WO2000064465A1 - Death domain containing receptors - Google Patents

Death domain containing receptors Download PDF

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Publication number
WO2000064465A1
WO2000064465A1 PCT/US2000/010741 US0010741W WO0064465A1 WO 2000064465 A1 WO2000064465 A1 WO 2000064465A1 US 0010741 W US0010741 W US 0010741W WO 0064465 A1 WO0064465 A1 WO 0064465A1
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antibody
polypeptide
seq
amino acid
sequence
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PCT/US2000/010741
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English (en)
French (fr)
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Guo-Liang Yu
Jian Ni
Reiner L. Gentz
Patrick J. Dillon
Vishva M. Dixit
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Human Genome Sciences, Inc.
The Regents Of The University Of Michigan
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Priority to JP2000613455A priority Critical patent/JP2003522118A/ja
Priority to CA002371114A priority patent/CA2371114A1/en
Priority to EP00926218A priority patent/EP1178815A4/de
Priority to AU44782/00A priority patent/AU4478200A/en
Publication of WO2000064465A1 publication Critical patent/WO2000064465A1/en

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • A61P37/06—Immunosuppressants, e.g. drugs for graft rejection
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C07K14/4701—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals not used
    • C07K14/4747—Apoptosis related proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K16/00—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies
    • C07K16/18—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans
    • C07K16/28—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants
    • C07K16/2878—Immunoglobulins [IGs], e.g. monoclonal or polyclonal antibodies against material from animals or humans against receptors, cell surface antigens or cell surface determinants against the NGF-receptor/TNF-receptor superfamily, e.g. CD27, CD30, CD40, CD95
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2317/00—Immunoglobulins specific features
    • C07K2317/20—Immunoglobulins specific features characterized by taxonomic origin
    • C07K2317/21—Immunoglobulins specific features characterized by taxonomic origin from primates, e.g. man
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2799/00—Uses of viruses
    • C12N2799/02—Uses of viruses as vector
    • C12N2799/021—Uses of viruses as vector for the expression of a heterologous nucleic acid
    • C12N2799/026—Uses of viruses as vector for the expression of a heterologous nucleic acid where the vector is derived from a baculovirus

Definitions

  • the present invention relates to a novel member of the tumor necrosis factor family of receptors. More specifically, isolated nucleic acid molecules are provided encoding human Death Domain Containing Receptors (DR3 and DR3-V1). Death Domain Containing Receptor 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 and antagonists of DR3 activity.
  • 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 intra- cellular response.
  • 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-Iigands, the "TNF-receptor” superfamily.
  • TNF- ⁇ lymphotoxin- ⁇
  • LT- ⁇ lymphotoxin- ⁇
  • LT- ⁇ lymphotoxin- ⁇
  • FasL CD40L
  • CD27L CD30L
  • 4-1BBL 4-1BBL
  • OX40L nerve growth factor
  • NGF nerve growth factor
  • 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 (A. Meager, 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. (A. Meager, supra).
  • TNF and LT- ⁇ are capable of binding to two TNF receptors (the 55- and 75-kd TNF receptors).
  • a large number of biological effects elicited by TNF and LT- ⁇ , acting through their receptors, include hemorrhagic necrosis of transplanted tumors, cytotoxicity, a role in endotoxic shock, inflammation, immunoregulation, proliferation and anti-viral responses, as well as protection against the deleterious effects of ionizing radiation.
  • 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 (B. Beutler and C. Von
  • Apoptosis or programmed cell death, is a physiologic process essential to the normal development and homeostasis of multicellular organisms (H. whilr, Science 261, 1445-1449 (1995)). Derangements of apoptosis contribute to the pathogenesis of several human diseases including cancer, neurodegenerative disorders, and acquired immune deficiency syndrome (C.B. Thompson, Science 261, 1456-1462 (1995)). Recently, much attention has focused on the signal transduction and biological function of two cell surface death receptors, Fas/APO-1 and TNFR-1 (J.L. Cleveland etal, Cell81, 479-482 (1995); A. Fraser et al, Cell 85, 781-784 (1996); S.
  • TNFR-1 can signal an array of diverse biological activities-many of which stem from its ability to activate NF-kB (L.A. Tartaglia et al, 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, Id. ; H. Hsu et al,
  • 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 novel members of the TNF receptor family.
  • the present invention provides for isolated nucleic acid molecules comprising, or alternatively consisting of, nucleic acid sequences encoding the amino acid sequences shown in SEQ ED NO:2 and SEQ ED NO:4 or the amino acid sequence encoding the cDNAs deposited as ATCC Deposit No. 97456 on
  • the present invention also provides vectors and host cells for recombinant expression of the nucleic acid molecules described herein, as well as to methods of making such vectors and host cells and for using them for production of DR3 or DR3 Variant 1 (DR3-V1) (formerly named DDCR) polypeptides or peptides by recombinant techniques.
  • DR3-V1 DR3-V1
  • DDCR DR3 Variant 1
  • the invention further provides an isolated DR3 or DR3-V1 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 DR3 or DR3-V1 protein.
  • diagnostic assays such as quantitative and diagnostic assays for detecting levels of DR3 or DR3-V1 protein.
  • a diagnostic assay in accordance with the invention for detecting over- expression of DR3 or DR3-V1, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors.
  • 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 v. 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, which involves administering to a cell which expresses the DR3 polypeptide an effective amount of an agonist capable of increasing DR3 mediated signaling.
  • DR3 mediated signaling is increased to treat and/or prevent a disease wherein decreased apoptosis is exhibited.
  • the present invention is directed to a method for inhibiting apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the DR3 polypeptide an effective amount of an antagonist capable of decreasing DR3 mediated signaling.
  • DR3 mediated signaling is decreased to treat and/or prevent 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.
  • the method involves contacting cells which express the DR3 or DR3-V1 polypeptide with a candidate compound 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 absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway.
  • a cell expressing the DR3 or DR3-V1 polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.
  • FIG. 1A-1C shows the nucleotide and deduced amino acid sequence of DR3-V1. It is predicted that amino acids 1-35 constitute the signal peptide, amino acids 36-212 constitute the extracellular domain, amino acids 213-235 constitute the transmembrane domain, amino acids 236-428 constitute the intracellular domain, and amino acids 353-419 the death domain.
  • FIG. 2A-2B shows the nucleotide and deduced amino acid sequence of DR3. It is predicted that amino acids 1-24 constitute the signal peptide, amino acids 25-201 constitute the extracellular domain, amino acids 202-224 constitute the transmembrane domain, amino acids 225-417 constitute the intracellular domain, and amino acids 342-408 constitute the death domain.
  • FIG. 3A-3D shows the regions of similarity between the amino acid sequences of the DR3-V1, human tumor necrosis factor receptor 1, and Fas receptor (SEQ ED NOs: 5 and 6).
  • FIG. 4 shows an analysis of the DR3-V1 amino acid sequence.
  • Alpha, beta, turn and coil regions; hydrophilicity and hydrophobicity; amphipathic regions; flexible regions; antigenic index and surface probability are shown.
  • amino acid residues 1-22, 33-56, 59-82, 95-1 12, 122-133, 161-177, 179-190, 196-205 in SEQ ED NO : 2 correspond to the shown highly antigenic regions of the DR3-V1 protein.
  • the present invention provides isolated nucleic acid molecules comprising, or alternatively consisting of, a nucleic acid sequence encoding the DR3-V1 or DR3 polypeptide whose amino acid sequence is shown in SEQ ID NO:2 and SEQ ED NO: 4, respectively, or a fragment of the polypeptide.
  • the DR3-V1 and DR3 polypeptides of the present invention share sequence homology with human TNF
  • the nucleotide sequence shown in SEQ ED NOT was obtained by sequencing the HTTNB61 clone, which was deposited on March 1, 1996 at the American Type Culture Collection, 10801 University Boulevard. , Manassas, VA 20110-2209, USA, and given Accession Number 97456.
  • the deposited cDNA is contained in the pBluescript SK(-) plasmid (Stratagene, La olla, CA).
  • the nucleotide sequence shown in SEQ ED NO: 3 was obtained by sequencing a cDNA obtained from a HUVEC library, which was deposited on October 10, 1996 at the American Type Culture Collection, 10801 University Boulevard. , Manassas, VA 20110-2209, USA, and given Accession Number 97757.
  • the deposited cDNA is contained in the pBluescript SK(-) plasmid (Stratagene, LaJolla, CA).
  • 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.), 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.
  • isolated polypeptide or protein is intended a polypeptide or protein removed from its native environment.
  • recombinantly produced polypeptides and proteins expressed in host cells are considered isolated for purposes of the invention, as are native or recombinant polypeptides which have been substantially purified by any suitable technique such as, for example, the single-step purification method disclosed in Smith and Johnson, Gene 67:31-40
  • nucleic acid molecule of the present invention encoding a DR3 -VI or DR3 polypeptide may be obtained using standard cloning and screening procedures, such as those for cloning cDNAs using mRNA as starting material.
  • nucleic acid molecule described in SEQ ED NO: 1 was discovered in a cDNA library derived from cells of a human testis tumor.
  • nucleic acid molecule described in SEQ ID NO:3 was discovered in a human HUVEC cDNA library.
  • genes of the present invention have also been identified in cDNA libraries of the following tissues: fetal liver, fetal brain, tonsil and leukocyte. Furthermore, multiple forms of DR3 transcript are seen in Northern Blots and PCR reactions indicating that multiple variants of the transcript exists, possibly due to alternate splicing of the message.
  • the DR3-V1 (formerly called DDCR) gene contains an open reading frame encoding a protein of about 428 amino acid residues whose initiation codon is at position 198-200 of the nucleotide sequence shown in SEQ ID NOT, with a leader sequence of about 35 amino acid residues, and a deduced molecular weight of about 47 kDa.
  • DDCR DDCR-V1
  • DR3-V1 polypeptide of the invention shares the greatest degree of homology with human TNF Rl.
  • the DR3-V1 polypeptide shown in SEQ ID NO:2 is about 20% identical and about 50% similar to human TNF Rl.
  • the DR3 gene contains an open reading frame encoding a protein of about 417 amino acid residues whose initiation codon is at position 1 -3 of the nucleotide sequence shown in SEQ ID NO:3, with a leader sequence of about 24 amino acid residues, and a deduced molecular weight of about 43 kDa.
  • the DR3 polypeptide of the invention shares the greatest degree of homology with human TNF Rl.
  • the DR3 polypeptide shown in SEQ ID NO: 3 is about 20% identical and about 50% similar to human TNF Rl.
  • the present invention also provides the mature form(s) of the
  • proteins secreted by mammalian cells have a signal or secretory leader sequence which is cleaved from the mature protein once export of the growing protein chain across the rough endoplasmic reticulum has been initiated.
  • Most mammalian cells and even insect cells cleave secreted proteins with the same specificity.
  • cleavage of a secreted protein is not entirely uniform, which results in two or more mature species of the protein.
  • the cleavage specificity of a secreted protein is ultimately determined by the primary structure of the complete protein, that is, it is inherent in the amino acid sequence of the polypeptide.
  • the present invention provides a nucleotide sequence encoding the mature DR3-V1 or DR3 polypeptides having the amino acid sequence encoded by the cDNAs contained in the host identified as ATCC Deposit No. 97456 or 97757, respectively, and as shown in SEQ ID NO: 2 and SEQ ED NO :4.
  • the mature DR3-V1 or DR3 protein having the amino acid sequence encoded by the cDNAs contained in the host identified as ATCC Deposit No. 97456 or 97757, respectively is meant the mature form(s) of the DR3-V1 or DR3 protein produced by expression in a mammalian cell (e.g.
  • the mature DR3-V1 or DR3 having the amino acid sequence encoded by the cDNAs contained in ATCC Deposit No. 97456 or 97757, respectively, may or may not differ from the predicted "mature" DR3-V1 protein shown in SEQ ED NO: 2 (amino acids from about 36 to about 428) or DR3 protein shown in SEQ ED NO:4 (amino acids from about 24 to about 417) depending on the accuracy of the predicted cleavage site based on computer analysis.
  • the leader sequence for the DR3-V1 protein is predicted to consist of amino acid residues 1- 35 in SEQ ED NO: 2, while the predicted mature DR3-V1 protein consists of residues 36-428.
  • the leader sequence for the DR3 protein is predicted to consist of amino acid residues 1-24 in SEQ ED NO:4, while the predicted mature DR3 protein consists of residues 25-
  • the actual DR3-V1 polypeptide encoded by the deposited cDNA comprises about 428 amino acids, but may be anywhere in the range of 410-440 amino acids; and the actual leader sequence of this protein is about 35 amino acids, but may be anywhere in the range of about 25 to about 45 amino acids.
  • the actual DR3 polypeptide encoded by the deposited cDNA comprises about 417 amino acids, but may be anywhere in the range of 400-430 amino acids; and the actual leader sequence of this protein is about 24 amino acids, but may be anywhere in the range of about 14 to about 34 amino acids.
  • nucleic acid molecules of the present invention may be in the form of RNA, such as mRNA, or in the form of DNN including, for instance, cD ⁇ A and genomic D ⁇ A obtained by cloning or produced synthetically.
  • the D ⁇ A may be double-stranded or single-stranded.
  • Single-stranded D ⁇ A 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.
  • nucleic acid contained in a clone that is a member of a library e.g., a genomic or cD A 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 according to the present invention may be produced naturally, recombinantly, or synthetically. Isolated nucleic acid molecules of the present invention include DR3-V1
  • DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in SEQ ED NO: l and further include DNA molecules which comprise, or alternatively consist of, a sequence substantially different than all or part of the ORF whose initiation codon is at position 198-200 of the nucleotide sequence shown in SEQ ID NO: 1 but which, due to the degeneracy of the genetic code, still encode the DR3-V1 polypeptide or a fragment thereof.
  • ORF open reading frame
  • Isolated nucleic acid molecules of the present invention also include DR3 DNA molecules comprising, or alternatively consisting of, an open reading frame (ORF) shown in SEQ ID NO: 3 and further include DNA molecules which comprise, or alternatively consist of, a sequence substantially different than all or part of the ORF whose initiation codon is at position 1-3 of the nucleotide sequence shown in SEQ ED NO: 3 but which, due to the degeneracy of the genetic code, still encode the DR3 polypeptide or a fragment thereof.
  • ORF open reading frame
  • the invention provides isolated nucleic acid molecules encoding the DR3-V1 polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid deposited as ATCC Deposit No. 97456 on March 1, 1996.
  • the invention provides isolated nucleic acid molecules encoding the DR3 polypeptide having an amino acid sequence encoded by the cDNA contained in the plasmid deposited as ATCC Deposit No. 97757 on October 10, 1996.
  • these nucleic acid molecules will encode the mature polypeptide encoded by the above-described deposited cDNAs.
  • the invention further provides an isolated nucleic acid molecule having the nucleotide sequence shown in SEQ ED NO: 1 or SEQ ED NO: 3 or the nucleotide sequence of the DR3-V1 or
  • DR3 cDNA contained in the above-described deposited plasmids, or a nucleic acid molecule having a sequence complementary to one of the above sequences.
  • isolated DNA molecules and fragments thereof are useful, for example, as DNA probes for gene mapping by in situ hybridization with chromosomes, and for detecting expression of the DR3 -V 1 or DR3 gene in human tissue (including testis tumor tissue) by Northern blot analysis.
  • DR3 expression has been detected in a wide range of tissues and cell types including endothelial cells, liver cells, hepatocellular tumor, lymph nodes, Hodgkin's lymphoma, tonsil, bone marrow, spleen, heart, thymus, pericardium, healing wound (skin), brain, pancreas tumor, burned skin, U937 cells, testis, colon cancer (metasticized to liver), pancreas, rejected kidney, adipose, ovary, olfactory epithelium, striatum depression, HeLa cells, LNCAP (upon treatment with +30 nM androgen), 8 week embryo tissues, 9 week embryo tissues, fetal brain tissues, fetal kidney tissues, fetal heart tissues, fetal thymus tissues, fetal lung tissues, fetal liver tissues, fetal spleen tissues, T-cell helper II, activated T-cell (16 hr), activated T-cell (24 hr), primary dend
  • the present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of the isolated nucleic acid molecules described herein.
  • a fragment of an isolated nucleic acid molecule having the nucleotide sequence of one of the deposited cDNAs or the nucleotide sequence shown in SEQ ED NO: 1 or SEQ ED NO:3 is intended fragments at least about 15 nt, and more preferably at least about 20 nt, still more preferably at least about 30 nt, and even more preferably, at least about 40 nt in length which are useful as diagnostic probes and primers as discussed herein.
  • nt are also useful according to the present invention as are fragments corresponding to most, if not all, of the nucleotide sequence of one of the deposited cDNAs or as shown in SEQ ED NO: 1 or SEQ ED NO:3.
  • a fragment at least 20 nt in length is intended fragments which include 20 or more contiguous bases from the nucleotide sequence of one of the deposited cDNAs or the nucleotide sequence as shown in SEQ ED NO: 1 or SEQ ID NO:3.
  • the present invention is further directed to polynucleotides comprising, or alternatively consisting of, fragments of isolated nucleic acid molecules which encode subportions of DR3-V1 and DR3.
  • the invention provides polynucleotides comprising, or alternatively consisting of, the nucleotide sequences of a member selected from the group consisting of nucleotides 198-257, 208-267, 218-277, 228-287, 238-297, 248-307, 258-317, 268-327, 278-337, 288-347, 298-357, 308-367, 318-377, 328-387, 338-397, 348-407, 358-417, 368-427, 378-437, 388-447, 398-457, 408-469, 428-487, 458-517, 478-537, 498-557, 518-577, 538-597, 558-617, 578-637, 598-657, 638-697
  • the present invention is further directed to polynucleotides comprising, or alternatively consisting of, isolated nucleic acid molecules which encode domains of DR3-V1 and DR3.
  • the invention provides polynucleotides comprising, or alternatively consisting of, nucleic acid molecules which encode beta- sheet regions of DR3-V1 protein set out in Table 2.
  • polynucleotides include nucleic acid molecules which encode a polypeptide comprise, or alternatively consist of, one, two, three, four, five or more amino acid sequences selected from the group consisting of amino acid residues from about 24 to about 32, amino acid residues from about 53 to about 58, amino acid residues from about 133 to about 142, amino acid residues from about 202 to about 234, amino acid residues from about 281 to about 288, amino acid residues from about 304 to about 312, and amino acid residues from about
  • Preferred nucleic acid fragments of the present invention include nucleic acid molecules encoding one, two, three, four, five, or more amino acids sequences selected from the group consisting of: a polypeptide comprising, or alternatively consisting of, the DR3-V1 extracellular domain (amino acid residues from about 36 to about 212 in SEQ ID NO:2); a polypeptide comprising, or alternatively consisting of, the DR3-V1 transmembrane domain (amino acid residues from about 213 to about 235 in SEQ ID NO: 2; a polypeptide comprising, or alternatively consisting of, the DR3-V1 intracellular domain (amino acid residues from about 236 to about 428 in SEQ ID NO:2; and a polypeptide comprising, or alternatively consisting of, the DR3-V1 death domain (amino acid residues from about 353 to about 419 in SEQ ED NO:2).
  • Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the invention also provides polynucleotides comprising, or alternatively consisting of, nucleic acid molecules encoding: amino acid residues from about 1 to about 215 of SEQ ID NO: 2; amino acid residues from about 30 to about 215 of SEQ ID NO:2; amino acid residues from about 215 to about 240 of SEQ ED NO:2; amino acid residues from about 240 to about 428 of SEQ ED NO:2; and amino acid residues from about 350 to about 420 of SEQ ID NO:2.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • nucleic acid fragments of the present invention further include nucleic acid molecules encoding epitope-bearing portions of the DR3-V1 protein.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding: a polypeptide comprising, or alternatively consisting of, amino acid residues from about 1 to about 22 in SEQ ID NO: 2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 33 to about 56 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 59 to about 82 in SEQ ED NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 95 to about 112 in SEQ ID NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues from about 122 to about 133 in SEQ ED NO:2; a polypeptide comprising, or alternatively consisting of, amino acid residues
  • SEQ ED NO:2 SEQ ED NO:2; and a polypeptide comprising, or alternatively consisting of, amino acid residues from about 196 to about 205 in SEQ ID NO:2.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids.
  • the inventors have determined that the above polypeptide fragments are antigenic regions of the DR3-V1 protein.
  • nucleic acid fragments of the present invention also include nucleic acid molecules encoding epitope-bearing portions of the DR3 protein.
  • nucleic acid fragments of the present invention include nucleic acid molecules encoding the corresponding regions to those epitope-bearing regions of the DR3-V1 protein disclosed above. Methods for determining other such epitope-bearing portions of the DR3 protein are described in detail below.
  • 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, the complement of a polynucleotide fragment described herein, or the cDNA plasmids contained in ATCC Deposit 97456 or ATCC Deposit 97757.
  • stringent hybridization conditions is intended overnight incubation at 42° C in a solution comprising, or alternatively consisting of: 50% formamide, 5x SSC (750mM NaCl, 75mM trisodium citrate), 50 mM sodium phosphate (pH 7.6), 5x Denhardt's solution, 10%) dextran sulfate, and 20 ⁇ g/ml denatured, sheared salmon sperm DNN followed by washing the filters in 0. lx SSC at about 65 °C.
  • 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 nt of the reference polynucleotide.
  • nt nucleotides
  • nt nucleotides
  • 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., the deposited cDNAs or the nucleotide sequence as shown in SEQ ID NO: 1 or SEQ ED NO:3).
  • a polynucleotide which hybridizes only to a poly A sequence such as the 3 ' terminal poly(A) tract of the DR3-V1 cDNA shown in SEQ ED NO: 1), or to a complementary stretch of T (or U) resides, 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 generated from an oligo-dT primed cDNA library).
  • nucleic acid molecules of the present invention which encode the DR3-V1 or DR3 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 corresponds to an epitope derived of influenza hemagglutinin protein, which has been described by Wilson et al, Cell 37:161 (1984), for instance.
  • the present invention further relates to variants of the nucleic acid molecules of the present invention, which encode for fragments, analogs or derivatives of the DR3-V1 or DR3 polypeptide.
  • Variants may occur naturally, such as an allelic variant.
  • allelic variant is intended one of several alternate forms of a gene occupying a given locus on a chromosome of an organism. Gene II, Lewin, B., ed., John Wiley & Sons, New York (1985).
  • Non- naturally occurring variants may be produced using art-known mutagenesis techniques.
  • variants include those produced by nucleotide substitutions, deletions, or additions which may involve one or more nucleotides.
  • the variants may be altered in coding or non-coding regions or both. Alterations in the coding regions may produce conservative or non-conservative amino acid substitutions, deletions, or additions.
  • nucleic acid molecules that are at least 80% identical, and more preferably at least 85%, 90%,
  • DR3 death domain or (1) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c), (d), (e), (f), (g), (h), (i), (j) or (k) above.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • polynucleotide having a nucleotide sequence at least, for example, 95% "identical" to a reference nucleotide sequence encoding a DR3-V1 or DR3 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 DR3-V1 or DR3.
  • 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 mismatches 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 DR3-V1 or DR3 encoding nucleotide sequence shown respectively in SEQ ED NO: 2 and SEQ ED NO:4 or any DR3-V1 or DR3 polynucleotide fragment (e.g., a polynucleotide encoding the amino acid sequence of any of the DR3-V1 or DR3 N- and/or C- terminal deletions described herein), variant, derivative or analog, as described herein.
  • nucleic acid molecule is at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to, for instance, the encoding nucleotide sequence shown in SEQ ED NO: 2 or SEQ ED NO:4, or to the nucleotide sequence of the deposited cDNAs, can be determined conventionally using known computer programs such as the Bestfit program
  • 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 a reference 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 ⁇ l (Comp. App. Biosci. (5:237-245 (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.
  • the present application is directed to nucleic acid molecules at least 80%), 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ED NO:3 or to the nucleic acid sequence of the deposited cDNAs, irrespective of whether they encode a polypeptide having DR3 functional activity.
  • nucleic acid molecules at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequences disclosed herein, (e.g., nucleic acid sequences encoding a polypeptide having the amino acid sequence of an N- and/or C-terminal deletion disclosed herein, such as, for example, a nucleic acid molecule encoding amino acids 30 to 200, 30 to 215, 215 to 240, 240 to 428, 350 to 420, or 2 to 428 of SEQ ED NO:2), irrespective of whether they encode a polypeptide having DR3 functional activity.
  • nucleic acid sequences disclosed herein e.g., nucleic acid sequences encoding a polypeptide having the amino acid sequence of an N- and/or C-terminal deletion disclosed herein, such as, for example, a nucleic acid molecule encoding amino acids 30 to 200, 30 to 215, 215 to 240, 240 to 428, 350
  • nucleic acid molecule does not encode a polypeptide having DR3 functional 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 DR3 functional activity include, inter alia, (1) isolating the DR3 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 DR3-V1 or DR3 gene, as described in Verma et al,
  • nucleic acid molecules having sequences at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the nucleic acid sequence shown in SEQ ID NO: 1, SEQ ED NO: 3 or to the nucleic acid sequence of the deposited cDNAs which do, in fact, encode a polypeptide having DR3 functional activity.
  • a polypeptide having DR3 functional activity is intended polypeptides exhibiting activity similar, but not necessarily identical, to an activity of the DR3 proteins of the invention (either the full-length protein or, preferably, the mature protein), as measured in a particular biological assay.
  • a DR3-V1 or DR3 functional activity can routinely be measured by determining the ability of a DR3-V1 or DR3 polypeptide to bind a DR3-V1 or DR3 ligand (e.g., TNF- ⁇ - ⁇ , NF-kB, TRADD). Further, DR3 functional activity can be measured using the cell death assays performed essentially as previously described (A.M.
  • Nuclei of cells transfected with DR3 will exhibit apoptotic morphology as assessed by DAPI staining. Similar to TNFR-1 and Fas/APO-1 (M. Muzio etal, Cell 85, 811-821 (1996); M. P. Boldin et al, Cell 85, 803-815 (1996); M. Tewari et al, J Biol Chem 270, 3255-60 (1995)), DR3-induced apoptosis is blocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk. In addition, apoptosis induced by DR3 is also blocked by dominant negative versions of FADD (FADD-DN) or FLICE (FLICE-DN/MACHalC360S).
  • FADD-DN FADD-DN
  • FLICE FLICE-DN/MACHalC360S
  • DR3 polypeptides and fragments, variants derivatives, and analogs thereof, can be assayed by various methods.
  • 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, irnmunodiffusion 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” immunoassay
  • 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. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • 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. etal, 1995, Microbioi. Rev. 59:94-123.
  • physiological correlates of binding to its substrates can be assayed.
  • nucleic acid molecules having a sequence at least 80%, 85%, 90%, 92%, 95%, 96%, 97%,
  • nucleic acid sequence of the deposited cDNAs will encode polypeptides "having DR3 functional activity.”
  • degenerate variants of any of these nucleotide sequences all encode the same polypeptide, in many instances, 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 DR3 functional activity. This is because the skilled artisan is fully aware of amino acid substitutions that are either less likely or not likely to significantly effect protein function (e.g. , replacing one aliphatic amino acid with a second aliphatic amino acid), as further described below.
  • This invention is also related to the use of the DR3-V1 or DR3 polynucleotides to detect complementary polynucleotides such as, for example, as a diagnostic reagent. Detection of a mutated form of DR3-V1 or DR3 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 DR3-V1 or DR3 or a soluble form thereof, such as, for example, tumors or autoimmune disease.
  • RNA or cDNA may also be used in the same ways.
  • PCR primers complementary to the nucleic acid encoding DR3-V1 or DR3 can be used to identify and analyze DR3-V1 or DR3 expression and mutations.
  • 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 DR3-V1 or DR3 RNA or alternatively, radiolabeled DR3-V1 or DR3 antisense DNA sequences. Perfectly matched sequences can be distinguished from mismatched duplexes by RNase A digestion or by differences in melting temperatures.
  • Sequence differences between a reference gene and genes having mutations also may be revealed by direct DNA sequencing.
  • cloned 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 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 250: 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 etal, Proc. Natl. Acad. Sci. USA 85: 4397-4401 (1985)).
  • nuclease protection assays such as RNase and SI protection or the chemical cleavage method (e.g., Cotton etal, 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")
  • RFLP restriction fragment length polymorphisms
  • Southern blotting of genomic DNA In addition to more conventional gel-electrophoresis and DNA sequencing, mutations also can be detected by in situ analysis.
  • sequences of the present invention are also valuable for chromosome identification.
  • the sequence is specifically targeted to and can hybridize with a particular location on an individual human chromosome.
  • the mapping of DNAs to chromosomes according to the present invention is an important first step in correlating those sequences with genes associated with disease.
  • the cDN A herein disclosed is used to clone genomic DNA of a DR3-V1 or a DR3 gene. This can be accomplished using a variety of well known techniques and libraries, which generally are available commercially. The genomic DNA is then used for in situ chromosome mapping using well known techniques for this purpose.
  • sequences can be mapped to chromosomes by preparing PCR primers (preferably 15-25 bp) from the cDNA. Computer analysis of the 3' untranslated region of the gene is used to rapidly select primers that do not span more than one exon in the genomic DNA, thus complicating the amplification process. These primers are then used for PCR screening of somatic cell hybrids containing individual human chromosomes. Fluorescence in situ hybridization ("FISH”) of a cDNA clone to a metaphase chromosomal spread can be used to provide a precise chromosomal location in one step. This technique can be used with cDNA as short as 50 or 60.
  • FISH Fluorescence in situ hybridization
  • the present invention also relates to vectors which include DNA molecules of the present invention, host cells which are genetically engineered with vectors of the invention and the production of polypeptides of the invention 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.
  • Preferred among vectors, in certain respects, 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 SV40, 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) including, for instance, a promoter to direct mRNA transcription.
  • promoters 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 (UAA, UGA or UAG) 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 such as E. coli, Streptomyces and Salmonella typhimurium cells
  • Hosts for a great variety of expression constructs are well known, and those of skill will be enabled by the present disclosure readily to select a host for expressing a polypeptides in accordance with this aspect of the present invention.
  • vectors preferred for use in bacteria are pQ ⁇ 70, pQE60 and pQE-9, available from Qiagen; pBS vectors, Phagescript vectors, Bluescript vectors, pNH8A, pNH16a, pNH18A, pNH46N available from Stratagene; and ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5 available from Pharmacia.
  • eukaryotic vectors are pWL ⁇ EO, 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 constructs discussed above.
  • the host cell can be a higher eukaryotic cell, such as a mammalian 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.
  • 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.
  • 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., the DR3 coding sequence), and/or to include genetic material (e.g., heterologous polynucleotide sequences) that is operably associated with DR3-V1 or DR3 polynucleotides of the invention, and which activates, alters, and/or amplifies endogenous DR3-V1 or DR3 polynucleotides.
  • endogenous genetic material e.g., the DR3 coding sequence
  • genetic material e.g., heterologous polynucleotide sequences
  • heterologous control regions e.g., promoter and/or enhancer
  • endogenous DR3-V1 or DR3 polynucleotide sequences via homologous recombination (.vee, e.g., US Patent Number
  • the polypeptide may be expressed in a modified form, such as a fusion protein, and may include not only secretion signals but also additional heterologous functional regions.
  • a region of additional amino acids, particularly charged amino acids may 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.
  • region also may be added to the polypeptide to facilitate purification. Such regions may 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 counterpart 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 0232 262).
  • Fc portion proves to be a hindrance to use in therapy and diagnosis, for example when the fusion protein is to be used as antigen for immunizations.
  • human proteins such as, hTL-5-receptor has been fused with Fc portions for the purpose of high-throughput screening assays to identify antagonists of hEL-5. See, D. Bennett et al, Journal of Molecular Recognition, Vol. 8:52-58 (1995) and K.
  • the DR3 and DR3-V1 polypeptides can be recovered and purified from recombinant cell cultures 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. Well known techniques for refolding protein may be employed to regenerate active conformation when the polypeptide is denatured during isolation and/or purification.
  • HPLC high performance liquid chromatography
  • Polypeptides of the 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 may be 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.
  • DR3-V1 or DR3 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 DR3.
  • applications in treatment and/or prevention of tumors, resistance to parasites, bacteria and viruses, to induce proliferation of T-cells, endothelial cells and certain hematopoietic cells, to treat and/or prevent restenosis, graft vs. host disease, to regulate anti-viral responses and to prevent certain autoimmune diseases after stimulation of DR3 by an agonist.
  • Additional applications relate to the prognosis, diagnosis, prevention and/or treatment of disorders of cells, tissues and organisms. These aspects of the invention are discussed further below.
  • the invention further provides an isolated DR3-V1 or DR3 polypeptide having the amino acid sequence shown in SEQ ED NO: 2 and SEQ ID NO:4, respectively, or a fragment thereof. It will be recognized in the art that some amino acid sequence of DR3-V1 or DR3 can be varied without significant effect of the structure or function of the protein. If such differences in sequence are contemplated, it should be remembered that there will be critical areas on the protein which determine activity. Such areas will usually comprise residues which make up the ligand binding site or the death domain, or which form tertiary structures which affect these domains.
  • the invention further includes variations of the DR3-V1 or DR3 protein which show substantial DR3 functional activity or which include regions of DR3 -V 1 or DR3 such as the protein fragments discussed below.
  • Such mutants include deletions, insertions, inversions, repeats, and type substitutions.
  • guidance concerning which amino acid changes are likely to be phenotypically silent can be found in J.U. Bowie et al, Science 247: 1306-1310 (1990).
  • substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids are substitutions of charged amino acids with another charged amino acid and with neutral or negatively charged amino acids. The latter results in proteins with reduced positive charge to improve the characteristics of the DR3-V1 or DR3 protein. The prevention of aggregation is highly desirable.
  • the replacement of amino acids can also change the selectivity of binding 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.
  • the DR3-V1 or DR3 receptor of the 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 1). TABLE 1. Conservative Amino Acid Substitutions.
  • the number of amino acid substitutions a skilled artisan would make depends on many factors, including those described above. Generally speaking, the number of substitutions for any given DR3-V1 or DR3 polypeptide will not be more than 50, 40, 30, 25, 20, 15, 10, 5 or 3.
  • Amino acids in the DR3-V1 or DR3 protein of the 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, or in vitro proliferative activity. 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.
  • polypeptides of the present invention are preferably provided in an isolated form, and preferably are substantially purified.
  • a recombinantly produced version of the DR3-V1 or DR3 polypeptide is substantially purified by the one- step method described in Smith and Johnson, Gene (57:31-40 (1988).
  • polypeptides of the present invention also include the polypeptide encoded by the deposited cDNAs including the leader, the mature polypeptide encoded by the deposited the cDNAs minus the leader (i.e., the mature protein), the polypeptide of SEQ ED NO:2 or SEQ ED NO:4 including the leader, the polypeptide of SEQ ID NO: 2 or SEQ ID NO:4 minus the leader, the extracellular domain, the transmembrane domain, the intracellular domain, soluble polypeptides comprising, or alternatively consisting of, all or part of the extracellular and intracellular domains but lacking the transmembrane domain as well as polypeptides which are at least 80%> identical, more preferably at least 80%> or 85% identical, still more preferably at least 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the polypeptide encoded by the deposited cDNAs, to the polypeptide of SEQ ID NO:2 or SEQ ID NO:4, and also include portions
  • a polypeptide having an amino acid sequence at least 95% identical to a reference amino acid sequence 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 of the reference amino acid of a DR3-V1 or DR3.
  • 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% of the total amino acid residues in the reference sequence may be inserted into the reference sequence.
  • amino acid sequence shown in SEQ ED NO:2 or SEQ ED NO:4 can be determined conventionally using known computer programs such as the Bestfit program (Wisconsin Sequence Analysis Package, Version 8 for Unix, Genetics
  • 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%> of the total number of amino acid residues 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. 6:237-245 (1990)).
  • the percent identity is corrected by calculating the number of residues of the query sequence that are N- and C-terminal of the subject sequence, which are not matched/aligned with a corresponding subject residue, as a percent of the total bases of the query sequence.
  • a determination of whether a residue 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 final percent identity score is what is used for the purposes of this embodiment. Only residues to the N- and C-termini of the subject sequence, which are not matched/aligned with the query sequence, are considered for the purposes of manually adjusting the percent identity score. That is, only query residue positions outside the farthest N- and C-terminal residues of the subject sequence. For example, a 90 amino acid residue subject sequence is aligned with a 100 residue query sequence to determine percent identity.
  • the deletion occurs at the N-terminus of the subject sequence and therefore, the FASTDB alignment does not show a matching/alignment of the first 10 residues at the N-terminus.
  • 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 of the subject sequence which are not matched/aligned with the query.
  • the DR3-V1 polypeptide is a 428 residue protein exhibiting three main structural domains.
  • the ligand binding domain was identified within amino acid residues from about 36 to about
  • the transmembrane domain was identified within amino acid residues from about 213 to about 235 in SEQ ID NO:2.
  • the intracellular domain was identified within amino acid residues from about 236 to about 428 in SEQ ED NO:2.
  • the intracellular domain includes a death domain at amino acid residues from about 353 to about 419.
  • Further preferred fragments of the polypeptide shown in SEQ ID NO: 2 include the mature protein from amino acid residues about 36 to about 428 and soluble polypeptides comprising, or alternatively consisting of, all or part of the extracellular and intracellular domains but lacking the transmembrane domain.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention also provides polypeptides comprising, or alternatively consisting of, one, two, three, four, five or more amino acid sequences selected from the group consisting of amino acid residues from about 1 to about 215 of
  • SEQ ID NO:2 amino acid residues from about 30 to about 215 of SEQ ED NO:2; amino acid residues from about 215 to about 240 of SEQ ID NO:2; amino acid residues from about 240 to about 428 of SEQ ID NO:2; and amino acid residues from about 350 to about 420 of SEQ ID NO:2.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or
  • the DR3 polypeptide is a 417 residue protein exhibiting three main structural domains.
  • the ligand binding domain was identified within amino acid residues from about 25 to about 201 in SEQ ED NO:4.
  • the transmembrane domain was identified within amino acid residues from about 202 to about 224 in SEQ ED NO:4.
  • the intracellular domain was identified within amino acid residues from about 225 to about 417 in SEQ ID NO:4.
  • the intracellular domain includes a death domain at amino acid residues from about 342 to about 408.
  • polypeptide shown in SEQ ED NO:4 include the mature protein from amino acid residues about 25 to about 417 and soluble polypeptides comprising, or alternatively consisting of, all or part of the extracellular and intracellular domains but lacking the transmembrane domain.
  • “about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the full length polypeptides encoded by the DR3-V1 and DR3 cDNA differ only in the amino acid sequence of the leader peptide.
  • the first 24 amino acids of the polypeptide shown in SEQ ID NO: 1 The first 24 amino acids of the polypeptide shown in SEQ ID NO: 1
  • DR3 cDNA encode an identical mature protein having the same biological activity.
  • the invention further provides DR3-V1 or DR3 polypeptides encoded by the deposited cDNAs including the leader and DR3-V1 or DR3 polypeptide fragments selected from the mature protein, the extracellular domain, the transmembrane domain, the intracellular domain, and the death domain.
  • polypeptides of the 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 molecular weight markers on SDS-PAGE gels or on molecular sieve gel filtration columns using methods well known to those of skill in the art.
  • the invention provides a peptide or polypeptide comprising, or alternatively consisting of, an epitope-bearing portion of a polypeptide described herein.
  • Antigenic epitope-bearing peptides and polypeptides of the invention are therefore useful to raise antibodies, including monoclonal antibodies, that bind specifically to a polypeptide of the invention See, for instance, Wilson et al , Cell 37 767-778 (1984) at 777
  • Antigenic epitope-bearing peptides and polypeptides of the invention preferably contain a sequence of at least seven, more preferably at least nine and most preferably between at least about 15 to about 30 amino acids contained within the amino acid sequence of a polypeptide of the invention In this context "about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) amino acids Polynucleotides encoding these antigenic epitope-b earing peptides are also encompassed by the invention
  • Non-limiting examples of antigenic polypeptides or peptides that can be used to generate DR3 -specific antibodies include a polypeptide comprising, or alternatively consisting of, amino acid residues from about 1 to about 22 in SEQ ID NO 2, a polypeptide comprising, or alternatively consisting of, one, two, three, four, five or more amino acid sequences selected from the group consisting of amino acid residues from about 33 to about 56 in SEQ ID NO 2, a polypeptide comprising, or alternatively consisting of, amino acid residues from about 59 to about 82 in SEQ ID NO 2, a polypeptide comprising, or alternatively consisting of, amino acid residues from about 95 to about 112 in SEQ ED NO 2, a polypeptide comprising, or alternatively consisting of, amino acid residues from about 122 to about 133 in SEQ ID NO 2, a polypeptide comprising, or alternatively consisting of, amino acid residues from about 161 to about 177 in SEQ ID NO 2, a polypeptide comprising,
  • the epitope-bearing peptides and polypeptides of the invention may be produced by any conventional means.
  • R.A. Houghten 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
  • DR3-V1 or DR3 polypeptides of the present invention can be combined with parts of the constant domain of immunoglobulins (IgG), resulting in chimeric polypeptides.
  • IgG immunoglobulins
  • These 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 DR3-V1 or DR3 protein or protein fragment alone (Fountoulakis et al, J Biochem 270:3958-3964 (1995)).
  • the present invention thus encompasses polypeptides comprising, or alternatively consisting of, an epitope of the polypeptide having an amino acid sequence of SEQ ID NO:2 or SEQ ID NO:4, or an epitope of the polypeptide sequence encoded by a polynucleotide sequence contained in the plasmid deposited as ATCC Deposit No.
  • the present invention further encompasses polynucleotide sequences encoding an epitope of a polypeptide sequence of the invention (such as, for example, the sequence disclosed in SEQ ID NO: 1 or SEQ
  • polynucleotide sequences of the complementary strand of a polynucleotide sequence encoding an epitope of the invention polynucleotide sequences which hybridize to the complementary strand under stringent hybridization conditions or lower stringency hybridization conditions defined supra.
  • Polynucleotides encoding these antigenic epitope-bearing peptides are also encompassed by the invention.
  • 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. Natl. Acad. Sci.
  • 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.
  • 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, J.G Sutcliffe 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.
  • 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. 4,631,211).
  • 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.
  • 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)).
  • 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).
  • 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., 66:2347-2354 (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 adsorption to the peptide on a solid support and elution of the selected antibodies according to methods well known in the art.
  • the polypeptides of the present invention comprising an immunogenic or antigenic epitope can be fused to other polypeptide sequences.
  • the polypeptides of the 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.
  • Such fusion proteins may facilitate purification and may increase half-life in vivo. This has been shown 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. See, e.g., EP 394,827; Traunecker et al, Nature, 337:84-86 (1988).
  • IgG Fusion proteins that have a disulfide-linked dimeric structure due to the IgG portion disulfide bonds have also been found to be more efficient in binding and neutralizing other molecules than monomeric polypeptides or fragments thereof alone. See, e.g., Fountoulakis et al, J.
  • 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 of the 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; 5,811,238; 5,830,721; 5,834,252; and 5,837,458, and Patten et al , Curr. Opinion Biotechnol.
  • alteration of polynucleotides corresponding to SEQ ID NO: l or SEQ ID NO: 3 and the polypeptides encoded by these polynucleotides may be achieved by DNA shuffling.
  • DNA shuffling involves the assembly of two or more
  • polynucleotides of the invention 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. As mentioned above, even if deletion of one or more amino acids from the
  • N-terminus of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind DR3-V1 ligand) may still be retained.
  • other functional activities e.g., biological activities, ability to multimerize, ability to bind DR3-V1 ligand
  • the ability of shortened DR3-V1 muteins to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptides generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the N-terminus.
  • Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that an immunologic activities.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the DR3-V1 amino acid sequence shown in SEQ ED NO:2, up to the arginine residue at position number 423 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n 1-428 of SEQ ID NO: 2, where nl is an integer from 2 to 423 corresponding to the position of the amino acid residue in SEQ ED NO:2.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues E-2 to P-428; E-3 to P-428; T-4 to P-428; Q-5 to P-428; Q-6 to P-428; G-7 to P-428; E-8 to P-428; A-9 to P-428; P-10 to P-428; R-l 1 to P-428; G-12 to P-428; Q-13 to P-428; L-14 to P-428; R-l 5 to P-428; G-16 to P-428; E-17 to P-428; S-18 to
  • P-428 A- 19 to P-428; A-20 to P-428; P-21 to P-428; V-22 to P-428 P-23 to P-428 Q-24 to P-428; A-25 to P-428; L-26 to P-428; L-27 to P-428 L-28 to P-428 V-29 to P-428; L-30 to P-428; L-31 to P-428; G-32 to P-428 A-33 to P-428 R-34 to P-428; A-35 to P-428; Q-36 to P-428; G-37 to P-428 G-38 to P-428 T-39 to P-428; R-40 to P-428; S-41 to P-428; P-42 to P-428 R-43 to P-428 C-44 to P-428; D-45 to P-428; C-46 to P-428; A-47 to P-428 G-48 to P-428 D-49 to P-428; F-50 to P-428; H-51 to P-428; K-52 to P-428
  • P-428 M-211 to P-428; F-212 to P-428; W-213 to P-428; V-214 to P-428; Q-215 to P-428; V-216 to P-428; L-217 to P-428; L-218 to P-428; A-219 to P-428; G-220 to P-428; L-221 to P-428; V-222 to P-428; V-223 to P-428; P-224 to P-428; L-225 to P-428; L-226 to P-428; L-227 to P-428; G-228 to P-428; A-229 to P-428; T-230 to P-428; L-231 to P-428; T-232 to P-428; Y-233 to P-428; T-234 to P-428; Y-235 to P-428; R-236 to P-428; H-237 to P-428; C-238 to P-428; W-239 to P-428; P-240 to P-428; H-241 to P-428; K
  • 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 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 DR3 -V 1 polypeptide shown in SEQ ID NO : 2, up to the glutamine 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-ml of SEQ ED NO:2, where ml is an integer from 6 to 427 corresponding to the position of the amino acid residue in SEQ ID NO:2. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues M-l 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%>, 91%, 98%, or 99% identical to the polynucleotide sequences encoding 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 of an DR3-V1 polypeptide, which may be described generally as having residues nl-ml of SEQ ID NO:2, where nl and ml are integers as described above.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention. As mentioned above, even if deletion of one or more amino acids from the
  • N-terminus of an extracellular domain of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind DR3 -V 1 ligand) may still be retained.
  • other functional activities e.g., biological activities, ability to multimerize, ability to bind DR3 -V 1 ligand
  • the ability of shortened DR3-V1 extracellular domain muteins to induce and/or bind to antibodies which recognize the complete, mature or extracellular domain forms of the polypeptides generally will be retained when less than the majority of the residues of the complete, mature or extracellular domain polypeptide are removed from the N-terminus.
  • Whether a particular polypeptide lacking N-terminal residues of an extracellular domain of a polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a DR3-V1 extracellular domain mutein with a large number of deleted N-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six DR3-V1 extracellular domain 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 DR3-V1 extracellular domain amino acid sequence shown in SEQ ED NO:2, up to the cysteine residue at position number 206 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n2-212 of SEQ ED NO: 2, where n2 is an integer from 36 to 206 corresponding to the position of the amino acid residue in SEQ ED NO:2. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues Q-36 to
  • F-212 D-172 to F-212, C-173 to F-212, G-174 to F-212, T-175 to F-212, C-176 to F-212, L-177 to F-212, P-178 to F-212, G-179 to F-212, F-180 to F-212, Y-181 to F-212, E-182 to F-212, H-183 to F-212, G-184 to F-212, D-185 to F-212, G-l 86 to F-212, C-187 to F-212, V-188 to F-212, S-189 to F-212, C-190 to F-212, P-191 to F-212, T-192 to F-212, S-193 to F-212, T-194 to F-212,
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%>,
  • 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. Also as mentioned above, even if deletion of one or more amino acids from the C-terminus of an extracellular domain of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g., biological activities, ability to multimerize, ability to bind DR3-V1 ligand) may still be retained.
  • other functional activities e.g., biological activities, ability to multimerize, ability to bind DR3-V1 ligand
  • the ability of the shortened DR3-V1 extracellular domain mutein to induce and/or bind to antibodies which recognize the complete, mature or extracellular domain forms of the polypeptide generally will be retained when less than the majority of the residues of the complete, mature or extracellular domain of a polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of an extracellular domain of a polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a DR3-V1 extracellular domain mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six DR3-V1 extracellular domain amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the extracellular domain of the DR3-V1 polypeptide shown in SEQ ID NO: 2, up to the proline residue at position number 42, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues 36-m2 of SEQ ID NO:2, where m2 is an integer from 42 to 212 corresponding to the position of the amino acid residue in SEQ ID NO:2. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues Q-36 to F-212; Q-36 to M-211; Q-36 to Q-210; Q-36 to R-209; Q-36 to W-208; Q-36 to G-207; Q-36 to C-206 Q-36 to V-205; Q-36 to A-204; Q-36 to A-203; Q-36 to C-202; Q-36 to R-201 Q-36 to E-200; Q-36 to P-199; Q-36 to C- 198; Q-36 to S-197; Q-36 to G-196 Q-36 to L-195; Q-36 to T-194; Q-36 to S- 193; Q-36 to T-192; Q-36 to P-191 Q-36 to C-190; Q-36 to S-189; Q-36 to V- 188; Q-36 to C-
  • 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 of an DR3-V1 polypeptide, which may be described generally as having residues n2-m2 of SEQ YD NO:2, where n2 and m2 are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the DR3 amino acid sequence shown in SEQ ID NO:4, up to the arginine residue at position number 412 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n3-417 of SEQ ED NO:4, where n3 is an integer from 2 to 412 corresponding to the position of the amino acid residue in SEQ ED NO:4.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues E-2 to P-417; Q-3 to P-417; R-4 to P-417; P-5 to P-417; R-6 to P-417; G-7 to P-417; C-8 to P-417; A-9 to P-417; A-10 to P-417; V-11 to P-417; A-12 to P-417; A-13 to P-417; A-14 to P-417; L-15 to P-417; L-16 to P-417; L-17 to P-417; V-18 to
  • P-417 C-141 to P-417; L-142 to P-417; D-143 to P-417; C-144 to P-417; G-145 to P-417; A-146 to P-417; L-147 to P-417; H-148 to P-417; R-149 to P-417; H-150 to P-417; T-151 to P-417; R-152 to P-417; L-153 to P-417; L-154 to P-417; C-155 to P-417; S-156 to P-417; R-157 to P-417; R-158 to P-417; D-159 to P-417; T-160 to P-417; D-161 to P-417; C-162 to P-417; G-163 to P-417;
  • 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 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 ability of the shortened DR3 mutein to induce and/or bind to antibodies which recognize the complete or mature forms of the polypeptide generally will be retained when less than the majority of the residues of the complete or mature polypeptide are removed from the C-terminus. Whether a particular polypeptide lacking C-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art. It is not unlikely that a DR3 mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities. In fact, peptides composed of as few as six DR3 amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the DR3 polypeptide shown in SEQ ID NO:4, up to the arginine 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 l-m3 of SEQ ED NO:4, where m3 is an integer from 6 to 416 corresponding to the position of the amino acid residue in SEQ ED NO:4. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues M-l to G-416; M-l to R-415; M-l to Q-414; M-l to L-413; M-l to R-412; M-l to S-411; M-l to R-410; M-l to L-409; M-l to D-408; M-l to E-407; M-l to V-406; M-l to C-405; M-l to G-404; M-l to D-403; M-l to L-402; M-l 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%o identical to the polynucleotide sequences encoding 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 of an DR3 polypeptide, which may be described generally as having residues n3-m3 of SEQ ID NO: 4, where n3 and m3 are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention. As mentioned above, even if deletion of one or more amino acids from the
  • N-terminus of an extracellular domain of a protein results in modification of loss of one or more biological functions of the protein, other functional activities (e.g. , biological activities, ability to multimerize, ability to bind DR3 ligand) may still be retained.
  • other functional activities e.g. , biological activities, ability to multimerize, ability to bind DR3 ligand
  • the ability of shortened DR3 extracellular domain muteins to induce and/or bind to antibodies which recognize the complete, mature or extracellular domain forms of the polypeptides generally will be retained when less than the majority of the residues of the complete, mature or extracellular domain polypeptide are removed from the N-terminus.
  • Whether a particular polypeptide lacking N-terminal residues of a complete polypeptide retains such immunologic activities can readily be determined by routine methods described herein and otherwise known in the art.
  • the present invention further provides polypeptides having one or more residues deleted from the amino terminus of the DR3 extracellular domain amino acid sequence shown in SEQ ID NO:4, up to the cysteine residue at position number 195 and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues n4-201 of SEQ ED NO:4, where n4 is an integer from 25 to 195 corresponding to the position of the amino acid residue in SEQ ID NO:4.
  • Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues Q-25 to F-201; G-26 to F-201 ; G-27 to F-201; T-28 to F-201; R-29 to F-201; S-30 to F-201; P-31 to F-201; R-32 to F-201; C-33 to F-201; D-34 to F-201; C-35 to F-201; A-36 to F-201; G-37 to F-201; D-38 to F-201; F-39 to F-201; H-40 to F-201; K-41 to F-201; K-42 to F-201; 1-43 to F-201; G-44 to F-201; L-45 to F-201; F-46 to F-201; C-47 to F-201; C-48 to F-201; R-49 to F-201; G-50 to F-201; C
  • the present invention is also directed to nucleic acid molecules comprising, or alternatively consisting of, a polynucleotide sequence at least 80%>,
  • 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.
  • a DR3 extracellular domain mutein with a large number of deleted C-terminal amino acid residues may retain some biological or immunogenic activities.
  • peptides composed of as few as six DR3 extracellular domain amino acid residues may often evoke an immune response.
  • the present invention further provides polypeptides having one or more residues deleted from the carboxy terminus of the amino acid sequence of the DR3 extracellular domain polypeptide shown in SEQ ED NO: 4, up to the proline residue at position number 31, and polynucleotides encoding such polypeptides.
  • the present invention provides polypeptides comprising, or alternatively consisting of, the amino acid sequence of residues l-m4 of SEQ ID NO:4, where m4 is an integer from 31 to 201 corresponding to the position of the amino acid residue in SEQ ID NO:4. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the invention provides polynucleotides encoding polypeptides comprising, or alternatively consisting of, the amino acid sequence of a member selected from the group consisting of amino acid residues Q-25 to F-201; Q-25 to M-200; Q-25 to Q-199; Q-25 to R-198; Q-25 to W-197; Q-25 to G-196; Q-25 to C-195; Q-25 to V-194; Q-25 to A-193; Q-25 to A-192; Q-25 to C- 191 ; Q-25 to R- 190; Q-25 to E- 189; Q-25 to P- 188; Q-25 to C-187; Q-25 to
  • the invention also provides polypeptides having one or more amino acids deleted from both the amino and the carboxyl termini of a DR3 extracellular domain polypeptide, which may be described generally as having residues n4-m4 of SEQ ID NO: 4, where n4 and m4 are integers as described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • the present application is also directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99% identical to the DR3 polypeptide sequence set forth herein as nl-ml, n2-m2, n3-m3, and/or n4-m4.
  • the application is directed to proteins containing polypeptides at least 80%, 85%, 90%, 92%, 95%, 96%, 97%, 98% or 99%o identical to polypeptides having the amino acid sequence of the specific DR3
  • DR3 proteins of the invention comprise, or alternatively consist of, fusion proteins as described above wherein the DR3 polypeptides are those described as nl-ml, n2-m2, n3-m3, and/or n4-m4 herein.
  • the application is directed to nucleic acid molecules at least 80%, 85%, 90%, 92%, 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.
  • cysteine rich regions of DR3-V1 and DR3 are important for interactions between DR3-V1 and DR3 and their respective ligands. Accordingly, specific embodiments of the invention are directed to polynucleotides encoding polypeptides which comprise, or alternatively consist of, the amino acid sequence of amino acid residues 58 to 103, 106 to 136, 141 to 173, or 176 to 206 of SEQ ED NO:2. Additional embodiments of the invention are directed to polynucleotides encoding DR3-V1 or DR3 polypeptides which comprise, or alternatively consist of, any combination of 1, 2, 3, or all 4 of the cysteine rich regions described above. Polypeptides encoded by these polynucleotides are also encompassed by the invention.
  • the polynucleotide fragments of the invention encode a polypeptide which demonstrates a DR3 functional activity.
  • a polypeptide demonstrating a DR3-V1 or DR3 "functional activity" is meant, a polypeptide capable of displaying one or more known functional activities associated with a full-length (complete) DR3 -VI or DR3 protein.
  • Such functional activities include, but are not limited to, biological activity (e.g., ability to induce apoptosis), antigenicity (the ability to bind, or compete for binding with a DR3-V1 or DR3 polypeptide for binding, to an anti-DR3-Vl or anti-DR3 antibody), immunogenicity (ability to generate antibody which binds to a DR3-V1 or DR3 polypeptide), ability to form multimers with DR3-V1 or DR3 polypeptides of the invention, and ability to bind to a receptor or ligand for a DR3-V1 or DR3 polypeptide (e.g., TNF- ⁇ , TNF- ⁇ - ⁇ ).
  • biological activity e.g., ability to induce apoptosis
  • antigenicity the ability to bind, or compete for binding with a DR3-V1 or DR3 polypeptide for binding, to an anti-DR3-Vl or anti-DR3 antibody
  • immunogenicity ability to generate
  • DR3-V1 or DR3 polypeptides can be assayed by various methods.
  • 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 as
  • agglutination assays e.g., gel agglutination assays, hemagglutination as
  • 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. Many means are known in the art for detecting binding in an immunoassay and are within the scope of the present invention.
  • 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, Microbioi. Rev. 59:94-123 (1995).
  • physiological correlates of DR3-V1 or DR3 binding to its substrates can be assayed.
  • assays described herein may routinely be applied to measure the ability of DR3-V1 or DR3 polypeptides and fragments, variants derivatives and analogs thereof to elicit DR3-V1 or DR3 related biological activity (e.g., to induce apoptosis in vitro or in vivo).
  • the ability of polynucleotides and polypeptides of the invention to increase or decrease apoptosis can routinely be determined using techniques known in the art.
  • biological activity can routinely be measured using cell death assays performed essentially as previously described (Chinnaiyan et al. , Cell81:505-512 (1995); Boldmetal.J. Biol.
  • cysteine rich regions of DR3-V1 or DR3 is important for interactions between DR3-V1 or DR3 and its ligands. Accordingly, specific embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, the amino acid sequence of amino acid residues 58 to 103, 106 to 136, 141 to 173, or 176 to 206 of SEQ ID NO:2. Additional embodiments of the invention are directed to polypeptides which comprise, or alternatively consist of, any combination of 1, 2, 3, or all 4 of the cysteine rich regions described above. Polynucleotides encoding these polypeptides are also encompassed by the invention.
  • fragments of the invention are fragments characterized by structural or functional attributes of DR3-V1 or DR3.
  • 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) DR3-V1 or DR3 (SEQ ID NO:2 or SEQ ED NO:4).
  • Certain preferred regions are those set out in FIG. 4 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 or SEQ ID NO:4, such preferred regions include; Garnier- Robson predicted alpha-regions, beta-regions, turn-regions, and coil-regions; Chou-Fasman predicted alpha-regions, beta-regions, turn-regions, and coil- regions; Kyte-Doolittle predicted hydrophilic and 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 polynucleotides of the invention encode functional attributes of DR3-V1 or DR3.
  • 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 ("coil-regions”), hydrophilic regions, hydrophobic regions, alpha amphipathic regions, beta amphipathic regions, flexible regions, surface-forming regions and high antigenic index regions of DR3-V1 or DR3.
  • the data presented in columns VIII, IX, XIII, and XIV of Table 2 can be used to determine regions of DR3-V1 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 IV 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.
  • FIG. 4 Certain preferred regions in these regards are set out in FIG. 4, but may, as shown in Table 2, be represented or identified by using tabular representations of the data presented in FIG. 4.
  • the DNA* STAR computer algorithm used to generate FIG. 4 (set on the original default parameters) was used to present the data in FIG. 4 in a tabular format (See Table 2).
  • the tabular format of the data in FIG. 4 may be used to easily determine specific boundaries of a preferred region.
  • the above-mentioned preferred regions set out in FIG. 4 and in Table 2 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. 4 and in Table 2, such preferred regions include Garnier-Robson alpha-regions, beta-regions, turn-regions, and coil-regions (columns I, III, V, and VII in Table
  • fragments in this regard are those that comprise, or alternatively consist of, regions of DR3-V1 and DR3 that combine several structural features, such as several of the features set out above in Table 2.
  • the invention further provides for the proteins containing polypeptide sequences encoded by the polynucleotides of the invention.
  • the present invention is further directed to isolated polypeptides comprising, or alternatively consisting of, fragments of DR3-V1 and DR3.
  • the invention provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids 1-60, 11-70, 21-80, 31-90, 41-100, 51-110, 61-120,
  • the invention also provides isolated polypeptides comprising, or alternatively consisting of, the amino acid sequences of a member selected from the group consisting of amino acids 1-60, 11-70, 21-80, 31-90, 41-100, 51-110, 61-120, 71-130, 81-140, 91-150, 101-160, 111-170, 121-180, 131-190, 141-200, 151-210, 161-220, 171-230, 181-240, 191-250, 201-260, 211-270, 221-280,
  • the DR3-V1 or DR3 proteins of the invention may be in monomers or multimers (i.e., dimers, trimers, tetramers, and higher multimers). Accordingly, the present invention relates to monomers and multimers of the DR3-V1 or DR3 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.
  • homomer refers to a multimer containing only DR3-V1 or DR3 proteins of the invention (including DR3-V1 or DR3 fragments, variants, and fusion proteins, as described herein). These homomers may contain DR3-V1 or DR3 proteins having identical or different polypeptide sequences. In a specific embodiment, a homomer of the invention is a multimer containing only DR3-V1 or DR3 proteins having an identical polypeptide sequence. In another specific embodiment, a homomer of the invention is a multimer containing DR3-V1 or DR3 proteins having different polypeptide sequences.
  • the multimer of the invention is a homodimer (e.g., containing DR3-V1 or DR3 proteins having identical or different polypeptide sequences) or a homotrimer (e.g., containing DR3-V1 or DR3 proteins having identical or different polypeptide sequences).
  • 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 DR3 gene) in addition to the DR3-V1 or DR3 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
  • heteromultimers of the invention such as, for example, heterotrimers or heterotetramers
  • 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 DR3-V1 or DR3 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
  • 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.
  • covalent associations may involve one or more amino acid residues contained in the heterologous polypeptide sequence in aDR3-Vl or DR3 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 DR3-Vl-Fc or DR3-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 incorporated 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 incorporated 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 incorporated 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 incorporated 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 incorporated by reference in its entirety).
  • proteins of the invention can be chemically synthesized using techniques known in the art (e.g., see Creighton, 1983, Proteins: Structures and Molecular Principles, W.H. Freeman & Co., N.Y., and Hunkapiller, M. et al, Nature 310: 105-111 (1984)).
  • a peptide corresponding to a fragment of the DR3-V1 or DR3 polypeptides of the 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 DR3-V1 or DR3 polypeptide sequence.
  • Non-classical amino acids include, but are not limited to, to the D-isomers of the common amino acids,
  • 2,4-diaminobutyric acid ⁇ -amino isobutyric acid, 4-aminobutyric acid, Abu, 2- amino butyric acid, -Abu, ⁇ -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, ⁇ -alanine, fluoro-amino acids, designer amino acids such as ⁇ -methyl amino acids, Ca-methyl amino acids, Na-methyl amino acids, and amino acid analogs in general.
  • the amino acid can be D (dextrorotary) or L (levorotary)
  • 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. 13:4331 (1986); and Zoller etal, Nucl. Acids Res. 10:6481 (1982)), cassette mutagenesis (see, e.g., Wells et al. , Gene 34:315 (1985)), restriction selection mutagenesis (see, e.g. , Wells et al. , Philos. Trans. R. Soc. London SerA 317:4X 5 (1986)).
  • art-known mutagenesis techniques include, but are not limited to oligonucleotide mediated mutagenesis, alanine scanning, PCR mutagenesis, site directed mutagenesis (see, e.
  • the invention additionally, encompasses DR3-V1 and DR3 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 of the protein.
  • chemically modified derivatives of DR3-V1 or DR3 which may provide additional advantages such as increased solubility, stability and circulating time of the polypeptide, or decreased immunogenicity (see U. S. Patent No. 4, 179,337).
  • 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 of the 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, 3000, 3500, 4000, 4500, 5000, 5500, 6000, 6500, 7000, 7500, 8000, 8500, 9000, 9500, 10,000, 10,500, 11,000, 11,500, 12,000, 12,500, 13,000, 13,500, 14,000, 14,500, 15,000, 15,500, 16,000, 16,500, 17,000, 17,500, 18,000, 18,500, 19,000, 19,500, 20,000, 25,000, 30,000, 35,000, 40,000, 50,000, 55,000, 60,000, 65,000, 70,000, 75,000, 80,000, 85,000, 90,000, 95,000, or 100,000 kDa.
  • the polyethylene glycol may have a branched structure.
  • Branched polyethylene glycols are described, for example, in U.S. Patent No. 5,643,575; Morpurgo et al, Appl. Biochem. Biotechnol. 56:59-12 (1996); Vorobjev et al, Nucleosides Nucleotides 18.2145-2150 (1999); and Caliceti et al, Bioconjug. Chem. 70:638-646 (1999), the disclosures of each of which are incorporated herein by reference.
  • polyethylene glycol molecules should be attached to the protein with consideration of effects on functional or antigenic domains of the protein. There are a number of attachment methods available to those skilled in the art, e.g., EP 0 401 384, herein incorporated by reference
  • 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 lysine 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.
  • One or more reaction chemistries may be employed to attach polyethylene glycol to specific amino acid residues (e.g., lysine, histidine, aspartic acid, glutamic acid, or cysteine) of the protein or to more than one type of amino acid residue (e.g., lysine, histidine, aspartic acid, glutamic acid, cysteine and combinations thereof) of the protein.
  • polyethylene glycol as an illustration of the 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.
  • substantially selective derivatization of the 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 etal, Intern. J. of Hematol 68: 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 2 CF 3 ).
  • MPEG monmethoxy polyethylene glycol
  • ClSO 2 CH 2 CF 3 tresylchloride
  • polyethylene glycol is directly attached to amine groups of the -I l l- protein.
  • the invention includes protein-polyethylene glycol conjugates produced by reacting proteins of the 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- succinimidyl succinate, MPEG activated with l,l'-carbonyldiimidazole, MPEG-
  • the number of polyethylene glycol moieties attached to each protein of the invention may also vary.
  • the pegylated proteins of the 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, 7-9, 8-10, 9-11, 10-12, 11-13, 12-14, 13-15, 14-16, 15-17, 16-18, 17-19, or 18- 20 polyethylene glycol moieties per protein molecule. Methods for determining the degree of substitution are discussed, for example, in Delgado et al, Crit. Rev. 77?er ⁇ . Drug Carrier Sys. 9:249-304 (1992).
  • the present invention also relates to diagnostic assays such as quantitative and diagnostic assays for detecting levels of DR3-V1 or DR3 protein, or the soluble form thereof, in cells and tissues, including determination of normal and abnormal levels.
  • diagnostic assays such as quantitative and diagnostic assays for detecting levels of DR3-V1 or DR3 protein, or the soluble form thereof, in cells and tissues, including determination of normal and abnormal levels.
  • a diagnostic assay in accordance with the invention for detecting over-expression of DR3-V1 or DR3, or soluble form thereof, compared to normal control tissue samples may be used to detect the presence of tumors, for example.
  • Assay techniques that can be used to determine levels of a protein, such as an DR3 protein of the present invention, or a soluble form thereof, in a sample derived from a host are well-known to those of skill in the art.
  • Such assay methods include radioimmunoassays, competitive-binding assays, Western Blot analysis and
  • Assaying DR3-V1 or DR3 protein levels in a biological sample can occur using any art-known method.
  • Preferred for assaying DR3-V1 or DR3 protein levels in a biological sample are antibody-based techniques.
  • DR3- VI or DR3 protein expression in tissues can be studied with classical immunohistological methods. M. Jalkanen et al, J. Cell. Biol. 101:916-985 (1985); M. Jalkanen et al, J. Cell . Biol 105:3081-3096 (1987).
  • Other antibody-based methods useful for detecting DR3-V1 or DR3 protein gene expression include immunoassays, such as the enzyme linked immunosorbent assay (ELISA) and the radioimmunoassay (RIA).
  • ELISA enzyme linked immunosorbent assay
  • RIA radioimmunoassay
  • Suitable labels are known in the art and include enzyme labels, such as glucose oxidase, radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulphur ( 35 S), tritium ( 3 H), indium (' 12 In), and technetium ( 99m Tc), and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as glucose oxidase, radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulphur ( 35 S), tritium ( 3 H), indium (' 12 In), and technetium ( 99m Tc)
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • the present invention further relates to antibodies and T-cell antigen receptors (TCR) which immunospecifically bind a polypeptide, preferably an epitope, of the present invention (as determined by immunoassays well known in the art for assaying specific antibody-antigenbinding).
  • TCR T-cell antigen receptors
  • Antibodies of the invention include, but are not limited to, polyclonal, monoclonal, multispecific, human, humanized or chimeric antibodies, single chain antibodies, Fab fragments, F(ab') fragments, fragments produced by a Fab expression library, anti-idiotypic (anti-Id) antibodies (including, e.g., anti-Id antibodies to antibodies of the invention), and epitope-binding fragments of any of the above.
  • antibody refers to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules, i.e., molecules that contain an antigen binding site that immunospecifically binds an antigen.
  • the immunoglobulin molecules of the invention can be of any type (e.g., IgG, IgE, IgM, IgD, IgA and IgY), class (e.g., IgGl, EgG2, IgG3, lgG4, IgAl and IgA2) or subclass of immunoglobulin molecule.
  • the antibodies are human antigen-binding antibody fragments of the 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 of the 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 of the present invention may be monospecific, bispecific, trispecific or of greater multispecificity. Multispecific antibodies may be specific for different epitopes of a polypeptide of the present invention or may be specific for both a polypeptide of the present invention as well as for a heterologous epitope, such as a heterologous polypeptide or solid support material. See, e.g.,
  • Antibodies of the present invention may be described or specified in terms of the epitope(s) or portion(s) of a polypeptide of the present invention that they recognize or specifically bind.
  • 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 Figures.
  • Antibodies that specifically bind any epitope or polypeptide of the present invention may also be excluded. Therefore, the present invention includes antibodies that specifically bind polypeptides of the present invention, and allows for the exclusion of the same.
  • Antibodies of the 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 of a polypeptide of the present invention are included.
  • Antibodies that bind polypeptides with at least 95%>, at least 90%), at least 85%, at least 80%, at least 75%, at least 70%, at least 65%, at least 60%, at least 55%, and at least 50%> identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • 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%, and less than 50% identity (as calculated using methods known in the art and described herein) to a polypeptide of the present invention are also included in the present invention.
  • antibodies that 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.
  • binding affinities include those with a dissociation constantorKd lessthan 5X10- 2 M, 10 "2 M, 5X10 '3 M, 10 "3 M, 5X10 "4 M,
  • the invention also provides antibodies that competitively inhibit binding of an antibody to an epitope of the 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 of the present invention may act as agonists or antagonists of the polypeptides of the present invention.
  • the present invention includes antibodies which disrupt the receptor/ligand interactions with the polypeptides of the 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
  • receptor activation may be determined by techniques described herein or otherwise known in the art. For example, 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).
  • 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% of the activity in absence of the 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 of the 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 included in the invention.
  • antibodies may act as receptor agonists, i.e., potentiate or activate either all or a subset of the biological activities of the ligand-mediated receptor activation.
  • the antibodies may be specified as agonists, 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 or antagonists of the polypeptides of the present invention.
  • Antibodies of the present invention may be used, for example, but not limited to, 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 of the polypeptides of the 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 herein in its 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., PCT publications WO 92/08495; WO 91/14438; WO 89/12624; U.S. Patent No. 5,314,995; and EP 396,387.
  • the antibodies of the 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.
  • the derivative may contain one or more non-classical amino acids.
  • the antibodies of the 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. For example, a polypeptide of the 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
  • Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.
  • monoclonal antibodies can be produced using hybridoma techniques including those known in the art and taught, for example, in Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, vznd ed. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Cell Hybridomas 563-681 (Elsevier, N.Y., 1981) (said references incorporated by reference in their entireties).
  • the term "monoclonal antibody” as used herein is not limited to antibodies produced through hybridoma technology.
  • the term “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. Thus, the term “monoclonal antibody” is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies can be prepared using a wide variety of techniques known in the art including the use of hybridoma and recombinant and phage display technology.
  • mice can be immunized with a polypeptide of the invention or a cell expressing such peptide.
  • an immune response e.g., antibodies specific for the antigen are detected in the mouse serum
  • the mouse spleen is harvested and splenocytes isolated.
  • the splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example cells from cell line SP20 available from the ATCC.
  • Hybridomas are selected and cloned by limited dilution.
  • hybridoma clones are then assayed by methods known in the art for cells that secrete antibodies capable of binding a polypeptide of the invention.
  • Ascites fluid which generally contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.
  • 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 of the 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 of the invention.
  • Antibody fragments that recognize specific epitopes may be generated by known techniques.
  • Fab and F(ab')2 fragments of the 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.
  • the antibodies of the present invention can also be generated using various phage display methods known in the art.
  • phage display methods functional antibody domains are displayed on the surface of phage particles which carry the polynucleotide sequences encoding them.
  • phage can be utilized to display antigen-binding domains expressed from a repertoire or combinatorial antibody library (e.g., human or murine).
  • Phage expressing an antigen binding domain that binds the antigen of interest can be selected or identified with antigen, e.g., using labeled antigen or antigen bound or captured to a solid surface or bead.
  • Phage used in these methods are typically filamentous phage including fd and Ml 3 binding domains expressed from phage 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 of the present invention include those disclosed in Brinkman et al, J. Immunol. Methods 182:41-50 (1995); Ames et al, J.
  • 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, e.g., as described in detail below.
  • 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 PCT publication WO 92/22324; Mullinax et al, BioTechniques 12(6):864-869
  • a chimeric antibody is a molecule in which different portions of the antibody are derived from different animal species, such as antibodies having a variable region derived from a murine monoclonal antibody and a human immunoglobulin constant region. Methods for producing chimeric antibodies are known in the art.
  • Humanized antibodies are antibody molecules from non-human species antibody that binds the desired antigen having one or more complementarity determining regions (CDRs) from the non-human species and framework regions from a human immunoglobulin molecule.
  • CDRs complementarity determining regions
  • framework residues in the human framework regions will be substituted with the corresponding residue from the CDR donor antibody to alter, preferably improve, antigen binding.
  • framework substitutions are identified by methods well known in the art, e.g., by modeling of the interactions of the CDR and framework residues to identify framework residues important for antigen binding and sequence comparison to identify unusual framework residues at particular positions.
  • Antibodies can be humanized using a variety of techniques known in the art including, for example, CDR-grafting (EP 239,400; PCT publication WO 91/09967; U.S. Patent Nos.
  • 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 of the 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.
  • Completely human antibodies which recognize a selected epitope can be generated using a technique referred to as "guided selection.”
  • a selected non-human monoclonal antibody e.g. , a mouse antibody, is used to guide the selection of a completely human antibody recognizing the same epitope.
  • antibodies to the polypeptides of the invention can, in turn, be utilized to generate anti-idiotype antibodies that "mimic" polypeptides of the invention using techniques well known to those skilled in the art. (See, e.g., Greenspan & Bona, FASEB J. 7(5) :431 -444 (1989) and ⁇ issinoff J. Immunol.
  • antibodies which bind to and competitively inhibit polypeptide multimerization and/or binding of a polypeptide of the invention to a ligand can be used to generate anti-idiotypes that "mimic" the polypeptide multimerization and/or binding domain and, as a consequence, bind to and neutralize polypeptide and/or its ligand.
  • anti-idiotypes or Fab fragments of such anti-idiotypes can be used in therapeutic regimens to neutralize polypeptide ligand.
  • anti-idiotypic antibodies can be used to bind a polypeptide of the invention and/or to bind its ligands/receptors, and thereby block its biological activity.
  • 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 of the invention, preferably, an antibody that binds to a polypeptide having the amino acid sequence of SEQ ID NO:2 or 4.
  • the polynucleotides may be obtained, and the nucleotide sequence of the 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 17:242 (1994)), which, briefly, involves the synthesis of overlapping oligonucleotides containing portions of the 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 of the 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 into a
  • 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., C ot iaet al.J. Mol. Biol. 278:451-419 (1998) for a listing of human framework regions).
  • the polynucleotide generated by the combination of the framework regions and CDRs encodes an antibody that specifically binds a polypeptide of the invention.
  • one or more amino acid substitutions may be made within the framework regions, and, preferably, the amino acid substitutions improve binding of the 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 mAb and a human immunoglobulin constant region, e.g., humanized antibodies.
  • Single chain antibodies are formed by linking the heavy and light chain fragments of the 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 of the 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.
  • Recombinant expression of 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.
  • the vector for the production of the antibody molecule may be produced by recombinant DNA technology using techniques well known in the art.
  • the invention provides replicable vectors comprising a nucleotide sequence encoding an antibody molecule of the 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 of the 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 of the invention.
  • the invention includes host cells containing a polynucleotide encoding an antibody of the 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.
  • 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.
  • 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 theE. 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; pI ⁇ vectors (Inouye & Inouye, 1985, Nucleic Acids Res.
  • the antibody coding sequence may be cloned individually into non-essential regions (for example the polyhedrin gene) of the virus and placed under control of an AcNPV promoter (for example the polyhedrin promoter).
  • 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 of the viral genome 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. Natl. Acad. Sci. USA 57:355-359).
  • 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. (seeBittneret ⁇ /., 1987, Methods in Enzymol. 753:51-544).
  • a host cell strain may be chosen which modulates the expression of the 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 of the 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.
  • 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 herpes simplex virus thymidine kinase (Wigler et al, 1977, Cell 77:223), hypoxanthine-guanine phosphoribosyltransferase (Szybalska 8c 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 of the marker gene. Since the amplified region is associated with the antibody gene, production of the 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 cDN A 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 (or portion thereof, preferably at least 10, 20 or 50 amino acids of the polypeptide) 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 of the present invention may also be used in in vifro 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; Naramura et ⁇ /., Immunol. Lett. 39:91-99 (1994), U.S. Patent 5,474,981; Gillies et al, PNAS 59: 1428-1432 (1992); Fell et al, J. Immunol. 146:2446-2452 (1991), which are incorporated by reference in their entireties.
  • 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 of the 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.
  • the polypeptides may also be fused or conjugated to the above antibody portions to form multimers.
  • Fc portions fused to the polypeptides of the present invention can form dimers through disulfide bonding between the Fc portions.
  • the Fc portion may hinder therapy and diagnosis if the fusion protein is used as an antigen for immunizations.
  • human proteins such as hEL-5 receptor
  • Fc portions for the purpose of high-throughput screening assays to identify antagonists of hEL-5.
  • the 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, 91311), among others, many of which are commercially available. As described in Gentz et al, Proc. Natl. Acad. Sci. USA 56:821-824
  • 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 etal, Cell 37:161 (1984)) and the "flag" tag.
  • 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.
  • an antibody or fragment thereof may be conjugated to a therapeutic moiety such as a cytotoxin, 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,
  • 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/ -dichlorodiamine platinum (II) (DDP) cisplatin), anthracyclines (e.g., daunorubicin (formerly daunomycin) and doxorubicin), antibiotics (
  • antimetabolites e.g., methotrexate, 6-mercaptopurine, 6-thioguanine, cytarabine, 5-fluorouracil decarbazine
  • 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.
  • Antibodies may also be attached to solid supports, which are particularly useful for immunoassays or purification of the target antigen.
  • solid supports include, but are not limited to, glass, cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride or polypropylene.
  • the antibodies of the 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.
  • 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 of the antigen), transferring the protein sample from the polyacrylamide gel to a membrane such as nitrocellulose, PVDF or nylon, blocking the membrane in blocking solution (e.g., PBS with 3%> BSA or non-fat milk), washing the membrane in washing buffer (e.g.
  • a polyacrylamide gel e.g., 8%-20% SDS-PAGE depending on the molecular weight of the antigen
  • a membrane such as nitrocellulose, PVDF or nylon
  • blocking solution e.g., PBS with 3%> BSA or non-fat milk
  • washing buffer e.g.
  • a detectable compound conjugated to a detectable compound conjugated to a detectable compound may be added to the well.
  • the antibody may be coated to the well.
  • a second antibody conjugated to a detectable compound may be added following the addition of the 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 11.2.1.
  • 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 aradioimmunoassay comprising the incubation of labeled antigen (e.g., 3 H or 125 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 of the 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 of the 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 of the invention (including fragments, analogs and derivatives thereof as described herein). While not intending to be bound to theory, DR3 receptors are believed to induce programmed cell death by the association/cross-linking of death domains between different receptor molecules.
  • DR3 receptors are also known to be present in a number of tissues and on the surfaces of a number of cell types. These tissues and cell types include endothelial cells, liver cells, hepatocellular tumor, lymph nodes, Hodgkin's lymphoma, tonsil, bone marrow, spleen, heart, thymus, pericardium, healing wound (skin), brain, pancreas tumor, burned skin, U937 cells, testis, colon cancer (metasticized to liver), pancreas, rejected kidney, adipose, ovary, olfactory epithelium, striatum depression, HeLa cells, LNCAP (upon treatment with +30 nM androgen), HUVEC (human umbilical vein endothelial cells), 8 week embryo tissues, 9 week embryo tissues, fetal brain tissues, fetal kidney tissues, fetal heart tissues,
  • TNF- ⁇ - ⁇ activities are believed to be modulated, at least in part, through interaction with DR3 receptors.
  • Antibodies which act as both agonists and antagonists of receptor functions are known in the art.
  • Deng et al (Blood 92: 1981-1988 ( 1998)) describe a monoclonal antibody which binds to the human c-Mpl receptor and stimulates megakaryocytopoiesis.
  • the monoclonal antibody described in Deng et al. is thus a c-Mpl receptor agonist.
  • Antibodies which bind to DR3 receptors will have varying effects on these receptors. These effects differ based on the specific portions of the DR3 receptor to which the antibodies bind and the three-dimensional conformation of the antibody molecules themselves. Thus, antibodies which bind to the extracellular domain of a DR3 receptor can either stimulate or inhibit DR3 activities (e.g., the induction of apoptosis). Antibodies which stimulate DR3 receptor activities (e.g., by facilitating the association between DR3 receptor death domains) are DR3 agonists and antibodies which inhibit DR3 receptor activities (e.g., by blocking the binding of TNF- ⁇ - ⁇ and/or preventing the association between DR3 receptor death domains) are DR3 antagonists.
  • Agonists of the invention which stimulate the activity of DR3 death domains will induce apoptosis in endothelial cells.
  • agonists of the invention can inhibit the formation of blood and lymph vessels and, thus, can be used to prevent and/or treat diseases and conditions associated with hypervascularization and neovascularization.
  • diseases and conditions associated with angiogenesis which can be prevented and/or treated using agonists of the invention include hypertrophic and keloid scarring, proliferative diabetic retinopathy, arteriovenous malformations, atherosclerotic plaques, hemophilic joints, nonunion fractures, Osler-Weber syndrome, psoriasis, pyogenic granuloma, scleroderma, tracoma, menorrhagia, and vascular adhesions.
  • T-cell mediated immune responses as well as preventing and/or treating diseases and conditions associated with increased T-cell proliferation.
  • Diseases and conditions associated with T-cell mediated immune responses and increased T-cell proliferation include graft-v-host responses and diseases, inflammation, autoimmune diseases, and T-cell leukemias.
  • DR3 receptors can function, for examples, by either binding to DR3 receptors and blocking the binding of ligands which stimulate DR3 death domain activity (e.g., TNF- ⁇ - ⁇ ) or inhibiting DR3 receptor conformational changes associated with membrane signal transduction.
  • DR3 death domain activity e.g., TNF- ⁇ - ⁇
  • DR3 receptor conformational changes associated with membrane signal transduction e.g., TNF- ⁇ - ⁇
  • An example of a condition associated with decreased vascularization that can be treated using antagonists of the invention is delayed wound healing.
  • Antagonists of the invention can thus prevent and/or inhibit apoptosis from occurring in endothelial cells at wound sites and thereby promote wound healing in healing impaired individuals, as well as in individuals who heal at "normal” rates.
  • antagonists of the invention can be used to promote and/or accelerate wound healing.
  • NEDS acquired immune deficiency syndrome
  • related afflictions e.g., AIDS related complexes
  • T-cell immunodeficiencies e.g., radiation sickness
  • T-cell depletion due to radiation and/or chemotherapy e.g., radiation sickness
  • an antagonist of the invention when administered to an individual for the treatment and/or prevention of a disease or condition associated with decreased T-cell populations, the antagonist 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
  • the antibodies of the present invention may be used therapeutically in a number of ways, includes binding polynucleotides or polypeptides of the present invention locally or systemically in the body or by direct cytotoxicity of the antibody, e.g., as mediated by complement (CDC) or by effector cells (ADCC).
  • CDC complement
  • ADCC effector cells
  • 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 of the 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.
  • the proteins of the invention can also be expressed in transgenic animals.
  • 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.
  • techniques described herein or otherwise known in the art are used to express polypeptides of the invention in humans, as part of a gene therapy protocol. Any technique known in the art may be used to introduce the transgene
  • nucleic acids of the invention into animals to produce the founder lines of transgenic animals.
  • Such techniques include, but are not limited to, pronuclear microinjection (Paterson etal, Appl. Microbioi. 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
  • 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 355:810-813 (1997)), each of which is herein incorporated by reference in its entirety).
  • 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, / ' . 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. (Lasko et al, Proc. Natl. Acad. Sci. USA 59:6232-6236 (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 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.
  • 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 DR3-V1 or DR3 polypeptides, studying conditions and/or disorders associated with aberrant DR3-V1 or DR3 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., animal, including human) or an MHC compatible donor and 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 (using viral vectors, and preferably vectors that integrate the transgene into the cell genome) or transfection procedures, including, but not limited to, the use of plasmids, cosmids, YACs, naked DNA, electroporation, liposomes, etc.
  • 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 Alternatively, 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. (See, e.g., Anderson et al, US Patent Number 5,399,349; and Mulligan & Wilson, US Patent Number 5,460,959, each of which is incorporated by reference herein in its entirety).
  • TNF Tumor Necrosis Factor
  • the 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 (D. V. Goeddel et al. , "Tumor Necrosis Factors: Gene Structure and Biological Activities," Symp. Quant. Biol. 51:591-
  • TNF-family ligands induce such various cellular responses by binding to TNF-family receptors, including the DR3-V1 or DR3 of the present invention.
  • Cells which express the DR3-V1 or DR3 polypeptide and are believed to have a potent cellular response to DR3-V1 or DR3 ligands include lymphocytes, fibroblasts, macrophages, synovial cells, activated T-cells, lymphoblasts and epithelial cells.
  • 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.
  • Such cellular responses include not only normal physiological responses to TNF-family ligands, 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 (J.C. Ameisen, 7E>S 5: 1 197-1213 (1994); P.H. Krammeret /., Curr. Opin. Immunol. 6:219-289 (1994)).
  • DR3-V1 orDR3 polynucleotides, polypeptides, agonists or antagonists of the invention may be used in developing treatments and diagnostic/prognostic assays for any disorder mediated (directly or indirectly) by defective, or insufficient amounts of DR3.
  • DR3-V1 or DR3 polypeptides, agonists or antagonists may be administered to a patient (e.g., mammal, preferably human) afflicted with such a disorder.
  • a gene therapy approach may be applied to treat and/or prevent such disorders. Disclosure herein of DR3-V1 or DR3 nucleotide sequences permits the detection of defective DR3 genes, and the replacement thereof with normal DR3-encoding genes.
  • Defective genes may be detected in in vitro diagnostic assays, and by comparison of the DR3-V1 or DR3 nucleotide sequence disclosed herein with that of a DR3 gene derived from a patient suspected of harboring a defect in this gene.
  • cancers such as follicular lymphomas, carcinomas with p53 mutations, and hormone-dependent tumors, such as breast cancer, prostrate cancer, Karposi's sarcoma and ovarian cancer
  • autoimmune disorders such as multiple sclerosis, Sjogren's syndrome, Hashimoto's thyroiditis, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, and rheumatoid arthritis
  • viral infections such as herpes viruses, pox viruses and adenoviruses
  • 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., Hodgkin's disease and non-Hodgkin's disease), multiple myeloma, Waldenstrom's macroglobulinemia, heavy chain disease, and solid tumors including, but not limited to, sarcomas and carcinomas such as fibrosarcoma, myxosarcoma, liposarcoma, chondro
  • AEDS 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, Grave's disease Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus erythematosus, immune-related glomerulonephritis, autoimmune gastritis, thrombocytopenic purpura, and rheumatoid arthritis) myelodysplastic syndromes (such as aplastic anemia), graft vs.
  • autoimmune disorders such as, multiple sclerosis, Sjogren's syndrome, Grave's disease Hashimoto's thyroiditis, autoimmune diabetes, biliary cirrhosis, Behcet's disease, Crohn's disease, polymyositis, systemic lupus ery
  • DR3 polynucleotides, polypeptides, agonists, and/or antagonists are used to treat, prevent, diagnose and/or prognose the diseases and disorders listed above.
  • 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 the DR3-V1 or DR3 polypeptide an effective amount of DR3-V1 or DR3 ligand, analog or an agonist capable of increasing DR3-V1 or DR3 mediated signaling.
  • DR3-V1 or DR3 mediated signaling is increased to treat and/or prevent a disease wherein decreased apoptosis or decreased cytokine and adhesion molecule expression is exhibited .
  • An agonist can include soluble forms of DR3-V1 or DR3 and monoclonal antibodies directed against the DR3-V1 or DR3 polypeptide.
  • the present invention is directed to a method for inhibiting apoptosis induced by a TNF-family ligand, which involves administering to a cell which expresses the DR3-V1 or DR3 polypeptide an effective amount of an antagonist capable of decreasing DR3-V1 or DR3 mediated signaling.
  • an antagonist capable of decreasing DR3-V1 or DR3 mediated signaling.
  • DR3-V1 or DR3 mediated signaling is decreased to treat and/or prevent a disease wherein increased apoptosis or NF-kB expression is exhibited.
  • An antagonist can include soluble forms of DR3-V1 or DR3 and monoclonal antibodies directed against the DR3-V1 or DR3 polypeptide.
  • DR3-Vl-Fc and DR3-Fc proteins and soluble portions of the extracellular domains of DR3-V1 and DR3 proteins are useful in stimulating neovascularization and angiogenesis.
  • these polypeptides are useful, for example, for the treatment and/or prevention of diseases and conditions associated withhypovascularization (e.g. , Turner's syndrome, cardiovascular aging, bronchial stenosis, depression).
  • DR3-Fc proteins receptor/Fc fusion proteins, and nucleic acid molecules which encode such proteins.
  • These fusion proteins include those having amino acid sequences of the extracellular domains of the DR3 proteins of the invention.
  • portions of DR3 extracellular domains which are useful in the preparation of DR3 receptor/Fc fusion proteins include amino acids 1 to 199 in SEQ ID NO:4 and amino acids 1 to 210, 37 to 210, 50 to 210, and 100 to 210 in SEQ ID NO:2.
  • afflictions which can be treated and/or prevented by DR3-V1 and DR3 mediated stimulation of angiogenesis include soft tissue traumas (e.g., cuts and bruises), ulcers (e.g., peptic, skin and venous), and sclerodermas.
  • agonist is intended naturally occurring and synthetic compounds capable of enhancing or potentiating apoptosis.
  • antagonist is intended naturally occurring and synthetic compounds capable of inhibiting apoptosis. 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.
  • One such screening procedure involves the use of melanophores which are transfected to express the receptor of the present invention.
  • 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) activation of the receptor polypeptide of the present invention by contacting the melanophore cells which encode the receptor with both a TNF-family ligand and the candidate antagonist (or agonist). Inhibition or enhancement of the signal generated by the ligand indicates that the compound is an antagonist or agonist of the ligand/receptor signaling pathway.
  • Other screening techniques include the use of cells which express the receptor (for example, transfected CHO cells) in a system which measures extracellular pH changes caused by receptor activation, for example, as described in Science 246: 181-296 (October 1989).
  • compounds may be contacted with a cell which expresses the receptor polypeptide of the 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 receptor.
  • Another such screening technique involves introducing RNA encoding the receptor into Xenopus oocytes to transiently express the 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 of the receptor.
  • Another screening technique involves expressing in cells a construct wherein the 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 herein above described by detecting activation of the receptor or inhibition of activation of the receptor from the phospholipase signal.
  • Another method involves screening for compounds which inhibit activation of the receptor polypeptide of the present invention antagonists by determining inhibition of binding of labeled ligand to cells which have the receptor on the surface thereof.
  • Such a method involves transfecting a eukaryotic cell with DNA encoding the receptor such that the cell expresses the receptor on its surface and contacting the cell with a compound in the presence of a labeled form of a known ligand.
  • the ligand can be labeled, e.g., by radioactivity.
  • the amount of labeled ligand bound to the receptors is measured, e.g., by measuring radioactivity of the receptors. If the compound binds to the receptor as determined by a reduction of labeled ligand which binds to the receptors, the binding of labeled ligand to the receptor is inhibited.
  • a screening method for determining whether a candidate agonist or antagonist is capable of enhancing or inhibiting a cellular response to a TNF-family ligand.
  • the method involves contacting cells which express the DR3-V1 or DR3 polypeptide with a candidate compound 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 absence of the candidate compound, whereby an increased cellular response over the standard indicates that the candidate compound is an agonist of the ligand/receptor signaling pathway and a decreased cellular response compared to the standard indicates that the candidate compound is an antagonist of the ligand/receptor signaling pathway.
  • saying a cellular response is intended qualitatively or quantitatively measuring a cellular response to a candidate compound and/or a TNF-family ligand (e.g., determining or estimating an increase or decrease in T cell proliferation or tritiated thymidine labeling).
  • a cell expressing the DR3-V1 or DR3 polypeptide can be contacted with either an endogenous or exogenously administered TNF-family ligand.
  • Agonist 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 267: 1451-1458 (1995)).
  • agonists include polyclonal and monoclonal antibodies raised against the DR3-V1 or DR3 polypeptide, or a fragment thereof.
  • Such agonist antibodies raised against a T ⁇ F- family receptor are disclosed in L.A. Tartaglia et al, Proc. Natl. Acad. Sci. USA 55:9292-9296 (1991); and L.A. Tartaglia and D V. Goeddel, supra. See, also, PCT Application WO 94/09137.
  • Antagonist 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 (such as Adenovirus EIB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barr virus BHRFl, LMP-1, African swine fever virus LMW5-HL, and Herpes virus yl 34.5), calpain inhibitors, cysteine protease inhibitors, and tumor promoters (such as PMA, Phenobarbital, and - Hexachlorocyclohexane).
  • viral genes such as Adenovirus EIB, Baculovirus p35 and IAP, Cowpox virus crmA, Epstein-Barr virus BHRFl, LMP-1, African swine fever virus LMW5-HL, and Herpes virus yl 34.5
  • calpain inhibitors such as PMA, Phenobarbital, and
  • antagonists according to the present invention are nucleic acids corresponding to the sequences contained in DR3-V1 or DR3, or the complementary strand thereof, and/or to nucleotide sequences contained in the deposited cDNAs having ATCC Deposit No. 97456 and 97757.
  • antisense sequence is generated internally by the organism, in another embodiment, the antisense sequence is separately administered (see, for example, O'Connor, J., Neurochem. 56:560 (1991), and Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression, CRC Press, Boca Raton, FL (1988).
  • the 5 ' coding portion of a polynucleotide that encodes the mature polypeptide of the present invention may be used to design an antisense RNA oligonucleotide of from about 10 to 40 base pairs in length.
  • a DNA oligonucleotide is designed to be complementary to a region of the 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 DR3-V1 or DR3 antisense nucleic acid of the invention is produced intracellularly by transcription from an exogenous sequence.
  • a vector or a portion thereof is transcribed, producing an antisense nucleic acid (RNA) of the invention.
  • RNA antisense nucleic acid
  • Such a vector would contain a sequence encoding the DR3-V1 or DR3 antisense nucleic acid.
  • Such 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 of the sequence encoding DR3-V1 or DR3, 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 SV40 early promoter region
  • the ability to hybridize will depend on both the degree of complementarity and the length of the antisense nucleic acid Generally, the larger the hybridizing nucleic acid, the more base mismatches with a DR3-V1 or DR3 R ⁇ A it may contain and still form a stable duplex (or triplex as the case may be).
  • One skilled in the art can ascertain a tolerable degree of mismatch by use of standard procedures to determine the melting point of the hybridized complex. Oligonucleotides that are complementary to the 5' end of the message, e.g., the 5' untranslated sequence up to and including the AUG initiation codon, should work most efficiently at inhibiting translation.
  • oligonucleotides complementary to either the 5'- or 3 '- non- translated, non-coding regions of the DR3-V1 or DR3 shown in SEQ ID NO.2 and SEQ ID NO:4 could be used in an antisense approach to inhibit translation of endogenous DR3-V1 or DR3 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. Whether designed to hybridize to the 5 '-, 3 '- or coding region of DR3-V1 or DR3 mRNN 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 about 10 nucleotides, at least about 17 nucleotides, at least about 25 nucleotides or at least about 50 nucleotides. In this context "about” includes the particularly recited value and values larger or smaller by several (5, 4, 3, 2, or 1) nucleotides.
  • the 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,
  • 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 2-0-methylribonucleotide (Inoue et al, Nucl.
  • antisense nucleotides complementary to the DR3 -V 1 or DR3 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 RNN 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 rnR ⁇ A at site specific recognition sequences can be used to destroy DR3-V1 or DR3 rnR ⁇ As, the use of hammerhead ribozymes is preferred. Hammerhead ribozymes cleave mR ⁇ As at locations dictated by flanking regions that form complementary base pairs with the target mR ⁇ A.
  • hammerhead ribozymes The construction and production of hammerhead ribozymes is well known in the art and is described more fully in Haseloff and Gerlach, Nature 334:585-591 (1988). There are numerous potential hammerhead ribozyme cleavage sites within the nucleotide sequence of DR3-V1 (SEQ ED NO:2) or DR3 (SEQ ID NO: 4).
  • the ribozyme is engineered so that the cleavage recognition site is located near the 5' end of the DR3-V1 or DR3 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 DR3-V1 or DR3 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 DR3-V1 or DR3 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 DR3 gene and/or its promoter using targeted homologous recombination.
  • endogenous gene expression can also be reduced by inactivating or "knocking out" the DR3 gene and/or its promoter using targeted homologous recombination.
  • a mutant, non-functional polynucleotide of the invention flanked by DNA homologous to the endogenous polynucleotide sequence (either the coding regions or regulatory regions of the 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.
  • Such approaches are particularly suited in research and agricultural fields where modifications to embryonic stem cells can be used to generate animal offspring with an inactive targeted gene (see, e.g., Thomas & Capecchi 1987 and Thompson 1989, supra).
  • this approach can be routinely adapted for use in humans provided the recombinant DNA constructs are directly administered or targeted to the required site in vivo using appropriate viral vectors that 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.
  • antagonists according to the present invention include soluble forms of DR3-V1 or DR3 (e.g., fragments of the DR3-V1 shown in SEQ ED NO:2 and DR3 shown in SEQ ID NO:4) that include the ligand binding domain from the extracellular region of the full length receptor).
  • soluble forms of the DR3-V1 or DR3 which may be naturally occurring or synthetic, antagonize DR3 -V 1 or DR3 mediated signaling by competing with the cell surface bound forms of the receptor for binding to TNF-family ligands.
  • Antagonists of the present invention also include antibodies specific for TNF-family ligands and both DR3-Vl-Fc and DR3-Fc fusion proteins.
  • 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, TNF- ⁇ , lymphotoxin- ⁇ (LT- ⁇ , also known as TNF- ⁇ ), LT- ⁇ (found in complex heterotrimer LT- ⁇ 2- ⁇ ), FasL, TNF- ⁇ (International Publication No. WO 96/14328), TNF- ⁇ - ⁇ , TNF- ⁇ - ⁇ (International Publication No WO 00/08139), A1M-I (International Publication No.
  • WO 97/33899 AEM-II (International Publication No. WO 97/34911), APRIL (J. Exp. Med. 188(6): 1 185-1190), endokine- ⁇ (International Publication No. WO 98/07880), neutrokine- ⁇ (International Publication No. WO 98/18921), CD40L, CD27L, CD30L, 4-1BBL, OX40L and nerve growth factor (NGF).
  • AEM-II International Publication No. WO 97/34911
  • APRIL J. Exp. Med. 188(6): 1 185-1190
  • endokine- ⁇ International Publication No. WO 98/07880
  • neutrokine- ⁇ International Publication No. WO 98/18921
  • CD40L CD27L
  • CD30L CD30L
  • 4-1BBL 4-1BBL
  • OX40L nerve growth factor
  • Polyclonal and monoclonal antibody agonists or antagonists according to the present invention can be raised according to the methods disclosed herein and/or known in the art, such as, for example, those methods described in Tartaglia and Goeddel, J. Biol Chem. 267 (7) -.4304-4301(1992)); Tartaglia etal,
  • soluble forms of the receptor which may be naturally occurring or synthetic, antagonize DR3-V1 or DR3 mediated signaling by competing with the cell surface DR3-V1 or DR3 for binding to TNF-family ligands.
  • soluble forms of the receptor that include the ligand binding domain are novel cytokines capable of inhibiting apoptosis induced by TNF-family ligands. These are preferably expressed as dimers or trimers, since these have been shown to be superior to monomeric forms of soluble receptor as antagonists, e.g., IgG-Fc-TNF receptor family fusions.
  • DR3 is a death domain-containing molecule capable of triggering both apoptosis and NF-kB activation, two pathways dominant in the regulation of the immune system.
  • antibody or “monoclonal antibody” 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 methods using DR3-V1 or DR3 immunogens of the present invention.
  • DR3-V1 or DR3 immunogens include the full length DR3-V1 or DR3 polypeptide (which may or may not include the leader sequence) and DR3-V1 or DR3 polypeptide fragments such as the ligand binding domain, the transmembrane domain, the intracellular domain and the death domain.
  • Proteins and other compounds which bind the DR3-V1 or DR3 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 340:245-246 (1989)).
  • yeast two-hybrid system Fields and Song, Nature 340:245-246 (1989)
  • a modified version of the yeast two- hybrid system has been described by Roger Brent and his colleagues (J. Gyuris et al, Cell 75:791-803 (1993); A.S. Zervos et al, Cell 72:223-232 (1993)).
  • 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 the ligand/receptor signaling pathway.
  • TNF ligand family include, but are not limited to, the DR3-V1 or DR3 ligand, TNF- ⁇ , lymphotoxin- ⁇ (LT- ⁇ , also known as TNF- ⁇ ), (International Publication No. WO
  • the immune system of the recipient animal In rejection of an allograft, the immune system of the 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 of the 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 that 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.
  • Agonist 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 the DR3-V1 or DR3 polypeptide, and thereby are susceptible to compounds which enhance apoptosis.
  • the present invention further provides a method for creating immune privileged tissues.
  • Antagonist of the invention can further be used in the treatment and/or prevention of Inflammatory Bowel-Disease.
  • DR3 like TNFR1 , also activates the NF-kB transcription factor, which is very closely associated with the stimulation of cytokine (e.g., EL-8) and adhesion molecule (e.g. , ELAM) transcription.
  • soluble DR3, agonist or antagonist mABs may be used to diagnose, prognose, treat and/or prevent this form of cancer.
  • soluble DR3 or neutralizing mABs may be used to treat and/or prevent various chronic and acute forms of inflammation such as rheumatoid arthritis, osteoarthritis, psoriasis, septicemia, and inflammatory bowel disease.
  • DR3 polynucleotides, polypeptides, agonists or antagonists of the invention may be used to diagnose, prognose, treat and/or prevent cardiovascular disorders, including peripheral artery disease, such as limb ischemia.
  • 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 syphilis, and cardiovascular tuberculosis.
  • heart disease such as arrhythmias, carcinoid heart disease, high cardiac output, low cardiac
  • 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
  • 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
  • Myocardial ischemias include coronary disease, such as angina pectoris, coronary aneurysm, coronary arteriosclerosis, coronary thrombosis, coronary vasospasm, myocardial infarction and myocardial stunning.
  • 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.
  • 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, periventricularleukomalacia, vascular headache, cluster headache, migraine, and vertebrobasilar insufficiency.
  • Vasculitis includes aortitis, arteritis, Behcet's Syndrome, Churg-Strauss Syndrome, mucocutaneous lymph node syndrome, thromboangiitis obliterans, hypersensitivity vasculitis, Schoenlein- Henoch purpura, allergic cutaneous vasculitis, and Wegener's granulomatosis.
  • a DR3 polynucleotide, polypeptide, agonist, or antagonist of the invention is used to diagnose, prognose, treat and/or prevent thrombotic microangiopathies.
  • thrombotic thrombocytopenic purpura TTP
  • Increasing TTP-associated mortality rates have been reported by the U.S. Centers for Disease Control (Torok etal, Am. J. Hematol. 50:84 (1995)).
  • HUS HUS
  • a disorder known as childhood/diarrhea-associated HUS differs in etiology from adult HUS.
  • conditions characterized by clotting of small blood vessels may be diagnosed, prognosed, treated and/or prevented using DR3. 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 of the microvasculature in the heart has been reported in multiple sclerosis patients. As a further example, treatment, prevention, diagnosis and/or prognosis of systemic lupus erythematosus (SLE) is contemplated.
  • SLE systemic lupus erythematosus
  • DR3 polynucleotides, polypeptides, agonists or antagonists of the invention may be employed in combination with other agents useful in treating, preventing, diagnosing and/or prognosing a particular disorder.
  • agents useful in treating, preventing, diagnosing and/or prognosing a particular disorder For example, in an in vitro study reported by Laurence et al. (Blood 87:3245 (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.
  • a DR3 polynucleotide, polypeptide, agonist or antagonist, and an anti-FAS blocking antibody are both administered to a patient afflicted with a disorder characterized by thrombotic microanglopathy, such as TTP or HUS.
  • thrombotic microanglopathy 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 incorporated 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.
  • neovascularization including solid tumor growth and metastases, arthritis, some types of eye disorders, and psoriasis.
  • the present invention provides for treatment, prevention, diagnosis and/or prognosis of diseases or disorders associated with neovascularization by administration of the DR3 polynucleotides and/or polypeptides of the invention (including DR3 agonists and/or antagonists).
  • Malignant and metastatic conditions which can be diagnosed, prognosed, treated and/or prevented 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 diagnosed, prognosed, treated and/or prevented with the DR3 polynucleotides and polypeptides of the present invention (including DR3 agonists and DR3 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.
  • disorders which can be diagnosed, prognosed, treated and/or prevented with the DR3 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.
  • Polynucleotides and/or polypeptides of the invention, and/or agonists and/or antagonists thereof, are useful in the prognosis, diagnosis, treatment and/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, gliobalstoma, cancer associated with mutation or alteration of p53, brain tumor, bladder cancer, uterocervical cancer, colon cancer, colorectal cancer, non-small cell carcinoma of the lung, small cell carcinoma of the lung, stomach cancer, etc.), lymphoproliferative disorders (e.g., lymphadenopathy and lymphomas (e.g., EBV induced lymphoproliferations and Hodgkin's disease), microbial (e.g., viral, bacterial, etc.) infection (e.g., HIV-1 infection, HIV-2 infection, herpesvirus infection
  • osteomyelodysplasia e.g., aplastic anemia, etc.
  • liver disease e.g., acute and chronic hepatitis, liver injury, and cirrhosis
  • autoimmune disease e.g., multiple sclerosis, myasthenia gravis, rheumatoid arthritis, systemic lupus erythematosus, immune complex glomerulonephritis, autoimmune diabetes, autoimmune thrombocytopenic purpura, Grave's disease, Hashimoto's thyroiditis, inflammatory autoimmune diseases, etc.
  • cardiomyopathy e.g., dilated cardiomyopathy
  • diabetes diabetic complications (e.g., diabetic nephropathy, diabetic neuropathy, diabetic retinopathy), influenza, 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, wound healing (e.g., wounds, burns, and bone fractures), and regulating bone formation and treating and/or preventing osteoporosis.
  • 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 and/or anti-viral immune responses.
  • 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 of the 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 of the invention and/or agonists and/or antagonists thereof are useful as immunosuppressive agents, for example in the treatment and/or prevention of autoimmune disorders or in the prevention of transplant rejection.
  • polynucleotides and/or polypeptides of the invention are used to diagnose, prognose, treat and/or prevent chronic inflammatory, allergic or autoimmune conditions, such as those described herein or are otherwise known in the art.
  • 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 of the 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 of the antibody nucleic acids (Roller and Smithies,
  • 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, of the antibody. Delivery of the 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. , by infection using defective or attenuated retrovirals or other viral vectors (see U.S. Patent No. 4,980,286), or by direct injection of naked DNA, or by use of microparticle bombardment
  • 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 ⁇ lL); WO 92/22635 dated December 23, 1992 (Wilson et ⁇ l.); WO92/20316 dated November 26, 1992 (Findeis et ⁇ l.); WO93/14188 dated July 22, 1993 (Clarke et ⁇ l.), WO 93/20221 dated October 14, 1993 (Young)).
  • a specific receptor see, e.g., PCT Publications WO 92/06180 dated April 16, 1992 (Wu et ⁇ lL); WO 92/22635 dated December 23, 1992 (Wilson et ⁇ l.); WO92/20316 dated November 26, 1992 (Findeis et ⁇ l.); WO93/14188 dated July 22, 1993 (Clarke e
  • the 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. US A 86: 8932-8935; Zijlstra et ⁇ l, 1989, Nature 342:435-438).
  • viral vectors that contains nucleic acid sequences encoding an antibody of the invention are used.
  • a retroviral vector can be used (see Miller et ⁇ l., 1993, Meth. Enzymol. 217:581-599). These retroviral vectors have been to delete retroviral sequences that are not necessary for packaging of the 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. More detail about retroviral vectors can be found in Boesen et al, 1994, Biotherapy
  • 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, 1993, Current Opinion in Genetics and Development 3:499-503 present a review of adenovirus-based gene therapy. Bout et al, 1994, Human Gene Therapy 5:3-
  • 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 No. 5,436, 146).
  • 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 of the 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, etb. Enzymol. 277:599-618; Cohen etal, 1993, Meth.
  • the technique should provide for the stable transfer of the 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 purposes 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 of the present invention (see, e.g., PCT Publication WO 94/08598, dated April 28, 1994; Stemple and Anderson, 1992, Cell 71:913-985; Rheinwald,
  • the nucleic acid to be introduced for purposes of gene therapy comprises an inducible promoter operably linked to the coding region, such that expression of the nucleic acid is controllable by controlling the presence or absence of the 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.
  • 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 of the present invention.
  • administration of an "effective amount" of an agonist or antagonist is intended an amount of the compound that is sufficient to enhance or inhibit a cellular response to a TNF-family ligand and include polypeptides.
  • administration of an "effective amount” of an agonist or antagonists is intended an amount effective to enhance or inhibit DR3-V1 or DR3 mediated apoptosis.
  • an agonist according to the present invention can be co-administered with a TNF-family ligand.
  • 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 a DR3 polypeptide administered parenterally per dose will be in the range of about
  • this dose is at least 0.01 mg/kg/day, and most preferably for humans between about 0.01 and 1 mg/kg/day for the hormone.
  • the DR3 agonists or antagonists is typically administered at a dose rate of about 1 ⁇ g/kg/hour to about
  • 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 R A technique.
  • patient dosaging may be adjusted to achieve regular on-going trough blood levels, as measured by RIN on the order of from 50 to 1000 ng/ml, preferably 150 to 500 ng/ml.
  • a compound of the invention e.g., encapsulation in liposomes, microparticles, microcapsules, recombinant cells capable of expressing the compound, receptor- mediated endocytosis (see, e.g., Wu and Wu, 1987, J. Biol. Chem. 262:4429- 4432), construction of a nucleic acid as part of a retroviral or other vector, etc.
  • Methods of introduction include but are not limited to intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, epidural, and oral routes.
  • the compounds or compositions may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.) and may be administered together with other biologically active agents. Administration can be systemic or local.
  • Pulmonary administration can also be employed, e.g., by use of an inhaler or nebulizer, and formulation with an aerosolizing agent.
  • compositions comprising an 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 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 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.
  • the agonist can be "co-administered" either
  • pharmaceutically acceptable carrier is meant a non-toxic solid, semisolid or liquid filler, diluent, encapsulating material or formulation auxiliary of any type.
  • parenteral refers to modes of administration which include intravenous, intramuscular, intraperitoneal, intrasternal, subcutaneous and intraarticular injection and infusion.
  • 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.
  • DR3-V1 or DR3 polypeptide containing the transmembrane region can also be used when appropriately solubilized by including detergents, such as CHAPS or NP-40, with buffer.
  • a protein, including an antibody, of the invention care must be taken to use materials to which the protein does not absorb.
  • the compound or composition can be delivered in a vesicle, in particular a liposome (see Langer, 1990, Science 249: 1527-1533;
  • the compound or composition can be delivered in a controlled release system.
  • a pump may be used (see
  • polymeric materials can be used (see Medical Applications of Controlled Release, Langer and Wise (eds.), CRC Pres., Boca Raton, Florida (1974); Controlled Drug Bioavailability, Drug Product Design and Performance, Smolen and Ball (eds.), Wiley, New York (1984); Ranger and Peppas, J., 1983, Macromol. Sci. Rev. Macromol. Chem. 23:61; see also Levy etal, 1985, Science 228: 190; During et al, 1989, Ann. Neurol 25:351; Howard et al, 1989, .
  • a controlled release system can be placed in proximity of the therapeutic target, i. e. , the brain, thus requiring only a fraction of the systemic dose (see, e.g., Goodson, in Medical Applications of Controlled Release, supra, vol. 2, pp. 115-138 (1984)).
  • Other controlled release systems are discussed in the review by Langer
  • the nucleic acid can be administered in vivo to promote expression of its encoded protein, by constructing it as part of an appropriate nucleic acid expression vector and administering it so that it becomes intracellular, e.g., by use of a retroviral vector (see U.S. Patent No.
  • microparticle bombardment e.g., a gene gun; Biolistic, Dupont
  • coating lipids or cell-surface receptors or transfecting agents, or by administering it in linkage to a homeobox- like peptide which is known to enter the nucleus (see, e.g., Joliot et al, 1991, Proc. Natl. Acad. Sci. USA 55: 1864-
  • nucleic acid can be introduced intracellularly and incorporated within host cell DNA for expression, by homologous recombination.
  • compositions comprise a therapeutically effective amount of a compound, and a pharmaceutically acceptable carrier.
  • a pharmaceutically acceptable carrier for example
  • “pharmaceutically acceptable” means approved by a regulatory of the 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.
  • 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.
  • the composition if desired, 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. Such 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 compounds of the 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 amount of the compound of the invention which will be effective in the treatment, inhibition and/or prevention of a disease or disorder associated with aberrant expression and/or activity of a polypeptide of the invention can be determined by standard clinical techniques.
  • in vitro assays may optionally be employed to help identify optimal dosage ranges.
  • the precise dose to be employed in the formulation will also depend on the route of administration, and the seriousness of the disease or disorder, and should be decided according to the judgment of the practitioner and each patient's circumstances. Effective doses may be extrapolated from dose-response curves derived from in vitro or animal model test systems
  • the dosage administered to a patient is typically 0.1 mg/kg to 100 mg/kg of the patient's body weight.
  • the dosage administered to a patient is between 0.1 mg/kg and 20 mg/kg of the patient's body weight, more preferably 1 mg/kg to 10 mg/kg of the patient's body weight.
  • human antibodies have a longer half-life within the human body than antibodies from other species due to the immune response to the foreign polypeptides. Thus, lower dosages of human antibodies and less frequent administration is often possible.
  • the dosage and frequency of administration of antibodies of the invention may be reduced by enhancing uptake and tissue penetration (e.g., into the brain) of the antibodies by modifications such as, for example, lipidation.
  • 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 of the 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.
  • 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 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.
  • 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 of the 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 not limited to, other members of the TNF family, chemotherapeutic agents, antibiotics, steroidal and non-steroidal anti-inflammatories, conventional immunotherapeutic agents, cytokines 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 of the invention are administered in combination with other members of the TNF family.
  • TNF, TNF-related or TNF- like molecules that may be administered with the compositions of the invention include, but are not limited to, soluble forms of TNF- ⁇ , lymphotoxin- ⁇ (LT- ⁇ , also known as TNF- ⁇ ), LT- ⁇ (found in complex heterotrimer LT- ⁇ 2- ⁇ ), OPGL,
  • TNF- ⁇ International Publication No. WO 96/14328
  • TNF- ⁇ - ⁇ International Publication No. WO 00/08139
  • TNF- ⁇ - ⁇ International Publication No. WO 00/08139
  • AEM-I International Publication No. WO 97/33899
  • AEM-II International Publication No. WO 97/34911
  • endokine- ⁇ International Publication No. WO 98/07880
  • TR6 International Publication No. WO 98/30694
  • OPG International Publication No. WO 98/30694
  • 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).
  • CD40L CD40 ligand
  • AVRENDTM soluble form of CD40L
  • biologically active fragments, variants, or derivatives of CD40L e.g., 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 (Knoll), ASM-981 (Novartis), interleukin- 1 receptor (e.g., Immunex), interleukin- 4 receptor (e.g.
  • Immunex 1CM3 (ICOS), BMS- 188667 (Bristol-Myers Squibb), anti-TNF Ab (e.g., Therapeutic antibodies), CG-1088 (Celgene), anti-B7 Mab (e.g., Innogetics), MEDI-507 (BioTransplant), ABX-CBL (Abgenix).
  • ICM3 ICM3
  • BMS- 188667 Bristol-Myers Squibb
  • anti-TNF Ab e.g., Therapeutic antibodies
  • CG-1088 Celgene
  • anti-B7 Mab e.g., Innogetics
  • MEDI-507 BioTransplant
  • ABX-CBL Abgenix
  • 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 of the invention, include, but are not limited to, RETROVERTM (zidovudine/AZT), VIDEXTM (didanosine/ddl), HIVEDTM (zalcitabine/ddC), ZERITTM (stavudine/d4T), EPIVIRTM (lamivudine/3TC), and COMBIVERTM (zidovudine/lamivudine).
  • Non-nucleoside reverse transcriptase inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, VIRNMUNETM (nevirapine), RESCRIPTORTM (delavirdine), and SUST1VATM (efavirenz).
  • Protease inhibitors that may be administered in combination with the compositions ofthe invention, include, but are not limited to, CRIXIVANTM (indinavir), NORVERTM (ritonavir), INVERASETM (saquinavir), and VERACEPTTM (nelfinavir).
  • antiretro viral 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, prevent, and/or diagnose AEDS and/or to treat, prevent, and/or diagnose 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, PENTAMIDENETM, ATOVAQUONETM, ISONIAZIDTM REFAMPENTM, PYRAZENAMEDETM, ETHAMBUTOLTM, RIFABUTENTM
  • compositions of the invention are used in any combination with TRIMETHOPRIM- S ULFAMETHOXAZOLETM, DAP S ONETM PENTAMIDTNETM, and/or ATOVAQUONETM to prophylactically treat, prevent and/or diagnose an opportunistic Pneumocystis carinii pneumonia infection.
  • compositions of the invention are used in any combination with ISONIAZIDTM, REFAMPENTM, PYRAZENAMEDETM, and/or ETHAMBUTOLTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium avium complex infection.
  • compositions of the invention are used in any combination with REFABUTENTM, CLARITHROMYCENTM, and/or AZITHROMYCTNTM to prophylactically treat, prevent, and/or diagnose an opportunistic Mycobacterium tuberculosis infection.
  • compositions of the invention are used in any combination with GANCICLOVIRTM, FOSCARNETTM, and/or CIDOFOVIRTM to prophylactically treat, prevent, and/or diagnose an opportunistic cytomegalovirus infection.
  • compositions of the invention are used in any combination with
  • compositions of the invention are used in any combination with ACYCLOVIRTM and/or FAMCICOLVERTM to prophylactically treat, prevent, and/or diagnose an opportunistic herpes simplex virus type I and/or type II infection.
  • compositions ofthe invention are used in any combination with P YREMETHAMENETM and/or LEUCOVORTNTM to prophylactically treat, prevent, and/or diagnose an opportunistic Toxoplasma gondii infection.
  • compositions ofthe invention are used in any combination with LEUCOVORINTM and/or NEUPOGENTM to prophylactically treat, prevent, and/or diagnose 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 of the invention include, but are not limited to, amoxicillin, aminoglycosides, ⁇ -lactam (glycopeptide), ⁇ -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, glucortico steroids, and RAPAMUNETM
  • immunosuppressants may be used to prevent rejection of organ or bone marrow transplantation.
  • compositions ofthe 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.
  • the 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 ofthe invention are administered in combination with an NSAED.
  • 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 Chiroscience), 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 (Dong Wha), darbufelone mesylate
  • 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, methotrexate, anti-TNF antibody, ENBRELTM and/or suflasalazine.
  • compositions of the invention are administered in combination with methotrexate. In another embodiment, the compositions ofthe 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. In another embodiment, the compositions ofthe invention are administered in combination with ENBRELTM and methotrexate.
  • compositions ofthe invention are administered in combination with ENBRELTM, methotrexate and suflasalazine.
  • compositions of the invention are administered in combination with ENBRELTM, methotrexate and suflasalazine.
  • one or more antimalarials is combined with one of the above- recited combinations.
  • the compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), ENBRELTM, methotrexate and suflasalazine.
  • the compositions of the invention are administered in combination with an antimalarial (e.g., hydroxychloroquine), sulfasalazine, anti- TNF antibody, and methotrexate.
  • compositions of the invention are administered in combination with an antibiotic agent.
  • Antibiotic agents that may be administered with the compositions of the invention include, but are not limited to, tetracycline, metronidazole, amoxicillin, ⁇ -lactamases, aminoglycosides, macrolides, quinolones, fluoroquinolones, cephalosporins, erythromycin, ciprofloxacin, and streptomycin.
  • 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-acetamidocaproicacid, 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 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., fluorouracil,
  • 5-FU methotrexate, floxuridine, interferon ⁇ -2b, glutamic acid, plicamycin, mercaptopurine, and 6-thioguanine
  • 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 thiote
  • 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, EL-3, EL-4, IL-5, EL-6, IL-7, EL-10, EL-12, EL-13, EL-15, anti-CD40, CD40L, EFN- ⁇ and TNF- ⁇ .
  • the 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-682110; Platelet Derived Growth Factor-B (PDGF-B), as disclosed in European Patent Number EP-282317; Placental Growth Factor (PIGF), as disclosed in International Publication Number WO 92/06194; Placental Growth Factor-2 (P1GF-2), as disclosed in Hauser et al, Growth Factors, 4:259-268 (1993); Vascular Endothelial Growth Factor (VEGF), as disclosed in International Publication Number WO 90/13649; Vascular Endothelial Growth Factor-A (VEGF-A), as disclosed in European Patent Number EP-506477; Vascular Endothelial Growth Factor-2 (VEGF-2), as disclosed in International Publication Number WO 96
  • Vascular Endothelial Growth Factor-D VEGF-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,
  • compositions of the invention are administered in combination with other therapeutic or prophylactic regimens, such as, for example, radiation therapy.
  • the invention also provides a method of delivering compositions containing the polypeptides ofthe invention (e.g., compositions containing DR3 polypeptides or anti-DR3 antibodies associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs) to targeted cells, expressing the membrane-bound form of DR3 on their surface, or alternatively, anDR3 receptor on their surface.
  • DR3 polypeptides or anti-DR3 antibodies ofthe invention may be associated with heterologous polypeptides, heterologous nucleic acids, toxins, or prodrugs via hydrophobic, hydrophilic, ionic and/or covalent interactions.
  • the invention provides a method for the specific delivery of compositions ofthe invention to cells by administering polypeptides of the invention (e.g., DR3 or anti-DR3 antibodies) that are associated with heterologous polypeptides or nucleic acids.
  • polypeptides of the invention e.g., DR3 or anti-DR3 antibodies
  • the invention provides a method for delivering a therapeutic protein into the targeted cell.
  • the invention provides a method for delivering a single stranded nucleic acid (e.g., antisense or ribozymes) or double stranded nucleic acid (e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed) into the targeted cell.
  • a single stranded nucleic acid e.g., antisense or ribozymes
  • double stranded nucleic acid e.g., DNA that can integrate into the cell's genome or replicate episomally and that can be transcribed
  • the invention provides a method for the specific destruction of cells (e.g., the destruction of tumor cells) by administering polypeptides ofthe invention (e.g., DR3 polypeptides or anti-DR3 antibodies) in association with toxins or cytotoxic prodrugs.
  • polypeptides ofthe invention e.g., DR3 polypeptides or anti-DR3 antibodies
  • the invention provides a method for the specific destruction of cells expressing the membrane-bound form ofDR3 on their surface (e.g., spleen, bone marrow, kidney and PBLs) by administering anti-DR3 antibodies in association with toxins or cytotoxic prodrugs.
  • toxin compounds that bind and activate endogenous cytotoxic effector systems, radioisotopes, holotoxins, modified toxins, catalytic subunits of toxins, cytotoxins (cytotoxic agents), or any molecules or enzymes not normally present in or on the surface of a cell that under defined conditions cause the cell's death.
  • Toxins that may be used according to the methods ofthe invention include, but are not limited to, radioisotopes known in the art, compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseudomonas exotoxin A, diphtheria toxin, saporin, momordin, gelonin, pokeweed antiviral protein, ⁇ -sarcin and cholera toxin.
  • radioisotopes known in the art
  • compounds such as, for example, antibodies (or complement fixing containing portions thereof) that bind an inherent or induced endogenous cytotoxic effector system, thymidine kinase, endonuclease, RNAse, alpha toxin, ricin, abrin, Pseu
  • Toxin also includes a cytostatic or cytocidal agent, a therapeutic agent or a radioactive metal ion, e.g., ⁇ -emitters such as, for example, 213 Bi, or other radioisotopes such as, for example, 103 Pd, 133 Xe, 131 1, 68 Ge, "Co, 65 Zn, 85 Sr, 32 P, 35 S, 90 Y, 153 Sm, 153 Gd, 169 Yb, 51 Cr, 54 Mn, 75 Se, 113 Sn, 90 Yttrium, 117 Tin, 186 Rhenium, 166 Holmium, and 188 Rhenium; luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • ⁇ -emitters such as, for example, 213 Bi
  • radioisotopes such as, for example, 103 Pd, 133 Xe, 131 1, 68 Ge, "Co,
  • 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
  • cytotoxic prodrug is meant a non-toxic compound that is converted by an enzyme, normally present in the cell, into a cytotoxic compound.
  • Cytotoxic prodrugs that may be used according to the methods ofthe invention include, but are not limited to, glutamyl derivatives of benzoic acid mustard alkylating agent, phosphate derivatives of etoposide or mitomycin C, cytosine arabinoside, daunorubisin, and phenoxyacetamide derivatives of doxorubicin.
  • the invention also provides a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • a pharmaceutical pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical compositions of the invention.
  • Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration.
  • Labeled antibodies, and derivatives and analogs thereof, which specifically bind to a polypeptide of interest can be used for diagnostic purposes to detect, diagnose, or monitor diseases and/or disorders associated with the aberrant expression and/or activity of a polypeptide of the invention.
  • the invention provides for the detection of aberrant expression of a polypeptide of interest, comprising (a) assaying the expression of the polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of aberrant expression.
  • the invention provides a diagnostic assay for diagnosing a disorder, comprising (a) assaying the expression ofthe polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
  • a diagnostic assay for diagnosing a disorder comprising (a) assaying the expression ofthe polypeptide of interest in cells or body fluid of an individual using one or more antibodies specific to the polypeptide interest and (b) comparing the level of gene expression with a standard gene expression level, whereby an increase or decrease in the assayed polypeptide gene expression level compared to the standard expression level is indicative of a particular disorder.
  • the presence of a relatively high amount of transcript in biopsied tissue from an individual may indicate a predisposition for the development of the disease, or may provide a means for detecting the disease prior
  • Antibodies of the invention can be used to assay protein levels in a biological sample using classical immunohistological methods known to those of skill in the art (e.g., see Jalkanen, M. et al., J. Cell. Biol. 707:976-985 (1985); Jalkanen, M. etal, J. Cell. Biol. 105:3081-3096 (1987)).
  • Other antibody-based methods useful for detecting protein 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; radioisotopes, such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( U2 In), and technetium ( 99 Tc); luminescent labels, such as luminol; and fluorescent labels, such as fluorescein and rhodamine, and biotin.
  • enzyme labels such as, glucose oxidase
  • radioisotopes such as iodine ( 125 I, 121 I), carbon ( 14 C), sulfur ( 35 S), tritium ( 3 H), indium ( U2 In), and technetium ( 99 Tc)
  • luminescent labels such as luminol
  • fluorescent labels such as fluorescein and rhodamine, and biotin.
  • One aspect ofthe invention is the detection and diagnosis of a disease or disorder associated with aberrant expression of a polypeptide ofthe interest in an animal, preferably
  • diagnosis comprises: a) administering (for example, parenterally, subcutaneously, or intraperitoneally) to a subject an effective amount of a labeled molecule which specifically binds to the polypeptide of interest; b) waiting for a time interval following the administering for permitting the labeled molecule to preferentially concentrate at sites in the subject where the polypeptide is expressed (and for unbound labeled molecule to be cleared to background level); c) determining background level; and d) detecting the labeled molecule in the subject, such that detection of labeled molecule above the background level indicates that the subject has a particular disease or disorder associated with aberrant expression of the polypeptide of interest.
  • Background level can be determined by various methods including, comparing the amount of labeled molecule detected to a standard value previously determined for a particular system.
  • the size ofthe subject and the imaging system used will determine the quantity of imaging moiety needed to produce diagnostic images.
  • the quantity of radioactivity injected will normally range from about 5 to 20 millicuries of 99mTc.
  • the labeled antibody or antibody fragment will then preferentially accumulate at the location of cells which contain the specific protein. In vivo tumor imaging is described in S.W. Burchiel et al,
  • the time interval following the administration for permitting the labeled molecule to preferentially concentrate at sites in the subject and for unbound labeled molecule to be cleared to background level is 6 to 48 hours or 6 to 24 hours or 6 to 12 hours. In another embodiment the time interval following administration is 5 to 20 days or 5 to 10 days.
  • monitoring ofthe disease or disorder is carried out by repeating the method for diagnosing the disease or disease, for example, one month after initial diagnosis, six months after initial diagnosis, one year after initial diagnosis, etc.
  • Presence of the labeled molecule can be detected in the patient using methods known in the art for in vivo scanning. These methods depend upon the type of label used. Skilled artisans will be able to determine the appropriate method for detecting a particular label. Methods and devices that may be used in the diagnostic methods of the invention include, but are not limited to, computed tomography (CT), whole body scan such as position emission tomography (PET), magnetic resonance imaging (MRI), and sonography.
  • CT computed tomography
  • PET position emission tomography
  • MRI magnetic resonance imaging
  • sonography sonography
  • the molecule is labeled with a radioisotope and is detected in the patient using a radiation responsive surgical instrument (Thurston et al, U.S. Patent No. 5,441,050).
  • the molecule is labeled with a fluorescent compound and is detected in the patient using a fluorescence responsive scanning instrument.
  • the molecule is labeled with a positron emitting metal and is detected in the patent using positron emission-tomography.
  • the molecule is labeled with a paramagnetic label and is detected in a patient using magnetic resonance imaging (MRI).
  • MRI magnetic resonance imaging
  • the present invention provides kits that can be used in the above methods.
  • kits comprises an antibody of the invention, preferably a purified antibody, in one or more containers.
  • the kits ofthe present invention contain a substantially isolated polypeptide comprising an epitope which is specifically immunoreactive with an antibody included in the kit.
  • the kits of the present invention further comprise a control antibody which does not react with the polypeptide of interest.
  • kits of the present invention contain a means for detecting the binding of an antibody to a polypeptide of interest (e.g., the antibody may be conjugated to a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate).
  • a detectable substrate such as a fluorescent compound, an enzymatic substrate, a radioactive compound or a luminescent compound, or a second antibody which recognizes the first antibody may be conjugated to a detectable substrate.
  • the kit is a diagnostic kit for use in screening serum containing antibodies specific against proliferative and/or cancerous polynucleotides and polypeptides.
  • a kit may include a control antibody that does not react with the polypeptide of interest.
  • a kit may include a substantially isolated polypeptide antigen comprising an epitope which is specifically immunoreactive with at least one anti-polypeptide antigen antibody.
  • a kit includes means for detecting the binding of said antibody to the antigen (e.g.
  • the antibody may be conjugated to a fluorescent compound such as fluorescein or rhodamine which can be detected by flow cytometry
  • the kit may include a recombinantly produced or chemically synthesized polypeptide antigen
  • the polypeptide antigen ofthe kit may also be attached to a solid support
  • the detecting means of the above- described kit includes a solid support to which said polypeptide antigen is attached
  • a kit may also include a non-attached reporter-labeled anti-human antibody.
  • binding ofthe antibody to the polypeptide antigen can be detected by binding ofthe said reporter-labeled antibody.
  • the invention includes a diagnostic kit for use in screening serum containing antigens ofthe polypeptide of the invention
  • the diagnostic kit includes a substantially isolated antibody specifically immunoreactive with polypeptide or polynucleotide antigens, and means for detecting the binding of the polynucleotide or polypeptide antigen to the antibody
  • the antibody is attached to a solid support
  • the antibody may be a monoclonal antibody
  • the detecting means of the kit may include a second, labeled monoclonal antibody Alternatively, or in addition, the detecting means may include a labeled, competing antigen
  • test serum is reacted with a solid phase reagent having a surface-bound antigen obtained by the methods ofthe present invention
  • a solid phase reagent having a surface-bound antigen obtained by the methods ofthe present invention
  • reporter-labeled anti-human antibody to bind reporter to the reagent in proportion to the amount of bound anti-antigen antibody on the solid support.
  • the reagent is again washed to remove unbound labeled antibody, and the amount of reporter associated with the reagent is determined
  • the reporter is an enzyme which is detected by incubating the solid phase in the presence of a suitable fluorometric, luminescent or colorimetric substrate (Sigma, St. Louis, MO).
  • the solid surface reagent in the above assay is prepared by known techniques for attaching protein material to solid support material, such as polymeric beads, dip sticks, 96-well plate or filter material. These attachment methods generally include non-specific adsorption ofthe protein to the support or covalent attachment ofthe protein, typically through a free amine group, to a chemically reactive group on the solid support, such as an activated carboxyl, hydroxyl, or aldehyde group. Alternatively, streptavidin coated plates can be used in conjunction with biotinylated antigen(s).
  • the invention provides an assay system or kit for carrying out this diagnostic method.
  • the kit generally includes a support with surface-bound recombinant antigens, and a reporter-labeled anti-human antibody for detecting surface-bound anti-antigen antibody.
  • the DNA sequence encoding the mature DR3-V1 protein in the cDNA contained in ATCC No. 97456 is amplified using PCR oligonucleotide primers specific to the amino terminal sequences of the DR3-V1 protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
  • 5'-GCGCCATGGGGGCCCGGCGGCAG-3' contains anNcol site and 15 nucleotide starting from 290 nucleotide to 304 in S ⁇ Q ID ⁇ O: l.
  • 5'-GCGAAGCTTCTNGGACCCAGANCATCTGCC-3' contains a Hindlll site, a stop codon and 18 nucleotides complimentary to nucleotides from 822 to 840 in S ⁇ Q ID NO: 1.
  • Vector is pQ ⁇ 60. The protein is not tagged.
  • restriction sites are convenient to restriction enzyme sites in the bacterial expression vector pQ ⁇ 60, which are used for bacterial expression in these examples.
  • pQE60 encodes ampicillin antibiotic resistance (" Amp r ”) and contains a bacterial origin of replication ("ori"), an EPTG inducible promoter, a ribosome binding site ("RBS").
  • Amp r ampicillin antibiotic resistance
  • ori bacterial origin of replication
  • RBS ribosome binding site
  • Insertion ofthe DDCR protein DNA into the restricted pQE60 vector places the DR3-V1 protein coding region downstream of and operably linked to the vector's IPTG-inducible promoter and in-frame with an initiating AUG appropriately positioned for translation of DR3-V1 protein.
  • E. coli strain M15/rep4 containing multiple copies ofthe plasmid pREP4, which expresses lac repressor and confers kanamycin resistance ("Kan r "), is used in carrying out the illustrative example described herein.
  • This strain which is only one of many that are suitable for expressing DR3-V1 protein, is available commercially from Qiagen.
  • Transformants are identified by their ability to grow on LB plates in the presence of ampicillin and kanamycin. Plasmid DNA is isolated from resistant colonies and the identity ofthe cloned DNA confirmed by restriction analysis.
  • Clones containing the desired constructs are grown overnight ("O/N") in liquid culture in LB media supplemented with both ampicillin (100 ⁇ g/ml) and kanamycin (25 ⁇ g/ml).
  • the O/N culture is used to inoculate a large culture, at a dilution of approximately 1 : 100 to 1 :250.
  • the cells are grown to an optical density at 600nm("OD600")ofbetween0.4and0.6.
  • Isopropyl-B-D-thiogalactopyranoside (“EPTG”) is then added to a final concentration of 1 mM to induce transcription from lac repressor sensitive promoters, by inactivating the lacl repressor. Cells subsequently are incubated further for 3 to 4 hours.
  • Inclusion bodies are purified from the disrupted cells using routine collection techniques, and protein is solublized from the inclusion bodies into 8M urea.
  • the 8M urea solution containing the solublized protein is passed over a PD-10 column in 2X phosphate-buffered saline ("PBS"), thereby removing the urea, exchanging the buffer and refolding the protein.
  • PBS 2X phosphate-buffered saline
  • the protein is purified by a further step of chromatography to remove endotoxin. Then, it is sterile filtered.
  • the sterile filtered protein preparation is stored in 2X PBS at a concentration of 95 ⁇ /ml.
  • a typical mammalian expression vector contains the promoter element, which mediates the initiation of transcription of mRNA, the protein coding sequence, and signals required for the termination of transcription and polyadenylation ofthe transcript. Additional elements include enhancers, Kozak sequences and intervening sequences flanked by donor and acceptor sites for RNA splicing.
  • Suitable expression vectors for use in practicing the present invention include, for example, vectors such as pSVL and pMSG (Pharmacia, Uppsala, Sweden), pRSVcat (ATCC 37152), pSV2dhfr (ATCC 37146) and pBC12MI (ATCC67109).
  • Mammalian host cells that could be used include, human HeLa, 283, H9 and Jurkat cells, mouse NIH3T3 and C127 cells, Cos 1, Cos 7, and CV1 African green monkey cells, quail QC1-3 cells, mouse L cells, and Chinese hamster ovary (CHO) cells.
  • a gene of interest can be expressed in stable cell lines that contain the gene integrated into a chromosome.
  • the co-transfection with a selectable marker such as dhfr, gpt, neomycin, hygromycin allows the identification and isolation ofthe transfected cells.
  • the transfected gene can also be amplified to express large amounts ofthe encoded protein.
  • the DHFR dihydrofolate reductase
  • the mammalian cells are grown in increasing amounts of methotrexate for selection and the cells with the highest resistance are selected.
  • These cell lines contain the amplified gene(s) integrated into a chromosome. Chinese hamster ovary (CHO) cells are often used for the production of proteins.
  • the expression vectors pCl and pC4 contain the strong promoter (LTR) of the Rous Sarcoma Virus (Cullen et al, Molecular and Cellular Biology
  • the vectors contain in addition the 3' intron, the polyadenylation and termination signal ofthe rat preproinsulin gene.
  • the expression plasmid, pDR3-Vl HA is made by cloning a cDNA encoding DR3-V1 (ATCC No. 97456) into the expression vector pcDNAI/Amp (which can be obtained from Invitrogen, Inc.).
  • Expression plasmid, pDR3 HA is made by cloning a cDNA encoding DR3 (ATCC No. 97757) into the expression vector pcDNAI/Amp.
  • the expression vector pcDNAI/amp contains: (1) an E. coli origin of replication effective for propagation in E. coli and other prokaryotic cell; (2) an ampicillin resistance gene for selection of plasmid-containing prokaryotic cells;
  • an SV40 origin of replication for propagation in eukaryotic cells; (4) a CMV promoter, a polylinker, an SV40 intron, and a polyadenylation signal arranged so that a cDNA conveniently can be placed under expression control of the CMV promoter and operably linked to the SV40 intron and the polyadenylation signal by means of restriction sites in the polylinker.
  • a DNA fragment encoding the entire DR3 -V 1 or Dr3 precursor and a HA tag fused in frame to its 3' end is cloned into the polylinker region ofthe vector so that recombinant protein expression is directed by the CMV promoter.
  • the HA tag corresponds to an epitope derived from the influenza hemagglutinin protein described by Wilson etal, Cell 37:161 (1984). The fusion ofthe HA tag to the target protein allows easy detection of the recombinant protein with an antibody that recognizes the HA epitope.
  • the plasmid construction strategy is as follows: The DR3-V1 or DR3 cDNA of the deposit cDNA is amplified using primers that contained convenient restriction sites, much as described above regarding the construction of expression vectors for expression of DR3-V1 or
  • one ofthe primers contains a hemagglutinin tag ("HA tag") as described above.
  • Suitable primers for DR3-V1 include the following, which are used in this example, the 5' primer:
  • CGCGGATCCGCCATCATGGAGGAGACGCAGCAG 3' contains the underlined BamHl site, an ATG start codon and 5 codons thereafter.
  • Suitable primers for DR3 include the following, which are used in this example, the 5' primer:
  • 5'CGCGGATCCGCCATCATGGAGCAGCGGCCGCGG3'(SEQEDNO:10) contains the underlined BamHl site, an ATG start codon and 5 codons thereafter.
  • the 3' primer for both DR3 and DR3-V1, containing the underlined Xbal site, stop codon, hemagglutinin tag and last 14 nucleotide of 3' coding sequence (at the 3' end) has the following sequence:
  • the PCR amplified DNA fragment and the vector, pcDNAI/Amp, are digested with BamHl and Xbal and then ligated.
  • the ligation mixture is transformed into E. coli strain SURE (available from Stratagene Cloning Systems, 1 1099 North Torrey Pines Road, La Jolla, CA 92037) the transformed culture is plated on ampicillin media plates which then are incubated to allow growth of ampicillin resistant colonies. Plasmid DNA is isolated from resistant colonies and examined by restriction analysis and gel sizing for the presence of the DR3-V1 or DR3 -encoding fragment.
  • COS cells are transfected with an expression vector, as described above, using DEAE- DEXTRAN, as described, for instance, in Sambrook etal, Molecular Cloning: a Laboratory Manual, Cold Spring Laboratory Press, Cold Spring Harbor, NY
  • Cells are incubated under conditions for expression of DR3-V1 or DR3 by the vector.
  • DR3-V1 HA fusion protein or the DR3 HA fusion protein is detected by radiolabelling and immunoprecipitation, using methods described in, for example Harlow et al, Antibodies: a Laboratory Manual, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (1988). To this end, two days after transfection, the cells are labeled by incubation in media containing 35 S-cysteine for 8 hours. The cells and the media are collected, and the cells are washed and then lysed with detergent-containing REPA buffer: 150 mM
  • Plasmid pCl is used for the expression of DR3-V1 or DR3 (ATCC No. 97456 or ATCC No. 97757, respectively) protein.
  • Plasmid pCl is a derivative of the plasmid pSV2-dhfr (ATCC Accession No. 37146). Both plasmids contain the mouse DHFR gene under control of the SV40 early promoter. Chinese hamster ovary- or other cells lacking dihydrofolate activity that are transfected with these plasmids can be selected by growing the cells in a selective medium (alpha minus MEM, Life Technologies) supplemented with the chemotherapeutic agent methotrexate.
  • a selective medium alpha minus MEM, Life Technologies
  • MTX methotrexate
  • a second gene is linked to the DHFR gene it is usually co-amplified and over-expressed. It is state ofthe art to develop cell lines carrying more than 1,000 copies ofthe genes Subsequently, when the methotrexate is withdrawn, cell lines contain the amplified gene integrated into the chromosome(s).
  • Plasmid pCl contains for the expression ofthe gene of interest a strong promoter ofthe long terminal repeat (LTR) ofthe Rous Sarcoma Virus (Cullen et al. , Molecular and Cellular Biology 5:438-441 (March 1985)), plus a fragment isolated from the enhancer ofthe immediate early gene of human cytomegalovirus
  • CMV Chroshart et ⁇ l., Cell 47:521-530 (1985)
  • B ⁇ mHl followed by the 3' intron
  • polyadenylation site ofthe rat preproinsulin gene Downstream from the promoter are the following single restriction enzyme cleavage sites that allow the integration ofthe genes: B ⁇ mHl followed by the 3' intron and the polyadenylation site ofthe rat preproinsulin gene.
  • Other high efficient promoters can also be used for the expression, e.g., the human ⁇ -actin promoter, the SV40 early or late promoters or the long terminal repeats from other retroviruses, e.g., HIV and HTLVI.
  • the polyadenylation of the mRNA other signals, e.g., from the human growth hormone or globin genes can be used as well.
  • Stable cell lines carrying a gene of interest integrated into the chromosomes can also be selected upon co-transfection with a selectable marker such as gpt, G418 or hygromycin. It is advantageous to use more than one selectable marker in the beginning, e.g., G418 plus methotrexate.
  • the plasmid pC 1 is digested with the restriction enzyme BamHl and then dephosphorylated using calf intestinal phosphates by procedures known in the art.
  • the vector is then isolated from a 1% agarose gel.
  • the DNA sequence encoding DR3-V1 or DR3 in the deposited cDNA is amplified using PCR oligonucleotide primers specific to the amino acid carboxyl terminal sequence ofthe DR3-V1 or DR3 protein and to vector sequences 3' to the gene. Additional nucleotides containing restriction sites to facilitate cloning are added to the 5' and 3' sequences respectively.
  • the 5' oligonucleotide primer for DR3-V1 has the sequenc:
  • the 5' oligonucleotide primer for DR3 has the sequence: 5' CGCGGATCCGCC ATC ATGGAGC AGCGGCCGCGG 3 ' (SEQ ED NO: 13) containing the underlined BamHl restriction site, which encodes a start AUG, followed by the Kozak sequence and 18 nucleotides ofthe DR3 coding sequence set out in SEQ ID NO: 3 beginning with the first base ofthe ATG codon.
  • the 3' primer for both DR3 and DR3-V1 has the sequence: 5' CGCGGATCCTCACGGGCCGCGCTGCA 3' (SEQ ID NO: 14) containing the underlined BamHl restriction site followed by 17 nucleotides complementary to the last 14 nucleotides ofthe DR3-V1 or DR3 coding sequence set out in SEQ ID NO: 14
  • restriction sites are convenient to restriction enzyme sites in the CHO expression vectors pC 1.
  • the amplified DR3 or DR3-V1 DNA and the vector pCl both are digested with BamHl and the digested DNAs then ligated together. Insertion of the DR3 -V 1 or DR3 DNA into the BamHl restricted vector placed the DR3 -V 1 or DR3 coding region downstream of and operably linked to the vector's promoter. The sequence ofthe inserted gene is confirmed by DNA sequencing.
  • Chinese hamster ovary cells lacking an active DHFR enzyme are used for transfection. 5 ⁇ g ofthe expression plasmid CI are cotransfected with 0.5 ⁇ g of the plasmid pSVneo using the lipofecting method (Feigner et al, supra).
  • the plasmid pSV2-neo contains a dominant selectable marker, the gene neo from Tn5 encoding an enzyme that confers resistance to a group of antibiotics including
  • the cells are seeded in alpha minus MEM supplemented with 1 mg/ml G418. After 2 days, the cells are trypsinized and seeded in hybridoma cloning plates (Greiner, Germany) and cultivated from 10-14 days. After this period, single clones are trypsinized and then seeded in 6-well petri dishes using different concentrations of methotrexate (25 nM, 50 nM, 100 nM, 200 nM, 400 nM).
  • Clones growing at the highest concentrations of methotrexate are then transferred to new 6-well plates containing even higher concentrations of methotrexate (500 nM, 1 ⁇ M, 2 ⁇ M, 5 ⁇ M). The same procedure is repeated until clones grow at a concentration of 100 ⁇ M.
  • the expression ofthe desired gene product is analyzed by Western blot analysis and SDS-PAGE.
  • the 5' primer for DR3-V1 has the sequence: 5' CGCGGATCC GCCATCATGGAGGAGACGCAGCAG 3' (SEQ ID NO: 15) containing the underlined BamHl restriction enzyme site followed by a Kozak sequence and a number of bases ofthe sequence of DR3-V1 of SEQ ED NO: l .
  • An efficient signal for initiation of translation in eukaryotic cells, as described by M. Kozak, J. Mol. Biol. 196:941-950 (1987) is appropriately located in the vector portion ofthe construct.
  • the 5' primer for DR3 has the sequence:
  • the 5' end ofthe amplified fragment encoding DR3 provides an efficient signal peptide.
  • the 3' primer for both DR3 and DR3-V1 has the sequence: 5' GCGAGATCTAGTCTGGACCCAGAACATCTGCCTCC 3' (SEQ ED NO: 17) containing the underlined Xbal restriction followed by nucleotides complementary to the DR3-V1 or DR3 nucleotide sequence set out in SEQ ED NO: 1 or SEQ ID NO: 3, respectively, followed by the stop codon.
  • the amplified fragment is isolated from a 1% agarose gel using a commercially available kit ("Geneclean," BIO 101 Inc., La Jolla, Ca.) The fragment then is digested with BamHl and Aspl 8 and again is purified on a 1%> agarose gel.
  • the vector pA2 is used to express the DR3-V1 or DR3 protein in the baculovirus expression system, using standard methods, such as those described in Summers et al. , A Manual of Methods for Baculovirus Vectors and Insect Cell Culture Procedures, Texas Agricultural Experimental Station Bulletin No. 1555 (1987).
  • This expression vector contains the strong polyhedron promoter ofthe Autograph californica nuclear polyhedrosis virus (ACMNPV) followed by convenient restriction sites.
  • ACMNPV Autograph californica nuclear polyhedrosis virus
  • the ⁇ - galactosidase gene from E. coli is inserted in the same orientation as the polyhedron promoter and is followed by the polyadenylation signal of the polyhedron gene.
  • the polyhedron sequences are flanked at both sides by viral sequences for cell-mediated homologous recombination with wild-type viral DNA to generate viable virus that express the cloned polynucleotide.
  • baculovirus vectors could be used in place of p A2, such as pAc373, pVL941 and pAcIMl provided, as those of skill readily will appreciate, that construction provides appropriately located signals for transcription, translation, trafficking and the like, such as an in-frame AUG and a signal peptide, as required.
  • Such vectors are described in Luckow et al, Virology 770:31-39 (1989), among others.
  • the plasmid is digested with the restriction enzymesTi mHI andNb l and then is dephosphorylated using calf intestinal phosphatase, using routine procedures known in the art.
  • the DNA is then isolated from a 1% agarose gel using a commercially available kit ("Geneclean" BIO 101 Inc., La Jolla, Ca.). This vector DNA is designated herein "V2".
  • Fragment F2 and the dephosphorylated plasmid V2 are ligated together with T4 DNA ligase
  • E. coli HB101 cells are transformed with ligation mix and spread on culture plates.
  • Bacteria are identified that contain the plasmid with the human DDCR gene by digesting DNA from individual colonies using BamHl and Xbal and then analyzing the digestion product by gel electrophoresis. The sequence ofthe cloned fragment is confirmed by DNA sequencing. This plasmid is designated herein pBac DR3-V1 or pBac DR3.
  • plasmid pBac DR3-V1 or pBac DR3 is co-transfected with 1.0 ⁇ g of a commercially available linearized baculovirus DNA ("BaculoGoldTM baculovirus DNA", Pharmingen, San Diego, CA.), using the lipofection method described by Feigner et al, Proc. Natl. Acad. Sci. USA 54:7413-7417 (1987).
  • Lipofectin plus 90 ⁇ l Grace's medium are added, mixed and incubated for 15 minutes at room temperature. Then the transfection mixture is added drop-wise to Sf9 insect cells (ATCC CRL 1711) seeded in a 35 mm tissue culture plate with 1 ml Grace's medium without serum. The plate is rocked back and forth to mix the newly added solution. The plate is then incubated for 5 hours at 27 °C. After
  • the transfection solution is removed from the plate and 1 ml of Grace's insect medium supplemented with 10% fetal calf serum is added.
  • the plate is put back into an incubator and cultivation is continued at 27 °C for four days.
  • plaque assay After four days the supernatant is collected and a plaque assay is performed, as described by Summers and Smith, cited above. An agarose gel with "Blue Gal” (Life Technologies Inc., Gaithersburg) is used to allow easy identification and isolation of gal-expressing clones, which produce blue-stained plaques. (A detailed description of a "plaque assay” of this type can also be found in the user's guide for insect cell culture and baculovirology distributed by Life Technologies Inc., Gaithersburg, page 9-10).
  • the virus is added to the cells. After appropriate incubation, blue stained plaques are picked with the tip of an Eppendorf pipette. The agar containing the recombinant viruses is then resuspended in an Eppendorf tube containing 200 ⁇ l of Grace's medium. The agar is removed by a brief centrifugation and the supernatant containing the recombinant baculovirus is used to infect Sf9 cells seeded in 35 mm dishes. Four days later the supernatants of these culture dishes are harvested and then they are stored at 4°C. A clone containing properly inserted DR3-V1 or DR3 is identified by DNA analysis including restriction mapping and sequencing.
  • Sf9 cells are grown in Grace's medium supplemented with 10% heat- inactivated FBS. The cells are infected with the recombinant baculovirus V- DR3-V1 at a multiplicity of infection ("MOI") of about 2 (about 1 to about 3). Six hours later the medium is removed and is replaced with SF900 II medium minus methionine and cysteine (available from Life Technologies Inc., Gaithersburg). 42 hours later, 5 ⁇ Ci of 35 S-methionine and 5 ⁇ Ci 35 S cysteine
  • the cells are further incubated for 16 hours and then they are harvested by centrifugation, lysed and the labeled proteins are visualized by SDS-PAGE and autoradiography.
  • Northern blot analysis is carried out to examine DR3-V1 gene (ATCC No. 97456) expression in human tissues, using methods described by, among others, Sambrook et al, cited above.
  • a cDNA probe containing the entire nucleotide sequence ofthe DR3-V1 protein (SEQ ED NOT) is labeled with 32 P using the rec ⁇ primeTM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT1200-1. The purified labeled probe is then used to examine various human tissues for DR3-V1 mRNA.
  • DR3-V1 human immune system tissues
  • PBLs peripheral blood leukocytes
  • thymus thymus
  • spleen colon
  • small intestine DR3-V1 expression appears to be restricted to lymphocyte compartments, it can be envisaged that DR3-V1 plays a role in lymphocyte homeostasis.
  • Northern blot analysis is carried out to examine DR3 gene (ATCC No. 97757) expression in human tissues, using methods described by, among others, Sambrook et al, cited above.
  • a cDNA probe containing the entire nucleotide sequence of the DR3 protein (SEQ ID NO: l) is labeled with 3 P using the red ⁇ primeTM DNA labeling system (Amersham Life Science), according to manufacturer's instructions. After labeling, the probe is purified using a CHROMA SPIN- 100TM column (Clontech Laboratories, Inc.), according to manufacturer's protocol number PT 1200-1. The purified labeled probe is then used to examine various human tissues for DR3 mRNA.
  • MTN Multiple Tissue Northern
  • H human tissues
  • EM human immune system tissues
  • DR3 DR3 was detected in tissues enriched in lymphocytes including peripheral blood leukocytes (PBLs), thymus, spleen, colon, and small intestine.
  • PBLs peripheral blood leukocytes
  • TNFR-1 is ubiquitously expressed and Fas/APO-1 is expressed in lymphocytes, liver, heart, lung, kidney, and ovary (Watanabae- Fukunaga et al, J. Immunol 148: 1214-9 (1992)).
  • DR3 expression appears to be restricted to lymphocyte compartments, it can be envisaged that DR3 plays a role in lymphocyte homeostasis.
  • C Northern Blot analysis ofDR3 in various cell lines
  • CD 19+ CD4+ and CD8+ cells were isolated with cell type specific immunomagnetic beads (Drynal, Lake Success, NY)
  • RNA of adult tissues were purchased from Clonetech (Palo Alto, CA). Total RNA was extracted from cell lines (in exponential growth phase) and primary cells with TriReagent (Molecular Research Center, Inc., Cincinnati, OH).
  • RNA was fractionated in a 1% agarose gel containing formaldehyde cast in a Wide Mini-Sub Cell gel tray (Bio-Rad, Hercules, CA) as described (Sambrook, et al.) with slight modifications.
  • the formaldehyde concentration was reduced to 0.5M and the RNA was stained prior to electrophoresis with 100 ⁇ g/ml of ethidium bromide that was added to the loading buffer.
  • cDNA inserts labeled with 2 P-dCTP by random priming (Stratagene, La Jolla, CA), were denatured with 0.25M NaOH (10 min at 37°C) and added to the prehybridization solution. After 24-65 hr at 42 °C, the blots were washed under high stringency conditions (Sambrook, et al.) and exposed to X-ray films.
  • DR3 expression of DR3 was assessed by Northern blot in the following cell lines: TF274 (bone marrow stromal); MG63, TE85 (osteo sarcoma); K562
  • DR3 expression was detected in several lymphoblast cell lines. In the purified human hematopoietic cell populations, DR3 was weakly expressed in CD 19+ cells, and more highly expressed in monocytes. However the highest levels were observed in T cells (CD4+ or CD8+) upon stimulation with PMA and PHA, indicating that DR3 probably plays a role in the regulation of T cell activation.
  • DR3 contains a death domain
  • DAMs death domain-containing adapter molecules
  • GST fusion proteins were prepared from E. coli strain BL21(DE3)pLysS using standard published procedures and the recombinant proteins immobilized onto glutathione-agarose beads.
  • 35 S-Labeled FADD, REP and TRADD were prepared by in vitro transcription-translation using the TNT or T7 or SP6-coupled reticulocyte lysate system from Promega according to manufacturer's instructions, using pcDNA3 AU1-FADD (A.M. Chinnaiyan et al, Cell 81: 505- 12 (1995); M.P. BoXdmetal, J Biol Chem 270: 7795-8 (1995); F.C.
  • the gel was Coomassie stained to monitor equivalency of loading.
  • constructs encoding Flag-TNFR- 1 and Flag- ⁇ TNFR-1 were used.
  • the Flag-TNFR- 1 and Flag- ⁇ TNFR-1 constructs were described elsewhere (A.M. Chinnaiyan et al, J Biol Chem 277 : 4961 -4965 ( 1996)) .
  • the constructs encoding Flag-TNFR- 1 and Flag- ⁇ TNFR-1 were described elsewhere (A.M. Chinnaiyan et al, J Biol Chem
  • DR3 and ⁇ DR3 were cloned into the EBI Kodak FLAG plasmid (pCMVlFLAG) utilizing the signal peptide provided by the vector.
  • pCMVlFLAG EBI Kodak FLAG plasmid
  • 293 cells (2 x 10 6 /100mm plate) were grown in DMEM media containing 10%> heat-inactivated fetal bovine serum containing penicillin G, streptomycin, glutamine, and non-essential amino acids.
  • Cells were transfected using calcium phosphate precipitation with the constructs encoding the indicated proteins in combination with pcDNA3-CrmA (M. Tewari et al, JBiol Chem 270: 3255-60 (1995)) to prevent cell death and thus maintain protein expression.
  • Cells were lysed in 1 ml lysis buffer (50mM Hepes, 150mM NaCl, lmM EDTA, 1% NP-40, and a protease inhibitor cocktail). Lysates were immunoprecipitated with a control monoclonal antibody or anti-Flag antibody for at least 4 hrs, at 4°C as previously described (A.M.
  • FADD associated with the GST-Fas cytoplasmic domain
  • TRADD associated with the GST-TNFR-1 cytoplasmic domain
  • GST-DDCR associated specifically with TRADD, but not FADD or RIP.
  • GST-DDR3 truncated death domain mutant of DR3
  • DR3 and TRADD were transiently transfected with plasmids that direct the synthesis of myc-epitope tagged TRADD (myc-TRADD) and Flag-epitope tagged DR3 (Flag-DR3), Flag- TNFR-1 ormutants. Consistent with the in vitro binding study, TRADD specifically coprecipitated with DR3 and TNFR-1, but not with the death domain mutants, DDR3 and DTNFR-1. Thus, it appears that DR3, like TNFR-1, may activate downstream signaling cascades by virtue of its ability to recruit the adapter molecule TRADD.
  • TRADD Overexpression of TRADD induces apoptosis and NF-kB activation-two ofthe most important activities signaled by TNFR- 1 (H. Hsu et al, supra). Upon oligomerization of TNFR-1 by trimeric TNF, TRADD is recruited to the receptor signaling complex (H. Hsu et al, Cell 54:299-308 (1996)). TRADD can then recruit the following signal transducing molecules: 1) TRAF2, a TNFR-2- and CD40 - associated molecule (M. Rothe et al, Cell 78: 681-92 (1994); M.
  • Fas/APO-1 and TNFR-1 in mammalian cells mimics receptor activation (M. Muzio et al, Cell 85: 817-827 (1996); M. P. Boldin et al, Cell 85: 803-815 (1996)).
  • this system was utilized to study the functional role of DDCR.
  • Ectopic expression of DR3 in MCF7 breast carcinoma cells and 293 human embryonic kidney cells induced rapid apoptosis.
  • FADD-DN expression construct (A.M. Chinnaiyan et al, J Biol Chem 271: 4961-4965 (1996)) were transiently transfected with pCMV- ⁇ - galatosidase in the presence of a ten-fold excess of pcDNA3 expression constructs encoding the indicated proteins using lipofectamine (GEBCO-BRL). 293 cells were likewise transfected using the CaPO 4 method. The ICE family inhibitor z-VAD-fmk (Enzyme Systems Products, Dublin, CA) was added to the cells at a concentration of lO ⁇ M, 5 hrs after transfection.
  • DR3 or DDR3 was cotransfected with the pLantern expression construct (GEBCO-BRL) which encodes green fluorescent protein (photographic inset). Cells were visualized by fluorescence microscopy using a FITC range barrier filter cube. Nuclei of transfected cells were visualized by DAPI staining and the image overlaid. (Cell death assays were performed essentially as previously described (Chinnaiyan et al, Cell 57:505-12 (1995); Boldin, et al, J. Biol. Chem.
  • DR3 induces NF-kB activation which is inhibitable by REP-DN (Stangeret ⁇ /., Cell 81:513-23 (1995)) and TRAF2-DN (Hsu etal, Cell 57:495-504 (1995); Rothe et al, Cell 78:681-92 (1994); Rothe et al. Science 269.1424-1421 (1995)), 293 cells were co-transfected with the indicated molecules and an NF-kB luciferase reporter plasmid (Kotheetal, Cell 75:681-92
  • the cells displayed morphological alterations typical of cells undergoing apoptosis, becoming rounded, condensed and detaching from the dish.
  • plasmids encoding full-length DR3 or DDR3 were co-transfected with the pLantern reporter construct encoding green fluorescent protein.
  • Nuclei of cells transfected with DR3, but not DDR3, exhibited apoptotic morphology as assessed by DAPI staining. Similar to TNFR-1 and Fas/APO-1 (M. Muzio et ⁇ /., Cell 85: 817-827 (1996); M. P. Boldin et al, Cell 85: 803-815 (1996); M.
  • DR3-induced apoptosis was blocked by the inhibitors of ICE-like proteases, CrmA and z-VAD-fmk. Importantly, apoptosis induced by DR3 was also blocked by dominant negative versions of FADD (FADD-DN) or FLICE (FLICE-DN/MACHalC360S), which were previously shown to inhibit death signaling by Fas/APO- 1 and TNFR- 1 (M.
  • FADD-DN FADD-DN
  • FLICE-DN/MACHalC360S FLICE-DN/MACHalC360S
  • NF-kB activation we examined whether DR3 could activate NF-kB.
  • Transfection of a control vector or expression of Fas/APO-1 failed to induce NF-kB activation.
  • NF-kB was activated by ectopic expression of DR3 or TNFR-1, but not by the inactive signaling mutants DDR3 or DTNFR-1.
  • DR3 -induced NF-kB activation was blocked by dominant negative derivatives of REP (RIP-DN) and TRAF2 (TRAF2-DN), which were previously shown to abrogate TNF-induced NF-kB activation (H. Hsu et al, Cell 84: 299- 398 (1996); H.
  • DR3 is a death domain-containing molecule capable of triggering both apoptosis and NF-kB activation, two pathways dominant in the regulation ofthe immune system.
  • the experiments also demonstrate the internal signal transduction machinery of this novel cell death receptor.
  • TRADD multivalent adapter molecule
  • Another method of gene therapy according to the present invention involves operably associating the endogenous DR3 sequence with a promoter via homologous recombination as described, for example, in US Patent Number
  • Polynucleotide constructs are made which contain a promoter and targeting sequences, which are homologous to the 5 ' non-coding sequence of endogenous
  • the targeting sequence will be sufficiently near the 5 ' end of DR3 so the promoter will be operably linked to the endogenous sequence upon homologous recombination.
  • the promoter and the targeting sequences can be amplified using PCR.
  • the amplified promoter contains distinct restriction enzyme sites on the 5 ' and 3 ' ends.
  • the 3 ' end ofthe first targeting sequence contains the same restriction enzyme site as the 5' end of the amplified promoter and the 5' end of the second targeting sequence contains the same restriction site as the 3 ' end of the amplified promoter.
  • the amplified promoter and the amplified targeting sequences are digested with the appropriate restriction enzymes and subsequently treated with calf intestinal phosphatase.
  • the digested promoter and digested targeting sequences are added together in the presence of T4 DNA ligase.
  • the resulting mixture is maintained under conditions appropriate for ligation ofthe two fragments.
  • the construct is size fractionated on an agarose gel then purified by phenol extraction and ethanol precipitation.
  • the polynucleotide constructs are administered as naked polynucleotides via electroporation. However, the polynucleotide constructs may also be administered with transfection-facilitating agents, such as liposomes, viral sequences, viral particles, precipitating agents, etc.
  • DR3-V1 or DR3 in the cell.
  • Expression may be detected by immunological staining, or any other method known in the art.
  • Fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in DMEM.+ 10%> fetal calf serum. Exponentially growing or early stationary phase fibroblasts are trypsinized and rinsed from the plastic surface with nutrient medium. An aliquot ofthe cell suspension is removed for counting, and the remaining cells are subjected to centrifugation. The supernatant is aspirated and the pellet is resuspended in 5 ml of electroporation buffer (20 mM
  • the cells are recentrifuged, the supernatant aspirated, and the cells resuspended in electroporation buffer containing 1 mg/ml acetylated bovine serum albumin.
  • the final cell suspension contains approximately 3X10 6 cells/ml. Electroporation should be performed immediately following resuspension.
  • Plasmid DNA is prepared according to standard techniques. For example, to construct a plasmid for targeting to the DR3 locus, plasmid pUC 18 (MBI Fermentas, Amherst, NY) is digested with Hindlll. The CMV promoter is amplified by PCR with an Xbal site on the 5 ' end and a BamHl site on the 3 'end.
  • Two DR3 non-coding sequences are amplified via PCR: one DR3 non-coding sequence (DR3 fragment 1) is amplified with a Hindlll site at the 5' end and an Xb ⁇ l site at the 3 'end; the other DR3 non-coding sequence (DR3 fragment 2) is amplified with a B ⁇ mHl site at the 5 'end and a Hindlll site at the 3 'end.
  • the CMV promoter and DR3 fragments are digested with the appropriate enzymes (CMV promoter - Xb ⁇ l and B ⁇ mHl; DR3 fragment 1 - Xb ⁇ l; DR3 fragment 2 -
  • Plasmid DNA is added to a sterile cuvette with a 0.4 cm electrode gap (Bio-Rad). The final DNA concentration is generally at least 120 ⁇ g/ml. 0.5 ml ofthe cell suspension (containing approximately 1.5X10 6 cells) is then added to the cuvette, and the cell suspension and DNA solutions are gently mixed.
  • Electroporation is performed with a Gene-Pulser apparatus (Bio-Rad). Capacitance and voltage are set at 960 ⁇ F and 250-300 V, respectively. As voltage increases, cell survival decreases, but the percentage of surviving cells that stably incorporate the introduced DNA into their genome increases dramatically. Given these parameters, a pulse time of approximately 14-20 mSec should be observed.
  • Electroporated cells are maintained at room temperature for approximately 5 minutes, and the contents ofthe cuvette are then gently removed with a sterile transfer pipette. The cells are added directly to 10 ml of prewarmed nutrient media (DMEM with 15% calf serum) in a 10 cm dish and incubated at
  • the engineered fibroblasts are then injected into the host, either alone or after having been grown to confluence on cytodex 3 microcarrier beads.
  • the fibroblasts now produce the protein product.
  • the fibroblasts can then be introduced into a patient as described above.
  • the antibodies of the present invention can be prepared by a variety of methods. (See, Ausubel et al, eds., 1998, Current Protocols in Molecular Biology, John Wiley & Sons, NY, Chapter 2.) As one example of such methods, cells expressing DR3-V1 or DR3 are administered to an animal to induce the production of sera containing polyclonal antibodies. In a preferred method, a preparation of DR3-V1 or DR3 protein is prepared and purified to render it substantially free of natural contaminants. Such a preparation is then introduced into an animal in order to produce polyclonal antisera of greater specific activity. Monoclonal antibodies specific for protein DR3-V1 or DR3 are prepared using hybridoma technology.
  • an animal preferably a mouse
  • DR3-V1 or DR3 polypeptide or, more preferably, with a secreted DR3-V1 or DR3 polypeptide-expressing cell.
  • Such polypeptide- expressing cells are cultured in any suitable tissue culture medium, preferably 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.
  • tissue culture medium preferably 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.
  • a 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 ATCC.
  • 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 80:225-232 (1981).
  • the hybridoma cells obtained through such a selection are then assayed to identify clones which secrete antibodies capable of binding the DR3-V1 or DR3 polypeptide.
  • additional antibodies capable of binding to DR3-V1 or DR3 polypeptide can be produced in a two-step procedure using anti-idiotypic antibodies.
  • a method makes use ofthe fact that antibodies are themselves antigens, and therefore, it is possible to obtain an antibody which binds to a second antibody.
  • 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 DR3-V1 or DR3 protein-specific antibody can be blocked by DR3-V1 or DR3.
  • Such antibodies comprise anti-idiotypic antibodies to the DR3-V1 or DR3 protein-specific antibody and are used to immunize an animal to induce formation of further DR3-V1 or DR3 protein-specific antibodies.
  • an antibody is "humanized". Such antibodies can be produced using genetic constructs derived from hybridoma cells producing the monoclonal antibodies described above. Methods for producing chimeric and humanized antibodies are known in the art and are discussed infra. (See, for review, Morrison, Science 229: 1202 (1985); Oi etal, BioTechniques 4:214 (1986); Cabilly et al, U.S. Patent No.
  • Naturally occurring V-genes isolated from human PBLs are constructed into a large library of antibody fragments which contain reactivities against polypeptides of the present invention to which the donor may or may not have been exposed (see, e.g., U.S. Patent 5,885,793 incorporated herein in its entirety by reference).
  • a library of scFvs is constructed from the RNA of human PBLs as described in WO92/01047. To rescue phage displaying antibody fragments, approximately 10 9 E. coli harboring the phagemid are used to inoculate 50 ml of 2xTY containing 1 %> glucose and 100 ⁇ g/ml of ampicillin (2xTY-AMP-GLU) and grown to an O.D. of 0.8 with shaking.
  • M13 delta gene 111 is prepared as follows: M13 delta gene III helper phage does not encode gene III protein, hence the phage(mid) displaying antibody fragments have a greater avidity of binding to antigen. Infectious Ml 3 delta gene III particles are made by growing the helper phage in cells harboring a pUC19 derivative supplying the wild type gene III protein during phage morphogenesis. The culture is incubated for 1 hour at 37°C without shaking and then for a further hour at 37°C with shaking.
  • Cells are pelleted (EEC-Centra 8, 4000 revs/min for 10 min), resuspended in 300 ml 2xTY broth containing 100 ⁇ g ampicillin/ml and 25 ⁇ g kanamycin/ml (2xTY-AMP-KAN) and grown overnight, shaking at 37°C.
  • Phage particles are purified and concentrated from the culture medium by two PEG-precipitations (Sambrook et al. , 1990), resuspended in 2 ml PB S and passed through a 0.45 ⁇ m filter (Minisart NML; Sartorius) to give a final concentration of approximately 10 13 transducing units/ml (ampicillin-resistant clones).
  • Immunotubes (Nunc) are coated overnight in PBS with 4 ml of either 100 mg/ml or 10 mg/ml of a polypeptide ofthe present invention. Tubes are blocked with 2% Marvel-PBS for 2 hours at 37°C and then washed 3 times in PBS. Approximately 10 13 TU of phage are applied to the tube and incubated for 30 minutes at room temperature tumbling on an over and under turntable and then left to stand for another 1.5 hours. Tubes are washed 10 times with PBS, 0.1%> Tween-20 and 10 times with PBS.
  • Phage are eluted by adding 1 ml of 100 mM triethylamine and rotating 15 minutes on an under and over turntable after which the solution is immediately neutralized with 0.5 ml of TOM Tris-HCl, pH 7.4. Phage are then used to infect 10 ml of mid-log E. coli TGI by incubating eluted phage with bacteria for 30 minutes at 37°C. The E. coli are then plated on TYE plates containing 1%> glucose and 100 ⁇ g/ml ampicillin.
  • the resulting bacterial library is then rescued with delta gene 3 helper phage as described above to prepare phage for a subsequent round of selection This process is then repeated for a total of 4 rounds of affinity purification with tube-washing increased to 20 times with PBS, 0.1% Tween-20 and 20 times with PBS for rounds 3 and 4.
  • Eluted phage from the 3rd and 4th rounds of selection are used to infect E. coli HB 2151 and soluble scFv is produced (Marks et al, J. Mol. Biol. 222:581-597 (1991)) from single colonies for assay.
  • ELISAs are performed with microtitre plates coated with either 10 pg/ml of the polypeptide of the present invention in 50 mM bicarbonate pH 9.6. Clones positive in ELISA are further characterized by PCR fingerprinting (see, e.g., WO92/01047) and then by sequencing.
  • RNA is isolated from entire families or individual patients presenting with a phenotype of interest (such as a disease). cDNA is then generated from these RNA samples using protocols known in the art. (See, Sambrook et al, 1990) The cDNA is then used as a template for PCR, employing primers surrounding regions of interest in SEQ ID NOT . Suggested PCR conditions consist of 35 cycles at 95 °C for 30 seconds, 60-120 seconds at 52-58° C; and 60-120 seconds at 70°C, using buffer solutions described in Sidransky, D., etal, Science 252:106 (1991).
  • PCR products are then sequenced using primers labeled at their 5' end with T4 polynucleotide kinase, employing SequiTherm Polymerase. (Epicentre Technologies). The intron-exon borders of selected exons of DR3 are also determined and genomic PCR products analyzed to confirm the results. PCR products harboring suspected mutations in DR3 are then cloned and sequenced to validate the results ofthe direct sequencing.
  • PCR products of DR3 are cloned into T-tailed vectors as described in Holton, T.A. and Graham, M.W., Nucleic Acids Research, 19: 1 156 (1991) and sequenced with T7 polymerase (United States Biochemical). Affected individuals are identified by mutations in DR3 not present in unaffected individuals.
  • Genomic rearrangements are also observed as a method of determining alterations in the DR3 gene.
  • Genomic clones isolated using techniques known in the art are nick-translated with digoxigenindeoxy-uridine 5'-triphosphate
  • Hybridization with the labeled probe is carried out using a vast excess of human cot-1 DNA for specific hybridization to the DR3 genomic locus. Chromosomes are counterstained with 4,6-diamino-2-phenylidole and propidium iodide, producing a combination of C- and R-bands. Aligned images for precise mapping are obtained using a triple-band filter set (Chroma Technology, Brattleboro, VT) in combination with a cooled charge-coupled device camera (Photometries, Arlington, AZ) and variable excitation wavelength filters. (Johnson, C.
  • DR3 polypeptides can be detected in a biological sample, and if an increased or decreased level of DR3 is detected, this polypeptide is a marker for a particular phenotype. Methods of detection are numerous, and thus, it is understood that one skilled in the art can modify the following assay to fit their particular needs.
  • antibody-sandwich ELISAs are used to detect DR3 in a sample, preferably a biological sample.
  • Wells of a microtiter plate are coated with specific antibodies to DR3, at a final concentration of 0.2 to 10 ⁇ g/ml.
  • the antibodies are either monoclonal or polyclonal and are produced using technique known in the art.
  • the wells are blocked so that non-specific binding of DR3 to the well is reduced.
  • the coated wells are then incubated for > 2 hours at RT with a sample containing DR3.
  • serial dilutions of the sample should be used to validate results.
  • the plates are then washed three times with deionized or distilled water to remove unbounded DR3.
  • the present invention relates to a method for treating an individual in need of a decreased level of DR3 biological activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of DR3 antagonist.
  • Preferred antagonists for use in the present invention are DR3 -specific antibodies.
  • conditions caused by a decrease in the standard or normal expression level of DR3 in an individual can be treated by administering DR3, preferably in a soluble and/or secreted form.
  • the invention also provides a method of treatment of an individual in need of an increased level of DR3 polypeptide comprising administering to such an individual a pharmaceutical composition comprising an amount of DR3 to increase the biological activity level of DR3 in such an individual.
  • a patient with decreased levels of DR3 polypeptide receives a daily dose 0.1-100 ⁇ g/kg of the polypeptide for six consecutive days.
  • the polypeptide is in a soluble and/or secreted form.
  • the present invention also relates to a method for treating an individual in need of an increased level of DR3 biological activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of DR3 or an agonist thereof.
  • Antisense technology is used to inhibit production of DR3.
  • This technology is one example of a method of decreasing levels of DR3 polypeptide, preferably a soluble and/or secreted form, due to a variety of etiologies, such as cancer.
  • a patient diagnosed with abnormally increased levels of DR3 is administered intravenously antisense polynucleotides at 0.5, 1.0, 1.5, 2.0 and 3.0 mg/kg day for 21 days. This treatment is repeated after a 7-day rest period if the is determined to be well tolerated.
  • fibroblasts which are capable of expressing soluble and/or mature DR3 polypeptides, onto a patient.
  • fibroblasts are obtained from a subject by skin biopsy. The resulting tissue is placed in tissue-culture medium and separated into small pieces. Small chunks ofthe tissue are placed on a wet surface of a tissue culture flask, approximately ten pieces are placed in each flask. The flask is turned upside down, closed tight and left at room temperature over night. After 24 hours at room temperature, the flask is inverted and the chunks of tissue remain fixed to the bottom ofthe flask and fresh media (e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin) is added. The flasks are then incubated at 37°C for approximately one week.
  • fresh media e.g., Ham's F12 media, with 10% FBS, penicillin and streptomycin
  • pMV-7 (Kirschmeier, P.T. et al, DNA, 7:219-25 (1988)), flanked by the long terminal repeats of the Moloney murine sarcoma virus, is digested with EcoRI and Hindlll and subsequently treated with calf intestinal phosphatase.
  • the linear vector is fractionated on agarose gel and purified, using glass beads.
  • the cDNA encoding DR3 can be amplified using PCR primers which correspond to the 5' and 3' end encoding sequences respectively.
  • the 5' primer contains an EcoRI site and the 3' primer includes a Hindlll site.
  • Equal quantities ofthe Moloney murine sarcoma virus linear backbone and the amplified EcoRI and Hindlll fragment are added together, in the presence of T4 DNA ligase.
  • the resulting mixture is maintained under conditions appropriate for ligation ofthe two fragments.
  • the ligation mixture is then used to transform E. coli HBIOI, which are then plated onto agar containing kanamycin for the purpose of confirming that the vector contains properly inserted DR3.
  • the amphotropic p A317 or GP+am 12 packaging cells are grown in tissue culture to confluent density in Dulbecco's Modified Eagles Medium (DMEM) with 10% calf serum (CS), penicillin and streptomycin.
  • DMEM Dulbecco's Modified Eagles Medium
  • CS calf serum
  • the packaging cells now produce infectious viral particles containing the DR3 gene (the packaging cells are now referred to as producer cells).
  • Fresh media is added to the transduced producer cells, and subsequently, the media is harvested from a 10 cm plate of confluent producer cells.
  • the spent media, containing the infectious viral particles is filtered through a Millipore filter to remove detached producer cells and this media is then used to infect fibroblast cells.
  • fibroblasts Media is removed from a sub-confluent plate of fibroblasts and quickly replaced with the media from the producer cells. This media is removed and replaced with fresh media. If the titer of virus is high, then virtually all fibroblasts will be infected and no selection is required. If the titer is very low, then it is necessary to use a retroviral vector that has a selectable marker, such as neo or his. Once the fibroblasts have been efficiently infected, the fibroblasts are analyzed to determine whether DR3 protein is produced.

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WO2003013581A1 (en) * 2001-08-03 2003-02-20 Genset S.A. Agonists and antagonists of genceptin for the treatment of metabolic disorders
EP1361433A2 (de) * 2002-04-09 2003-11-12 Kabushiki Kaisha Hayashibara Seibutsu Kagaku Kenkyujo Verfahren zur erkennung die therapeutisches Wirkung TNF
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EP1667730A2 (de) * 2003-08-20 2006-06-14 University of Miami Zusammensetzungen und verfahren zur behandlung von entzündlicher lungenerkrankung
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US9017679B2 (en) 2005-08-30 2015-04-28 University Of Miami Immunomodulating tumor necrosis factor receptor 25 (TNFR25) agonists, antagonists, and immunotoxins
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US9603925B2 (en) 2013-01-09 2017-03-28 University Of Miami Compositions comprising TL1A-Ig fusion protein for the regulation of T regulatory cells, and methods for their use

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EP1178815A4 (de) 2003-02-05

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