• receptor activation can be determined by detecting the phosphorylation (e.g., tyrosine or serine/threonine) of the receptor or its substrate by immunoprecipitation followed by western blot analysis (for example, as described supra).
• phosphorylation e.g., tyrosine or serine/threonine
• antibodies are provided that inhibit ligand or receptor activity by at least 90%, at least 80%, at least 70%, at least 60%, or at least 50% ofthe activity in absence ofthe antibody.
• the invention also features receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
• receptor-specific antibodies which both prevent ligand binding and receptor activation as well as antibodies that recognize the receptor-ligand complex, and, preferably, do not specifically recognize the unbound receptor or the unbound ligand.
• neutralizing antibodies which bind the ligand and prevent binding ofthe ligand to the receptor, as well as antibodies which bind the ligand, thereby preventing receptor activation, but do not prevent the ligand from binding the receptor.
• antibodies which activate the receptor are also act as receptor agonists, i.e., potentiate or activate either all or a subset ofthe biological activities ofthe ligand-mediated receptor activation.
• the antibodies may be specified as agonists, TRAIL binding facilitators, antagonists or inverse agonists for biological activities comprising the specific biological activities of the peptides of the invention disclosed herein.
• the invention further relates to antibodies which act as agonists, TRAIL binding facilitators, or antagonists of the polypeptides of the present invention.
• the above antibody agonists or TRAIL binding facilitators can be made using methods known in the art. See, e.g., PCT publication WO 96/40281 ; U.S. Patent No. 5,81 1.097; Deng et al, Blood 92 (6) -A 98 - 988 (1998); Chen etal, Cancer Res.
• Antibodies of the present invention have uses that include, but are not limited to, methods known in the art to purify, detect, and target the polypeptides of the present invention including both in vitro and in vivo diagnostic and therapeutic methods.
• the antibodies have use in immunoassays for qualitatively and quantitatively measuring levels ofthe polypeptides ofthe present invention in biological samples. See, e.g., Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988) (incorporated by reference in the entirety).
• the antibodies of the present invention may be used either alone or in combination with other compositions.
• the antibodies may further be recombinantly fused to a heterologous polypeptide at the N- or C-terminus or chemically conjugated (including covalently and non-covalently conjugations) to polypeptides or other compositions.
• antibodies of the present invention may be recombinantly fused or conjugated to molecules useful as labels in detection assays and effector molecules such as heterologous polypeptides, drugs, or toxins. See, e.g., WO 92/08495; WO 91/14438; WO 89/12624; US
• Patent 5,314,995 and EP 0 396 387.
• the antibodies ofthe invention include derivatives that are modified, i.e, by the covalent attachment of any type of molecule to the antibody such that covalent attachment does not prevent the antibody from generating an anti-idiotypic response.
• the antibody derivatives include antibodies that have been modified, e.g.. by glycosylation, acetylation, pegylation, phosphorylation, amidation, derivatization by known protecting/blocking groups, proteolytic cleavage, linkage to a cellular ligand or other protein, etc. Any of numerous chemical modifications may be carried out by known techniques, including, but not limited to specific chemical cleavage, acetylation, formylation, metabolic synthesis of tunicamycin, etc. Additionally, the derivative may contain one or more non-classical amino acids.
• the antibodies ofthe present invention may be generated by any suitable method known in the art.
• Polyclonal antibodies to an antigen of interest can be produced by various procedures well known in the art.
• a polypeptide ofthe invention can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific for the antigen.
• adjuvants may be used to increase the immunological response, depending on the host species, and include but are not limited to, Freund's (complete and incomplete), mineral gels such as aluminum hydroxide, surface active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (bacille Calmette-Guerin) and Corynebacterium parvum. Such adjuvants are also well known in the art.
• the antibodies ofthe present invention are monoclonal antibodies.
• the term "monoclonal antibody" is not a limited to antibodies produced through hybridoma technology.
• monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and not the method by which it is produced. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technology.
• monoclonal antibodies can be prepared using hybridoma technology (Kohl ex etal, Nature 256:495 (1975); K ⁇ hler et ⁇ /., Ewr. J. Immunol
• Such cells may be cultured in any suitable tissue culture medium; however, it is preferable to culture cells in Earle's modified Eagle's medium supplemented with 10% fetal bovine serum (inactivated at about 56° C), and supplemented with about 10 g/1 of nonessential amino acids, about 1,000 U/ml of penicillin, and about 100 ⁇ g/ml of streptomycin.
• the splenocytes of such mice are extracted and fused with a suitable myeloma cell line.
• Any suitable myeloma cell line may be employed in accordance with the present invention; however, it is preferable to employ the parent myeloma cell line (SP2O), available from the American Type Culture Collection, Rockville, Maryland.
• SP2O parent myeloma cell line
• the resulting hybridoma cells are selectively maintained in HAT medium, and then cloned by limiting dilution as described by Wands et al. (Gastroenterology 50:225-232 (1981)). The hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the desired TRID antigen.
• the present invention provides methods of generating monoclonal antibodies as well as antibodies produced by the method comprising culturing a hybridoma cell secreting an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with an antigen ofthe invention with myeloma cells and then screening the hybridomas resulting from the fusion for hybridoma clones that secrete an antibody able to bind a polypeptide ofthe invention.
• Antibody fragments that recognize specific epitopes may be generated by known techniques.
• Fab and F(ab')2 fragments ofthe invention may be produced by proteolytic cleavage of immunoglobulin molecules, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
• F(ab')2 fragments contain the variable region, the light chain constant region and the CHI domain of the heavy chain.
• Hybridoma techniques include those known in the art and taught in Harlow et al., ANTIBODIES: A LABORATORY MANUAL, (Cold Spring Harbor Laboratory Press, 2nd ed. 1988); Hammerling, et al., in: MONOCLONAL ANTIBODIES AND T-CELL HYBRIDOMAS 563-681 (Elsevier, N.Y., 1981)
• Fab and F(ab')2 and other fragments ofthe antibodies ofthe present invention may be used according to the methods disclosed herein.
• Fab and F(ab')2 fragments may be produced by proteolytic cleavage, using enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
• enzymes such as papain (to produce Fab fragments) or pepsin (to produce F(ab')2 fragments).
• TRID protein-binding fragments can be produced through the application of recombinant DNA technology or through synthetic chemistry.
• additional antibodies capable of binding to the TRID antigen may be produced in a two-step procedure through the use of anti-idiotypic antibodies.
• TRID-protein specific antibodies are used to immunize an animal, preferably a mouse.
• the splenocytes of such an animal are then used to produce hybridoma cells, and the hybridoma cells are screened to identify clones which produce an antibody whose ability to bind to the TRID protein-specific antibody can be blocked by the TRID protein antigen.
• Such antibodies comprise anti-idiotypic antibodies to the TRID protein-specific antibody and can be used to immunize an animal to induce formation of further TRID protein-specific antibodies.
• chimeric monoclonal antibodies For in vivo use of anti-TRID in humans, it may be preferable to use "humanized" chimeric monoclonal antibodies. Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric antibodies are known in the art. See, for review. Morrison, Science 229:1202 (1985); Oi et al., BioTechniques 4:214 (1986); Cabilly etal., U.S. Patent No.4,816,567; Taniguchi et al., EP 171496; Morrison et al., EP 173494; Neuberger et al., WO 8601533;
• antibodies ofthe present invention can be produced through the application of recombinant DNA and phage display technology or through synthetic chemistry using methods known in the art.
• the antibodies of the present invention can be prepared using various phage display methods known in the art.
• phage display methods functional antibody domains are displayed on the surface of a phage particle which carries polynucleotide sequences encoding them.
• Phage with a desired binding property are selected from a repertoire or combinatorial antibody library (e.g. human or murine) by selecting directly with antigen, typically antigen bound or captured to a solid surface or bead.
• Phage used in these methods are typically filamentous phage including fd and Ml 3 with Fab, Fv or disulfide stabilized Fv antibody domains recombinantly fused to either the phage gene III or gene VIII protein.
• Examples of phage display methods that can be used to make the antibodies ofthe present invention include those disclosed in Brinkman U. et al J. Immunol Methods 752:41-50 (1995); Ames, R.S. et al J. Immunol. Methods 184:111-186 (1995); Kettleborough, CA. et al. Eur. J. Immunol 24:952-958 (1994); Persic, L. et al Gene 757:9-18 (1997); Burton, D.R. etal Advances in Immunology 57:191-280 (1994); PCT/GB91/01134; WO 90/02809; WO 91/10737; WO 92/01047; WO
• the antibody coding regions from the phage can be isolated and used to generate whole antibodies, including human antibodies, or any other desired antigen binding fragment, and expressed in any desired host including mammalian cells, insect cells, plant cells, yeast, and bacteria.
• techniques to recombinantly produce Fab, Fab' and F(ab')2 fragments can also be employed using methods known in the art such as those disclosed in WO 92/22324; Mullinax, R.L. et al.
• Antibodies can be humanized using a variety of techniques including CDR-grafting (EP 0 239 400; WO 91/09967; US Patent 5,530,101 ; and 5,585,089), veneering or resurfacing (EP 0 592 106; EP 0 519 596; Padlan E.A.,
• Human antibodies can also be produced using transgenic mice which are incapable of expressing functional endogenous immunoglobulins, but which can express human immunoglobulin genes.
• the human heavy and light chain immunoglobulin gene complexes may be introduced randomly or by homologous recombination into mouse embryonic stem cells.
• the human variable region, constant region, and diversity region may be introduced into mouse embryonic stem cells in addition to the human heavy and light chain genes.
• the mouse heavy and light chain immunoglobulin genes may be rendered non-functional separately or simultaneously with the introduction of human immunoglobulin loci by homologous recombination. In particular, homozygous deletion ofthe JH region prevents endogenous antibody production.
• the modified embryonic stem cells are expanded and microinjected into blastocysts to produce chimeric mice. The chimeric mice are then bred to produce homozygous offspring that express human antibodies.
• the transgenic mice are immunized in the normal fashion with a selected antigen, e.g., all or a portion of a polypeptide of the invention.
• Monoclonal antibodies directed against the antigen can be obtained from the immunized, transgenic mice using conventional hybridoma technology.
• the human immunoglobulin transgenes harbored by the transgenic mice rearrange during B-cell differentiation, and subsequently undergo class switching and somatic mutation.
• Lonberg and Huszar (1995, Int. Rev. Immunol. 13:65-93).
• antibodies to the TRID proteins ofthe invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" TRID using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5):437-444; (1989) and Nissinoff, J. Immunol. 747(8):2429-2438 (1991)).
• antibodies which bind to TRID and competitively inhibit TRID multimerization and/or binding to ligand can be used to generate anti- idiotypes that "mimic" the TRID mutimerization and/or binding domain and, as a consequence, bind to and neutralize TRID and/or its ligand.
• Such neutralizing anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize TRID ligand.
• anti-idiotypic antibodies can be used to bind TRID, or to bind TRID ligands/receptors, and thereby block
• antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide ofthe present invention may be specific for antigens other than polypeptides of the present invention.
• antibodies may be used to target the polypeptides of the present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides of the present invention to antibodies specific for particular cell surface receptors.
• Antibodies fused or conjugated to the polypeptides ofthe present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g. , Harbor et ⁇ l.
• the present invention further includes compositions comprising, or alternatively consisting of, TRID polypeptides ofthe present invention fused or conjugated to antibody domains other than the variable regions.
• the polypeptides ofthe present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
• the antibody portion fused to a polypeptide ofthe present invention may comprise the hinge region, CH 1 domain, CH2 domain, and
• polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in the art.
• the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
• Fc portions fused to the polypeptides ofthe present invention can form dimers through disulfide bonding between the Fc portions.
• Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and IgM. Methods for fusing or conjugating the polypeptides of the present invention to antibody portions are known in the art. See e.g., US Patents 5,336,603,
• the present invention includes antibodies which disrupt the ability of the proteins ofthe invention to multimerize.
• the present invention includes antibodies which allow the proteins ofthe invention to multimerize, but disrupts the ability ofthe proteins ofthe invention to bind one or more TRID receptor(s)/ligand(s) (e.g., TRAIL).
• the present invention includes antibodies which allow the proteins ofthe invention to multimerize. and bind TRID receptor(s)/ligand(s) (e.g., TRAIL), but blocks biological activity associated with the TRID/receptor/ligand complex.
• the invention further provides polynucleotides comprising a nucleotide sequence encoding an antibody of the invention and fragments thereof.
• the invention also encompasses polynucleotides that hybridize under stringent or lower stringency hybridization conditions, e.g., as defined supra, to polynucleotides that encode an antibody, preferably, that specifically binds to a polypeptide ofthe invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2.
• the polynucleotides may be obtained, and the nucleotide sequence ofthe polynucleotides determined, by any method known in the art.
• a polynucleotide encoding the antibody may be assembled from chemically synthesized oligonucleotides (e.g., as described in Kutmeier et al. BioTechniques 77:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions ofthe sequence encoding the antibody, annealing and ligation of those oligonucleotides, and then amplification of the ligated oligonucleotides by PCR.
• a polynucleotide encoding an antibody may be generated from nucleic acid from a suitable source. If a clone containing a nucleic acid encoding a particular antibody is not available, but the sequence ofthe antibody molecule is known, a nucleic acid encoding the immunoglobulin may be obtained from a suitable source (e.g., an antibody cDNA library, or a cDNA library generated from, or nucleic acid, preferably poly A+ RNA, isolated from, any tissue or cells expressing the antibody, such as hybridoma cells selected to express an antibody of the invention) by PCR amplification using synthetic primers hybridizable to the 3' and 5' ends of the sequence or by cloning using an oligonucleotide probe specific for the particular gene sequence to identify, e.g. , a cDNA clone from a cDNA library that encodes the antibody. Amplified nucleic acids generated by PCR may then be cloned
• nucleotide sequence and corresponding amino acid sequence of the antibody may be manipulated using methods well known in the art for the manipulation of nucleotide sequences, e.g., recombinant DNA techniques, site directed mutagenesis, PCR, etc. (see, for example, the techniques described in Sambrook et al, 1990, Molecular Cloning, A Laboratory Manual, 2d Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY and Ausubel et al, eds., 1998.
• the amino acid sequence of the heavy and/or light chain variable domains may be inspected to identify the sequences of the complementarity determining regions (CDRs) by methods that are well know in the art, e.g. , by comparison to known amino acid sequences of other heavy and light chain variable regions to determine the regions of sequence hypervariability.
• CDRs complementarity determining regions
• one or more of the CDRs may be inserted within framework regions, e.g., into human framework regions to humanize a non-human antibody, as described supra.
• the framework regions may be naturally occurring or consensus framework regions, and preferably human framework regions (see, e.g., Chothia et ⁇ /.,J Mol Biol.
• the polynucleotide generated by the combination ofthe framework regions and CDRs encodes an antibody that specifically binds a polypeptide ofthe invention.
• one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding ofthe antibody to its antigen. Additionally, such methods may be used to make amino acid substitutions or deletions of one or more variable region cysteine residues participating in an intrachain disulfide bond to generate antibody molecules lacking one or more intrachain disulfide bonds.
• Other alterations to the polynucleotide are encompassed by the present invention and within the skill of the art.
• a chimeric antibody is a molecule in which different portions are derived from different animal species, such as those having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region, e.g., humanized antibodies.
• Single chain antibodies are formed by linking the heavy and light chain fragments ofthe Fv region via an amino acid bridge, resulting in a single chain polypeptide.
• Techniques for the assembly of functional Fv fragments in E. coli may also be used (Skerra et al, 1988, Science 242:1038- 1041).
• the antibodies ofthe invention can be produced by any method known in the art for the synthesis of antibodies, in particular, by chemical synthesis or preferably, by recombinant expression techniques.
• an antibody of the invention or fragment, derivative or analog thereof, e.g., a heavy or light chain of an antibody of the invention, requires construction of an expression vector containing a polynucleotide that encodes the antibody. Once a polynucleotide encoding an antibody molecule or a heavy or light chain of an antibody, or portion thereof
• the vector for the production ofthe antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
• methods for preparing a protein by expressing a polynucleotide containing an antibody encoding nucleotide sequence are described herein. Methods which are well known to those skilled in the art can be used to construct expression vectors containing antibody coding sequences and appropriate transcriptional and translational control signals. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination.
• the invention thus, provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule ofthe invention, or a heavy or light chain thereof, or a heavy or light chain variable domain, operably linked to a promoter.
• Such vectors may include the nucleotide sequence encoding the constant region ofthe antibody molecule (see, e.g. , PCT Publication WO 86/05807; PCT Publication WO 89/01036; and U.S. Patent No. 5,122,464) and the variable domain of the antibody may be cloned into such a vector for expression of the entire heavy or light chain.
• the expression vector is transferred to a host cell by conventional techniques and the transfected cells are then cultured by conventional techniques to produce an antibody ofthe invention.
• the invention includes host cells containing a polynucleotide encoding an antibody ofthe invention, or a heavy or light chain thereof, operably linked to a heterologous promoter.
• vectors encoding both the heavy and light chains may be co-expressed in the host cell for expression of the entire immunoglobulin molecule, as detailed below.
• a variety of host-expression vector systems may be utilized to express the antibody molecules of the invention.
• Such host-expression systems represent vehicles by which the coding sequences of interest may be produced and subsequently purified, but also represent cells which may, when transformed or transfected with the appropriate nucleotide coding sequences, express an antibody molecule of the invention in situ.
• These include but are not limited to microorganisms such as bacteria (e.g., E. coli, B. subtilis) transformed with recombinant bacteriophage DNA, plasmid DNA or cosmid DNA expression vectors containing antibody coding sequences; yeast (e.g., Saccharomyces, Pichia) transformed with recombinant yeast expression vectors containing antibody coding sequences; insect cell systems infected with recombinant virus expression vectors (e.g.
• baculovirus containing antibody coding sequences
• plant cell systems infected with recombinant virus expression vectors e.g. , cauliflower mosaic virus, CaMV; tobacco mosaic virus, TMV
• recombinant plasmid expression vectors e.g., Ti plasmid
• mammalian cell systems e.g., COS, CHO, BHK, 293, 3T3 cells harboring recombinant expression constructs containing promoters derived from the genome of mammalian cells (e.g.
• metallothionein promoter or from mammalian viruses (e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter).
• mammalian viruses e.g., the adenovirus late promoter; the vaccinia virus 7.5K promoter.
• bacterial cells such as Escherichia coli, and more preferably, eukaryotic cells, especially for the expression of whole recombinant antibody molecule, are used for the expression of a recombinant antibody molecule.
• mammalian cells such as Chinese hamster ovary cells (CHO), in conjunction with a vector such as the major intermediate early gene promoter element from human cytomegalovirus is an effective expression system for antibodies (Foecking et al, 1986, Gene 45:101 ; Cockett et al , 1990, Bio/Technology 5:2).
• a number of expression vectors may be advantageously selected depending upon the use intended for the antibody molecule being expressed.
• vectors which direct the expression of high levels of fusion protein products that are readily purified may be desirable.
• Such vectors include, but are not limited, to the E. coli expression vector pUR278 (Ruther et al, 1983, EMBO J. 2:1791), in which the antibody coding sequence may be ligated individually into the vector in frame with the lac Z coding region so that a fusion protein is produced; pIN vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
• pGEX vectors may also be used to express foreign polypeptides as fusion proteins with glutathione S-transferase (GST).
• GST glutathione S-transferase
• fusion proteins are soluble and can easily be purified from lysed cells by adso ⁇ tion and binding to a matrix glutathione-agarose beads followed by elution in the presence of free glutathione.
• the pGEX vectors are designed to include thrombin or factor Xa protease cleavage sites so that the cloned target gene product can be released from the GST moiety.
• Autographa californica nuclear polyhedrosis virus (AcNPV) is used as a vector to express foreign genes.
• the virus grows in Spodoptera frugiperda cells.
• the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) ofthe virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
• a number of viral-based expression systems may be utilized.
• the antibody coding sequence of interest may be ligated to an adenovirus transcription/translation control complex, e.g. , the late promoter and tripartite leader sequence.
• This chimeric gene may then be inserted in the adenovirus genome by in vitro or in vivo recombination. Insertion in a non- essential region ofthe viral genome (e.g. , region El or E3) will result in a recombinant virus that is viable and capable of expressing the antibody molecule in infected hosts, (e.g. , see Logan & Shenk, 1984, Proc.
• Specific initiation signals may also be required for efficient translation of inserted antibody coding sequences. These signals include the ATG initiation codon and adjacent sequences. Furthermore, the initiation codon must be in phase with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translational control signals and initiation codons can be of a variety of origins, both natural and synthetic. The efficiency of expression may be enhanced by the inclusion of appropriate transcription enhancer elements, transcription terminators, etc. (see Bittner et al, 1987, Methods in Enzymol
• a host cell strain may be chosen which modulates the expression ofthe inserted sequences, or modifies and processes the gene product in the specific fashion desired. Such modifications (e.g., glycosylation) and processing (e.g. , cleavage) of protein products may be important for the function ofthe protein.
• Different host cells have characteristic and specific mechanisms for the post-translational processing and modification of proteins and gene products. Appropriate cell lines or host systems can be chosen to ensure the correct modification and processing of the foreign protein expressed. To this end, eukaryotic host cells which possess the cellular machinery for proper processing ofthe primary transcript, glycosylation, and phosphorylation ofthe gene product may be used.
• Such mammalian host cells include but are not limited to CHO, VERY, BHK, HeLa, COS, MDCK, 293, 3T3, WI38, and in particular, breast cancer cell lines such as, for example, BT483, Hs578T, HTB2, BT20 and T47D, and normal mammary gland cell line such as, for example, CRL7030 and Hs578Bst.
• cell lines which stably express the antibody molecule may be engineered.
• host cells can be transformed with DNA controlled by appropriate expression control elements (e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.), and a selectable marker.
• appropriate expression control elements e.g. , promoter, enhancer, sequences, transcription terminators, polyadenylation sites, etc.
• engineered cells may be allowed to grow for 1-2 days in an enriched media, and then are switched to a selective media.
• the selectable marker in the recombinant plasmid confers resistance to the selection and allows cells to stably integrate the plasmid into their chromosomes and grow to form foci which in turn can be cloned and expanded into cell lines.
• This method may advantageously be used to engineer cell lines which express the antibody molecule.
• Such engineered cell lines may be particularly useful in screening and evaluation of compounds that interact directly or indirectly with the antibody molecule.
• a number of selection systems may be used, including but not limited to the he ⁇ es simplex virus thymidine kinase (Wigler et al, 1977, Cell 77:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska & Szybalski, 192, Proc. Natl Acad. Sci. USA 48:202), and adenine phosphoribosyltransferase (Lowy et al , 1980, Cell 22:817) genes can be employed in tk-, hgprt- or aprt- cells, respectively.
• antimetabolite resistance can be used as the basis of selection for the following genes: dhfr, which confers resistance to methotrexate (Wigler et al , 1980, Natl. Acad. Sci. USA 77:357; O'Hare et al, 1981, Proc. Natl. Acad. Sci. USA 78: 1527); gpt, which confers resistance to mycophenolic acid (Mulligan & Berg, 1981. Proc. Natl. Acad. Sci.
• the expression levels of an antibody molecule can be increased by vector amplification (for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
• vector amplification for a review, see Bebbington and Hentschel, The use of vectors based on gene amplification for the expression of cloned genes in mammalian cells in DNA cloning, Vol. 3. (Academic Press, New York, 1987)).
• a marker in the vector system expressing antibody is amplifiable
• increase in the level of inhibitor present in culture of host cell will increase the number of copies ofthe marker gene. Since the amplified region is associated with the antibody gene, production ofthe antibody will also increase (Crouse et al . 1983, Mol. Cell. Biol.
• the host cell may be co-transfected with two expression vectors of the invention, the first vector encoding a heavy chain derived polypeptide and the second vector encoding a light chain derived polypeptide.
• the two vectors may contain identical selectable markers which enable equal expression of heavy and light chain polypeptides.
• a single vector may be used which encodes both heavy and light chain polypeptides. In such situations, the light chain should be placed before the heavy chain to avoid an excess of toxic free heavy chain (Proudfoot, 1986, Nature 322:52; Kohler, 1980. Proc. Natl Acad. Sci. USA 77:2197).
• the coding sequences for the heavy and light chains may comprise cDNA or genomic DNA.
• an antibody molecule of the invention may be purified by any method known in the art for purification of an immunoglobulin molecule, for example, by chromatography (e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography), centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• chromatography e.g., ion exchange, affinity, particularly by affinity for the specific antigen after Protein A, and sizing column chromatography
• centrifugation e.g., centrifugation, differential solubility, or by any other standard technique for the purification of proteins.
• the present invention encompasses antibodies recombinantly fused or chemically conjugated (including both covalently and non-covalently conjugations) to a polypeptide (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) of the present invention to generate fusion proteins.
• the fusion does not necessarily need to be direct, but may occur through linker sequences.
• the antibodies may be specific for antigens other than polypeptides
• antibodies may be used to target the polypeptides ofthe present invention to particular cell types, either in vitro or in vivo, by fusing or conjugating the polypeptides ofthe present invention to antibodies specific for particular cell surface receptors.
• Antibodies fused or conjugated to the polypeptides of the present invention may also be used in in vitro immunoassays and purification methods using methods known in the art. See e.g., Harbor et al , supra, and PCT publication WO 93/21232; EP 439,095; v mm et al., Immunol. Lett. 59:91-99 (1994); U.S.
• the present invention further includes compositions comprising the polypeptides of the present invention fused or conjugated to antibody domains other than the variable regions.
• the polypeptides of the present invention may be fused or conjugated to an antibody Fc region, or portion thereof.
• the antibody portion fused to a polypeptide ofthe present invention may comprise the constant region, hinge region, CHI domain, CH2 domain, and CH3 domain or any combination of whole domains or portions thereof.
• polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
• Fc portions fused to the polypeptides ofthe present invention can form dimers through disulfide bonding between the Fc portions.
• Higher multimeric forms can be made by fusing the polypeptides to portions of IgA and
• IgM Methods for fusing or conjugating the polypeptides ofthe present invention to antibody portions are known in the art. See, e.g., U.S. Patent Nos. 5,336,603; 5,622,929; 5,359,046; 5,349.053; 5,447,851 ; 5,1 12,946; EP 307,434; EP 367,166; PCT publications WO 96/04388; WO 91/06570; Ashkenazi etal, Proc. Natl. Acad. Sci. USA 88: 10535-10539 (1991); Zheng et al, J. Immunol.
• polypeptides of the present invention may be fused or conjugated to the above antibody portions to increase the in vivo half life ofthe polypeptides or for use in immunoassays using methods known in the art.
• polypeptides ofthe present invention may be fused or conjugated to the above antibody portions to facilitate purification.
• chimeric proteins consisting of the first two domains of the human CD4-polypeptide and various domains ofthe constant regions ofthe heavy or light chains of mammalian immunoglobulins. (EP 394,827; Traunecker et al. , Nature
• polypeptides ofthe present invention fused or conjugated to an antibody having disulfide- linked dimeric structures may also be more efficient in binding and neutralizing other molecules, than the monomeric secreted protein or protein fragment alone.
• the Fc part in a fusion protein is beneficial in therapy and diagnosis, and thus can result in, for example, improved pharmacokinetic properties.
• EP A 232,262 Alternatively, deleting the Fc part after the fusion protein has been expressed, detected, and purified, would be desired.
• the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
• human proteins such as hIL-5 receptor
• Fc portions for the pu ⁇ ose of high-throughput screening assays to identify antagonists of hIL-5.
• antibodies or fragments thereof of the present invention can be fused to marker sequences, such as a peptide to facilitates their purification.
• the marker amino acid sequence is a hexa-histidine peptide, such as the tag provided in a pQE vector (QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, C A, 9131 1), among others, many of which are commercially available.
• a pQE vector QIAGEN, Inc., 9259 Eton Avenue, Chatsworth, C A, 9131 1.
• hexa-histidine provides for convenient purification of the fusion protein.
• Other peptide tags useful for purification include, but are not limited to, the "HA" tag, which corresponds to an epitope derived from the influenza hemagglutinin protein (Wilson et al.
• the present invention further encompasses antibodies or fragments thereof conjugated to a diagnostic or therapeutic agent.
• the antibodies can be used diagnostically to, for example, monitor the development or progression of a tumor as part of a clinical testing procedure to, e.g., determine the efficacy of a given treatment and/or prevention regimens. Detection can be facilitated by coupling the antibody to a detectable substance. Examples of detectable substances include various enzymes, prosthetic groups, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, positron emitting metals using various positron emission tomographies, and nonradioactive paramagnetic metal ions. See, for example, U.S. Patent No.
• suitable enzymes include horseradish peroxidase, alkaline phosphatase, ⁇ -galactosidase, or acetylcholinesterase;
• suitable prosthetic group complexes include streptavidin/biotin and avidin/biotin;
• suitable fluorescent materials include umbelliferone, fluorescein, fluorescein isothiocyanate, rhodamine, dichlorotriazinylamine fluorescein, dansyl chloride or phycoerythrin;
• an example of a luminescent material includes luminol;
• bioluminescent materials include luciferase. luciferin, and aequorin; and
• suitable radioactive material include 125 I, ljl I, '"In or "Tc.
• an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cyto toxin, e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
• a cyto toxin e.g., a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion.
• a cytotoxin or cytotoxic agent includes any agent that is detrimental to cells.
• Examples include paclitaxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1 -dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, and puromycin and analogs or homologs thereof.
• Therapeutic agents include, but are not limited to, antimetabolites (e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine), alkylating agents (e.g., mechlorethamine, thioepa chlorambucil, melphalan, carmustine (BSNU) and lomustine (CCNU), cyclothosphamide, busulfan, dibromomannitol, streptozotocin, mitomycin C, and c/s-dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (e.g., dactinomycin (formerly actinomycin), bleomycin, mithramycin, and anthramycin (AMC)), and anti-mitotic agents (e.g
• the conjugates of the invention can be used for modifying a given biological response, the therapeutic agent or drug moiety is not to be construed as limited to classical chemical therapeutic agents.
• the drug moiety may be a protein or polypeptide possessing a desired biological activity.
• Such proteins may include, for example, a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin; a protein such as tumor necrosis factor, a-interferon, ⁇ -interferon, nerve growth factor, platelet derived growth factor, tissue plasminogen activator, a thrombotic agent or an anti-angiogenic agent, e.g., angiostatin or endostatin; or, biological response modifiers such as, for example, lymphokines, interleukin-1 ("IL-1 "), interleukin-2 (“IL-2”), interleukin-6 (“IL-6”), granulocyte macrophase colony stimulating factor (“GM-CSF”), granulocyte colony stimulating factor (“G-CSF”), or other growth factors.
• a toxin such as abrin, ricin A, pseudomonas exotoxin, or diphtheria toxin
• a protein such as tumor necrosis factor, a-
• Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification ofthe target antigen.
• solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, poly vinyl chloride or polypropylene.
• an antibody can be conjugated to a second antibody to form an antibody heteroconjugate as described by Segal in U.S. Patent No. 4,676,980, which is incorporated herein by reference in its entirety.
• An antibody, with or without a therapeutic moiety conjugated to it, administered alone or in combination with cytotoxic factor(s) and/or cytokine(s) can be used as a therapeutic.
• the antibodies ofthe invention may be assayed for immunospecific binding by any method known in the art.
• the immunoassays which can be used include but are not limited to competitive and non-competitive assay systems using techniques such as western blots, radioimmunoassays, ELISA (enzyme linked immunosorbent assay), "sandwich” immunoassays, immunoprecipitation assays, precipitin reactions, gel diffusion precipitin reactions, immunodiffusion assays, agglutination assays, complement-fixation assays, immunoradiometric assays, fluorescent immunoassays, protein A immunoassays, to name but a few.
• Immunoprecipitation protocols generally comprise lysing a population of cells in a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate atpH 7.2, 1%
• a lysis buffer such as RIPA buffer ( 1 % NP-40 or Triton X- 100, 1 % sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphate atpH 7.2, 1%
• Trasylol supplemented with protein phosphatase and/or protease inhibitors (e.g. , EDTA, PMSF, aprotinin, sodium vanadate), adding the antibody of interest to the cell lysate, incubating for a period of time (e.g. , 1 -4 hours) at 4° C, adding protein A and/or protein G sepharose beads to the cell lysate, incubating for about an hour or more at 4° C, washing the beads in lysis buffer and resuspending the beads in
• protein phosphatase and/or protease inhibitors e.g. , EDTA, PMSF, aprotinin, sodium vanadate
• the ability ofthe antibody of interest to immunoprecipitate a particular antigen can be assessed by, e.g. , western blot analysis.
• One of skill in the art would be knowledgeable as to the parameters that can be modified to increase the binding ofthe antibody to an antigen and decrease the background (e.g., pre-clearing the cell lysate with sepharose beads).
• immunoprecipitation protocols see, e.g., Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc., New
• Western blot analysis generally comprises preparing protein samples, electrophoresis of the protein samples in a polyacrylamide gel (e.g., 8%-20% SDS-PAGE depending on the molecular weight ofthe antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose.
• a polyacrylamide gel e.g., 8%-20% SDS-PAGE depending on the molecular weight ofthe antigen
• blocking the membrane in blocking solution e.g. , PBS with 3% BSA or non-fat milk
• washing buffer e.g. , PBS-Tween 20
• blocking the membrane with primary antibody the antibody of interest
• a secondary antibody which recognizes the primary antibody, e.g., an anti-human antibody
• an enzymatic substrate e.g., horseradish peroxidase or alkaline phosphatase
• radioactive molecule e.g. , 32 P or ,25 I
• ELISAs comprise preparing antigen, coating the well of a 96 well microtiter plate with the antigen, adding the antibody of interest conjugated to a detectable compound such as an enzymatic substrate (e.g.. horseradish peroxidase or alkaline phosphatase) to the well and incubating for a period of time, and detecting the presence ofthe antigen.
• a detectable compound such as an enzymatic substrate (e.g.. horseradish peroxidase or alkaline phosphatase)
• a detectable compound such as an enzymatic substrate (e.g.. horseradish peroxidase or alkaline phosphatase)
• a second antibody conjugated to a detectable compound may be added following the addition ofthe antigen of interest to the coated well.
• ELISAs see, e.g. , Ausubel et al., eds, 1994, Current Protocols in Molecular Biology, Vol. 1 , John Wiley & Sons, Inc., New York at 1 1.2.1.
• the binding affinity of an antibody to an antigen and the off-rate of an antibody-antigen interaction can be determined by competitive binding assays.
• a competitive binding assay is a radioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or ,25 I) with the antibody of interest in the presence of increasing amounts of unlabeled antigen, and the detection of the antibody bound to the labeled antigen.
• the affinity ofthe antibody of interest for a particular antigen and the binding off-rates can be determined from the data by scatchard plot analysis. Competition with a second antibody can also be determined using radioimmunoassays.
• the antigen is incubated with antibody of interest is conjugated to a labeled compound (e.g., 3 H or 125 I) in the presence of increasing amounts of an unlabeled second antibody.
• the present invention is further directed to antibody-based therapies which involve administering antibodies of the invention to an animal, preferably a mammal, and most preferably a human patient for treating and/or preventing one or more ofthe disorders or conditions described herein.
• Therapeutic compounds of the invention include, but are not limited to, antibodies of the invention (including fragments, analogs and derivatives thereof as described herein) and nucleic acids encoding antibodies ofthe invention (including fragments, analogs and derivatives thereof as described herein). While not intending to be bound to theory, TRID receptors are believed to inhibit programmed cell death by a process which involves the binding of TRID ligands (e.g., TRAIL) which are then not available to bind to receptors which - I m ⁇
• TRID ligands e.g., TRAIL
• agents e.g., antibodies which prevent binding of ligand to TRID will enhance programmed cell death.
• TRID receptors have been shown to bind TRAIL.
• TRID receptors are also known to be present in a number of tissues and on the surfaces of a number of cell types.
• TRAIL is a member ofthe TNF family of cytokines which has been shown to induce apoptotic cell death in a number of tumor cell lines and appears to mediate its apoptosis inducing effects through interaction with, e.g., DR4 and DR5 receptors. These death domain containing receptors are believed to form membrane-bound self-activating signaling complexes which initiate apoptosis through cleavage of caspases.
• TRAIL also binds to several receptors proposed to be "decoy" receptors, e.g., TRID, DcR2 (a receptor with a truncated death domain), DcRl (a GPI-anchored receptor), and OPG (a secreted protein which binds to another member of the TNF family, RANKL).
• TRID a receptor with a truncated death domain
• DcRl a GPI-anchored receptor
• OPG a secreted protein which binds to another member of the TNF family, RANKL.
• Antibodies which bind to TRID receptors are useful for treating and/or preventing diseases and conditions associated with increased or decreased apoptotic cell death. Further, these antibodies vary in the effect they have on TRID receptors. These effects differ based on the specific portions ofthe TRID receptor to which the antibodies bind, the three-dimensional conformation ofthe antibody molecules themselves, and/or the manner in which they interact with the TRID receptor. Thus, antibodies which bind to the extracellular domain of a TRID receptor can either stimulate or inhibit TRID activities (e.g. , the binding of TRAIL). Antibodies which stimulate TRID receptor's ability to bind TRAIL are TRAIL binding facilitators, and antibodies which inhibit TRID receptor activities
• TRID antagonists are TRID antagonists.
• TRID has an intracellular domain which may be involved in an intracellular signaling pathway.
• Agonists, including antibodies, are molecules which bind TRID in a manner which stimulates the intracellular signaling pathway.
• Antibodies ofthe invention which function as agonists and antagonists, and
• TRAIL binding facilitators of TRID receptors include antigen-binding antibody fragments such as Fab and F(ab') 2 fragments, Fd, single-chain Fvs (scFv), disulfide-linked Fvs (sdFv) and fragments comprising either a V L or V H domain, as well as polyclonal, monoclonal and humanized antibodies. Each of these antigen-binding antibody fragments and antibodies are described in more detail elsewhere herein.
• antibodies of the invention are useful for inhibiting TRID activity, thereby promoting apoptosis in cells which express TRID receptors (e.g. , cancer cells).
• Antibodies of this type are useful for prevention and/or treating diseases and conditions associated with increased cell survival and/or insensitivity to apoptosis-inducing agents (e.g.,
• TRAIL such as solid tissue cancers (e.g., skin cancer, head and neck tumors, breast tumors, endothelioma, lung cancer, osteoblastoma, osteoclastoma, and Kaposi's sarcoma) and leukemias.
• the invention encompasses anti-TRID antibodies that enhance the binding of TRAIL, denoted herein as TRAIL binding facilitators.
• TRAIL binding facilitators function by preventing apoptosis and are useful for preventing and/or treating diseases associated with increased apoptotic cell death. Examples of such diseases include diabetes mellitus, AIDS, neurodegenerative disorders, myelodysplastic syndromes, ischemic injury, toxin-induced liver disease, septic shock, cachexia and anorexia.
• an antagonist ofthe invention When an antagonist ofthe invention is administered to an individual for the treatment and/or prevention of a disease or condition associated with increased T-cell populations or increased cell proliferation (e.g., cancer), the antagonist may be co-administered with another agent which induces apoptosis (e.g. , TRAIL) or otherwise inhibits cell proliferation (e.g., an anti-cancer drug).
• another agent which induces apoptosis e.g. , TRAIL
• an anti-cancer drug e.g., an anti-cancer drug
• TRAIL binding facilitators of the invention are also useful for enhancing T-cell mediated immune responses, as well as preventing and/or treating diseases and conditions associated with decreased T-cell proliferation.
• Antibodies ofthe invention which enhance the binding of TRID receptor ligands to TRID receptors can inhibit T-cell apoptosis. The inhibition of apoptosis can, for example, either result in an increase in the expansion rate of in vivo T-cell populations or prevent a decrease in the size of such populations.
• TRAIL binding facilitators ofthe invention can be used to prevent and/or treat diseases or conditions associated with decreased or decreases in T-cell populations.
• TRAIL binding facilitator ofthe invention When a TRAIL binding facilitator ofthe invention is administered to an individual for the treatment and/or prevention of a disease or condition associated with decreased T-cell populations, the TRAIL binding facilitator may be co-administered with an agent which activates and/or induces lymphocyte proliferation (e.g., a cytokine).
• an agent which activates and/or induces lymphocyte proliferation e.g., a cytokine
• TRID antibodies are thus useful for treating and/or preventing malignancies, abnormalities, diseases and/or conditions involving tissues and cell types which express TRID receptors. Further, malignancies, abnormalities, diseases and/or conditions which can be treated and/or prevented by the induction of programmed cell death in cells which express TRID receptors can be treated and/or prevented using TRID receptor antagonists of the invention. Similarly, malignancies, abnormalities, diseases and/or conditions which can be treated and/or prevented by inhibiting programmed cell death in cells which express TRID receptors can be treated and/or prevented using TRID receptor TRAIL binding facilitators of the invention.
• antibodies ofthe present invention may be used therapeutically in a number of ways.
• antibodies which bind polynucleotides or polypeptides ofthe present invention can be administered to an individual (e.g., a human) either locally or systemically. Further, these antibodies can be administered alone, in combination with another therapeutic agent, or associated with or bound to a toxin.
• TRID antibodies may be utilized in combination with other monoclonal or chimeric antibodies, or with lymphokines, tumor necrosis factors or TNF-related molecules (e.g., TNF- ⁇ , TNF- ⁇ , TNF- ⁇ - ⁇ , TNF- ⁇ - ⁇ , and TRAIL), or hematopoietic growth factors (e.g. , IL-2, IL-3 and IL-7).
• TNF- ⁇ , TNF- ⁇ , TNF- ⁇ - ⁇ , TNF- ⁇ - ⁇ , TNF- ⁇ - ⁇ , and TRAIL hematopoietic growth factors
• antagonistic TRID antibodies may be administered in conjunction with TRAIL when one seeks to induce programmed cell death in cells which express TRID receptors ofthe invention. Combination therapies of this nature, as well as other combination therapies, are discussed below in more detail.
• the antibodies of the invention may be administered alone or in combination with other types of treatments (e.g. , radiation therapy, chemotherapy, hormonal therapy, immunotherapy and anti-tumor agents). Generally, administration of products of a species origin or species reactivity (in the case of antibodies) that is the same species as that ofthe patient is preferred. Thus, in a preferred embodiment, human antibodies, fragments derivatives, analogs, or nucleic acids, are administered to a human patient for therapy or prophylaxis.
• polypeptides or polynucleotides of the present invention It is preferred to use high affinity and/or potent in vivo inhibiting and/or neutralizing antibodies against polypeptides or polynucleotides of the present invention, fragments or regions thereof, for both immunoassays directed to and therapy of disorders related to polynucleotides or polypeptides, including fragments thereof, of the present invention.
• Such antibodies, fragments, or regions will preferably have an affinity for polynucleotides or polypeptides, including fragments thereof.
• Preferred binding affinities include those with a dissociation constant or Kd less than 5X10 "6 M, 10 "6 M, 5X10 '7 M, 10 "7 M, 5X 10 “8 M, 10- S M, 5X10- 9 M, 10- 9 M, 5X10-'°M, 10-'°M, 5X10-"M, 10-"M, 5X10- ,2 M, 10-' 2 M, 5X10 " ' 3 M, 10 "I3 M, 5X10 " ' 4 M, 10 "14 M, 5X10 " ' 5 M, and 10 " ' 5 M.
• TRID is expressed in hematopoeitic tissues and other normal human tissues.
• substantially altered (increased or decreased) levels of TRID gene expression can be detected in immune system tissue or other cells or bodily fluids (e.g., sera and plasma) taken from an individual having such a disorder, relative to a "standard" TRID gene expression level, that is, the TRID expression level in immune system tissues or bodily fluids from an individual not having the immune system disorder.
• the invention provides a diagnostic method useful during diagnosis of an immune system disorder, which involves measuring the expression level ofthe gene encoding the TRID protein in immune system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard TRID gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disorder.
• the invention provides a diagnostic method useful during diagnosis of an immune system disorder, including cancers which involves measuring the expression level ofthe gene encoding the TRID protein in immune system tissue or other cells or body fluid from an individual and comparing the measured gene expression level with a standard TRID gene expression level, whereby an increase or decrease in the gene expression level compared to the standard is indicative of an immune system disorder.
• a diagnosis of a disorder in the immune system including diagnosis of a tumor has already been made according to conventional methods
• the present invention is useful as a prognostic indicator, whereby patients exhibiting altered
• saying the expression level ofthe gene encoding a TRID protein is intended qualitatively or quantitatively measuring or estimating the level of TRID or the level ofthe mRNA encoding TRID in a first biological sample either directly (e.g. , by determining or estimating absolute protein level or mRNA level) or relatively (e.g., by comparing to the TRID protein level or mRNA level in a second biological sample).
• the TRID protein level or mRNA level in the first biological sample is measured or estimated and compared to a standard TRID protein level or mRNA level, the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder ofthe immune system.
• a standard TRID protein level or mRNA level the standard being taken from a second biological sample obtained from an individual not having the disorder or being determined by averaging levels from a population of individuals not having a disorder ofthe immune system.
• biological sample any biological sample obtained from an individual, body fluid, cell line, tissue culture, or other source which contains
• TRID protein or mRNA include body fluids (such as sera, plasma, urine, synovial fluid and spinal fluid) which contain free extracellular domain(s) (or soluable form(s)) of a TRID protein, immune system tissue, and other tissue sources found to express complete or extracellular domain of TRID. Methods for obtaining tissue biopsies and body fluids from mammals are well known in the art. Where the biological sample is to include mRNA, a tissue biopsy is the preferred source.
• the invention also contemplates the use of a gene ofthe present invention for diagnosing mutations in the TRID gene. For example, if a mutation is present in one ofthe genes ofthe present invention, conditions would result from a lack of production of the receptor polypeptides of the present invention. Further, mutations which enhance receptor polypeptide activity would lead to diseases associated with an over expression of the receptor polypeptide, e.g., endotoxic shock. Mutations in the genes can be detected by comparing the sequence ofthe defective gene with that of a normal one. Subsequently one can verify that a mutant gene is associated with a disease condition or the susceptibility to a disease condition. That is, a mutant gene which leads to the overexpression of TRID would be associated with an inability of TRAIL to inhibit tumor growth.
• immune system disorders which may be diagnosed by the foregoing assays include hypersensitivity, allergy, infectious disease, graft-host disease, immunodeficiency, autoimmune diseases and the like.
• Nucleic acids used for diagnosis may be obtained from a patient's cells, such as from blood, urine, saliva and tissue biopsy among other tissues.
• the genomic DNA may be used directly for detection or may be amplified enzymatically by using PCR (Saiki et al., Nature,
• RNA or cDNA may also be used for the same pu ⁇ ose.
• PCR primers complementary to the nucleic acid ofthe instant invention can be used to identify and analyze mutations in the human genes of the present invention. For example, deletions and insertions can be detected by a change in the size of the amplified product in comparison to the normal genotype. Point mutations can be identified by hybridizing amplified DNA to radiolabeled RNA or alternatively, radiolabeled antisense DNA sequences of the present invention. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures. Such a diagnostic would be particularly useful for prenatal or even neonatal testing.
• Sequence differences between the reference gene and "mutants" may be revealed by the direct DNA sequencing method.
• cloned DNA segments may be used as probes to detect specific DNA segments.
• the sensitivity of this method is greatly enhanced when combined with PCR.
• a sequencing primary used with double stranded PCR product or a single stranded template molecule generated by a modified PCR product
• the sequence determination is performed by conventional procedures with radiolabeled nucleotides or by automatic sequencing procedures with fluorescent tags
• Sequence changes at the specific locations may be revealed by nuclease protection assays, such as RNase and S 1 protection or the chemical cleavage method (for example, Cotton et al , PNAS, 85 4397-4401 (1985))
• TRID protein levels in a biological sample can occur using antibody-based techniques
• TRID protein expression in tissues can be studied with classical immunohistological methods (Jalkanen, M , et al , J Cell Biol 101 976-985 (1985), Jalkanen, M . etal J Cell Biol 105 3087-3096
• antibody-based methods useful for detecting TRID gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA)
• ELISA enzyme linked immunosorbent assay
• RIA radioimmunoassay
• Suitable antibody assay labels include enzyme labels, such as, glucose oxidase, and radioisotopes, such as iodine ( 125 I, 121 I), carbon ( l4 C), sulfur ( 35 S), tritium ( 3 H), indium (' ,2 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin
• TRID proteins can also be detected in vivo by imaging
• Antibody labels or markers for in vivo imaging of TRID proteins include those detectable by X-radiography, NMR or ESR
• suitable labels include radioisotopes such as barium or cesium, which emit detectable radiation but are not overtly harmful to the subject
• Suitable markers for NMR and ESR include those with a detectable characteristic spin, such as deuterium, which may be inco ⁇ orated mto the antibody by labeling of nutrients for the relevant hybridoma
• a TRID-specific antibody or antibody fragment which has been labeled with an appropriate detectable imaging moiety such as a radioisotope (for example, l31 I, " 2 In, 99m Tc), a radio-opaque substance, or a material detectable by nuclear magnetic resonance, is introduced (for example, parenterally, subcutaneously or intraperitoneally) into the mammal to be examined for immune system disorder.
• a radioisotope for example, l31 I, " 2 In, 99m Tc
• a radio-opaque substance for example, parenterally, subcutaneously or intraperitoneally
• the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99 Tc.
• the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain TRID protein.
• In vivo tumor imaging is described in S.W. Burchiel et al., "Immunopharmacokinetics of Radiolabeled Antibodies and Their Fragments" (Chapter 13 in Tumor Imaging: The Radiochemical Detection of Cancer, S.W. Burchiel and B. A. Rhodes, eds., Masson Publishing Inc. (1982)).
• Tumor Necrosis Factor (TNF) family ligands are known to be among the most pleiotropic cytokines, inducing a large number of cellular responses, including cytotoxicity, anti-viral activity, immunoregulatory activities, and the transcriptional regulation of several genes (Goeddel, D.V. etal. , "Tumor Necrosis Factors: Gene Structure and Biological Activities," Symp. Quant. Biol. 51:591- 609 (1986), Cold Spring Harbor; Beutler, B., and Cerami, A., Annu. Rev Biochem. 57:505-518 (1988); Old, L.J., Sci. Am. 255:59-75 (1988); Fiers, W.,
• TNF-family ligands induce such various cellular responses by binding to TNF-family receptors.
• Cells which express a TRID polypeptide and are believed to have a potent cellular response to TNFR ligands include lymphocytes, endothelial cells, keratinocytes, and prostate tissue.
• a cellular response to a TNF-family ligand is intended any genotypic, phenotypic, and/or morphologic change to a cell, cell line, tissue, tissue culture or patient that is induced by a TNF-family ligand.
• TRID polynucleotides, polynucleotides, TRAIL binding facilitators, agonists, and/or antagonists of the invention may be administered to a patient (e.g. , mammal, preferably human) afflicted with any disease or disorder mediated (directly or indirectly) by defective, or deficient levels of, TRID.
• a gene therapy approach may be applied to treat such diseases or disorders.
• TRID polynucleotide sequences are used to detect mutein TRID genes, including defective genes.
• Mutein genes may be identified in in vitro diagnostic assays, and by comparison ofthe TRID nucleotide sequence disclosed herein with that of a TRID gene obtained from a patient suspected of harboring a defect in this gene. Defective genes may be replaced with normal TRID -encoding genes using techniques known to one skilled in the art.
• the TRID polypeptides, polynucleotides, TRAIL binding facilitators, agonists, and/or antagonists ofthe present invention are used as research tools for studying the phenotypic effects that result from inhibiting TRAIL/TRID interactions on various cell types.
• TRID polypeptides and antagonists e.g. monoclonal antibodies to TRID
• in vitro assays for detecting TRAIL or TRID or the interactions thereof. It has been reported that certain ligands of the TNF family (of which
• TRAIL is a member) bind to more than one distinct cell surface receptor protein.
• a receptor protein designated DR4 reportedly binds TRAIL, but is distinct from the TRID ofthe present invention (Pan et al, Science 276: 1 1-113, (1997); hereby inco ⁇ orated by reference).
• a purified TRID polypeptide, TRAIL binding facilitator, agonist, and/or antagonist is used to inhibit binding of TRAIL to endogenous cell surface TRAIL receptors.
• soluble TRID polypeptides ofthe present invention may be employed to inhibit the interaction of TRAIL not only with cell surface TRID, but also with TRAIL receptor proteins distinct from TRID.
• TRAIL binding facilitators, agonists, and/or antagonists ofthe invention are used to inhibit a functional activity of TRAIL, in in vitro or in vivo procedures.
• TRID By inhibiting binding of TRAIL to cell surface receptors, TRID also inhibits biological effects that result from the binding of TRAIL to endogenous receptors.
• Various forms of TRID may be employed, including, for example, the above-described
• TRID fragments, derivatives, and variants that are capable of binding TRAIL.
• a soluble TRID is employed to inhibit a functional activity of TRAIL, e.g. , to inhibit TRAIL-mediated apoptosis of cells susceptible to such apoptosis.
• TRID is administered to a mammal (e.g., a human) to treat a TRAIL-mediated disorder.
• TRAIL-mediated disorders include conditions caused (directly or indirectly) or exacerbated by TRAIL.
• cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, including, but not limited to colon cancer, cardiac tumors, pancreatic cancer, melanoma, retinoblastoma, glioblastoma, lung cancer, intestinal cancer, testicular cancer, stomach cancer, neuroblastoma, myxoma, myoma, lymphoma, endothelioma, osteoblastoma, osteoclastoma, osteosarcoma, chondrosarcoma, adenoma, breast cancer, prostate cancer, Kaposi's sarcoma and ovarian cancer); autoimmune disorders (such as multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis. biliary cirrhosis,
• TRID polynucleotides, polypeptides, and/or antagonists of the invention are used to inhibit growth, progression, and/or metasis of cancers, in particular those listed above and in the following paragraph.
• Additional diseases or conditions associated with increased cell survival include, but are not limited to, progression, and/or metastases of malignancies and related disorders such as leukemia (including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)) and chronic leukemias (e.g., chronic myelocytic (granulocytic) leukemia and chronic lymphocytic leukemia)), polycythemia vera, lymphomas (e.g.
• leukemia including acute leukemias (e.g., acute lymphocytic leukemia, acute myelocytic leukemia (including myeloblastic, promyelocytic, myelomonocytic, monocytic, and erythroleukemia)
• chronic leukemias e.g., chronic mye
• sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma, endothehosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma, basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous gland carcinoma, papillary carcinoma, papillary adenocarcinomas, cysta
• sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenic sarcoma, chordo
• AIDS dementia
• neurodegenerative disorders such as Alzheimer's disease, Parkinson's disease, Amyotrophic lateral sclerosis, Retinitis pigmentosa, Cerebellar degeneration and brain tumor or prior associated disease
• autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus and immune- related glomerulonephritis and rheumatoid arthritis
• myelodysplastic syndromes such as aplastic anemia
• graft v graft v.
• ischemic injury such as that caused by myocardial infarction, stroke and reperfusion injury
• liver injury such as hepatitis related liver injury, ischemia reperfusion injury, cholestosis (bile duct injury) and liver cancer
• toxin-induced liver disease such as that caused by alcohol
• TRID polynucleotides, polypeptides TRAIL binding facilitators, and/or agonists are used to treat the diseases and disorders listed above.
• TRID polynucleotides, polypeptides, TRAIL binding facilitators, and/or TRID agonists of the invention are used to treat AIDS and pathologies associated with AIDS.
• Another embodiment of the present invention is directed to the use of TRID to reduce TRAIL-mediated death of T cells in HIV-infected patients.
• T cell apoptosis The role of T cell apoptosis in the development of AIDS has been the subject of a number of studies (see. for example, Meyaard et al, Science 257:217-219, 1992; Groux et al, J Exp. Med., 775:331, 1992; and Oyaizu et al, in Cell Activation and Apoptosis in HIV Infection, Andrieu and Lu, Eds., Plenum Press, New York, 1995, pp. 101-1 14). Fas-mediated apoptosis has been implicated in the loss of T cells in HIV individuals (Katsikis et al, J. Exp. Med. 757:2029-2036, 1995).
• HIV-induced apoptotic cell death has been demonstrated not only in vitro but also, more importantly, in infected individuals (Ameisen, J.C., AIDS 5: 1 197-1213 (1994) ; Finkel, T.H., and Banda, N.K., Curr. Opin. Immunol. (5:605-615(1995); Muro- Cacho, CA. et al, J. Immunol 154:5555-5566 (1995)).
• apoptosis and CD4 + T-lymphocyte depletion is tightly correlated in different animal models of AIDS (Brunner, T., et al, Nature 575:441-444 (1995); Gougeon, M.L., et al, AIDS Res. Hum. Retroviruses 9:553-563 (1993)) and, apoptosis is not observed in those animal models in which viral replication does not result in AIDS (Gougeon, M.L. et al., AIDS Res. Hum. Retroviruses 9:553-563 (1993)). Further data indicates that uninfected but primed or activated T lymphocytes from HIV- infected individuals undergo apoptosis after encountering the TNF-family ligand FasL.
• a method for treating HIV * individuals involves administering soluble TRID (e.g. , the extracellular domain) and/or
• activated human T-cells are believed to be induced to undergo programmed cell death (apoptosis) upon triggering through the CD3/T-cell receptor complex, a process termed activated-induced cell death
• AICD AICD of CD4 * T-cells isolated from HIV-infected asymptomatic individuals has been reported (Groux et al, supra). Thus. AICD may play a role in the depletion of CD4 + T-cells and the progression to AIDS in HIV-infected individuals.
• the present invention provides a method of inhibiting TRAIL-mediated T-cell death in HIV patients, comprising administering a TRID polypeptide of the invention (preferably, a soluble TRID polypeptide) and/or TRID agonist of the invention to the patients. Modes of administration and dosages are discussed in detail below.
• the patient is asymptomatic when treatment with TRID commences.
• peripheral blood T-cells may be extracted from an HIV patient, and tested for susceptibility to TRAIL-mediated cell death by procedures known in the art.
• a patient's blood or plasma is contacted with TRID polypeptides ofthe invention ex vivo.
• the TRID polypeptides of the invention may be bound to a suitable chromatography matrix by procedures known in the art.
• the patient's blood or plasma flows through a chromatography column containing TRID bound to the matrix, before being returned to the patient.
• the immobilized TRID polypeptide binds TRAIL, thus removing TRAIL protein from the patient's blood.
• a TRID polypeptide and/or agonist of the invention is administered in combination with other inhibitors of T-cell apoptosis.
• Fas-mediated apoptosis also has been implicated in loss of T-cells in HIV individuals (Katsikis et al, J. Exp. Med. 181 :2029-2036, 1995).
• a patient susceptible to both Fas ligand mediated and TRAIL mediated T-cell death may be treated with both an agent that blocks TRAIL/TRAIL receptor interactions and an agent that blocks Fas-ligand/Fas interactions.
• Suitable agents for blocking binding of Fas-ligandto Fas include, but are not limited to, soluble Fas polypeptides; multimeric forms of soluble Fas polypeptides (e.g...
• dimers of sFas/Fc dimers of sFas/Fc
• anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis
• anti-Fas-ligand antibodies that block binding of Fas-ligand to Fas
• muteins of Fas-ligand that bind Fas but do not transduce the biological signal that results in apoptosis.
• the antibodies employed according to this method are monoclonal antibodies.
• suitable agents for blocking Fas-ligand/Fas interactions including blocking anti-Fas monoclonal antibodies, are described in International application publication number WO 95/10540. hereby inco ⁇ orated by reference.
• TRID polypeptides or polynucleotides encoding TRID of the invention may be used to treat cardiovascular disorders, including peripheral artery disease, such as limb ischemia.
• Cardiovascular disorders include cardiovascular abnormalities, such as arterio-arterial fistula, arteriovenous fistula, cerebral arteriovenous malformations, congenital heart defects, pulmonary atresia, and Scimitar Syndrome.
• Congenital heart defects include aortic coarctation, cor triatriatum, coronary vessel anomalies, crisscross heart, dextrocardia, patent ductus arteriosus, Ebstein's anomaly, Eisenmenger complex, hypoplastic left heart syndrome, levocardia, tetralogy of fallot, transposition of great vessels, double outlet right ventricle, tricuspid atresia, persistent truncus arteriosus, and heart septal defects, such as aortopulmonary septal defect, endocardial cushion defects, Lutembacher's Syndrome, trilogy of Fallot, thrombotic microangiopathies (e.g.
• Cardiovascular disorders also include heart disease, such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac output, cardiac tamponade, endocarditis (including bacterial), heart aneurysm, cardiac arrest, congestive heart failure, congestive cardiomyopathy, paroxysmal dyspnea, cardiac edema, heart hypertrophy, congestive cardiomyopathy, left ventricular hypertrophy, right ventricular hypertrophy, post-infarction heart rupture, ventricular septal rupture, heart valve diseases, myocardial diseases, myocardial ischemia, pericardial effusion, pericarditis (including constrictive and tuberculous), pneumopericardium, postpericardiotomy syndrome, pulmonary heart disease, rheumatic heart disease, ventricular dysfunction, hyperemia, cardiovascular pregnancy complications, Scimitar Syndrome, cardiovascular s
• Arrhythmias include sinus arrhythmia, atrial fibrillation, atrial flutter, bradycardia, extrasystole, Adams-Stokes Syndrome, bundle-branch block, sinoatrial block, long QT syndrome, parasystole, Lown-Ganong-Levine Syndrome, Mahaim-type pre-excitation syndrome, Wolff-Parkinson-White syndrome, sick sinus syndrome, tachycardias, and ventricular fibrillation.
• Tachycardias include paroxysmal tachycardia, supraventricular tachycardia, accelerated idioventricular rhythm, atrioventricular nodal reentry tachycardia, ectopic atrial tachycardia, ectopic junctional tachycardia, sinoatrial nodal reentry tachycardia, sinus tachycardia, Torsades de Pointes, and ventricular tachycardia.
• Heart valve disease include aortic valve insufficiency, aortic valve stenosis, hear murmurs, aortic valve prolapse, mitral valve prolapse, tricuspid valve prolapse, mitral valve insufficiency, mitral valve stenosis, pulmonary atresia, pulmonary valve insufficiency, pulmonary valve stenosis, tricuspid atresia, tricuspid valve insufficiency, and tricuspid valve stenosis.
• Myocardial diseases include alcoholic cardiomyopathy, congestive cardiomyopathy, hypertrophic cardiomyopathy, aortic subvalvular stenosis, pulmonary subvalvular stenosis, restrictive cardiomyopathy, Chagas cardiomyopathy, endocardial fibroelastosis, endomyocardial fibrosis, Kearns Syndrome, myocardial reperfusion injury, and myocarditis.
• Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
• Cardiovascular diseases also include vascular diseases such as aneurysms, angiodysplasia, angiomatosis, bacillary angiomatosis, Hippel-Lindau Disease,
• Klippel-Trenaunay- Weber Syndrome Sturge-Weber Syndrome, angioneurotic edema, aortic diseases, Takayasu's Arteritis, aortitis, Leriche's Syndrome, arterial occlusive diseases, arteritis, enarteritis, polyarteritis nodosa, cerebrovascular disorders, diabetic angiopathies, diabetic retinopathy, embolisms, thrombosis, erythromelalgia, hemorrhoids, hepatic veno-occlusive disease, hypertension, hypotension, ischemia, peripheral vascular diseases, phlebitis, pulmonary veno- occlusive disease, Raynaud's disease, CREST syndrome, retinal vein occlusion, Scimitar syndrome, superior vena cava syndrome, telangiectasia, atacia telangiectasia, hereditary hemorrhagic telangiect
• Aneurysms include dissecting aneurysms, false aneurysms, infected aneurysms, ruptured aneurysms, aortic aneurysms, cerebral aneurysms, coronary aneurysms, heart aneurysms, and iliac aneurysms.
• Arterial occlusive diseases include arteriosclerosis, intermittent claudication, carotid stenosis, fibromuscular dysplasias, mesenteric vascular occlusion, Moyamoya disease, renal artery obstruction, retinal artery occlusion, and thromboangiitis obliterans.
• Cerebrovascular disorders include carotid artery diseases, cerebral amyloid angiopathy, cerebral aneurysm, cerebral anoxia, cerebral arteriosclerosis, cerebral arteriovenous malformation, cerebral artery diseases, cerebral embolism and thrombosis, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, cerebral hemorrhage, epidural hematoma, subdural hematoma, subaraxhnoid hemorrhage, cerebral infarction, cerebral ischemia (including transient), subclavian steal syndrome, periventricular leukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
• Embolisms include air embolisms, amniotic fluid embolisms, cholesterol embolisms, blue toe syndrome, fat embolisms, pulmonary embolisms, and thromoboembolisms.
• Thrombosis include coronary thrombosis, hepatic vein thrombosis, retinal vein occlusion, carotid artery thrombosis, sinus thrombosis, Wallenberg's syndrome, and thrombophlebitis.
• Ischemia includes cerebral ischemia, ischemic colitis, compartment syndromes, anterior compartment syndrome, myocardial ischemia, reperfusion injuries, and peripheral limb ischemia.
• Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein-
• Henoch pu ⁇ ura, allergic cutaneous vasculitis, and Wegener's granulomatosis Henoch pu ⁇ ura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
• TRID polynucleotides, polypeptides, TRAIL binding facilitators, and/or agonists of the invention are used to treat and/or prevent thrombotic microangiopathies.
• One such disorder is thrombotic thrombocytopenic pu ⁇ ura (TTP) (Kwaan, H.C., Semin. Hematol 24:11 (1987); Thompson et al,
• Plasma of TTP patients thus is thought to contain one or more factors that directly or indirectly induce apoptosis.
• TRAIL is present in the serum of TTP patients, and is likely to play a role in inducing apoptosis of microvascular endothelial cells.
• HUS hemolytic-uremic syndrome
• the invention is directed to use of TRID to treat and/or prevent the condition that is often referred to as "adult HUS" (even though it can strike children as well).
• a disorder known as childhood/diarrhea-associated HUS differs in etiology from adult HUS.
• conditions characterized by clotting of small blood vessels may be treated and/or prevented using TRID Such conditions include, but are not limited to, those described herein. For example, cardiac problems seen in about 5-10% of pediatric AIDS patients are believed to involve clotting of small blood vessels. Breakdown ofthe microvasculature in the heart has been reported in multiple sclerosis patients.
• a patient's blood or plasma is contacted TRID polynucleotides and/or polypeptides of the invention may be bound to a suitable chromatography matrix by procedures known in the art.
• the patient's blood or plasma flows through a chromatography column containing TRID polynucleotides and/or polypeptides of the invention bound to the matrix, before being returned to the patient.
• the immobilized TRID binds TRAIL, thus removing TRAIL protein from the patient's blood.
• TRID polynucleotides and/or polypeptides ofthe invention may be administered in vivo to a patient afflicted with a thrombotic microangiopathy.
• a soluble form of TRID polypeptide ofthe invention is administered to the patient.
• the present invention provides a method for treating and/or preventing a thrombotic microangiopathy, involving use of an effective amount of TRID.
• a TRID polypeptide may be employed in in vivo or ex vivo procedures, to inhibit TRAIL-mediated damage to (e.g. , apoptosis of) microvascular endothelial cells.
• TRID polynucleotides and/or polypeptides of the invention may be employed in combination with other agents useful in treating and/or preventing a particular disorder.
• other agents useful in treating and/or preventing a particular disorder For example, in an in vitro study reported by Laurence et al. (Blood 57:3245 (1996)), some reduction of TTP plasma-mediated apoptosis of microvascular endothelial cells was achieved by using an anti-Fas blocking antibody, aurintricarboxylic acid, or normal plasma depleted of cryoprecipitate.
• a patient may be treated with a polynucleotide and/or polypeptide of the invention in combination with an agent that inhibits Fas-ligand-mediated apoptosis of endothelial cells, such as, for example, an agent described above.
• an agent that inhibits Fas-ligand-mediated apoptosis of endothelial cells such as, for example, an agent described above.
• TRID polynucleotides and/or polypeptides ofthe invention and an anti-FAS blocking antibody are both administered to a patient afflicted with a disorder characterized by thrombotic microangiopathy, such as TTP or HUS.
• a disorder characterized by thrombotic microangiopathy such as TTP or HUS.
• Examples of blocking monoclonal antibodies directed against Fas antigen (CD95) are described in International patent application publication number WO 95/10540, hereby inco ⁇ orated by reference.
• angiogenesis is stringently regulated and spatially and temporally delimited. Under conditions of pathological angiogenesis such as that characterizing solid tumor growth, these regulatory controls fail. Unregulated angiogenesis becomes pathologic and sustains progression of many neoplastic and non-neoplastic diseases.
• a number of serious diseases are dominated by abnormal neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis. See, e.g. , reviews by Moses et al, Biotech. 9:630-634 (1991); Folkman et al, N. Engl J. Med, 555:1757-1763 (1995); Auerbach et al, J. Microvasc. Res. 29:401-41 1 (1985); Folkman, Advances in Cancer Research, eds. Klein and Weinhouse, Academic Press, New York, pp.
• the present invention provides for treatment of diseases or disorders associated with neovascularization by administration ofthe TRID polynucleotides and/or polypeptides ofthe invention (including TRID agonists and/or antagonists).
• Malignant and metastatic conditions which can be treated with the polynucleotides and polypeptides of the invention include, but are not limited to those malignancies, solid tumors, and cancers described herein and otherwise known in the art (for a review of such disorders, see Fishman et al., Medicine, 2d Ed., J. B. Lippincott Co., Philadelphia (1985)).
• ocular disorders associated with neovascularization which can be treated with the TRID polynucleotides and polypeptides of the present invention (including TRID agonists and TRID antagonists) include, but are not limited to: neovascular glaucoma, diabetic retinopathy, retinoblastoma, retrolental fibroplasia, uveitis, retinopathy of prematurity macular degeneration, corneal graft neovascularization, as well as other eye inflammatory diseases, ocular tumors and diseases associated with choroidal or iris neovascularization. See, e.g. , reviews by Waltman et al, Am. J. Ophthal 55:704-710 (1978) and Gartner et al, Surv.
• disorders which can be treated with the TRID polynucleotides and polypeptides of the present invention include, but are not limited to, hemangioma, arthritis, psoriasis, angiofibroma, atherosclerotic plaques, delayed wound healing, granulations, hemophilic joints, hypertrophic scars, nonunion fractures, Osler- Weber syndrome, pyogenic granuloma, scleroderma, trachoma, and vascular adhesions.
• the immune system ofthe recipient animal In rejection of an allograft, the immune system ofthe recipient animal has not previously been primed to respond because the immune system for the most part is only primed by environmental antigens. Tissues from other members ofthe same species have not been presented in the same way that, for example, viruses and bacteria have been presented.
• immunosuppressive regimens are designed to prevent the immune system from reaching the effector stage.
• the immune profile of xenograft rejection may resemble disease recurrence more than allograft rejection.
• the immune system In the case of disease recurrence, the immune system has already been activated, as evidenced by destruction of the native islet cells. Therefore, in disease recurrence the immune system is already at the effector stage.
• Antagonist of the present invention are able to suppress the immune response to both allografts and xenografts because lymphocytes activated and differentiated into effector cells will express TRID polypeptides, and thereby are susceptible to compounds which enhance TRID activity.
• the present invention further provides a method for creating immune privileged tissues.
• TRID antagonists or agonists ofthe invention may be useful for treating and/or preventing inflammatory diseases, such as rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in the diagnosis and treatment or prevention of a wide range of diseases and/or conditions.
• diseases and conditions include, but are not limited to, cancer (e.g., immune cell related cancers, breast cancer, prostate cancer, ovarian cancer, follicular lymphoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-small cell carcinoma ofthe lung, small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy), microbial (e.g., viral, bacterial, etc.) infection (e.g.,
• HIV-1 infection HIV-2 infection
• he ⁇ esvirus infection including, but not limited to, HSV-1, HSV-2, CMV, VZV, HHV-6, HHV-7, EBV
• adenovirus infection poxvirus infection
• human papilloma virus infection hepatitis infection
• hepatitis infection e.g. , HAV, HBV, HCV, etc.
• Helicobacter pylori infection invasive Staphylococcia, etc.
• parasitic infection nephritis
• bone disease e.g., osteoporosis
• atherosclerosis pain
• cardiovascular disorders e.g.
• neovascularization e.g., hypovascularization or reduced circulation
• ischemic disease e.g., myocardial infarction, stroke, etc.
• AIDS allergy, inflammation, neurodegenerative disease (e.g. , Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis, pigmentary retinitis, cerebellar degeneration, etc.), graft rejection (acute and chronic), graft vs.
• osteomyelodysplasia e.g., aplastic anemia, etc.
• joint tissue destruction in rheumatism liver disease (e.g., acute and chronic hepatitis, liver injury, and cirrhosis)
• autoimmune disease e.g., multiple sclerosis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopemc pu ⁇ ura,
• cardiomyopathy e.g., dilated cardiomyopathy
• diabetes e.g., diabetic complications
• asthma e.g., psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.
• diabetic complications e.g., diabetic nephropathy, diabetic neuropathy, diabetic retinopathy
• influenza e.g., asthma, psoriasis, glomerulonephritis, septic shock, and ulcerative colitis.
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in promoting angiogenesis, and wound healing (e.g., wounds, burns, and bone fractures), and regulating hematopoiesis.
• Polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are also useful as an adjuvant to enhance immune responsiveness to specific antigen, anti-viral immune responses,. More generally, polynucleotides and/or polypeptides of the invention and/or agonists and/or antagonists thereof are useful in regulating (i.e., elevating or reducing) immune response.
• polynucleotides and/or polypeptides ofthe invention may be useful in preparation or recovery from surgery, trauma, radiation therapy, chemotherapy, and transplantation, or may be used to boost immune response and/or recovery in the elderly and immunocompromised individuals.
• polynucleotides and/or polypeptides ofthe invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment or prevention of autoimmune disorders.
• polynucleotides and/or polypeptides ofthe invention are used to treat or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.
• the present invention is directed to a method for enhancing apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses a TNFR polypeptide an effective amount of an antagonist of the TRID polypeptide, capable of inhibiting TRID expression or its ligand binding ability (e.g. , to TRAIL).
• an antagonist of the TRID polypeptide capable of inhibiting TRID expression or its ligand binding ability (e.g. , to TRAIL).
• TNFR mediated signaling is increased to treat a disease wherein decreased apoptosis is exhibited.
• Antagonist can include monoclonal antibodies directed against the TRID polypeptide.
• antagonist is intended naturally occurring and synthetic compounds capable of enhancing or potentiating apoptosis.
• agonist is intended naturally occurring and synthetic compounds capable of inhibiting apoptosis. Whether any candidate "antagonist” or “agonist” ofthe present invention can enhance or inhibit apoptosis can be determined using art-known TNF-family ligand/receptor cellular response assays, including those described in more detail below.
• One such screening procedure involves the use of melanophores which are transfected to co-express a TNFR receptor which binds a TRAIL such as DR4 or
• TRID receptor polypeptide of the present invention Such a screening technique is described in PCT WO 92/01810, published February 6, 1992. Such an assay may be employed, for example, for screening for a compound which inhibits (or enhances) the activity ofthe receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptors with both a TNF-family ligand and the candidate antagonist (or agonist). Inhibition or enhancement ofthe signal generated by the ligand indicates that the compound is an antagonist or agonist of TRID activity.
• the TRID polypeptide and its agonists inhibit activation of the TNFR receptor, e.g., TRAIL receptor, whereas antagonists will increase activation.
• TRAIL receptor and TRID for example, transfected CHO cells
• compounds may be contacted with a cell which expresses a TRAIL receptor polypeptide and TRID ofthe present invention and a second messenger response, e.g. , signal transduction or pH changes, may be measured to determine whether the potential compound activates or inhibits the TRAIL receptor.
• Another such screening technique involves introducing RNA encoding the receptors into Xenopus oocytes to transiently express TRID and a TRAIL receptor.
• the receptor oocytes may then be contacted with the receptor ligand and a compound to be screened, followed by detection of inhibition or activation of a calcium signal in the case of screening for compounds which are thought to inhibit activation ofthe receptor.
• Another screening technique involves expressing in cells a construct wherein the TRAIL receptor is linked to a phospholipase C or D.
• Such cells include endothelial cells, smooth muscle cells, embryonic kidney cells, etc.
• the screening may be accomplished as hereinabove described by detecting activation ofthe receptor or inhibition of activation ofthe receptor from the phospholipase signal in the presence of TRID either co-expressed or added in soluble form along with the candidate compound.
• Another method involves screening for compounds which inhibit activation of a TRAIL receptor polypeptide in the presence ofthe TRID polypeptide ofthe present invention, either co-expressed or in soluble form.
• Agonists ofthe present invention are identified by determining inhibition of binding of labeled ligand to cells which have the TRAIL receptor on the surface thereof.
• Such a method involves transfecting a eukaryotic cell with DNA encoding a TRAIL binding receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labelled TRAIL and TRID.
• TRAIL can be labeled, e.g., by radioactivity.
• the amount of labeled TRAIL bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the TRID receptor as determined by an increase of labeled
• the compound which binds to the TRAIL receptor, the compound is a TRID antagonist.
• a screening method for determining whether a candidate TRID antagonist or agonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand (e.g. , apoptosis induced by TRAIL).
• a TNF-family ligand e.g. , apoptosis induced by TRAIL
• the method involves contacting cells which express a TNFR polypeptide with a candidate compound, TRID, and a TNF-family ligand, assaying a cellular response, and comparing the cellular response to a standard cellular response, the standard being assayed when contact is made with the ligand in the presence of TRID but in absence ofthe candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an antagonist and a decreased cellular response compared to the standard indicates that the candidate compound is an agonist.
• assaying a cellular response is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and/or a TNF-family ligand (e.g.
• a cell expressing the TNFR polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.
• Antagonist according to the present invention include naturally occurring and synthetic compounds such as, for example, TNF family ligand peptide fragments, transforming growth factor, neurotransmitters (such as glutamate, dopamine, N-methyl-D-aspartate), tumor suppressors (p53), cytolytic T cells and antimetabolites.
• Preferred agonist include chemotherapeutic drugs such as, for example, cisplatin, doxorubicin, bleomycin, cytosine arabinoside, nitrogen mustard, methotrexate and vincristine. Others include ethanol and -amyloid peptide. (Science 2(57:1457-1458 (1995)).
• Further preferred antagonist includes polyclonal and monoclonal antibodies raised against the TRID polypeptide, or a fragment thereof.
• Agonists according to the present invention include naturally occurring and synthetic compounds such as, for example, the CD40 ligand, neutral amino acids, zinc, estrogen, androgens, viral genes.
• viruses such as Adenovirus EIB, Baculovirus p35 and IAP, Cowpox virus crmA , Epstein-Barr virus BHRF1, LMP-1, African swine fever virus LMW5-HL, and He ⁇ esvirus yl 34.5
• calpain inhibitors such as PMA, Phenobarbital, and - Hexachlorocyclohexane.
• Agonists include polyclonal and monoclonal antagonist antibodies raised against TRAIL polypeptides or a fragment thereof.
• Other potential antagonists include antisense molecules.
• antagonists according to the present invention are nucleic acids corresponding to the sequences contained in TRID, or the complementary strand thereof, and/or to nucleotide sequences contained in ATCC Deposit No. 97798.
• antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, Okano H. et al, J. Neurochem. 56:560 (1991), and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
• Antisense technology can be used to control gene expression through antisense DNA or RNA or through triple-helix formation. Antisense techniques are discussed, for example, in Okano, J. Neurochem. 56:560 (1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC
• a DNA oligonucleotide is designed to be complementary to a region ofthe gene involved in transcription thereby preventing transcription and the production of the receptor.
• the antisense RNA oligonucleotide hybridizes to the mRNA in vivo and blocks translation of the mRNA molecule into receptor polypeptide.
• the oligonucleotides described above can also be delivered to cells such that the antisense RNA or DNA may be expressed in vivo to inhibit production of the receptor.
• the TRID antisense nucleic acid ofthe invention is produced intracellularly by transcription from an exogenous sequence. For example, a vector or a portion thereof, is transcribed, producing an antisense nucleic acid (RNA) of the invention.
• Such a vector would contain a sequence encoding the TRID antisense nucleic acid.
• a vector can remain episomal or become chromosomally integrated, as long as it can be transcribed to produce the desired antisense RNA.
• Such vectors can be constructed by recombinant DNA technology methods standard in the art.
• Vectors can be plasmid, viral, or others know in the art, used for replication and expression in vertebrate cells. Expression ofthe sequence encoding TRID, or fragments thereof, can be by any promoter known in the art to act in vertebrate, preferably human cells. Such promoters can be inducible or constitutive.
• Such promoters include, but are not limited to, the S V40 early promoter region (Bernoist and Chambon, Nature 29:304-310 (1981), the promoter contained in the 3' long terminal repeat of Rous sarcoma virus (Yamamoto et al, Cell 22:181-191 (1980), the he ⁇ es thymidine promoter (Wagner et al. , Proc. Natl Acad. Sci. U. S.A. 75: 1441 - 1445 (1981), the regulatory sequences ofthe metallothionein gene (Brinster, etal, Nature 296:39-42 (1982)), etc.
• the antisense nucleic acids of the invention comprise, or alternatively consist of, a sequence complementary to at least a portion of an RNA transcript of a TRID gene.
• absolute complementarity although preferred, is not required.
• a sequence "complementary to at least a portion of an RNA,” referred to herein, means a sequence having sufficient complementarity to be able to hybridize with the RNA, forming a stable duplex; in the case of double stranded TRID antisense nucleic acids, a single strand of the duplex DNA may thus be tested, or triplex formation may be assayed.
• the ability to hybridize will depend on both the degree of complementarity and the length ofthe antisense nucleic acid
• One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point ofthe hybridized complex.
• Oligonucleotides that are complementary to the 5' end ofthe message e.g. , the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation.
• sequences complementary to the 3' untranslated sequences of mRNAs have been shown to be effective at inhibiting translation of mRNAs as well. See generally, Wagner.
• oligonucleotides complementary to either the 5'- or 3'- non- translated, non-coding regions ofthe TRID shown in SEQ ID NO: 1 could be used in an antisense approach to inhibit translation of endogenous TRID mRNA.
• Oligonucleotides complementary to the 5' untranslated region of the mRNA should include the complement of the AUG start codon.
• Antisense oligonucleotides complementary to mRNA coding regions are less efficient inhibitors of translation but could be used in accordance with the invention.
• antisense nucleic acids should be at least six nucleotides in length, and are preferably oligonucleotides ranging from 6 to about 50 nucleotides in length. In specific aspects the oligonucleotide is at least 10 nucleotides, at least 17 nucleotides, at least 25 nucleotides or at least 50 nucleotides.
• the polynucleotides of the invention can be DNA or RNA or chimeric mixtures or derivatives or modified versions thereof, single-stranded or double- stranded.
• the oligonucleotide can be modified at the base moiety, sugar moiety. or phosphate backbone, for example, to improve stability of the molecule, hybridization, etc.
• the oligonucleotide may include other appended groups such as peptides (e.g., for targeting host cell receptors in vivo), or agents facilitating transport across the cell membrane (see, e.g. , Letsinger et al, Proc. Natl Acad. Sci. U.S.A. 55:6553-6556 (1989); Lemaitre et al, Proc.
• oligonucleotide may be conjugated to another molecule, e.g., a peptide, hybridization triggered cross-linking agent, transport agent, hybridization-triggered cleavage agent, etc.
• the antisense oligonucleotide may comprise at least one modified base moiety which is selected from the group including, but not limited to, 5-fluorouracil, 5-bromouracil, 5-chlorouracil, 5-iodouracil, hypoxanthine, xantine, 4-acetylcytosine, 5-(carboxyhydroxylmethyl) uracil, 5-carboxymethylaminomethyl-
• 2-thiouridine 5-carboxymethylaminomethyluracil, dihydrouracil, beta-D- galactosylqueosine, inosine, N6-isopentenyladenine, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, N6-adenine, 7-methylguanine, 5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil, beta-
• D-mannosylqueosine 5 ⁇ -methoxycarboxymethyluracil, 5-methoxyuracil, 2-methylthio-N6-isopentenyladenine, uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine, 2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5-methyluracil, uracil-5-oxyacetic acidmethylester, uracil-5-oxyacetic acid (v), 5-methyl-2-thiouracil, 3-(3-amino-3-N-2-carboxypropyl) uracil, (acp3)w, and 2,6-diaminopurine.
• the antisense oligonucleotide may also comprise at least one modified sugar moiety selected from the group including, but not limited to, arabinose, 2-fluoroarabinose, xylulose, and hexose.
• the antisense oligonucleotide comprises at least one modified phosphate backbone selected from the group including, but not limited to, a phosphorothioate, a phosphorodithioate, a phosphoramidothioate, a phosphoramidate, a phosphordiamidate, a methylphosphonate, an alkyl phosphotriester, and a formacetal or analog thereof.
• the antisense oligonucleotide is an ⁇ -anomeric oligonucleotide.
• An ⁇ -anomeric oligonucleotide forms specific double-stranded hybrids with complementary RNA in which, contrary to the usual ⁇ -units, the strands run parallel to each other (Gautier et al, Nucl. Acids Res. 75:6625-6641 (1987)).
• the oligonucleotide is a 20-0-methylribonucleotide (Inoue et al, Nucl. Acids Res. 75:6131-6148 (1987)), or a chimeric RNA-DNA analogue (Inoue el al, FEBS ett. 275:327-330 (1987)).
• Polynucleotides ofthe invention may be synthesized by standard methods known in the art, e.g. by use of an automated DNA synthesizer (such as are commercially available from Biosearch, Applied Biosystems, etc.).
• an automated DNA synthesizer such as are commercially available from Biosearch, Applied Biosystems, etc.
• phosphorothioate oligonucleotides may be synthesized by the method_of Stein et al. (Nucl. Acids Res. 16:3209 (1988))
• methylphosphonate oligonucleotides can be prepared by use of controlled pore glass polymer supports (Sarin et al, Proc. Natl. Acad. Sci. U.S.A. 55:7448-7451 (1988)), etc.
• antisense nucleotides complementary to the TRID coding region sequence could be used, those complementary to the transcribed untranslated region are most preferred.
• Potential antagonists according to the invention also include catalytic RNA, or a ribozyme (See, e.g. , PCT International Publication WO 90/1 1364, published October 4, 1990; Sarver et al, Science 247: 1222-1225 (1990). While ribozymes that cleave mRNA at site specific recognition sequences can be used to destroy TRID mRNAs, the use of hammerhead ribozymes is preferred.
• Hammerhead ribozymes cleave mRNAs at locations dictated by flanking regions that form complementary base pairs with the target mRNA. The sole requirement is that the target mRNA have the following sequence of two bases: 5'-UG-3'.
• the construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 554:585-591 (1988).
• ribozyme there are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of TRID (SEQ ID NO: l).
• the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the TRID mRNA; i.e., to increase efficiency and minimize the intracellular accumulation of non-functional mRNA transcripts.
• the ribozymes of the invention can be composed of modified oligonucleotides (e.g.. for improved stability, targeting, etc.) and should be delivered to cells which express TRID in vivo.
• DNA constructs encoding the ribozyme may be introduced into the cell in the same manner as described above for the introduction of antisense encoding DNA.
• a preferred method of delivery involves using a DNA construct "encoding" the ribozyme under the control of a strong constitutive promoter, such as, for example, pol III or pol II promoter, so that transfected cells will produce sufficient quantities of the ribozyme to destroy endogenous TRID messages and inhibit translation. Since ribozymes unlike antisense molecules, are catalytic, a lower intracellular concentration is required for efficiency.
• Endogenous gene expression can also be reduced by inactivating or "knocking out” the TRID gene and/or its promoter using targeted homologous recombination.
• endogenous gene expression can also be reduced by inactivating or "knocking out" the TRID gene and/or its promoter using targeted homologous recombination.
• a mutant, non-functional polynucleotide ofthe invention flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions ofthe gene) can be used, with or without a selectable marker and/or a negative selectable marker, to transfect cells that express polypeptides of the invention in vivo.
• techniques known in the art are used to generate knockouts in cells that contain, but do not express the gene of interest. Insertion of the DNA construct, via targeted homologous recombination, results in inactivation ofthe targeted gene.
• soluble forms of TRID i.e., TRID fragments that include the ligand binding domain from the extracellular region of the full-length receptor.
• soluble forms of the receptor which may be naturally occurring or synthetic, antagonize TNFR mediated signaling by competing with the cell surface TNFR for binding to TNF- family ligands.
• soluble forms ofthe TRID receptor that include the ligand binding domain are novel cytokines capable of inhibiting apoptosis induced by TNF-family ligands.
• Fas B a soluble form of the mouse Fas receptor
• Fas ligand acts physiologically to limit apoptosis induced by Fas ligand
• Proteins and other compounds which bind the extracellular domains are also candidate agonist and antagonist according to the present invention.
• binding compounds can be "captured” using the yeast two-hybrid system (Fields and Song, Nature 540:245-246 (1989)).
• yeast two-hybrid system Fields and Song, Nature 540:245-246 (1989)
• a modified version of the yeast two- hybrid system has been described by Roger Brent and his colleagues (Gyuris, J. et al, Cell 75:791-803 (1993); Zervos, A.S. et al, Cell 72:223-232 (1993)).
• TNF-family ligand By a "TNF-family ligand" is intended naturally occurring, recombinant, and synthetic ligands that are capable of binding to a member of the TNF receptor family and inducing and/or blocking the ligand/receptor signaling pathway.
• TNF ligand family include, but are not limited to, soluble forms of TNF- ⁇ , lymphotoxin-alpha (LT- ⁇ , also known as TNF- ⁇ ), LT- ⁇ (found in complex heterotrimer LT- ⁇ 2- ⁇ ), OPGL, FasL, TRAIL, CD27L, CD30L, CD40L, 4-1BBL, DcR3, OX40L, TNF- ⁇ (International Publication No. WO 96/14328), AIM-I (International Publication No. WO 97/33899), AIM-II (International
• DR4 International Publication No. WO 98/32856
• TR6 International Publication No. WO 98/30694
• TR7 International Publication No. WO 98/41629
• TRANK International Publication No. WO 98/56892
• TRIO International Publication No. WO 98/54202
• 312C2 International Publication No. WO 98/06842
• TR12 and soluble forms CD154, CD70, and CD153.
• TNF- ⁇ has been shown to protect mice from infection with he ⁇ es simplex virus type l (HSV-l) (Rossol-Vothet ⁇ /., J.Ge 7. Virol. 72:143-147 (1991)).
• HSV-l he ⁇ es simplex virus type l
• the mechanism ofthe protective effect of TNF- ⁇ is unknown but appears to involve neither interferons nor NK cell killing.
• One member ofthe family has been shown to mediate HSV- 1 entry into cells (Montgomery et al. , Eur. Cytokine Newt. 7: 159
• TRID antagonists ofthe present invention include both TRID amino acid sequences and antibodies capable of preventing mediated viral entry into cells. Such sequences and antibodies can function by either competing with cell surface localized for binding to virus or by directly blocking binding of virus to cell surface receptors.
• polyclonal and monoclonal antibody agonist or antagonist according to the present invention can be raised according to the methods disclosed in Tartaglia, L.A., and Goeddel, D.V., J. Biol Chem. 267 (7) -.4304-4301 (1992); Tartaglia, L.A.
• antibody or “monoclonal antibody” (mAb) as used herein is meant to include intact molecules as well as fragments thereof (such as, for example, Fab and F(ab') 2 fragments) which are capable of binding an antigen.
• Fab and F (ab') 2 fragments lack the Fc fragment of intact antibody, clear more rapidly from the circulation, and may have less non-specific tissue binding of an intact antibody (Wahl et al, J. Nucl. Med. 24:316-325 (1983)).
• Antibodies according to the present invention may be prepared by any of a variety of standard methods, such as those described above and known in the art, using TRID receptor immunogens ofthe present invention.
• TRID receptor immunogens include the TRID protein shown in SEQ ID NO:2 (which may or may not include a leader sequence) and polypeptide fragments of the receptor comprising, or alternatively consisting of. the ligand binding, extracellular, transmembrane, the intracellular domains of TRID, or any combination thereof.
• nucleic acids comprising sequences encoding antibodies or functional derivatives thereof, are administered to treat, inhibit and/or prevent a disease or disorder associated with aberrant expression and/or activity of a polypeptide ofthe invention, by way of gene therapy.
• Gene therapy refers to therapy performed by the administration to a subject of an expressed or expressible nucleic acid.
• the nucleic acids produce their encoded protein that mediates a therapeutic effect. Any of the methods for gene therapy available in the art can be used according to the present invention. Exemplary methods are described below.
• the compound comprises nucleic acid sequences encoding an antibody, said nucleic acid sequences being part of expression vectors that express the antibody or fragments or chimeric proteins or heavy or light chains thereof in a suitable host.
• nucleic acid sequences have promoters operably linked to the antibody coding region, said promoter being inducible or constitutive, and, optionally, tissue- specific.
• nucleic acid molecules are used in which the antibody coding sequences and any other desired sequences are flanked by regions that promote homologous recombination at a desired site in the genome, thus providing for intrachromosomal expression ofthe antibody nucleic acids (Koller and Smithies, 1989, Proc. Natl. Acad. Sci.
• the expressed antibody molecule is a single chain antibody; alternatively, the nucleic acid sequences include sequences encoding both the heavy and light chains, or fragments thereof, ofthe antibody.
• nucleic acids into a patient may be either direct, in which case the patient is directly exposed to the nucleic acid or nucleic acid- carrying vectors, or indirect, in which case, cells are first transformed with the nucleic acids in vitro, then transplanted into the patient. These two approaches are known, respectively, as in vivo or ex vivo gene therapy.
• the nucleic acid sequences are directly administered in vivo, where it is expressed to produce the encoded product. This can be accomplished by any of numerous methods known in the art, e.g. , by constructing them as part of an appropriate nucleic acid expression vector and administering it so that they become intracellular, e.g.
• nucleic acid-ligand complexes can be formed in which the ligand comprises a fusogenic viral peptide to disrupt endosomes. allowing the nucleic acid to avoid lysosomal degradation.
• the nucleic acid can be targeted in vivo for cell specific uptake and expression, by targeting a specific receptor (see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et al); WO 92/22635 dated December 23, 1992 (Wilson et al); WO92/20316 dated November 26, 1992
• nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination (Koller and Smithies, 1989, Proc. Natl. Acad. Sci. USA 86:8932-8935; Zijlstra et al, 1989, Nature 342:435-438).
• viral vectors that contains nucleic acid sequences encoding an antibody ofthe invention are used.
• a retroviral vector can be used (see Miller et al, 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been to delete retroviral sequences that are not necessary for packaging ofthe viral genome and integration into host cell DNA.
• the nucleic acid sequences encoding the antibody to be used in gene therapy are cloned into one or more vectors, which facilitates delivery of the gene into a patient.
• retroviral vectors More detail about retroviral vectors can be found in Boesen et al, 1994, Biotherapy 6:291-302, which describes the use of a retroviral vector to deliver the mdrl gene to hematopoietic stem cells in order to make the stem cells more resistant to chemotherapy.
• Other references illustrating the use of retroviral vectors in gene therapy are: Clowes et al, 1994, J. Clin. Invest. 93:644-651 ; Kiem et al, 1994, Blood 83 : 1467-1473; Salmons and Gunzberg, 1993, Human Gene Therapy 4: 129- 141 ; and Grossman and Wilson, 1993, Curr. Opin. in Genetics and Devel. 3: 1 10- 1 14.
• Adenoviruses are other viral vectors that can be used in gene therapy.
• Adenoviruses are especially attractive vehicles for delivering genes to respiratory epithelia. Adenoviruses naturally infect respiratory epithelia where they cause a mild disease. Other targets for adenovirus-based delivery systems are liver, the central nervous system, endothelial cells, and muscle. Adenoviruses have the advantage of being capable of infecting non-dividing cells. Kozarsky and Wilson,
• adenovirus vectors are used.
• Adeno-associated virus has also been proposed for use in gene therapy (Walsh etal, 1993, Proc. Soc. Exp. Biol. Med.204:289-300; U.S. Patent
• Another approach to gene therapy involves transferring a gene to cells in tissue culture by such methods as electroporation, lipofection, calcium phosphate mediated transfection, or viral infection.
• the method of transfer includes the transfer of a selectable marker to the cells. The cells are then placed under selection to isolate those cells that have taken up and are expressing the transferred gene. Those cells are then delivered to a patient.
• the nucleic acid is introduced into a cell prior to administration in vivo ofthe resulting recombinant cell.
• introduction can be carried out by any method known in the art, including but not limited to transfection, electroporation, microinjection. infection with a viral or bacteriophage vector containing the nucleic acid sequences, cell fusion, chromosome-mediated gene transfer, microcell-mediated gene transfer, spheroplast fusion, etc. Numerous techniques are known in the art for the introduction of foreign genes into cells (see, e.g., Loeffler and Behr, 1993, Meth.
• the technique should provide for the stable transfer ofthe nucleic acid to the cell, so that the nucleic acid is expressible by the cell and preferably heritable and expressible by its cell progeny.
• Recombinant blood cells e.g. , hematopoietic stem or progenitor cells
• Recombinant blood cells are preferably administered intravenously.
• the amount of cells envisioned for use depends on the desired effect, patient state, etc., and can be determined by one skilled in the art.
• Cells into which a nucleic acid can be introduced for pu ⁇ oses of gene therapy encompass any desired, available cell type, and include but are not limited to epithelial cells, endothelial cells, keratinocytes, fibroblasts, muscle cells, hepatocytes; blood cells such as T-lymphocytes, B-lymphocytes, monocytes, macrophages, neutrophils, eosinophils, megakaryocytes, granulocytes; various stem or progenitor cells, in particular hematopoietic stem or progenitor cells, e.g. , as obtained from bone marrow, umbilical cord blood, peripheral blood, fetal liver, etc.
• the cell used for gene therapy is autologous to the patient.
• nucleic acid sequences encoding an antibody are introduced into the cells such that they are expressible by the cells or their progeny, and the recombinant cells are then administered in vivo for therapeutic effect.
• stem or progenitor cells are used. Any stem and/or progenitor cells which can be isolated and maintained in vitro can potentially be used in accordance with this embodiment ofthe present invention (see, e.g., PCT Publication WO 94/08598, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71 :973-985; Rheinwald, 1980, Meth. Cell Bio. 27-4:229; and Pittelkow and Scott. 1986, Mayo Clinic Proc. 61:111).
• the nucleic acid to be introduced for pu ⁇ oses of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression ofthe nucleic acid is controllable by controlling the presence or absence ofthe appropriate inducer of transcription.
• the invention provides methods of treatment, inhibition and prophylaxis by administration to a subject of an effective amount of a compound or pharmaceutical composition of the invention, preferably an antibody of the invention.
• the compound is substantially purified (e.g., substantially free from substances that limit its effect or produce undesired side- effects).
• the subject is preferably an animal, including but not limited to animals such as cows, pigs, horses, chickens, cats, dogs, etc., and is preferably a mammal, and most preferably human.
• Formulations and methods of administration that can be employed when the compound comprises a nucleic acid or an immunoglobulin are described above; additional appropriate formulations and routes of administration can be selected from among those described herein below.
• the agonist or antagonists described herein can be administered in vitro, ex vivo, or in vivo to cells which express the receptor ofthe present invention.
• administration of an "effective amount" of an agonist or antagonist is intended an amount ofthe compound that is sufficient to enhance or inhibit a cellular response to a TNF-family ligand and include polypeptides.
• an "effective amount" of an agonist or antagonists is intended an amount effective to enhance or inhibit TRID activity.
• a TRID antagonist according to the present invention can be co- administered with a TNF-family ligand.
• effective amounts of an agonist or antagonist can be determined empirically and may be employed in pure form or in pharmaceutically acceptable salt, ester or prodrug form.
• the agonist or antagonist may be administered in compositions in combination with one or more pharmaceutically acceptable excipients.
• the total pharmaceutically effective amount of TRID polypeptide administered parenterally per dose will be in the range of about 1 ⁇ g/kg/day to 10 mg/kg/day of patient body weight, although, as noted above, this will be subject to therapeutic discretion. More preferably, this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day.
• the TRID agonist or antagonist is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about 50 ⁇ g/kg/hour, either by 1-4 injections per day or by continuous subcutaneous infusions, for example, using a mini-pump. An intravenous bag solution may also be employed.
• Dosaging may also be arranged in a patient specific manner to provide a predetermined concentration of an agonist or antagonist in the blood, as determined by the RIA technique.
• patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIA, on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
• Pharmaceutical compositions of the present invention for parenteral injection can comprise pharmaceutically acceptable sterile aqueous or nonaqueous solutions, dispersions, suspensions or emulsions as well as sterile powders for reconstitution into sterile injectable solutions or dispersions just prior to use.
• TRID polypeptides containing the transmembrane region can also be used when appropriately solubilized by including detergents, such as CHAPS or NP-40, with buffer.
• the compounds or pharmaceutical compositions of the invention are preferably tested in vitro, and then in vivo for the desired therapeutic or prophylactic activity, prior to use in humans.
• in vitro assays to demonstrate the therapeutic or prophylactic utility of a compound or pharmaceutical composition include, the effect of a compound on a cell line or a patient tissue sample.
• the effect ofthe compound or composition on the cell line and/or tissue sample can be determined utilizing techniques known to those of skill in the art including, but not limited to. rosette formation assays and cell lysis assays.
• in vitro assays which can be used to determine whether administration of a specific compound is indicated, include in vitro cell culture assays in which a patient tissue sample is grown in culture, and exposed to or otherwise administered a compound, and the effect of such compound upon the tissue sample is observed.
• compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
• pharmaceutical compositions are provided comprising a TRID agonist or antagonist and a pharmaceutically acceptable carrier or excipient, which may be administered orally, rectally, parenterally, intracistemally, intravaginally, intraperitoneally, topically (as by powders, ointments, drops or transdermal patch), bucally, or as an oral or nasal spray.
• a TRID antagonist and a TNF-family ligand clinical side effects can be reduced by using lower doses of both the ligand and the antagonist.
• the antagonist can be "co-administered" either before, after, or simultaneously with the TNF-family ligand, depending on the exigencies of a particular therapeutic application.
• pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
• pharmaceutically acceptable means approved by a regulatory agency ofthe Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, and more particularly in humans.
• parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
• carrier refers to a diluent, adjuvant, excipient, or vehicle with which the therapeutic is administered.
• Such pharmaceutical carriers can be sterile liquids, such as water and oils, including those of petroleum, animal, vegetable or synthetic origin, such as peanut oil, soybean oil, mineral oil, sesame oil and the like. Water is a preferred carrier when the pharmaceutical composition is administered intravenously. Saline solutions and aqueous dextrose and glycerol solutions can also be employed as liquid carriers, particularly for injectable solutions. Suitable pharmaceutical excipients include starch, glucose, lactose, sucrose, gelatin, malt, rice, flour, chalk, silica gel, sodium stearate, glycerol monostearate, talc, sodium chloride, dried skim milk, glycerol, propylene, glycol. water, ethanol and the like.
• compositions can also contain minor amounts of wetting or emulsifying agents, or pH buffering agents.
• These compositions can take the form of solutions, suspensions, emulsion, tablets, pills, capsules, powders, sustained- release formulations and the like.
• the composition can be formulated as a suppository, with traditional binders and carriers such as triglycerides.
• Oral formulation can include standard carriers such as pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, etc. Examples of suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E.W. Martin.
• compositions will contain a therapeutically effective amount of the compound, preferably in purified form, together with a suitable amount of carrier so as to provide the form for proper administration to the patient.
• the formulation should suit the mode of administration.
• the composition is formulated in accordance with routine procedures as a pharmaceutical composition adapted for intravenous administration to human beings.
• compositions for intravenous administration are solutions in sterile isotonic aqueous buffer.
• the composition may also include a solubilizing agent and a local anesthetic such as lignocaine to ease pain at the site ofthe injection.
• the ingredients are supplied either separately or mixed together in unit dosage form, for example, as a dry lyophilized powder or water free concentrate in a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
• a hermetically sealed container such as an ampule or sachette indicating the quantity of active agent.
• the composition is to be administered by infusion, it can be dispensed with an infusion bottle containing sterile pharmaceutical grade water or saline.
• an ampule of sterile water for injection or saline can be provided so that the ingredients may be mixed prior to administration.
• the compounds ofthe invention can be formulated as neutral or salt forms.
• Pharmaceutically acceptable salts include those formed with anions such as those derived from hydrochloric, phosphoric, acetic, oxalic, tartaric acids, etc., and those formed with cations such as those derived from sodium, potassium, ammonium, calcium, ferric hydroxides, isopropylamine, triethylamine, 2- ethylamino ethanol, histidine, procaine. etc.
• the TRID polypeptide is also suitably administered by sustained-release systems.
• sustained-release compositions include semipermeable polymer matrices in the form of shaped articles, e.g., films, or mirocapsules.
• Sustained-release matrices include polylactides (U.S. Pat. No. 3,773,919, EP 58,481), copolymers of L-glutamic acid and gamma-ethyl-L- glutamate (Sidman, U. et al., Biopolymers 22:541-556 (1983)), poly (2- hydroxy ethyl methacrylate) (R. Langer et al., J. Biomed. Mater. Res.
• Sustained- release TRID polypeptide compositions also include liposomally entrapped TRID polypeptides. Liposomes containing TRID polypeptides are prepared by methods known per se: DE 3,218,121 ; Epstein et al., Proc. Natl. Acad. Sci. (USA) 82:3688-3692 (1985); Hwang et al., Proc. Natl. Acad. Sci.
• the liposomes are ofthe small (about 200-800 Angstroms) unilamellar type in which the lipid content is greater than about 30 mol. percent cholesterol, the selected proportion being adjusted for the optimal TNFR polypeptide therapy.
• the carrier suitably contains minor amounts of additives such as substances that enhance isotonicity and chemical stability.
• Such materials are non- toxic to recipients at the dosages and concentrations employed, and include buffers such as phosphate, citrate, succinate, acetic acid, and other organic acids or their salts; antioxidants such as ascorbic acid; low molecular weight (less than about ten residues) polypeptides, e.g.
• polyarginine or tripeptides such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as poly vinylpyrrolidone; amino acids, such as glycine, glutamic acid, aspartic acid, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; counterions such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, or PEG.
• proteins such as serum albumin, gelatin, or immunoglobulins
• hydrophilic polymers such as poly vinylpyrrolidone
• amino acids such as glycine, glutamic acid, aspartic acid, or arginine
• monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, manose, or dextrins
• the TRID polypeptide is typically formulated in such vehicles at a concentration of about 0.1 mg/ml to 100 mg/ml, preferably 1-10 mg/ml, at a pH of about 3 to 8. It will be understood that the use of certain of the foregoing excipients, carriers, or stabilizers will result in the formation of TRID polypeptide salts.
• TRID polypeptides to be used for therapeutic administration must be sterile. Sterility is readily accomplished by filtration through sterile filtration membranes (e.g., 0.2 micron membranes).
• Therapeutic TRID polypeptide compositions generally are placed into a container having a sterile access port, for example, an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle.
• TRID polypeptides ordinarily will be stored in unit or multi-dose containers, for example, sealed ampoules or vials, as an aqueous solution or as a lyophilized formulation for reconstitution.
• a lyophilized formulation 10-ml vials are filled with 5 ml of sterile-filtered 1% (w/v) aqueous TRID polypeptide solution, and the resulting mixture is lyophilized.
• the infusion solution is prepared by reconstituting the lyophilized TRID polypeptide using bacteriostatic Water- for-Injection.
• compositions ofthe invention may be administered alone or in combination with other therapeutic agents.
• therapeutic agents that may be administered in combination with the compositions of the invention include but are not limited to, other members ofthe TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines, chemokines and/or growth factors.
• Combinations may be administered either concomitantly, e.g., as an admixture, separately but simultaneously or concurrently; or sequentially. This includes presentations in which the combined agents are administered together as a therapeutic mixture, and also procedures in which the combined agents are administered separately but simultaneously, e.g.. as through separate intravenous lines into the same individual.
• Administration "in combination” further includes the separate administration of one of the compounds or agents given first, followed by the second.
• compositions ofthe invention are administered in combination with other members ofthe TNF family.
• TNF, TNF-related or TNF- like molecules that may be administered with the compositions ofthe invention include, but are not limited to, soluble forms of TNF-alpha, lymphotoxin-alpha
• LT-alpha also known as TNF-beta
• LT-beta found in complex heterotrimer LT- alpha2-beta
• OPGL FasL
• CD27L CD30L
• CD40L 4-1BBL
• DcR3 OX40L
• TNF-gamma International Publication No. WO 96/14328
• International Publication No. WO 96/14328 International Publication No. WO 96/14328
• TNF- ⁇ - ⁇ TNF- ⁇ - ⁇
• TRAIL AIM-II
• compositions ofthe invention are administered in combination with CD40 ligand (CD40L), a soluble form of CD40L (e.g., AVRENDTM), biologically active fragments, variants, or derivatives of CD40L, anti-CD40L antibodies (e.g., agonistic or antagonistic antibodies), and/or anti-
• CD40 antibodies e.g., agonistic or antagonistic antibodies.
• compositions of the invention are administered in combination with one, two, three, four, five, or more of the following compositions: tacrolimus (Fujisawa), thalidomide (e.g. , Celgene), anti- Tac(Fv)-PE40 (e.g., Protein Design Labs), inolimomab (Biotest), MAK-195F
• ASM-981 Novartis
• interleukin- 1 receptor e.g. , Immunex
• interleukin- 4 receptor e.g. , Immunex
• ICM3 ICM3
• BMS-188667 Bristol-Myers Squibb
• anti-TNF Ab e.g. , Therapeutic antibodies
• CG-1088 Celgene
• anti-B7 monoclonal antibody e.g., Innogetics
• MEDI-507 BioTransplant
• ABX-CBL Abgenix
• a patient susceptible to both Fas ligand (Fas-L) mediated and TRAIL mediated cell death may be treated with both an agent that inhibits TRAIL/TRAIL-R interactions and an agent that inhibits Fas-L/Fas interactions.
• Suitable agents for blocking binding of Fas-L to Fas include, but are not limited to, soluble Fas polypeptides; oligomeric forms of soluble Fas polypeptides (e.g., dimers of sFas/Fc); anti-Fas antibodies that bind Fas without transducing the biological signal that results in apoptosis; anti-Fas-L antibodies that block binding of Fas-L to Fas; and muteins of Fas-L that bind Fas but do not transduce the biological signal that results in apoptosis.
• the antibodies employed according to this method are monoclonal antibodies. Examples of suitable agents for blocking Fas-L/Fas interactions, including blocking anti-Fas monoclonal antibodies, are described in WO 95/10540, hereby inco ⁇ orated by reference.
• compositions ofthe invention are administered in combination with antiretroviral agents, nucleoside reverse transcriptase inhibitors, non-nucleoside reverse transcriptase inhibitors, and/or protease inhibitors.
• Nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, RETROVIRTM (zidovudine/AZT), VIDEXTM (didanosine/ddl), HIVIDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVIRTM (zidovudine/lamivudine).
• RETROVIRTM zidovudine/AZT
• VIDEXTM didanosine/ddl
• HIVIDTM zalcitabine/ddC
• ZERITTM stavudine/d4T
• EPIVIRTM lamvudine/3TC
• COMBIVIRTM zidovudine/lamivudine
• Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, VIRAMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUSTIVATM (efavirenz).
• Protease inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVIRTM (ritonavir),
• antiretroviral agents nucleoside reverse transcriptase inhibitors, non- nucleoside reverse transcriptase inhibitors, and/or protease inhibitors may be used in any combination with compositions of the invention to treat AIDS and/or to prevent or treat HIV infection.
• compositions ofthe invention may be administered in combination with anti-opportunistic infection agents.
• Anti-opportunistic agents that may be administered in combination with the compositions ofthe invention, include, but are not limited to, TRIMETHOPRIM-SULFAMETHOXAZOLETM, DAPSONETM, PENTAMIDINETM, ATOVAQUONETM, ISONIAZIDTM,
• RIFAMPINTM PYRAZIN AMIDETM, ETHAMBUTOLTM, RIFABUTINTM, CLARITHROMYCINTM, AZITHROMYCINTM, GANCICLOVIRTM, FOSCARNETTM, CIDOFOVIRTM, FLUCONAZOLETM, ITRACONAZOLETM.
• KET O C ONAZ O L ETM A C Y C L O V I RTM , F AM C I C O L V I RTM .
• compositions of the invention are used in any combination with TRI METH OPRI M - SULFAMETHOXAZOL ETM , DAP S ONETM . PENTAMIDINETM, and/or ATOVAQUONETM to prophylactically treat and/or prevent an opportunistic Pneumocystis carinii pneumonia infection.
• compositions ofthe invention are used in any combination with ISONIAZIDTM, RIFAMPINTM, PYRAZINAMIDETM, and/or ETHAMBUTOLTM to prophylactically treat and/or prevent an opportunistic Mycobacterium avium complex infection.
• compositions ofthe invention are used in any combination with RIFABUTINTM, CLARITHROMYCINTM, and/or AZITHROMYCINTM to prophylactically treat and/or prevent an opportunistic Mycobacterium tuberculosis infection.
• compositions ofthe invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat and/or prevent an opportunistic cytomegalovirus infection.
• compositions of the invention are used in any combination with FLUCONAZOLETM, ITRACONAZOLETM, and/or KETOCONAZOLETM to prophylactically treat and/or prevent an opportunistic fungal infection.
• compositions ofthe invention are used in any combination with ACYCLOVIRTM and/or FAMCICOLVIRTM to prophylactically treat and/or prevent an opportunistic he ⁇ es simplex virus type
• compositions ofthe invention are used in any combination with PYRIMETHAMINETM and/or LEUCOVORINTM to prophylactically treat and/or prevent an opportunistic Toxoplasma gondii infection.
• compositions of the invention are used in any combination with LEUCOVORINTM and/or
• NEUPOGENTM to prophylactically treat and/or prevent an opportunistic bacterial infection.
• compositions of the invention are administered in combination with an antiviral agent.
• Antiviral agents that may be administered with the compositions ofthe invention include, but are not limited to, acyclovir, ribavirin, amantadine, and remantidine.
• compositions of the invention are administered in combination with an antibiotic agent.
• Antibiotic agents that may be administered with the compositions ofthe invention include, but are not limited to, amoxicillin, aminoglycosides, beta-lactam (glycopeptide), beta-lactamases, Clindamycin, chloramphenicol, cephalosporins, ciprofloxacin, ciprofloxacin, erythromycin, fluoroquinolones, macrolides, metronidazole, penicillins, quinolones, rifampin, streptomycin, sulfonamide, tetracyclines, trimethoprim, trimethoprim-sulfamthoxazole, and vancomycin.
• compositions ofthe invention include, but are not limited to, steroids, cyclosporine, cyclosporine analogs, cyclophosphamide methylprednisone, prednisone, azathioprine, FK-506, 15-deoxyspergualin, and other immunosuppressive agents that act by suppressing the function of responding T-cells.
• compositions ofthe invention are administered in combination with immunosuppressants.
• Immunosuppressants preparations that may be administered with the compositions ofthe invention include, but are not limited to, ORTHOCLONETM (OKT3), SANDIMMUNETM/NEORALTM/ SANGDYATM (cyclosporin), PROGRAFTM (tacrolimus), CELLCEPTTM (mycophenolate), Azathioprine, glucorticosteroids, and RAPAMUNETM
• immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
• compositions of the invention are administered alone or in combination with one or more intravenous immune globulin preparations.
• Intravenous immune globulin preparations that may be administered with the compositions ofthe invention include, but not limited to, GAMMARTM, IVEEGAMTM, SANDOGLOBULINTM, GAMMAGARD S/DTM, and GAMIMUNETM.
• compositions of the invention are administered in combination with intravenous immune globulin preparations in transplantation therapy (e.g., bone marrow transplant).
• transplantation therapy e.g., bone marrow transplant.
• the compositions of the invention are administered alone or in combination with an anti-inflammatory agent.
• Anti- inflammatory agents that may be administered with the compositions of the invention include, but are not limited to, glucocorticoids and the nonsteroidal anti-inflammatories, aminoarylcarboxylic acid derivatives, arylacetic acid derivatives, arylbutyric acid derivatives, arylcarboxylic acids, arylpropionic acid derivatives, pyrazoles, pyrazolones, salicylic acid derivatives, thiazinecarboxamides, e-acetamidocaproic acid, S-adenosylmethionine, 3-amino- 4-hydroxybutyric acid, amixetrine, bendazac, benzydamine, bucolome, difenpiramide, ditazol, emorfazone, guaiazulene, nabumetone, nimesulide, orgotein, oxaceprol, paranyline, perisoxal, pifoxime, proquazone, proxazole, and ten
• compositions of the invention are administered in combination with steroid therapy.
• Steroids that may be administered in combination with the compositions ofthe invention include, but are not limited to, oral corticosteroids, prednisone, and methylprednisolone (e.g., IV methylprednisolone).
• compositions ofthe invention are administered in combination with prednisone.
• the compositions of the invention are administered in combination with prednisone and an immunosuppressive agent.
• Immunosuppressive agents that may be administered with the compositions ofthe invention and prednisone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.
• compositions of the invention are administered in combination with methylprednisolone.
• the compositions ofthe invention are administered in combination with methylprednisolone and an immunosuppressive agent.
• Immunosuppressive agents that may be administered with the compositions of the invention and methylprednisolone are those described herein, and include, but are not limited to, azathioprine, cylophosphamide, and cyclophosphamide IV.
• compositions of the invention are administered in combination with an antimalarial.
• Antimalarials that may be administered with the compositions ofthe invention include, but are not limited to, hydroxychloroquine, chloroquine, and/or quinacrine.
• compositions of the invention are administered in combination with an NSAID.
• the compositions of the invention are administered in combination with one, two, three, four, five, ten, or more ofthe following drugs: NRD-101 (Hoechst Marion Roussel), diclofenac (Dimethaid), oxaprozin potassium (Monsanto), mecasermin (Chiron), T-614 (Toyama), pemetrexed disodium (Eli Lilly), atreleuton (Abbott), valdecoxib (Monsanto), kornac (Byk Gulden), campath, AGM-1470 (Takeda), CDP-571 (Celltech
• CM-101 CarboMed
• ML-3000 Merckle
• CB-2431 KS Biomedix
• CBF-BS2 KS Biomedix
• IL-lRa gene therapy Valentis
• JTE-522 Japan Tobacco
• paclitaxel Angiotech
• DW-166HC Denong Wha
• darbufelone mesylate Warner-Lambert
• soluble TNF receptor 1 soluble TNF receptor 1
• IPR- 6001 Institute for Pharmaceutical Research
• trocade Hoffman-La Roche
• compositions of the invention are administered in combination with one, two, three, four, five or more of the following drugs: methotrexate, sulfasalazine, sodium aurothiomalate, auranofin, cyclosporine, penicillamine, azathioprine, an antimalarial drug (e.g. , as described herein), cyclophosphamide, chlorambucil, gold, ENBRELTM (Etanercept), anti- TNF antibody, and prednisolone.
• the compositions of the invention are administered in combination with an antimalarial.
• compositions ofthe invention are administered in combination with methotrexate. In another embodiment, the compositions of the invention are administered in combination with anti-TNF antibody. In another embodiment, the compositions ofthe invention are administered in combination with methotrexate and anti-TNF antibody. In another embodiment, the compositions ofthe invention are administered in combination with suflasalazine. In another specific embodiment, the compositions of the invention are administered in combination with methotrexate, anti-TNF antibody, and suflasalazine. In another embodiment, the compositions of the invention are administered in combination ENBRELTM.
• compositions ofthe invention are administered in combination with ENBRELTM and methotrexate. In another embodiment, the compositions ofthe invention are administered in combination with ENBRELTM, methotrexate and suflasalazine. In another embodiment, the compositions of the invention are administered in combination with ENBRELTM, methotrexate and suflasalazine. In other embodiments, one or more antimalarials is combined with one of the above- recited combinations. In a specific embodiment, the compositions of the invention are administered in combination with an antimalarial (e.g. , hydroxychloroquine), ENBRELTM, methotrexate and suflasalazine. In another specific embodiment, the compositions of the invention are administered in combination with an antimalarial (e.g. , hydroxychloroquine), sulfasalazine, anti- TNF antibody, and methotrexate.
• an antimalarial e.g. , hydroxychloroqu
• compositions ofthe invention are administered in combination with a chemotherapeutic agent.
• Chemotherapeutic agents that may be administered with the compositions ofthe invention include, but are not limited to, antibiotic derivatives (e.g. , doxorubicin, bleomycin, daunorubicin, and dactinomycin); antiestrogens (e.g. , tamoxifen); antimetabolites (e.g.
• cytotoxic agents e.g., carmustine, BCNU, lomustine, CCNU, cytosine arabinoside, cyclophosphamide, estramustine, hydroxyurea, procarbazine, mitomycin, busulfan, cis-platin, and vincristine sulfate
• hormones e.g.
• medroxyprogesterone estramustine phosphate sodium, ethinyl estradiol, estradiol, megestrol acetate, methyltestosterone, diethylstilbestrol diphosphate, chlorotrianisene, and testolactone
• nitrogen mustard derivatives e.g., mephalen, chorambucil. mechlorethamine (nitrogen mustard) and thiotepa
• steroids and combinations e.g. , bethamethasone sodium phosphate
• others e.g., dicarbazine, asparaginase, mitotane, vincristine sulfate, vinblastine sulfate, and etoposide).
• compositions of the invention are administered in combination with cytokines.
• Cytokines that may be administered with the compositions ofthe invention include, but are not limited to, IL-2, IL-3,
• IL-4 IL-5.
• IL-6 IL-7, IL-10, IL-12.
• IL-13 IL-15.
• compositions of the invention are administered in combination with angiogenic proteins.
• Angiogenic proteins that may be administered with the compositions ofthe invention include, but are not limited to,. Glioma Derived Growth Factor (GDGF), as disclosed in European Patent Number EP-399816; Platelet Derived Growth Factor-A (PDGF-A), as disclosed in European Patent Number EP-6821 10; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (P1GF), as disclosed in International Publication Number
• GDGF Glioma Derived Growth Factor
• PDGF-A Platelet Derived Growth Factor-A
• PDGF-B Platelet Derived Growth Factor-B
• P1GF Placental Growth Factor
• VEGF Vascular Endothelial Growth Factor
• VEGF-A Vascular Endothelial Growth Factor-A
• VEGF-B Vascular Endothelial Growth Factor B-186
• VEGF-D Vascular Endothelial Growth Factor-D
• VEGF-D Vascular Endothelial Growth Factor-D
• VEGF-E Vascular Endothelial Growth Factor-E
• compositions of the invention are administered in combination with Fibroblast Growth Factors.
• Fibroblast Growth Factors that may be administered with the compositions ofthe invention include, but are not limited to, FGF-1, FGF-2, FGF-3, FGF-4, FGF-5, FGF-6, FGF-7,

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• 2000
  • 2000-05-18 WO PCT/US2000/013515 patent/WO2000071150A1/en not_active Application Discontinuation
  • 2000-05-18 JP JP2000619452A patent/JP2003502287A/ja not_active Withdrawn
  • 2000-05-18 EP EP00932514A patent/EP1191940A4/en not_active Withdrawn
  • 2000-05-18 CA CA002374674A patent/CA2374674A1/en not_active Abandoned
  • 2000-05-18 AU AU50224/00A patent/AU5022400A/en not_active Abandoned

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